Microbial consortia for treating vaginal conditions

Abstract

Provided are compositions of microbial consortia comprising a defined population of isolated lactobacilli configured to promote engraftment in a female genitourinary tract of a subject. Further provided herein are methods of administering the microbial consortia to the genitourinary tract of the subject.

Description

MICROBIAL CONSORTIA FOR TREATING VAGINAL CONDITIONSRELATED APPLICATIONSThis application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 599,555, filed November 15, 2023, entitled “Vaginal Bacterial Compositions”, the contents of which are incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTINGThe contents of the electronic sequence listing (F089570001WO00-SEQ- GJM.xml; Size: 42,018 bytes; and Date of Creation: November 15, 2024) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0001] The vagina is a fibromuscular tubular tract leading from the uterus to the exterior of the body in females. A healthy vagina is colonized by a mutually symbiotic flora of microorganisms, in particular of the genus Lactobacillus, that protects its host from vaginal infections. The acidity of a healthy vagina of a woman of child-bearing age (pH about 3.5- 4.5) is thought to be due to the degradation of secreted glycogen / glucose to lactic acid by lactobacilli. Acidic conditions are unfavorable for the growth of many pathogenic microorganisms and pathobionts, including bacteria, protozoa, and viruses. However, an imbalance in the vaginal microbiota may result in overgrowth of pathogenic microorganisms and pathobionts, resulting in dysbiosis, inflammation, and / or infections.

[0002] Lactobacillus -containing products (comprising, e.g., Lactobacillus acidophilus, Lactobacillus rhamnosus, or Lactobacillus gasseri) for intravaginal or oral use have been available for many years. These products include vaginal suppositories containing lyophilized Lactobacillus acidophilus or other Lactobacillus species (e.g., Lactobacillus rhamnosus, or Lactobacillus gasseri) of human origin as well as various nutritional supplements.

[0003] A need exists for the identification of specific bacteria and bacterial consortia capable of colonizing the human female genitourinary tract and a better understanding of thein vivo environment of the genitourinary tract and overall health profile of subjects receiving such isolates.SUMMARY OF THE INVENTION

[0004] Provided herein are compositions of microbial consortia comprising two or more bacterial isolates capable of engraftment of the human female genitourinary tract, e.g., of a female subject that exhibits, e.g., a dysbiosis of the genitourinary tract.

[0005] A microbial consortium of the present disclosure is designed, in some embodiments, to include specific Lactobacillus isolates (e.g., Lactobacillus crispatus and / or Lactobacillus jensenii isolates) having a diverse set of functional and genomic characteristics. For example, a microbial consortium of the disclosure may include two or more Lactobacillus isolates, wherein each isolate has different functional characteristics (e.g., different growth rates, different acidification rates, different carbohydrate utilization profiles, different rates of engraftment). Additionally, the two or more Lactobacillus isolates may further comprise different genomic characteristics. This difference in genomic characteristics can allow for the microbial consortium comprising the two or more Lactobacillus isolates to encompass a preselected portion of a species-level pangenome (e.g., all of the genes of a species-level pangenome that are linked to engraftment) even though any single Lactobacillus isolate might not encompass the preselected portion by itself.

[0006] A microbial consortium having a diverse set of functional and genomic characteristics as described herein can engraft in the female genitourinary tract of a diverse set of subjects, each of whom may have different vaginal environments (e.g., different resident microbial communities, e.g., different resident dysbiotic microbial communities). Subjects differ with respect to age, racial and national background, hormonal status (e.g., different stages of menstrual cycle, menopausal), presence of comorbidities, lifestyle, diet and geographic factors, among others. Each of these differences between subjects can affect the resident microbial community (e.g., including the composition of dysbiotic microbial communities and bacterial species) and the local environment (e.g., pH, levels of H2O2, levels of glycogen, levels of estrogen, etc.) in the genitourinary tract. The inventors of the present disclosure have identified that a microbial consortium having a diverse set of functional and genomic characteristics as described herein is able to be highly adaptable to such differentenvironments present in a diverse set of subjects and to successfully engraft within the genitourinary tract of these diverse subjects. In some embodiments, the microbial consortia of the present disclosure can successfully engraft without the use of antibiotics such as metronidazole (e.g., as a pre-treatment step prior to administration of the microbial consortia).

[0001] Some aspects of the present disclosure provide a microbial consortium comprising a defined population of isolated lactobacilli, the defined population comprising:(a) a Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770;(b) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772;(c) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771, and(d) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

[0002] Some aspects of the disclosure provide a microbial consortium comprising a defined population of isolated lactobacilli, the defined population comprising:(a) a Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value;(b) a Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value;(c) a Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value; and(d) a Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value.

[0003] Some aspects of the disclosure provide a method of increasing the relative abundance of lactobacilli in the female genitourinary tract of a subject comprisingadministering any one of the microbial consortia described herein to the genitourinary tract of the subject.

[0004] Some aspects of the disclosure provide a composition comprising Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770.

[0005] Some aspects of the disclosure provide a composition comprising Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772.

[0006] Some aspects of the disclosure provide a composition comprising Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771.

[0007] Some aspects of the disclosure provide a composition comprising Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

[0008] Some aspects of the disclosure provide a composition comprising two or more Lactobacillus bacteria selected from the group consisting of:(a) a Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770;(b) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772;(c) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771, and(d) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

[0009] Some aspects of the disclosure provide a composition comprising Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value.

[0010] Some aspects of the disclosure provide a composition comprising Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value.

[0011] Some aspects of the disclosure provide a composition comprising Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value.

[0012] Some aspects of the disclosure provide a composition comprising Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value.

[0013] Some aspects of the disclosure provide a composition comprising two or more Lactobacillus bacteria selected from the group consisting of:(a) Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value;(b) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value;(c) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value; and(d) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value.

[0014] In one embodiment, the disclosure provides a microbial consortium, comprising (i) a Lactobacillus jensenii isolate and (ii) at least two phenotypically distinct Lactobacillus crispatus isolates.

[0015] In another embodiment the disclosure provides a microbial consortium, comprising (i) a Lactobacillus jensenii isolate and (ii) at least three phenotypically distinct Lactobacillus crispatus isolates.

[0016] In yet another embodiment, the disclosure provides the disclosure provides a microbial consortium, comprising (i) a Lactobacillus jensenii isolate and (ii) at least two phenotypically distinct Lactobacillus crispatus isolates, wherein the at least two phenotypically distinct Lactobacillus crispatus isolates are each characterized by a different carbon substrate utilization profile. In various embodiments, the at least two phenotypicallydistinct Lactobacillus crispatus isolates is characterized as utilizing both mannitol and glucose. In certain embodiments, a first phenotypic ally distinct Lactobacillus crispatus isolate is characterized as utilizing mannitol to a lesser degree than a second phenotypically distinct Lactobacillus crispatus isolate.

[0017] The disclosure provides in another embodiment a microbial consortium, comprising (i) a Lactobacillus jensenii isolate and (ii) at least three phenotypically distinct Lactobacillus crispatus isolates, wherein the at least two phenotypically distinct Lactobacillus crispatus isolates are each characterized by a different carbon substrate utilization profile. In various embodiments, the at least three phenotypically distinct Lactobacillus crispatus isolates is characterized as utilizing both mannitol and glucose. In certain embodiments, a first phenotypically distinct Lactobacillus crispatus isolate is characterized as utilizing mannitol to a lesser degree than a second phenotypically distinct Lactobacillus crispatus isolate.

[0018] In other embodiments, the disclosure provides a microbial consortium comprising (i) a Lactobacillus jensenii isolate and (ii) at least two phenotypically distinct Lactobacillus crispatus isolates, wherein the at least two phenotypically distinct Lactobacillus crispatus isolates have different growth and / or acidification rates during logarithmic growth phase on media comprising mannitol or glucose as carbon substrates. In certain embodiments, the growth rate is determined according to the steps of Table 7. In other embodiments, the acidification rate is determined according to the steps of Table 8.

[0019] In various embodiments, each isolate of a microbial consortium has engraftment potential in recipient subjects. The engraftment potential can be assessed according to the various methods, including a method of detecting engraftment potential in a recipient subject of a microbial consortium, said method comprising detecting the presence of at least one bacterial isolate in the recipient at least 24 hours after administration of the microbial consortium to the recipient, e.g., to the vaginal lumen. In various embodiments for measuring engraftment, the microbial consortium comprises (i) a Lactobacillus jensenii isolate and (ii) at least two phenotypically distinct Lactobacillus crispatus isolates. In other embodiments, the microbial consortium comprises (i) a Lactobacillus jensenii isolate and (ii) at least three phenotypically distinct Lactobacillus crispatus isolates. In certain embodiments, measuring engraftment can include detecting at least two or three bacterial isolates after at least 24 hours after administration of the microbial consortium to the recipient. The detecting step in other embodiments can also be after at least 48 hours, 72hours, 7 day, 4 weeks, 8 weeks, and 16 weeks after administration of the microbial consortium to the recipient.

[0020] In various embodiments, the detecting step may comprise nucleic acid sequencing of the nucleic acid molecule, a genome or a genome region of at least one bacterial isolate. The sequencing can be whole genome sequencing.

[0021] When detecting engraftment, in certain embodiment, the detection may comprise measuring a relative abundance of the least one isolate of at least 1%, 5%, 10%, 15%, or 20% of a sample taken from the vaginal lumen after administration of the microbial consortium.

[0022] In various embodiments, the relative abundance of each isolate in the microbial consortium is ratiometrically equal.

[0023] In other embodiments, the percentage relative abundance of Lactobacillus in the consortium that are Lactobacillus jensenii is in the range of 15%-60% and the remaining balance of Lactobacillus are Lactobacillus crispatus. In still other embodiments, the percentage of Lactobacillus in the consortium that are Lactobacillus jensenii is in the range of 25% to 40% and the remaining balance of Lactobacillus are Lactobacillus crispatus. In other embodiments, the percentage of Lactobacillus in the consortium that are Lactobacillus jensenii is in the range of 20% to 30% and the remaining balance of Lactobacillus are Lactobacillus crispatus.

[0024] In various embodiments, the Lactobacillus isolates originated from vaginal secretions obtained from at least 2 female donors, or from at least 3 female donors. In various embodiments, the vaginal secretions comprise microbes from a eubiotic vaginal microbiome.

[0025] In other embodiments of the microbial consortia, the Lactobacillus jensenii isolate originates from a eubiotic vaginal microbiome comprising Lactobacillus jensenii and Lactobacillus crispatus, wherein a first of the Lactobacillus crispatus isolates originates from a eubiotic vaginal microbiome comprising Lactobacillus jensenii and Lactobacillus crispatus, and wherein a second of the Lactobacillus crispatus isolates originates from a eubiotic vaginal microbiome comprising Lactobacillus crispatus and lacking detectable Lactobacillus jensenii.

[0026] In embodiments, at least two of the at least three phenotypically distinct Lactobacillus crispatus isolates have different growth rates and / or acidification rates on one or more carbon substrates during logarithmic growth phase. In various embodiments thegrowth rate is determined according to the steps of Table 7 and the acidification rate is determined according to the steps of Table 8.

[0027] In various embodiments, at least two of the lactobacilli of the microbial consortia have different carbon utilization profiles. The carbon utilization profile can be selected from the group consisting of: a glycogen utilization profile; a glucose utilization profile; a fructose utilization profile; a mannose utilization profile; a maltodextrin utilization profile; and a mannitol utilization profile. In certain embodiments, the carbon utilization profile is a glycogen utilization profile. In certain other embodiments, the carbon utilization profile is a mannitol utilization profile.

[0028] In other embodiments, at least two of the lactobacilli of the microbial consortia have different growth rates, acidification rates, lactic acid production rates, and / or carbon utilization profiles. The carbon utilization profile can be selected from the group consisting of: a glycogen utilization profile; a glucose utilization profile; a fructose utilization profile; a mannose utilization profile; a maltodextrin utilization profile; and a mannitol utilization profile. In certain embodiments, the carbon utilization profile is a glycogen utilization profile. In certain other embodiments, the carbon utilization profile is a mannitol utilization profile. In certain embodiments, the microbial consortium has a growth rate that improves upon the lowest performing individual isolate of the consortium when grown in modified MRS medium, modified MRS + maltodextrin, modified MRS + fructose, modified MRS + mannose, or modified MRS + mannitol. In other embodiments, the microbial consortium has an acidification rate that improves upon the lowest performing individual isolate of the microbial consortium when grown in modified MRS medium, modified MRS + maltodextrin, or modified MRS + glycogen.

[0029] In various embodiments, the total bacterial content in the microbial consortium is in the range of 106to 1011CFUs. In other embodiments, the total bacterial content in the consortium is in the range of 107to 109CFUs. In still other embodiments, the total content of at least one lactobacillus isolate present in the microbial consortium is in the range of 2.0 X106-3.0 X 106to 2.0 X 1011- 3.0 X 1011CFUs. In yet other embodiments, the total content of at least one lactobacillus isolate present in the consortium is in the range of 2.0 X 107-3.0 X107to 2.0 X 109-3.0 X 109CFUs. The at least one lactobacillus isolate can be Lactobacillus jensenii.

[0030] In certain embodiments, the consortium has a growth rate greater than a two- member consortium comprising the Lactobacillus jensenii isolate and any one of the at leasttwo Lactobacillus crispatus isolates. The consortium also may have an acidification rate greater than a two-member consortium comprising the Lactobacillus jensenii isolate and any one of the at least two Lactobacillus crispatus isolates.

[0031] In other aspects, the disclosure provides a composition comprising a microbial consortium described herein, and a pharmaceutically acceptable buffer, diluent, or excipient. The composition may be in a dosage form, e.g., a suppository, a tablet, a capsule, a film, a gel, or a cream. In one embodiment, the dosage form is a capsule.

[0032] The composition in various embodiments may comprise a total bacterial content in the microbial consortium in the range of 106to 1011CFUs. In other embodiments, the total bacterial content in the consortium is in the range of 107to 109CFUs. In still other embodiments, the total content of at least one lactobacillus isolate present in the microbial consortium is in the range of 2.0 X 106-3.0 X 106to 2.0 X 1011- 3.0 X 1011CFUs. In yet other embodiments, the total content of at least one lactobacillus isolate present in the consortium is in the range of 2.0 X 107-3.0 X 107to 2.0 X 109-3.0 X 109CFUs. The at least one lactobacillus isolate can be Lactobacillus jensenii.

[0033] In another aspect, the disclosure relates to a kit comprising a microbial consortium of described herein or a composition comprising a microbial consortium described herein, and further comprising a device or applicator suitable for delivery of the microbial consortium or the composition to the vaginal lumen, and optionally instructions for use. In embodiments, kit may include an antiseptic wash and / or cleanse solution, which can be included in a device in some embodiments, such as, but not limited to a syringe or a presoaked gauze. In embodiments, the antiseptic wash and / or cleanse solution can comprise chlorhexidine.

[0034] In another aspect, the disclosure provides a method comprising administering the microbial consortium to the urogenital tract of a subject, e.g., a subject having a dysbiotic vaginal microbiome. The microbial consortium or composition can be administered as a capsule. In embodiments, the method results in populating the urogenital tract of the subject with lactobacilli and / or increasing the relative abundance of lactobacilli in the female urogenital tract of the subject, each of which may be assessed by detecting the presence of at least one bacterial isolate of the microbial consortium in the recipient at least 24 hours after administration of said microbial consortium to the recipient. In other embodiments, the detecting step can be performed after at least 48 hours, 72 hours, 7 day, 4 weeks, 8 weeks, and 16 weeks after administration of the microbial consortium to the recipient. In otherembodiments, the detecting step can comprise nucleic acid sequencing of a nucleic acid molecule, a genome or a genome region of at least one bacterial isolate. The sequencing may be whole genome sequencing. In some embodiments, the subject does not exhibit vaginal clinical symptoms, such as vaginal discharge, vaginal itching, vaginal irritation, and / or vaginal malodor. In other embodiments, the subject does not exhibit bacterial vaginosis (BV) clinical symptoms as diagnosed by Amsel criteria or Nugent score.

[0035] In another aspect, the disclosure provides a method comprising administering the microbial consortium to the urogenital tract of a subject, e.g., a subject having a dysbiotic vaginal microbiome, which is converted to a eubiotic vaginal microbiome after administering the microbial consortium. In embodiments, the eubiotic vaginal microbiome is stable for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least up to 1 year after administering the microbial consortium. In still other embodiments the eubiotic vaginal microbiome is stable for at least 1 menstrual cycle, at least 2 menstrual cycles, at least 3 menstrual cycles, at least 4 menstrual cycles, at least 5 menstrual cycles, at least 6 menstrual cycles, and at least 7 menstrual cycles, at least 8 menstrual cycles, at least 9 menstrual cycles, at least 10 menstrual cycles, at least 11 menstrual cycles, and at least 12 menstrual cycles, after administering the microbial consortium.

[0036] In other aspects, the disclosure provides a method of preparing a microbial consortium, the method comprising: (i) obtaining a Lactobacillus jensenii isolate; (ii) obtaining at least two phenotypically distinct Lactobacillus crispatus isolates; and (iii) combining the Lactobacillus jensenii isolate and the at least two phenotypically distinct Lactobacillus crispatus isolates in a mixture to form the microbial consortium.

[0037] In some embodiments, the method of preparing a microbial consortium comprises (i) obtaining a Lactobacillus jensenii isolate; (ii) obtaining at least three phenotypically distinct Lactobacillus crispatus isolates; and (iii) combining the Lactobacillus jensenii isolate and the at least three phenotypically distinct Lactobacillus crispatus isolates in a mixture to form the microbial consortium. In some embodiments, the microbial consortia formed is a 3 -isolate microbial consortium comprising a Lactobacillus jensenii isolate and two phenotypically distinct Lactobacillus crispatus isolates. In other embodiments, the microbial consortia formed is a 4-isolate microbial consortium comprising a Lactobacillus jensenii isolate and three phenotypically distinct Lactobacillus crispatus isolates.

[0038] In embodiments, the Lactobacillus jensenii isolate and each of the Lactobacillus crispatus isolates of the prepared consortia are ratiometrically equally represented in the mixture. The consortia in various embodiments may comprise a total bacterial content in the range of 106to 1011CFUs. In other embodiments, the total bacterial content in the consortium is in the range of 107to 109CFUs. In still other embodiments, the total content of at least one lactobacillus isolate present in the microbial consortium is in the range of 2.0 X106-3.0 X 106to 2.0 X 1011- 3.0 X 1011CFUs. In yet other embodiments, the total content of at least one lactobacillus isolate present in the consortium is in the range of 2.0 X 107-3.0 X107to 2.0 X 109-3.0 X 109CFUs. The at least one lactobacillus isolate can be Lactobacillus jensenii.

[0039] In various embodiments, the Lactobacillus jensenii isolate is derived from a first eubiotic vaginal donor as a Lactobacillus jensenii isolate that engrafts in a recipient subject following a vaginal microbiome transfer of an SCVMP; and wherein at least one Lactobacillus crispatus isolate is derived from a second eubiotic vaginal donor as a Lactobacillus crispatus isolate that engrafts in a recipient subject following a vaginal microbiome transfer of an SCVMP.

[0040] In certain embodiments, the Lactobacillus jensenii isolate comprises one or more carbohydrate utilization genes comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-10, or a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or up to 100% of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-10.

[0041] In other embodiment, a first of the Lactobacillus crispatus isolates comprises one or more carbohydrate utilization genes comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11-14, or a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or up to 100% of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11-14.

[0042] In another embodiment, a second of the Lactobacillus crispatus isolates comprises one or more carbohydrate utilization genes comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-18, or a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or up to 100% of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-18.

[0043] In still another embodiment, a third of the Lactobacillus crispatus isolates comprises one or more carbohydrate utilization genes comprising a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 19-22, or a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or up to 100% of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 19-22.

[0044] In another aspect, the disclosure provides a method of treating a subject having vaginal dysbiosis, comprising administering a therapeutically effective amount of the microbial consortium described herein, a composition described herein, or kit described herein, thereby treating the vaginal dysbiosis. In embodiments, the microbial consortium is a 3-isolate microbial consortium comprising a Lactobacillus jensenii isolate and two phenotypically distinct Lactobacillus crispatus isolates. In other embodiments, the microbial consortium is a 4-isolate microbial consortium a Lactobacillus jensenii isolate and three phenotypically disctinct Lactobacillus crispatus isolates.

[0045] In still another aspect, the disclosure provides a method of treating a subject having bacterial vaginosis (BV), comprising administering a therapeutically effective amount of the consortium described herein, a composition described herein, or kit described herein, thereby treating the bacterial vaginosis (BV). In embodiments, the microbial consortium is a 3-isolate microbial consortium comprising a Lactobacillus jensenii isolate and two phenotypically distinct Lactobacillus crispatus isolates. In other embodiments, the microbial consortium is a 4-isolate microbial consortium a Lactobacillus jensenii isolate and three phenotypically disctinct Lactobacillus crispatus isolates.

[0046] In another aspect, the disclosure provides a method for manufacturing a microbial consortium described herein comprising independently culturing each isolate of the microbial consortium in a bioreactor, harvesting the isolates, and combining the isolates in ratiometrically equal amounts to result in the microbial consortium. The bioreactor can be a 10L, 50L, 500L, 1000L, or up to 3000L bioreactor. The culturing step can result in at least 1g, 25g, 50g, 500g, 1kg, 10kg, or 50kg of lyophilized / dry weight of each isolate.

[0047] In some embodiments, the present disclosure provides a microbial consortium comprising a defined population of isolated lactobacilli configured to promote engraftment in a female genitourinary tract of a subject, the defined population comprising: a Lactobacillus jensenii isolate; a Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jenseniv, and at least one additional Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii.

[0048] In some embodiments, the Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii is capable of engrafting the female genitourinary tract of the subject for at least a pre-determined period of time.

[0049] In some embodiments, the defined population of isolated lactobacilli comprises: a relative abundance of 15% to 35% of the total viable cells are Lactobacillus jensenii bacteria; a relative abundance of 15% to 35% of the total viable cells are Lactobacillus crispatus bacteria that thrive in the presence of Lactobacillus jenseniv, and a relative abundance of 30% to 70% of the total viable cells are additional Lactobacillus crispatus bacteria that thrive independent of the presence of Lactobacillus jensenii.

[0050] In some embodiments, the defined population of isolated lactobacilli comprises: a relative abundance of about 25% of the total viable cells are Lactobacillus jensenii bacteria; a relative abundance of about 25% of the total viable cells are Lactobacillus crispatus bacteria that thrive in the presence of Lactobacillus jenseniv, and a relative abundance of about 50% of the total viable cells are additional Lactobacillus crispatus bacteria that thrive independent of the presence of Lactobacillus jensenii.

[0051] In some embodiments, wherein: the Lactobacillus jensenii isolate comprises a plurality of Lactobacillus jensenii bacteria; the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii comprises a plurality of Lactobacillus crispatus bacteria that thrives in the presence of Lactobacillus jenseniv, and the at least one additional Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii comprises a plurality of additional Lactobacillus crispatus bacteria that thrives independent of the presence of Lactobacillus jensenii.

[0052] Some aspects of the present disclosure provide a microbial consortium comprising a defined population of isolated Lactobacilli configured to promote engraftment in a female genitourinary tract of a subject, the defined population comprising: a Lactobacillus jensenii isolate; a Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jenseniv, and at least two additional Lactobacillus crispatus isolate, wherein the at least two additional Lactobacillus crispatus isolates have different growth rates, and wherein the at least two additional Lactobacillus crispatus isolates thrive independent of the presence of Lactobacillus jensenii.

[0053] In some embodiments, wherein a first of the at least two additional Lactobacillus crispatus isolates has a growth rate of 0.27-0.29 hours- 1 and a second of the at least twoadditional Lactobacillus crispatus isolates has a growth rate of 0.24-0.26 hours- 1 over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time); or a first of the at least two additional Lactobacillus crispatus isolates has a growth rate of 0.35-0.37 hours- 1 and a second of the at least two additional Lactobacillus crispatus isolates has a growth rate of 0.31-0.33 hours-1 over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0054] In some embodiments, at least two of the Lactobacillus crispatus isolates differ in their growth rates, glycogen utilization rate, rate of producing lactic acid, acidification rate, and / or time to engraftment in the female genitourinary tract.

[0055] In some embodiments, the defined population of isolated lactobacilli comprises: a relative abundance of 15% to 35% of the total viable cells are Lactobacillus jensenii bacteria; a relative abundance of 15% to 35% of the total viable cells are Lactobacillus crispatus bacteria that thrive in the presence of Lactobacillus jensenii', and a relative abundance of 15% to 35% of the total viable cells are additional Lactobacillus crispatus bacteria.

[0056] In some embodiments, the defined population of isolated lactobacilli comprises: a relative abundance of about 25% of the total viable cells are Lactobacillus jensenii bacteria; a relative abundance of about 25% of the total viable cells are Lactobacillus crispatus bacteria that thrive in the presence of Lactobacillus jenseniv, and a relative abundance of about 50% of the total viable cells are additional Lactobacillus crispatus bacteria.

[0057] In some embodiments, the Lactobacillus jensenii isolate comprises a plurality of Lactobacillus jensenii bacteria; the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii comprises a plurality of Lactobacillus crispatus bacteria that thrive in the presence of Lactobacillus jensenii', and each of the at least two additional Lactobacillus crispatus isolates comprises a plurality of Lactobacillus crispatus bacteria.

[0058] Some aspects of the present disclosure provide a microbial consortium comprising a defined population of isolated lactobacilli configured to promote engraftment in a female genitourinary tract of a subject, the defined population comprising:

[0059] a Lactobacillus jensenii isolate; a Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii', and at least two additional Lactobacillus crispatus isolates, wherein the at least two additional Lactobacillus crispatus isolates have different acidification rates, and wherein the at least two additional Lactobacillus crispatus isolates thrive independent of the presence of Lactobacillus jensenii.

[0060] In some embodiments, a first of the at least two additional Lactobacillus crispatus isolates has an acidification rate of about -0.006 hours- 1 and a second of the at least two additional Lactobacillus crispatus isolates has an acidification rate of about -0.007 hours- 1 over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time); or a first of the at least two additional Lactobacillus crispatus isolates has an acidification rate of -0.008 to -0.006 hours- 1 and a second of the at least two additional Lactobacillus crispatus isolates has an acidification rate of -0.012 to -0.010 hours- 1 over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0061] In some embodiments, at least two of the Lactobacillus crispatus isolates differ in their growth rates, capability of utilizing glycogen, rate of producing lactic acid, and / or time to engraftment in the female genitourinary tract.

[0062] In some embodiments, the defined population of isolated lactobacilli comprises: a relative abundance of 15% to 35% of the total viable cells are Lactobacillus jensenii bacteria; a relative abundance of 15% to 35% of the total viable cells are Lactobacillus crispatus bacteria that thrives in the presence of Lactobacillus jensenii', a relative abundance of 30% to 70% of the total viable cells are the at least two additional Lactobacillus crispatus bacteria.

[0063] In some embodiments, the defined population of isolated lactobacilli comprises:a relative abundance of about 25% of the total viable cells are Lactobacillus jensenii bacteria; a relative abundance of about 25% of the total viable cells are Lactobacillus crispatus bacteria that thrives in the presence of Lactobacillus jensenii', and a relative abundance of about 50% of the total viable cells are Lactobacillus crispatus bacteria.

[0064] In some embodiments, the Lactobacillus jensenii isolate comprises a plurality of Lactobacillus jensenii bacteria; the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii comprises a plurality of Lactobacillus crispatus bacteria that thrive in the presence of Lactobacillus jensenii', and each of the at least two additional Lactobacillus crispatus isolates comprises a plurality of Lactobacillus crispatus bacteria.

[0065] In some embodiments, the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii is capable of engrafting the female genitourinary tract of the subject for at least a pre-determined period of time in the presence of Lactobacillus jensenii.

[0066] In some embodiments, each of the lactobacilli are characterized by a genome having a distinct set of single nucleotide polymorphisms (SNPs), a distinct haplotype signature and / or a distinct set of single nucleotide variants (SNV).

[0067] In some embodiments, the distinct set of SNPs define a SNP profile.

[0068] In some embodiments, each Lactobacillus comprises a core genome, wherein the distinct set of SNVs is contained within the core genome.

[0069] In some embodiments, the genome of a Lactobacilli comprises a number of SNVs such that the core genome of the Lactobacilli, when aligned with a core genome of a reference Lactobacilli, has a percent identity greater than a threshold value, the threshold value defining a distinction between two different Lactobacilli isolates.

[0070] In some embodiments, the genome of each Lactobacillus crispatus isolate comprises a number of SNVs such that the genome of the Lactobacillus crispatus isolate, when aligned with a genome of a reference Lactobacillus crispatus isolate, has a percent identity greater than or equal to a threshold value, the threshold value defining a distinction between two different Lactobacillus crispatus isolates.

[0071] In some embodiments, the percent identity is greater than or equal to 99.81%.

[0072] In some embodiments, the percent identity is greater than or equal to 99.90%.

[0073] In some embodiments, the percent identity is greater than or equal to 99.96%.

[0074] In some embodiments, at least two of the lactobacilli differ in their growth rates.

[0075] In some embodiments, the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their growth rates.

[0076] In some embodiments, at least two Lactobacillus crispatus differ in their growth rates.

[0077] In some embodiments, the growth rates are growth rates in vitro (e.g., in culture) and / or in vivo.

[0078] In some embodiments, the growth rate of the Lactobacillus jensenii is 0.32-0.36 hours- 1 over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time); or 0.41-0.43 hours- 1 over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0079] In some embodiments, the growth rate of the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii is 0.35-0.41 hours- 1 over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time); or 0.39-0.41 hours- 1 over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0080] In some embodiments, the growth rate of a first additional Lactobacillus crispatus isolate is 0.27-0.29 hours-1 over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time); or 0.35- 0.37 hours-1 over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0081] In some embodiments, the growth rate of a second additional Lactobacillus crispatus isolate is 0.24-0.26 hours-1 over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of(final OD600 divided by initial OD600) divided by (final time minus initial time); or 0.31- 0.33 hours-1 over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0082] In some embodiments, at least two of the lactobacilli differ in their acidification rates.

[0083] In some embodiments, the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their acidification rates.

[0084] In some embodiments, at least two Lactobacillus crispatus differ in their acidification rates. In some embodiments, the acidification rates are acidification rates in vitro (e.g., in culture) and / or in vivo.

[0085] In some embodiments, the acidification rate of the Lactobacillus jensenii is - 0.005 to -0.003 over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time); or -0.009 to -0.007 over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0086] In some embodiments, the acidification rate of the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii is -0.009 to -0.007 over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time); or -0.012 to -0.010 over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0087] In some embodiments, the acidification rate of a first additional Lactobacillus crispatus isolate is -0.007 to -0.005 hours-1 over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time); or -0.008 to -0.006 hours-1 over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0088] In some embodiments, the acidification rate of a second additional Lactobacillus crispatus isolate is -0.008 to -0.006 hours- 1 over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time); or -0.012 to -0.010 hours-1 over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0089] In some embodiments, at least two of the lactobacilli differ in their capability of utilizing glycogen. In some embodiments, the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their capability of utilizing glycogen. In some embodiments, at least two Lactobacillus crispatus differ in their capability of utilizing glycogen. In some embodiments, the capability of utilizing glycogen is a capability of utilizing glycogen in vitro (e.g., in culture) and / or in vivo.

[0090] In some embodiments, at least two of the lactobacilli differ in their rate of producing lactic acid. In some embodiments, the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their rate of producing lactic acid. In some embodiments, at least two Lactobacillus crispatus differ in their rate of producing lactic acid. In some embodiments, the rate of producing lactic acid is a rate of producing lactic acid in vitro (e.g., in culture) and / or in vivo.

[0091] In some embodiments, at least two of the lactobacilli differ in their time to engraftment in the female genitourinary tract. In some embodiments, the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their time to engraftment in the female genitourinary tract. In some embodiments, at least two Lactobacillus crispatus differ in their time to engraftment in the female genitourinary tract. In some embodiments, the time to engraftment in the female genitourinary tract is determined in vivo.

[0092] In some embodiments, at least one of the lactobacilli isolates is capable of engrafting the female genitourinary tract of the subject within 24 hours after administration to the female genitourinary tract of the subject. In some embodiments, at least one of the lactobacilli isolates is capable of persisting within the female genitourinary tract of the subject for at least 4 days after administration to the female genitourinary tract of the subject.

[0093] In some embodiments, 2, 3 or 4 of the lactobacilli isolates are capable of persisting within the female genitourinary tract of the subject for at least 4, 5, 6, or 7 days after administration to the female genitourinary tract of the subject, optionally wherein 2, 3 or4 of the lactobacilli isolates are capable of persisting within the female genitourinary tract of the subject for at least 1 menstrual cycle after administration to the female genitourinary tract of the subject.

[0094] In some embodiments, the lactobacilli of the consortium: are capable of competing with the resident microbiota within the female genitourinary tract of the subject, optionally, without at least partial removal the resident microbiota, e.g., by administration to the genitourinary tract of an antimicrobial agent, such as, e.g., an antibiotic; or are capable of engrafting in the presence of the resident microbiota.

[0095] In some embodiments, each of the lactobacilli of the consortium were obtained from a healthy (e.g., ZzzctoZz(zcz7Zzz5-dominated) human donor. In some embodiments, the lactobacilli comprised in the defined population were obtained from at least two different healthy (e.g., L(zctoZ?(zcz7Zzz5-dominated) human donors, optionally wherein the lactobacilli comprised in the defined population were obtained from three different healthy (e.g., L(zctoZ?(zcz7Zzz5-dominated) human donors. In some embodiments, the Lactobacillus jensenii isolate and the Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii were obtained from different healthy (e.g., lactobacillus-dominated) human donors. In some embodiments, the at least two additional Lactobacillus crispatus isolates were obtained from the same healthy (e.g., lactobacillus-dominated) human donor.

[0096] In some embodiments, the at least two Lactobacillus crispatus isolates thrive independent of the presence of Lactobacillus jensenii each of the lactobacilli of the consortium are individually propagated, optionally using a large-scale culture method.

[0097] Some aspects of the disclosure provide a microbial consortium comprising a defined population of isolated lactobacilli configured to promote engraftment in a female genitourinary tract of a subject, the defined population comprising: a Lactobacillus jensenii isolate; and two or three Lactobacillus crispatus isolates, wherein at least two of the two or three Lactobacillus crispatus isolates have different growth rates, acidification rates, and / or rates of lactic acid production.

[0098] Some aspects of the disclosure provide a pharmaceutical composition comprising any one of the microbial consortia described herein and a pharmaceutically acceptable buffer, diluent or excipients.

[0099] Some aspects of the disclosure provide a method of increasing the relative abundance of lactobacilli in the female genitourinary tract of a subject comprisingadministering any one of the microbial consortia described herein to the genitourinary tract of the subject.

[0100] In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 90% (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, or 20%) of the total bacterial population prior to administration of the microbial consortium.

[0101] In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 70% (e.g., greater than 80%, 85%, 90%, 95%, or 98%) of the total bacterial population following administration of the microbial consortium.

[0102] In some embodiments, the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 5 percentage points (e.g., at least 10, 15, 20, 25, 30, 40, 50, or 60 percentage points) following administration of the microbial consortium.

[0103] In some embodiments, any one of the microbial consortia described herein is for use in therapy.

[0104] In some embodiments, any one of the microbial consortia described herein is for use in a method of treating dysbiosis of the female genitourinary tract of a subject, said method comprising administering the microbial consortium to the genitourinary tract of the subject, optionally wherein the subject is asymptomatic.

[0105] In some embodiments, the any one of the microbial consortia described herein is for use in a method of treating inflammation of the female genitourinary tract of a subject, said method comprising administering the microbial consortium to the genitourinary tract of the subject, optionally wherein the subject is asymptomatic.

[0106] Some aspects of the present disclosure provide a method of assembling a microbial consortium comprising two or more isolates belonging to one or more bacterial species, the method comprising: obtaining genetic sequencing data obtained from a purported sample of each of the two or more isolates; obtaining genetic sequencing data obtained from a reference sample of each of the isolates; using at least one computer processor to:align at least a core portion of the genetic sequencing data from the purported sample to the genetic sequencing data from the reference sample; calculate, using the aligned genetic sequencing data, a percent identity value describing a percent of the aligned genetic sequencing data that is identical; and determine that the purported sample and the reference sample comprise the same set of bacteria by determining that the percent identity value is greater than or equal to a threshold value defining a distinction between different isolates of the same bacterial species; and assembling the microbial consortium using the purported sample.

[0107] In some embodiments, the method further comprises determining the threshold value by: obtaining genetic sequencing data obtained from two or more samples of each of the two or more isolates; aligning, using at least one computer processor, at least a core portion of the obtained genetic sequencing data for pairs of the two or more isolates; calculating, using the at least one computer processor, a percent identity between each of the aligned pairs; and determining the threshold value by determining, using the at least one computer processor, a largest value of the calculated percent identities.

[0108] In some embodiments, the bacterial species comprises a species of Lactobacilli.

[0109] In some embodiments, the bacterial species comprises Lactobacillus crispatus.

[0110] In some embodiments, determining that the purported sample and the reference sample comprise the same set of bacteria comprises determining that the percent identity is greater than or equal to a threshold value of 99.81%. In some embodiments, determining that the purported sample and the reference sample comprise the same set of bacteria comprises determining that the percent identity is greater than or equal to a threshold value of 99.90%. In some embodiments, determining that the purported sample and the reference sample comprise the same set of bacteria comprises determining that the percent identity is greater than or equal to a threshold value of 99.96%.BRIEF DESCRIPTION OF THE DRAWINGS

[0111] FIG. 1 provides a schematic overview of isolation of bacteria from donor vaginal microbiomes.

[0112] FIG. 2 provides a selection scheme for identification of individual lactobacilli for inclusion in an exemplary microbial consortium.

[0113] FIGs. 3A-C provide a graph showing that genomic assemblies can be useful in distinguishing one Lactobacillus crispatus from another on the basis of percent identity across a segment of bacterial genomes.

[0114] FIGs. 4A-B show the relative abundance of L. crispatus bacteria in samples taken from the vaginal tract over multiple visits from a single donor

[0115] FIGs. 5A-D show the relative abundance of L. crispatus bacteria in samples taken from example recipient subjects following administration of the donor-obtained material from FIG. 4.

[0116] FIG. 6 shows a schematic overview of selection based on pangenome analysis.

[0117] FIGs. 7A-7B show graphs of growth (FIG. 7A) and pH (FIG. 7B) over time for tested isolates.

[0118] FIGs. 8A-8D show growth rate comparisons between single bacteria and the exemplary 4-member consortium (MC-1).

[0119] FIGs. 9A-9D show acidification change (pH change) comparisons between single bacteria and the exemplary 4-member consortium (MC-1).

[0120] FIGs. 10A-10H show D-lactic acid and L-lactic acid production comparisons between single bacteria and the exemplary 4-member consortium (MC-1).

[0121] FIGs. 11A-11B compares the percentage of D- / L-lactic acid produced across time in single bacteria and the exemplary 4-member consortium (MC-1).

[0122] FIGs. 12A-12D show the abundance of L. crispatus and L. jensenii within the exemplary 4-member (MC-1) consortium grown on MRS agar plates and relative abundance by species using qPCR over time.

[0123] FIG. 13 is a diagram depicting a flowchart of an illustrative process 1300 for assembling a microbial consortium, according to some embodiments of the technology described herein.

[0124] FIG. 14 depicts an illustrative implementation of a computer system that may be used in connection with some embodiments of the technology described herein.

[0125] FIG. 15 shows a schematic of the isolates contained in the exemplary 4-member consortium (MC-1).

[0126] FIGs. 16A-16L show the growth and growth rate of individual isolates and the exemplary 4-member consortium (MC-1) grown in MRS medium modified to include 20mg / mL maltodextrin (FIGs. 16A-B), glycogen (FIGs. 16C-D), glucose (FIGs. 16E-F), fructose (FIGs. 16G-H), mannose (FIGs. 161- J), or mannitol (FIGs. 16K-L).

[0127] FIGs. 17A-17L show the acidification of individual isolates and the exemplary4-member (MC-1) consortium grown in MRS medium modified to include 20mg / mL maltodextrin (FIGs. 17A-B), glycogen (FIGs. 17C-D), glucose (FIGs. 17E-F), fructose (FIGs. 17G-H) , mannose (FIGs. 171- J), or mannitol (FIGs. 17K-L).

[0128] FIGs. 18A-18F show the relative abundance (measured by qPCR) of the individual isolates of the 4-member consortium (MC-1) over time when grown in MRS medium modified to include 20mg / mL maltodextrin (FIGs. 18A), glycogen (FIGs. 18B), glucose (FIGs. 18C), fructose (FIGs. 18D), mannose (FIGs. 18E), or mannitol (FIGs. 18F).

[0129] FIGs. 19A-19D show comparisons of the exemplary 4-member consortium (MC- 1) and competitor strains in growth (FIG. 19A-B) and acidification (FIG. 19C-D).

[0130] FIGs. 20A-20C shows the growth comparison of individual isolates and the exemplary 4-member consortium (MC-1) (FIG. 20A) and the growth comparison of 2- member consortia and the exemplary 4-member consortium (FIG. 20B). A comparison of growth rates is shown after 8h for all individual isolates, 2-member and 4-member consortia (FIG. 20C).

[0131] FIGs. 21A-21C show the acidification comparison of individual isolates and the exemplary 4-member consortium (MC-1) (FIG. 21 A) and the acidification comparison of 2- member consortia and the exemplary 4-member consortium (FIG. 21B). A comparison of acidification rates is shown (FIG. 21C) after 8h for all individual isolates, 2-member and 4- member consortia.

[0132] FIGs. 22A-22B show the growth comparison of 3-member consortia and the exemplary 4-member consortium (MC-1) (FIG. 22A) and comparison of growth rates after 8h for 3-member and 4-member consortia (MC-1) (FIG. 22B).

[0133] FIGs. 23A-23B show the acidification comparison of 3-member consortia and the exemplary 4-member consortium (MC-1) (FIG. 23A) and comparison of acidification rates after 8h for 3-member and 4-member consortia (MC-1) (FIG. 23B).

[0134] FIGs. 24A-24C show the relative abundance in percentages of each isolate in the2-member consortia measured on plates (CFUs / ml based).

[0135] FIGs. 25A-25B show the relative abundance in percentages of each isolate in the3-member consortia measured by qPCR (CFUs / ml based).DETAILED DESCRIPTION

[0136] A healthy vaginal microbiome is primarily composed of bacterial species from the genus Lactobacillus (also referred to as “LactoZzacz7 / M5-dominated”), which provides protection against harmful pathogens and helps maintain a healthy or “eubiotic” vaginal environment. For purposes herein, a “eubiotic” vaginal environment or microbiome excludes a dysbiotic vaginal environment or microbiome. An important function of Lactobacillus in the vaginal microbiome is the maintenance of a healthy acidified state in the range of about pH 3.5-4.5 resulting from the metabolic activity of Lactobacillus on secreted glycogen / glucose in the vagina to produce lactic acid. Acidic conditions are unfavorable for the growth of many pathogenic microorganisms and pathobionts, including bacteria, protozoa, and viruses. When vaginal dysbiosis (i.e., “dysbiotic” vaginal environment) occurs, characterized by a decline in Lactobacillus and an increase in microbial diversity (including in some cases vaginal pathogens), it can elevate the risk of various genital tract conditions such as bacterial vaginosis, inflammation, aerobic vaginitis, vulvovaginal candidiasis, sexually transmitted infections, and complications during pregnancy, including preterm birth. If clinical symptoms are present, such as, e.g., vaginal discharge, vaginal itching, vaginal irritation, and / or vaginal malodor. Antibiotics and antifungals are frequently the first-choice treatments for treating these conditions, however, such treatments can become ineffective and cause conditions to recur.

[0137] Recently, live biotherapeutic products have emerged as an alternative category of therapeutic agents with a wide range of applications, including for potentially addressing vaginal microbiome issues. The United States Food and Drug Administration (FDA) defines live biotherapeutic products as biological products that contain living organisms, such as bacteria, and are intended to prevent, treat, or cure human diseases or conditions, excluding vaccines. Live biotherapeutic products particularly containing Lactobacillus species (e.g., Lactobacillus acidophilus, Lactobacillus rhamnosus, or Lactobacillus gasseri) for intravaginal or oral use have been available for many years. These products include vaginalsuppositories containing lyophilized Lactobacillus acidophilus or other Lactobacillus species (e.g., Lactobacillus rhamnosus, or Lactobacillus gasseri) of human origin as well as various nutritional supplements. However, more effective live biotherapeutic products for vaginal health and for resolving vaginal dysbiosis are much needed. In particular, a need exists for the development of effective consortia of bacterial isolates that may be administered as a live biotherapeutic product for the treatment and resolution of vaginal dysbiosis, vaginal infections, and for improving the overall health profile of subjects receiving such isolates.

[0138] Provided herein are microbial consortia comprising a defined population comprising two or more different bacteria capable of engraftment of the human female genitourinary tract, e.g., of a female subject that exhibits, e.g., a dysbiosis of the genitourinary tract. The microbial consortia of the disclosure, in some embodiments, comprise a defined population of isolated lactobacilli configured to promote engraftment in a female genitourinary tract of a subject, the defined population comprising: a Lactobacillus jensenii isolate; a Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii', and at least one additional Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii (e.g., two different Lactobacillus crispatus isolate, e.g., two different Lactobacillus crispatus isolate having different acidification rates). The genitourinary tract includes the reproductive tract, and it is thought that a link exists between the vaginal and endometrial spaces (e.g., that they represent interconnected biogeographical niches) and the exchange of, e.g., microorganisms and immune molecules between the niches (Chen C et al., The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases, Nature Communications vol 8, 875 (2017)).

[0139] Preparations of lactic acid-producing bacteria (e.g., including Lactobacilli) known in the art generally are not sufficiently capable of colonizing and engrafting in vivo, e.g., in the human female genitourinary tract of a diverse set of female subjects, e.g., female subjects exhibiting dysbiosis or infection of the vaginal microbial niche. Bacterial intervention strategies in the art that have attempted to revert dysbiosis in the genitourinary tract by changing the microbial environment to Lactobacillus dominance have focused on the selection of individual strains of Lactobacillus that are formulated into a probiotic supplement or live biotherapeutic product (LBP) that is then orally or locally administered, sometimes following an antibiotic treatment. However, these strategies lack convincing data showing the reversion of dysbiosis or infection in part due to poor engraftment potential of the strains (Vieira-Baptista et al 2022; van de Wijgert and Verwijs 2020; Thanaboonyawat etal 2023). The probiotic supplements have often consisted of non-vaginal Lactobacillus species such as L. rhamnosus, which are not suitable for durable vaginal engraftment, and most supplements use a single species or strain approach. With respect to LBPs, the only extensively studied vaginal LBP is L. crispatus CTV-05. In a recent double-blind randomized clinical study, the LBP containing L. crispatus CTV-05 did not impact reproductive outcomes in women with abnormal vaginal microbiota undergoing assisted reproductive procedures (Haahr et al. Human Reproduction, Volume 38, Issue Supplement_l, June 2023), emphasizing that a single-strain product is not effective in affecting the resident microbial communities of genitourinary tract of a diverse set of female subjects.

[0140] Four main issues have hampered the discovery of lactic acid-producing bacteria (e.g., including Lactobacilli) capable of colonizing and engrafting in vivo: (1) the source of the bacteria, (2) the process of identifying and isolating suitable bacteria, (3) the route of administration of the bacteria, and (4) the combination of the plurality of bacteria, e.g., the specific combination of bacterial isolates.

[0141] The inventors realized that in order to identify lactic acid-producing bacteria (e.g., including Lactobacilli) that are capable of colonizing and engrafting the female reproductive tract in vivo the following conditions are favorable:

[0142] First, the preparations of lactic acid-producing bacteria (e.g., including Lactobacilli) are sourced from a human female donor exhibiting healthy (non-dysbiotic) vaginal microbiota; i.e., the lactic acid-producing bacteria that will be administered to a human female subject are derived from a microbial niche (the donor female’s urogenital tract microbiota) that is adapted to survive and thrive in this environment; the lactic acidproducing bacteria are not sourced from food or other animal sources. Provided herein are methods that include bacterial preparations derived from healthy human female donors.

[0143] Second, bacteria or bacterial isolates capable of colonizing and engrafting in vivo in human subjects are identified after the donor preparation (comprising the lactic acidproducing bacteria) has been administered to a female recipient, e.g., after a predetermined period of time, not prior to the administration to a subject which is typically described in the art. Generally, the art describes methods that include first taking a (vaginal) sample from a healthy individual, then isolating and propagating individual isolates in vitro (in culture), followed by in vitro testing to assess the ability to colonize (e.g., adherence testing to vaginal epithelia cells (VEC) or HeLa cells, and to produce anti-microbial agents, e.g., hydrogen peroxide) in vitro, and finally administering an isolate(s) to a recipient. This has thedisadvantage that any in vitro testing comprises a reductionist simulation of the in vivo environment and results have been mixed as to the extent of predictability of success, using such in vitro methods.

[0144] Provided herein are microbial consortia comprising defined populations of isolated lactobacilli that were generated using methods that include engrafting bacterial preparations derived from healthy human female donors in human females (e.g., females who exhibit a dysbiosis), followed by taking a (vaginal) sample from the female subject after a predetermined amount of time, then determining which species and isolates were successful in colonizing and engrafting in the female subject’s urogenital tract in vivo, e.g., by comparing the constituents (bacterial taxa and isolates) of the donor female with those of the female subject (e.g., by nucleic acid sequencing), and then upon obtaining the identification of isolates that are successful in colonizing and engrafting the microbial niche in vivo, return to the healthy donor or a stored sample from the donor, from which the original material was prepared (or optionally the recipient’s sample) to isolate those specific isolates, propagate and formulate into bacterial preparations and / or (pharmaceutical) compositions. These methods thus remove the need for cumbersome and labor-intensive in vitro testing procedures and the uncertainty inherent in the current methods of having to predict from in vitro assays the ability to colonize an environment in vivo, thereby increasing the likelihood of identifying relevant, highly adapted, and successful isolates capable of colonizing and engrafting in vivo. The forward approach of first administering a donor sample comprising a microbial consortium and subsequently identifying the specific species and isolates that were capable of engrafting and improving the dysbiosis in the recipient's vaginal tract, not only provides a higher chance of identifying therapeutically effective isolates that are capable of improving dysbiosis, but also allows for identification of isolate consortia, e.g., preferred combinations of bacterial isolates, which represent a sub-population of the donor sample, which provide for the therapeutic effect.

[0145] A healthy vaginal flora is characterized by an acidic environment inhabited predominantly by lactic acid bacteria, primarily, species of Lactobacillus (residing in the vaginal microbial niche). The microbial composition in healthy women can differ, though it is typically dominated by Lactobacillus species such as L. crispatus, L. iners, L. gasseri, L. jensenii, and mixtures thereof. A healthy vagina of a women of child-bearing age is estimated to be dominated by 106-109colony forming units of lactic acid-producing bacteria (e.g., Lactobacillus} per gram of fluid. The species distribution differs between women of differentgeographical background, race (e.g., Asian, white women, black, Hispanic), age, lifestyle and the like. The composition of the vaginal flora is also influenced by which specific isolates the woman has inherited from her mother and / or which isolates have migrated from her digestive tract to the urogenital tract. Healthy, fertile women present with a pH of about 3 to 5.5 (more specifically between pH 3.5 and 4.5) in the vagina, primarily as a result of lactic acid production. Vaginal pH undergoes physiological changes from birth to menopause. The increase of vaginal pH is detrimental for the survival of Lactobacillus bacteria, but not for other microorganisms. The vaginal lactobacilli are believed to have a protective effect against vaginal colonization by pathogenic microorganisms (e.g., yeast (Candida albicans'), Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis, and viruses, e.g., HIV, HSV-2, and various anaerobes) and prevent the vaginal establishment of, for instance, bacteria that are present in the colon, such as Gardnerella vaginalis, Mobiluncus, Bacteroides, Prevotella and Escherichia coli.

[0146] Several factors may contribute to the disturbance of the vaginal flora. Factors may include, a) use of antibiotics to kill pathogenic bacteria which can lead to significantly reduced levels of lactobacilli in the vagina; b) hormonal changes, in particular changes in estrogen levels, which are observed in several phases of a woman's life (e.g., puberty, pregnancy, childbearing age, pre- and post-menopause); estrogen levels are thought to be associated with Lactobacillus levels (dominance) in the vagina; c) sexual intercourse, which can be associated with pH increases (semen generally is alkaline) that may disturb the vaginal flora, because bacteria other than lactobacilli may start to flourish once the vaginal pH increases; d) use of medications, e.g., chemotherapeutic s or antimycotics; e) use of birth control products; f) during menstruation; g) insufficient hygiene (e.g., promoting undesirable spread of the microorganisms from rectum to the urogenital area); h) general health status, such as having comorbidities, e.g., being diabetic; and (i) use of excessive hygienic measures in particular the use of vaginal douches.

[0147] Disturbance of the vaginal flora may lead to vaginal dysbiosis and vaginal disorders, e.g., candidiasis and bacterial vaginosis, which are two common vaginal disorders that affect women worldwide. Bacterial vaginosis is believed to be the result of displaced vaginal lactic acid-producing bacteria which are replaced by a range of unwanted species such as Gardnerella vaginalis, Bacteroides, Mobiluncus, Prevotella, and Mycoplasma hominis. Vaginal infections are most often associated with one or more of: Escherichia, Enterococcus, Pseudomonas, Proteus, Klebsiella, Streptococcus, Staphylococcus,Gardnerella, Ureaplasma, Bacteroides, Peptococcus, Neisseria, Serratia, Sneathia, Megasphera, Corynebacterium, Clostridium, and Candida.

[0148] Vaginal lactobacilli predominance is thought to play an important role in resistance to infection via production of lactic acid and acidification of the vagina and by production of other antimicrobial products, such as, e.g., hydrogen peroxide. The presence of lactobacilli in the vagina has been linked to decreased frequencies of bacterial vaginosis, yeast vaginitis and sexually transmitted pathogens, including Neisseria gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis and viruses, e.g., HIV and HSV. Lactobacillus dominance varies among ethnic groups (they are thought to be very predominant in Asian and white women but less so in black and Hispanic women, though they still often represent the majority).

[0149] Studies have shown that L(zctoZ?(zcz7Zzz5-depleted communities can be transient, lasting just a few days, while in other instances the depleted communities persist for many weeks. Some women with L(zctoZ?(zcz7Zzz5-depleted communities remain asymptomatic, e.g., with respect to vaginal health conditions, and healthy. However, such women may be at higher risk for infections and STDs.

[0150] The female urogenital (also known as genitourinary) tract consists of interconnected biogeographical niches, such as the vaginal niche and the endometrial niche. Bacteria from the vaginal microbial community can migrate through the cervix to remote sites of the urogenital tract. Dysbiosis in the vaginal microbial community can result in a dysbiosis at remote sites. Dysbiosis at these sites has been associated with a range of diseases and conditions, including urinary tract infection (UTI), pelvic inflammatory disease (PID), and bacterial vaginosis (BV). In some embodiments, administration of a microbial consortium comprising individual and unique lactobacilli (e.g., L. jensenii and / or L. crispatus bacteria) described herein to the vagina includes resolving dysbiosis in remote sites of the genitourinary tract.Lactobacillus jensenii

[0151] Lactobacillus jensenii is a Gram-positive, rod-shaped, aerotolerant species of Lactobacillus and is a naturally-occurring component of the vaginal microflora. In vitro studies have demonstrated that L. jensenii inhibit the release of pro-inflammatory mediators from vaginal epithelial cells, promoting anti-microbial defense while not inducing immune mediated inflammation (Witkin SS and Linhares IM, Why do lactobacilli dominate thehuman vaginal microbiota? BJOG 2017; 124:606-611). L. jensenii contains two D-lactate dehydrogenase genes and one L-lactate dehydrogenase gene for the production of lactic acid.

[0152] In some embodiments, the Lactobacillus jensenii isolate is obtained from a bacterial sample deposited as DSM Deposit Number 34770 (“DSM 34770”) with DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D- 38124 Braunschweig, on October 4, 2023. The aforementioned deposit was made pursuant to the terms of the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedures.

[0153] In some embodiments, the Lactobacillus jensenii isolate is derived or propagated from a bacterial sample deposited as DSM 34770. In some embodiments, the genome of the Lactobacillus jensenii isolate, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%).

[0154] In some embodiments, L. jensenii (e.g., L. jensenii isolates) can be obtained from a donor (e.g., a human donor) from vaginal secretions. In some embodiments, L. jensenii (e.g., L. jensenii isolates) can be obtained using a method as described in International Patent Publication WO 2022 / 185121, published on September 9, 2022, the content of which is incorporated herein by reference. For example, vaginal fluid can be collected from a donor in a menstrual cup or using a vaginal swab and plated on a selective culture medium (e.g., diffuse media, such as broth, and / or solid media, such as agar) to allow individual bacterial isolates to colonize and propagate. Non-limiting examples of selective culture media include De Man-Rogosa-Sharpe (MRS) media, Rogosa, M17, Elliker, and Thioglycolate. Individual colonies (e.g., bacterial isolates) can then be selected and propagated by inoculation into large-scale cultures. A large-scale culture method, as used herein, refers to a method of multiplying individual bacterial isolates by letting them reproduce in at least 1 L of a selective diffuse medium (e.g., broth). In some embodiments, a large-scale culture is about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 15 L, about 20 L, about 25 L, about 30 L, about 35 L, about 40 L, about 45 L, about 50 L, about 100 L, about 500 L, about 1,000 L, about 2,000 L, about 3,000 L, about 4,000 L, or about 5,000 L or more of a selective diffuse medium (e.g., broth). In some embodiments, a large-scale culture is between I L - 50L, between 1 L- 45 L, between 5 L - 45 L, between 5 L - 40 L, between 10 L - 40 L, between 10 L - 35 L, between 15 L - 35 L, between 15 L - 30 L, between 20 L - 30 L, between 20 L - 25 L, between 1 L - 5 L, between1 L - 10 L, between 5 L - 10 L, between 5 L - 15 L, between 10 L - 15 L, between 10 L - 20 L, between 15 L - 20 L, between 15 L - 25 L, between 20 L - 25 L, between 20 L - 30 L, between 25 L - 30 L, between 25 L - 35 L, between 30 L - 35 L, between 30 L - 40 L, between 35 L - 40 L, between 35 L - 45 L, between 40 L - 45 L, between 40 L - 50 L, between 45 L - 50 L, between 50 L - 250 L, between 100 L - 5,000 L, between 100 L - 2,000 L, between 100 L - 1,000 L, between 500 L - 5,000 L, between 500 L - 3,000 L, between 500 L - 1,500 L, or between 1,000 L - 10,000 L of a selective diffuse medium (e.g., broth).

[0155] In some embodiments, a microbial consortium of the present disclosure can include a L. jensenii isolate. In some embodiments, a growth rate is an in vitro growth rate that is calculated after 8 hours of bacterial growth in culture. In some embodiments, a growth rate is determined for a particular bacteria or consortia after an inoculation of MRS broth at 37 °C with 5 million CFU / mL. Growth rates may be determined according to the following equation: Growth rate equals natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has a growth rate of about 0.2 hours'1, about 0.25 hours'1, about 0.3 hours'1, about 0.35 hours'1, about 0.4 hours'1, about 0.45 hours'1, or about 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has a growth rate of between 0.2 - 0.25 hours'1, between 0.2 - 0.3 hours'1, between 0.2 - 0.35 hours'1, between 0.2 - 0.4 hours'1, between 0.2 - 0.45 hours'1, between 0.2 - 0.5 hours'1, between 0.25 - 0.3 hours'1, between 0.25 - 0.35 hours'1, between 0.25 - 0.4 hours'1, between 0.25 - 0.45 hours'1, between 0.25 - 0.5 hours'1, between 0.3 - 0.35 hours'1, between 0.3 - 0.4 hours'1, between 0.3 - 0.45 hours'1, between 0.3 - 0.5 hours'1, between 0.35 - 0.4 hours'1, between 0.35 - 0.45 hours'1, between 0.35 - 0.5 hours'1, between 0.4 - 0.45 hours'1, between 0.4 - 0.5 hours'1, or between 0.45 - 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has a growth rate of about 0.35 hours'1(e.g., 0.34 hours'1) whengrown in a selective diffuse medium (e.g., MRS) over a period of 8 hours in culture (starting pH of 4.7), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0156] In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has a growth rate after 8 hours of growth in culture of about 0.2 hours'1, about 0.25 hours'1, about 0.3 hours'1, about 0.35 hours'1, about 0.4 hours'1, about 0.45 hours'1, or about 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has a growth rate after 8 hours of growth in culture of between 0.2 - 0.25 hours'1, between 0.2 - 0.3 hours'1, between 0.2 - 0.35 hours'1, between 0.2 - 0.4 hours'1, between 0.2 - 0.45 hours'1, between 0.2 - 0.5 hours'1, between 0.25 - 0.3 hours'1, between 0.25 - 0.35 hours'1, between 0.25 - 0.4 hours'1, between 0.25 - 0.45 hours'1, between 0.25 - 0.5 hours'1, between 0.3 - 0.35 hours'1, between 0.3 - 0.4 hours'1, between 0.3 - 0.45 hours'1, between 0.3 - 0.5 hours'1, between 0.35 - 0.4 hours'1, between 0.35 - 0.45 hours'1, between 0.35 - 0.5 hours'1, between 0.4 - 0.45 hours'1, between 0.4 - 0.5 hours'1, or between 0.45 - 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has a growth rate after 8 hours of growth in culture of about 0.4 hours'1(e.g., 0.42 hours'1) when grown in a selective diffuse medium (e.g., MRS) over a period of 8 hours in culture (starting pH of 5.2), wherein the growth rate is calculated using the following equation: natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0157] In some embodiments, an acidification rate is an in vitro acidification rate that is calculated after 24 hours of bacterial growth in culture. In some embodiments, an acidification rate is determined for a particular bacteria or consortia after an inoculation of MRS broth at 37 °C with 5 million CFU / mL. Acidification rates may be determined according to the following equation: Acidification rate equals natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, anL. jensenii included in a microbial consortium of the present disclosure has an acidification rate of about -0.003 hours'1, about -0.004 hours'1, about -0.006 hours'1, about -0.007 hours'1, about -0.008 hours'1, about -0.009 hours'1, about -0.010 hours'1, about -0.011 hours'1, or about -0.12 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).. In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has an acidification rate between -0.003 to -0.005 hours' \ between -0.003 to -0.006 hours'1, between -0.003 to -0.007 hours'1, between -0.003 to - 0.008 hours'1, between -0.003 to -0.009 hours'1, between -0.003 to -0.010 hours'1, between - 0.003 to -0.011 hours'1, between -0.003 to -0.012 hours'1, between -0.004 to -0.006 hours'1, between -0.004 to -0.007 hours'1, between -0.004 to -0.008 hours'1, between -0.004 to -0.009 hours'1, between -0.004 to -0.010 hours'1, between -0.004 to -0.011 hours'1, between -0.004 to -0.012 hours'1, between -0.005 to -0.007 hours'1, between -0.005 to -0.008 hours'1, between - 0.005 to -0.009 hours'1, between -0.005 to -0.010 hours'1, between -0.005 to -0.011 hours'1, between -0.001 to -0.012 hours'1, between -0.006 to -0.008 hours'1, between -0.006 to -0.009 hours'1, between -0.006 to -0.010 hours'1, between -0.006 to -0.011 hours'1, between -0.006 to -0.12 hours'1, between -0.007 to -0.009 hours'1, between -0.007 to -0.010 hours'1, between - 0.007 to -0.011 hours'1, between -0.007 to -0.012 hours'1, between -0.008 to -0.010 hours'1, between -0.008 to -0.011 hours'1, between -0.008 to -0.012 hours'1, between -0.009 to -0.011 hours'1, between -0.009 to -0.012 hours'1, or between -0.010 to -0.012 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has an acidification rate of about -0.004 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time)..

[0158] In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has an acidification rate of about -0.003 hours'1, about -0.004 hours'1, about -0.006 hours'1, about -0.007 hours'1, about -0.008 hours'1, about -0.009 hours'1, about - 0.010 hours'1, about -0.011 hours'1, or about -0.12 hours'1when grown in a selective diffusemedium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has an acidification rate between -0.003 to -0.005 hours'1, between -0.003 to -0.006 hours'1, between -0.003 to - 0.007 hours'1, between -0.003 to -0.008 hours'1, between -0.003 to -0.009 hours'1, between - 0.003 to -0.010 hours'1, between -0.003 to -0.011 hours'1, between -0.003 to -0.012 hours'1, between -0.004 to -0.006 hours'1, between -0.004 to -0.007 hours'1, between -0.004 to -0.008 hours'1, between -0.004 to -0.009 hours'1, between -0.004 to -0.010 hours'1, between -0.004 to -0.011 hours'1, between -0.004 to -0.012 hours'1, between -0.005 to -0.007 hours'1, between - 0.005 to -0.008 hours'1, between -0.005 to -0.009 hours'1, between -0.005 to -0.010 hours'1, between -0.005 to -0.011 hours'1, between -0.001 to -0.012 hours'1, between -0.006 to -0.008 hours'1, between -0.006 to -0.009 hours'1, between -0.006 to -0.010 hours'1, between -0.006 to -0.011 hours'1, between -0.006 to -0.12 hours'1, between -0.007 to -0.009 hours'1, between - 0.007 to -0.010 hours'1, between -0.007 to -0.011 hours'1, between -0.007 to -0.012 hours'1, between -0.008 to -0.010 hours'1, between -0.008 to -0.011 hours'1, between -0.008 to -0.012 hours'1, between -0.009 to -0.011 hours'1, between -0.009 to -0.012 hours'1, or between - 0.010 to -0.012 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. jensenii included in a microbial consortium of the present disclosure has an acidification rate of about -0.008 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0159] In some embodiments, an L. jensenii of the present disclosure thrives. The term “thrives”, as used herein, refers to a bacteria (e.g., an L. jensenii isolate) that grows or propagates at a higher rate relative to a control bacteria when grown under the same conditions. A control bacteria can be, for example, a different isolate of the same species.

[0160] In some embodiments, an L. jensenii isolate is said to “thrive” if the L. jensenii isolate can grow and propagate in vitro or in vivo. In some embodiments, an L. jensenii isolate is said to “thrive” if the L. jensenii isolate has a growth rate that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, at least 20%, or at least 25% higher than the growth rate of a control L. jensenii (under the same conditions). In some embodiments, an L. jensenii isolate is said to “thrive” if the L. jensenii isolate has a growth rate of between 1% - 5%, between 1% - 10%, between 5% - 10%, between 5% - 15%, between 10% - 15%, between 10% - 20%, between 15% - 20%, between 15% - 25%, between 20% - 25%, between 1% - 25%, between 1% - 20%, between 5% - 25% , between 5% - 20%, or between 5% - 15% higher than the growth rate of a control L. jensenii (under the same conditions).Lactobacillus crispatus

[0161] Lactobacillus crispatus is a Gram-positive, rod-shaped, aerotolerant species of Lactobacillus and is a naturally-occurring component of the vaginal microflora. A vaginal microbiota dominated by L. crispatus is associated with a healthy vagina, as L. crispatus has been shown to provide a protective effect against sexually transmitted infections (STIs), bacterial vaginosis (BV), and vulvovaginal candidiasis (VVC) due to its ability to produce lactic acid and bacteriocin (Chee WJY et al., Vaginal microbiota and the potential of Lactobacillus derivatives in maintaining vaginal health, Microbial Cell Factories vol 19, 203 (2020)). L. crispatus contains two L-lactate dehydrogenase genes and one D-lactate dehydrogenase gene for the production of lactic acid.

[0162] In some embodiments, a Lactobacillus crispatus isolate of the present disclosure is obtained from a bacterial sample deposited as DSM Deposit Number 34771 (“DSM 34771”). In some embodiments, a Lactobacillus crispatus isolate of the present disclosure is obtained from a bacterial sample deposited as DSM Deposit Number 34772 (“DSM 34772”). In some embodiments, a Lactobacillus crispatus isolate of the present disclosure is obtained from a bacterial sample deposited as DSM Deposit Number 34773 (“DSM 34773”). DSM 34771, DSM 34772, and DSM 34773 were deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on October 4, 2023. The aforementioned deposits were made pursuant to the terms of the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedures.

[0163] In some embodiments, L. crispatus (e.g., L. crispatus isolates) can be obtained from a donor (e.g., a human donor) from vaginal secretions. In some embodiments, L. crispatus (e.g., L. crispatus isolates) can be obtained using a method as described in International Patent Publication WO 2022 / 185121, published on September 9, 2022, thecontent of which is incorporated herein by reference. For example, vaginal fluid can be collected from a donor in a menstrual cup or using a vaginal swab and plated on a selective culture medium (e.g., diffuse media, such as broth, and / or solid media, such as agar) to allow individual bacterial isolates to colonize and propagate. Non-limiting examples of selective culture media include De Man-Rogosa-Sharpe (MRS), Rogosa, M17, Elliker, and Thioglycolate. Individual colonies can then be selected and propagated by inoculation into large-scale cultures. A large-scale culture method, as used herein, refers to a method of multiplying individual bacterial isolates by letting them reproduce in at least 1 L of a selective diffuse medium (e.g., broth). In some embodiments, the large-scale culture is about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 15 L, about 20 L, about 25 L, about 30 L, about 35 L, about 40 L, about 45 L, about 50 L, about 100 L, about 500 L, about 1,000 L, about 2,000 L, about 3,000 L, about 4,000 L, or about 5,000 L, or more of a selective diffuse medium (e.g., broth). In some embodiments, the large-scale culture is between I L - 50L, between 1 L- 45 L, between 5 L - 45 L, between 5 L - 40 L, between 10 L - 40 L, between 10 L - 35 L, between 15 L - 35 L, between 15 L - 30 L, between 20 L - 30 L, between 20 L - 25 L, between 1 L - 5 L, between 1 L - 10 L, between 5 L - 10 L, between 5 L - 15 L, between 10 L - 15 L, between 10 L - 20 L, between 15 L - 20 L, between 15 L - 25 L, between 20 L - 25 L, between 20 L - 30 L, between 25 L - 30 L, between 25 L - 35 L, between 30 L - 35 L, between 30 L - 40 L, between 35 L - 40 L, between 35 L - 45 L, between 40 L - 45 L, between 40 L - 50 L, or between 45 L - 50 L, between 50 L - 250 L, between 100 L - 5,000 L, between 100 L -2,000 L, between 100 L - 1,000 L, between 500 L - 5,000 L, between 500 L - 3,000 L, between 500 L - 1,500 L, or between 1,000 L - 10,000 L of a selective diffuse medium (e.g., broth).

[0164] In some embodiments, a microbial consortium of the present disclosure can include one or more different L. crispatus isolates. In some embodiments, a microbial consortium of the present disclosure can include 1, 2, 3, 4, 5, or more different L. crispatus isolates.

[0165] In some embodiments, a growth rate is an in vitro growth rate that is calculated after 8 hours of bacterial growth in culture. In some embodiments, a growth rate is determined for a particular bacteria or consortia after an inoculation of MRS broth at 37 °C with 5 million CFU / mL. Growth rates may be determined according to the followingequation: Growth rate equals natural logarithm (Ln) of (final OD600 divided by initial OD600) divided by (final time minus initial time).

[0166] In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of about 0.2 hours'1, about 0.25 hours'1, about 0.3 hours'1, about 0.35 hours'1, about 0.4 hours'1, about 0.45 hours'1, or about 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 4.7. In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of between 0.2 - 0.25 hours'1, between 0.2 - 0.3 hours'1, between 0.2 - 0.35 hours'1, between 0.2 - 0.4 hours'1, between 0.2 - 0.45 hours'1, between 0.2 - 0.5 hours'1, between 0.25 - 0.3 hours'1, between 0.25 - 0.35 hours'1, between 0.25 - 0.4 hours'1, between 0.25 - 0.45 hours'1, between 0.25 - 0.5 hours'1, between 0.3 - 0.35 hours'1, between 0.3 - 0.4 hours'1, between 0.3 - 0.45 hours'1, between 0.3 - 0.5 hours'1, between 0.35 - 0.4 hours'1, between 0.35 - 0.45 hours'1, between 0.35 - 0.5 hours'1, between 0.4 - 0.45 hours'1, between 0.4 - 0.5 hours'1, or between 0.45 - 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 4.7. In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of about 0.3 hours'1(e.g., 0.28 hours'1) when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 4.7. In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of about 0.4 hours'1(e.g., 0.38 hours'1) when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 4.7. In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of about 0.25 hours'1when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 4.7.

[0167] In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of about 0.2 hours'1, about 0.25 hours'1, about 0.3 hours'1, about 0.35 hours'1, about 0.4 hours'1, about 0.45 hours'1, or about 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 5.2. In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has a growth rate of between 0.2 - 0.25 hours'1, between 0.2 - 0.3 hours'1, between 0.2 - 0.35 hours'1, between 0.2 - 0.4 hours'1, between 0.2 - 0.45 hours'1, between 0.2 - 0.5 hours'1, between 0.25 - 0.3 hours'1, between 0.25 - 0.35 hours'1, between 0.25 - 0.4 hours'1, between 0.25 - 0.45 hours'1, between 0.25 - 0.5 hours'1, between 0.3 - 0.35 hours'1, between 0.3 - 0.4 hours'1, between 0.3 - 0.45 hours'1, between 0.3 - 0.5 hours'1, between 0.35 - 0.4 hours'1, between 0.35 - 0.45 hours'1, between 0.35 - 0.5 hours'1, between 0.4 - 0.45 hours'1, between 0.4 - 0.5 hours'1, orbetween 0.45 - 0.5 hours'1when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 5.2. In some embodiments, an L. crispatus of the present disclosure has a growth rate of about 0.35 (e.g., 0.36) when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 5.2. In some embodiments, an L. crispatus of the present disclosure has a growth rate of about 0.4 hours'1when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 5.2. In some embodiments, an L. crispatus of the present disclosure has a growth rate of about 0.3 hours'1(e.g., 0.32 hours'1) when grown in a selective diffuse medium (e.g., MRS) at a starting pH of 5.2.

[0168] In some embodiments, an acidification rate is an in vitro acidification rate that is calculated after 24 hours of bacterial growth in culture. In some embodiments, an acidification rate is determined for a particular bacteria or consortia after an inoculation of MRS broth at 37 °C with 5 million CFU / mL. Acidification rates may be determined according to the following equation: Acidification rate equals natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0169] In some embodiments, an L. crispatus of the present disclosure has an acidification rate of about -0.003 hours'1, about -0.004 hours'1, about -0.006 hours'1, about - 0.007 hours'1, about -0.008 hours'1, about -0.009 hours'1, about -0.010 hours'1, about -0.011 hours'1, or about -0.12 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate between -0.003 to - 0.005 hours'1, between -0.003 to -0.006 hours'1, between -0.003 to -0.007 hours'1, between - 0.003 to -0.008 hours'1, between -0.003 to -0.009 hours'1, between -0.003 to -0.010 hours'1, between -0.003 to -0.011 hours'1, between -0.003 to -0.012 hours'1, between -0.004 to -0.006 hours'1, between -0.004 to -0.007 hours'1, between -0.004 to -0.008 hours'1, between -0.004 to -0.009 hours'1, between -0.004 to -0.010 hours'1, between -0.004 to -0.011 hours'1, between - 0.004 to -0.012 hours'1, between -0.005 to -0.007 hours'1, between -0.005 to -0.008 hours'1, between -0.005 to -0.009 hours'1, between -0.005 to -0.010 hours'1, between -0.005 to -0.011 hours'1, between -0.001 to -0.012 hours'1, between -0.006 to -0.008 hours'1, between -0.006 to -0.009 hours'1, between -0.006 to -0.010 hours'1, between -0.006 to -0.011 hours'1, between - 0.006 to -0.12 hours'1, between -0.007 to -0.009 hours'1, between -0.007 to -0.010 hours'1, between -0.007 to -0.011 hours'1, between -0.007 to -0.012 hours'1, between -0.008 to -0.010hours'1, between -0.008 to -0.011 hours'1, between -0.008 to -0.012 hours'1, between -0.009 to -0.011 hours'1, between -0.009 to -0.012 hours'1, or between -0.010 to -0.012 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of about -0.006 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of about -0.007 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of about -0.008 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of between -0.006 to -0.008 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 4.7), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0170] In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of about -0.003 hours'1, about -0.004 hours'1, about -0.006 hours'1, about -0.007 hours'1, about -0.008 hours'1, about -0.009 hours'1, about - 0.010 hours'1, about -0.011 hours'1, or about -0.12 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate between -0.003 to -0.005 hours'1, between -0.003 to -0.006 hours'1, between -0.003 to -0.007 hours'1, between -0.003 to -0.008 hours'1, between -0.003 to -0.009 hours'1, between - 0.003 to -0.010 hours'1, between -0.003 to -0.011 hours'1, between -0.003 to -0.012 hours'1, between -0.004 to -0.006 hours'1, between -0.004 to -0.007 hours'1, between -0.004 to -0.008 hours'1, between -0.004 to -0.009 hours'1, between -0.004 to -0.010 hours'1, between -0.004 to -0.011 hours'1, between -0.004 to -0.012 hours'1, between -0.005 to -0.007 hours'1, between - 0.005 to -0.008 hours'1, between -0.005 to -0.009 hours'1, between -0.005 to -0.010 hours'1, between -0.005 to -0.011 hours'1, between -0.001 to -0.012 hours'1, between -0.006 to -0.008 hours'1, between -0.006 to -0.009 hours'1, between -0.006 to -0.010 hours'1, between -0.006 to -0.011 hours'1, between -0.006 to -0.12 hours'1, between -0.007 to -0.009 hours'1, between - 0.007 to -0.010 hours'1, between -0.007 to -0.011 hours'1, between -0.007 to -0.012 hours'1, between -0.008 to -0.010 hours'1, between -0.008 to -0.011 hours'1, between -0.008 to -0.012 hours'1, between -0.009 to -0.011 hours'1, between -0.009 to -0.012 hours'1, or between - 0.010 to -0.012 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of about -0.007 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time). In some embodiments, an L. crispatus included in a microbial consortium of the present disclosure has an acidification rate of about -0.0011 hours'1when grown in a selective diffuse medium (e.g., MRS) over a period of 24 hours in culture (starting pH of 5.2), wherein the acidification rate is calculated using the following equation: natural logarithm (Ln) of (final pH divided by initial pH) divided by (final time minus initial time).

[0171] In some embodiments, an L. crispatus of the present disclosure thrives in the presence of L. jensenii bacteria. The term “thrives”, as used herein, refers to a bacteria (e.g., an L. crispatus isolate) that grows or propagates at a higher rate relative to a control bacteria when grown under the same conditions. A control bacteria can be, for example, a different isolate of the same species.

[0172] In some embodiments, an L. crispatus isolate is said to “thrive” if the L. crispatus isolate can grow and propagate in vitro or in vivo. In some embodiments, an L. crispatus isolate is said to “thrive” if the L. crispatus isolate has a growth rate that is at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% higher than the growth rate of a control L. crispatus (under the same conditions). In some embodiments, an L. crispatus isolate is said to “thrive” if the L. crispatus isolate has a growth rate of between 1% - 5%, between 1% - 10%, between 5% - 10%, between 5% - 15%, between 10% - 15%, between 10% - 20%, between 15% - 20%, between 15% - 25%, between 20% - 25%, between 1% - 25%, between 1% - 20%, between 5% - 25% , between 5% - 20%, or between 5% - 15% higher than the growth rate of a control L. crispatus (under the same conditions).

[0173] In some embodiments, the L. crispatus that thrives in the presence of L. jensenii has a growth rate that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% higher when grown in the presence of L. jensenii than the growth rate of a control L. crispatus grown in the presence of L. jensenii (under the same conditions). In some embodiments, the L. crispatus that thrives in the presence of L. jensenii has a growth rate of between 1% - 5%, between 1% - 10%, between 5% - 10%, between 5% - 15%, between 10% - 15%, between 10% - 20%, between 15% - 20%, between 15% - 25%, between 20% - 25%, between 1% - 25%, between 1% - 20%, between 5% - 25% , between 5% - 20%, or between 5% - 15% higher when grown in the presence of L. jensenii than the growth rate of a control L. crispatus grown in the presence of L. jensenii (under the same conditions). In some embodiments, a microbial consortium of the present disclosure contains one or more L. crispatus bacteria that thrive in the presence of L. jensenii. In some embodiments, a microbial consortium of the present disclosure contains 1, 2, 3, 4, or 5 L. crispatus bacteria that thrive in the presence of L. jensenii.

[0174] In some embodiments, the Lactobacillus crispatus isolate that thrives in the presence of L. jensenii bacteria is obtained from a bacterial sample deposited as DSM Deposit Number 34772.

[0175] In some embodiments, the Lactobacillus crispatus isolate that thrives in the presence of L. jensenii bacteria is derived or propagated from a bacterial sample deposited as DSM 34770. In some embodiments, the genome of the Lactobacillus crispatus isolate that thrives in the presence of L. jensenii bacteria, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%).

[0176] Aspects of this disclosure provide, in part, an L. crispatus bacteria that thrives in the presence of L. jensenii that is capable of engrafting the female genitourinary tract of a subject for at least a pre-determined period of time in the presence of an L. jensenii bacteria.

[0177] The engraftment of an L. crispatus that thrives in the presence of L. jensenii in the female genitourinary tract can persist over a pre-determined period of time in the presence of L. jensenii. In some embodiments, the engraftment of an L. crispatus that thrives in the presence of L. jensenii persists for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months. 1 year, 2 years, or more in the presence of L. jensenii. In some embodiments, the engraftment of an L. crispatus that thrives in the presence of L. jensenii persists between 1-7 days, between 1-2 weeks, between 1-3 weeks, between 1-4 weeks, between 2-4 weeks, between 1-2 months, between 2-4 months, between 4-6 months, between 6-8 months, between 8-10 months, between 10-12 months, between 1-6 months, between 6-12 months, between 3-9 months, between 6 months - 1 year, between 6 months - 2 years, or between 1-2 years in the presence of L. jensenii. The duration for which engraftment of an L. crispatus that thrives in the presence of L. jensenii persists in the presence of L. jensenii varies depending on several factors known to those of skill in the art, such as the specific medical condition being treated, individual variability (e.g., lifestyle and diet), and underlying causes of the condition.

[0178] In some embodiments, an L. crispatus isolate of the present disclosure thrives independent of the presence of L. jensenii. In other words, an L. crispatus isolate can, in some embodiments, thrive regardless of whether an L. jensenii is present. A bacteria (e.g. an L. crispatus isolate) that thrives independent of another bacteria (e.g., an L. jensenii isolate) is one that maintains a similar growth rate when grown in the presence or absence of the another bacteria (e.g., an L. jensenii isolate).

[0179] In some embodiments, the Lactobacillus crispatus isolate that thrives independent of the presence of L. jensenii bacteria is obtained from a bacterial sample deposited as DSM Deposit Number 34771. In some embodiments, the Lactobacillus crispatus isolate that thrives independent of the presence of L. jensenii bacteria is obtained from a bacterial sample deposited as DSM Deposit Number 34773.

[0180] In some embodiments, the Lactobacillus crispatus isolate that thrives independent of the presence of L. jensenii bacteria is derived or propagated from a bacterial sample deposited as DSM 34771. In some embodiments, the genome of the Lactobacilluscrispatus isolate that thrives independent of the presence of L. jensenii bacteria, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%).

[0181] In some embodiments, the Lactobacillus crispatus isolate that thrives independent of the presence of L. jensenii bacteria are derived or propagated from a bacterial sample deposited as DSM 34773. In some embodiments, the genome of the Lactobacillus crispatus isolate that thrives independent of the presence of L. jensenii bacteria, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%).

[0182] In some embodiments, a microbial consortium of the present disclosure contains one or more L. crispatus bacteria that thrive independent of the presence of L. jensenii. In some embodiments, a microbial consortium of the present disclosure contains 1, 2, 3, 4, or 5 L. crispatus bacteria that thrive independent of the presence of L. jensenii. In some embodiments, a microbial consortium of the present disclosure contains two L. crispatus bacteria (e.g., two different L. crispatus bacteria) that thrive independent of the presence of L. jensenii.

[0183] In some embodiments, each L. crispatus bacteria that thrives independent of the presence of L. jensenii is capable of engrafting the female genitourinary tract of the subject for at least a pre-determined period of time. The engraftment of an L. crispatus isolate that thrives independent of the presence of L. jensenii in the female genitourinary tract can persist over a pre-determined period of time. In some embodiments, the engraftment of an L. crispatus isolate that thrives independent of the presence of L. jensenii persists for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months. 1 year, 2 years, or more. In some embodiments, the engraftment of an L. crispatus isolate that thrives independent of the presence of L. jensenii persists between 1-7 days, between 1-2 weeks, between 1-3 weeks, between 1-4 weeks, between 2-4 weeks, between 1-2 months, between 2-4 months, between 4-6 months, between 6-8 months, between 8-10 months, between 10-12 months, between 1-6 months, between 6-12 months, between 3-9 months, between 6 months - 1 year, between 6 months - 2 years, or between 1-2 years. The duration for which engraftment of an L. crispatus that thrives independent of the presence of L. jensenii persists varies depending on several factors known to those of skill in the art, suchas the specific medical condition being treated, individual variability (e.g., lifestyle and diet), and underlying causes of the condition.

[0184] In some embodiments, the one or more L. crispatus isolates comprises a first and a second L. crispatus isolate. In some embodiments, the first L. crispatus isolates has a growth rate of about 0.28 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the first L. crispatus isolates has a growth rate of about 0.36 hours'1(e.g., when grown in selective medium starting at pH 5.2) In some embodiments, the first L. crispatus isolate has an acidification rate of about -0.006 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the first L. crispatus isolate has an acidification rate of about -0.007 hours'1(e.g., when grown in a selective medium starting at pH 5.2). In some embodiments, the second L. crispatus isolates has a growth rate of about 0.38 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the second L. crispatus isolate has a growth rate of about 0.4 hours'1(e.g., when grown in a selective medium starting at pH 5.2). In some embodiments, the second L. crispatus isolate has an acidification rate of about -0.008 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the second L. crispatus isolate has acidification rate of about -0.011 hours'1(e.g., when grown in a selective medium starting at pH 5.2).

[0185] In some embodiments, the one or more L. crispatus isolates comprises a first, a second, and a third L. crispatus isolate. In some embodiments, the first L. crispatus isolate has a growth rate of about 0.28 hours'1(e.g., when grown in a selective medium starting at pH 4.7) . In some embodiments, the first L. crispatus isolate has a growth rate of about 0.36 hours'1(e.g., when grown in selective medium starting at pH 5.2) In some embodiments, the first L. crispatus isolate has an acidification rate of about -0.006 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the first L. crispatus isolate has an acidification rate of about -0.007 hours'1(e.g., when grown in a selective medium starting at pH 5.2). In some embodiments, the second L. crispatus isolate has a growth rate of about 0.38 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the second L. crispatus isolate has a growth rate of about 0.4 hours'1(e.g., when grown in a selective medium starting at pH 5.2). In some embodiments, the second L. crispatus isolate has an acidification rate of about -0.008 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the second L. crispatus isolate has acidification rate of about -0.011 hours'1(e.g., when grown in a selective medium startingat pH 5.2). In some embodiments, the third L. crispatus isolate has a growth rate of about 0.25 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the third L. crispatus isolate has a growth rate of about 0.32 hours'1(e.g., when grown in a selective medium starting at pH 5.2). In some embodiments, the third L. crispatus isolate has an acidification rate of about -0.007 hours'1(e.g., when grown in a selective medium starting at pH 4.7). In some embodiments, the third L. crispatus isolate has acidification rate of about -0.011 hours'1(e.g., when grown in a selective medium starting at pH 5.2).

[0186] In some embodiments, different lactobacilli (e.g., L. crispatus, L. jensenii) included in microbial consortia of the present disclosure differ in their respective growth rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%. For example, the growth rate of an L. jensenii isolate may differ by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% relative to an L. crispatus isolate. In some embodiments, a first lactobacilli isolate (e.g., L. crispatus isolate, L. jensenii isolate) and a second lactobacilli isolate differ in their growth rates by between 1-2%, between 1-3%, between 1-4%, between1-5%, between 1-6%, between 1-7%, between 1-8%, between 1-9%, between 1-10%, between 1-11%, between 1-12%, between 1-13%, between 1-14%, between 1-15%, between2-3%, between 2-4%, between 2-5%, between 2-6%, between 2-7%, between 2-8%, between 2-9%, between 2-10%, between 2-11%, between 2-12%, between 2-13%, between 2-14%, between 2-15%, between 3-4%, between 3-5%, between 3-6%, between 3-7%, between 3- 8%, between 3-9%, between 3-10%, between 3-11%, between 3-12%, between 3-13%, between 3-14%, between 3-15%, between 4-5%, between 4-6%, between 4-7%, between 4- 8%, between 4-9%, between 4-10%, between 4-11%, between 4-12%, between 4-13%, between 4-14%, between 4-15%, between 5-6%, between 5-7%, between 5-8%, between 5- 9%, between 5-10%, between 5-11%, between 5-12%, between 5-13%, between 5-14%, between 5-15%, between 6-7%, between 6-8%, between 6-9%, between 6-10%, between 6- 11%, between 6-12%, between 6-13%, between 6-14%, between 6-15%, between 7-8%, between 7-9%, between 7-10%, between 7-11%, between 7-12%, between 7-13%, between 7-14%, between 7-15%, between 8-9%, between 8-10%, between 8-11%, between 8-12%, between 8-13%, between 8-14%, between 8-15%, between 9-10%, between 9-11%, between 9-12%, between 9-13%, between 9-14%, between 9-15%, between 10-11%, between 10-12%, between 10-13%, between 10-14%, between 10-15%, between 11-12%, between 11- 13%, between 11-14%, between 11-15%, between 12-13%, between 12-14%, between 12- 15%, between 13-14%, between 13-15%, or between 14-15%.

[0187] In some embodiments, the growth rate of a lactobacilli isolate refers to its growth rate at a pH of around 3, around 3.5, around 4, around 4.5, around 5, around 5.5, around 6, or around 6.5. In some embodiments, the growth rate of a lactobacilli isolate refers to its growth rate at a pH of between 3-6.5, between 3-5.5, between 3-5, between 3-4.5, between 3-4, between 3.5-5.5, between 3.5-5, or between 3.5-4.5. In some embodiments, the growth rate of a lactobacilli isolate refers to its growth rate at a pH of about 4.7 or 5.2. In some embodiments, the growth rate of a lactobacilli isolate refers to its growth rate at a temperature of around 5°C, around 10°C, around 15°C, around 20°C, around 25°C, around 30°C, around 35°C, around 40°C, around 45°C, around 50°C, or around 55°C. In some embodiments, the growth rate of a lactobacilli isolate refers to its growth rate at a temperature of between 5 °C - 15°C, between 10°C - 20°C, between 15°C - 25°C, between 20°C - 30°C, between 25°C - 35°C, between 30°C - 40°C, between 35°C - 44°C, between 40°C - 50°C, or between 45°C- 55°C. In some embodiments, the growth rate of a lactobacilli isolate refers to its growth rate at a temperature of about 37 °C.

[0188] In some embodiments, a Lactobacillus isolate of the present disclosure (e.g., L. jensenii isolate, L. crispatus isolate) is capable of utilizing glycogen. A bacteria that is capable of utilizing glycogen is one that possesses the enzymatic machinery necessary to break down glycogen (e.g., glycogen phosphorylase), enabling the bacteria to use glycogen as a source of energy and carbon for its metabolic processes. Lactobacilli that are capable of utilizing glycogen into glucose can then utilize glucose through fermentation pathways to produce lactic acid, which contributes to the maintenance of an acidic environment in the vagina (Amabebe E and Anumba DOC, The Vaginal Microenvironment: The Physiologic Role of lactobacilli, Front. Med. Vol. 8 (2018)). Methods to identify bacteria capable of utilizing glycogen are known to those in the art and may include, for example, culturing bacteria in a glycogen-rich agar or broth, performing a reducing sugar assay or growth inhibition assay, or evaluating polysaccharide breakdown products using electrospray ionization.

[0189] In some embodiments, a Lactobacillus isolate of the present disclosure (e.g., L. jensenii isolate, L. crispatus isolate) is capable of utilizing a variety of carbohydrate substrates in order to grow and propagate. A Lactobacillus bacteria may be capable ofutilizing D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, Methyl a D- glucopyranoside, N-acetylglucosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose (sucrose), D-trehalose, starch, glycogen, and / or gentiobiose in order to grow and propagate. In some embodiments, a L. jensenii bacteria is capable of utilizing D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D- mannitol, Methyl a D-glucopyranoside, N-acetylglucosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose (sucrose), D- trehalose, Starch, Glycogen, and / or Gentiobiose in order to grow and propagate. In some embodiments, a L. crispatus bacteria is capable of utilizing D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, Methyl a D-glucopyranoside, N-acetylglucosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-cellobiose, D-maltose, D-lactose, D- saccharose (sucrose), D-trehalose, Starch, Glycogen, and / or Gentiobiose in order to grow and propagate. A microbial consortia comprising one or more Lactobaccilus bacteria (e.g., L. jensenii, L. crispatus) can grow and propagate in the presence of D-ribose, D-galactose, D- glucose, D-fructose, D-mannose, D-mannitol, Methyl a D-glucopyranoside, N- acetylglucosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-cellobiose, D- maltose, D-lactose, D-saccharose (sucrose), D-trehalose, Starch, Glycogen, and / or Gentiobiose.

[0190] A Lactobacillus bacteria may include one or more genes that encode proteins that allow for the utilization of a variety of carbohydrate substrates. For example, a Lactobacillus bacteria may include one or more genes that encode proteins that allow for the utilization of D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, Methyl a D- glucopyranoside, N-acetylglucosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D- cellobiose, D-maltose, D-lactose, D-saccharose (sucrose), D-trehalose, Starch, Glycogen, and / or Gentiobiose.

[0191] In some embodiments, a Lactobacillus bacteria (e.g., a L. crispatus bacteria) includes the pulA gene (which encodes for pullanase) which allows for glycogen utilization in the bacteria. In some embodiments, a Lactobacillus bacteria (e.g., a L. crispatus bacteria) includes a full-length pulA gene (which encodes for pullanase) which allows for glycogen utilization in the bacteria. In some embodiments, a Lactobacillus bacteria (e.g., a L. crispatus bacteria) includes a pulA gene having a deletion or insertion in the pulA gene which renders the resulting protein at least partially dysfunctional.

[0192] Bacteria that possess the enzyme lactate dehydrogenase are capable of producing lactic acid (D-lactate and / or L-lactate), which results in the acidification of the environment and inhibits the growth of pathogenic bacteria and yeast in the vagina. Techniques to evaluate lactic acid production rates are known to those of skill in the art and may involve measuring the pH of a lactobacilli sample (e.g., a culture of lactobacilli) or by using a lactate assay kit (e.g., a colorimetric or fluorometric lactate assay kit) over time. In some embodiments, lactobacilli (e.g., L. crispatus, L. jensenii) of the present disclosure produces around 1 g / L, around 2 g / L, around 3 g / L, around 4 g / L, around 5 g / L, around 6 g / L, around 7 g / L, around 8 g / L, around 9 g / L, around 10 g / L, around 11 g / L, around 12 g / L, around 13 g / L, around 14 g / L, or around 15 g / L of lactic acid (D-lactate and / or L-lactate). In some embodiments, lactobacilli (e.g., L. crispatus, L. jensenii) of the present disclosure produces between 1-2 g / L, between 1-3 g / L, between 1-4 g / L, between 1-5 g / L, between 1-6 g / L, between 1-7 g / L, between 1-8 g / L, between 1-9 g / L, between 1-10 g / L, between 1-11 g / L, between 1-12 g / L, between 1-13 g / L, between 1-14 g / L, between 1-15 g / L, between 2-3 g / L , between 2-4 g / L, between 2-5 g / L, between 2-6 g / L, between 2-7 g / L, between 2-8 g / L, between 2-9 g / L, between 2-10 g / L, between 21-11 g / L, between 2-12 g / L, between 1-13 g / L, between 2-14 g / L, between 2-15 g / L, between 3-4 g / L, between 3-5 g / L, between 3-6 g / L, between 3-7 g / L, between 3-8 g / L, between 3-9 g / L, between 3-10 g / L, between 3-11 g / L, between 3-12 g / L, between 3-13 g / L, between 3-14 g / L, between 3-15 g / L, between 4-5 g / L, between 4-6 g / L, between 4-7 g / L, between 4-8 g / L, between 4-9 g / L, between 4-10 g / L, between 4-11 g / L, between 4-12 g / L, between 4-13 g / L, between 4-14 g / L, between 4-15 g / L, between 5-6 g / L, between 5-7 g / L, between 5-8 g / L, between 5-9 g / L, between 5-10 g / L, between 5-11 g / L, between 5-12 g / L, between 5-13 g / L, between 5-14 g / L, between 5-15 g / L, between 6-7 g / L, between 6-8 g / L, between 6-9 g / L, between 6-10 g / L, between 6-11 g / L, between 6-12 g / L, between 6-13 g / L, between 6-14 g / L, between 6-15 g / L, between 7-8 g / L, between 7-9 g / L, between 7-10 g / L, between 7-11 g / L, between 7-12 g / L, between 7-13 g / L, between 7-14 g / L, between 7-15 g / L, between 8-9 g / L, between 8-10 g / L, between 8-11 g / L, between 8-12 g / L, between 8-13 g / L, between 8-14 g / L, between 8-15 g / L, between 9-10 g / L, between 9-11 g / L, between 9-12 g / L, between 9-13 g / L, between 9-14 g / L, between 9-15 g / L, between 10-11 g / L, between 10-12 g / L, between 10-13 g / L, between 10-14 g / L, between 10-15 g / L, between 11-12 g / L, between 11-13 g / L, between 11-14 g / L, between 11-15 g / L, between 12-13 g / L, between 12-14 g / L, between 12-15 g / L, between 13-14 g / L, between 13-15 g / L, or between 14-15 g / L of lactic acid (D-lactate and / or L-lactate).

[0193] In some embodiments, lactobacilli (e.g., L. crispatus, L. jensenii) of the present disclosure produces lactic acid (D-lactate and / or L-lactate) at a rate of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1 gram of lactic acid (D-lactate and / or L-lactate) per gram of biomass per hour (g / g / h). In some embodiments, lactobacilli (e.g., L. crispatus, L. jensenii) of the present disclosure produces lactic acid (D-lactate and / or L-lactate) at a rate of between 0.1-0.2 g / g / h, between 0.1-0.3 g / g / h, between 0.1-0.4 g / g / h, between 0.1-0.5 g / g / h, between 0.1-0.6 g / g / h, between 0.1-0.7 g / g / h, between 0.1-0.8 g / g / h, between 0.1-0.9 g / g / h, between 0.1- 1.0 g / g / h, between 0.2-0.3 g / g / h, between 0.2-0.4 g / g / h, between 0.2-0.5 g / g / h, between 0.2-0.6 g / g / h, between 0.2-0.7 g / g / h, between 0.2-0.8 g / g / h, between 0.2-0.9 g / g / h, between 0.3-0.4 g / g / h, between 0.3-0.5 g / g / h, between 0.3-0.6 g / g / h, between 0.3-0.7 g / g / h, between 0.3-0.8 g / g / h, between 0.3-0.9 g / g / h, between 0.3- 1.0 g / g / h, between 0.4-0.5 g / g / h, between 0.4-0.6 g / g / h, between 0.4-0.7 g / g / h, between 0.4-0.8 g / g / h, between 0.4-0.9 g / g / h, between 0.4- 1.0 g / g / h, between 0.5-0.6 g / g / h, between 0.5-0.7 g / g / h, between 0.5-0.8 g / g / h, between 0.5-0.9 g / g / h, between 0.5-1.0 g / g / h, between 0.6-0.7 g / g / h, between 0.6-0.8 g / g / h, between 0.6-0.9 g / g / h, between 0.6-1.0 g / g / h, between 0.7-0.8 g / g / h, between 0.7-0.9 g / g / h, between 0.7- 1.0 g / g / h, between 0.8-0.9 g / g / h, between 0.8-1.0 g / g / h, or between 0.9-1.0 g / g / h.

[0194] Lactic acid can be composed of two enantiomers of lactate, D-lactate and L- lactate, and can be quantified using chiral chromatography (e.g., chiral high performance liquid chromatography). In some embodiments, D-lactate represents 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of total lactate. In some embodiments, D-lactate represents between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 55%, between 10% and 60%, between 10% and 65%, between 10% and 70%, between 10% and 75%, between 10% and 80%, between 10% and 85%, between 10% and 90%, between 10% and 95%, between 10% and 100%, between 25% and 30%, between 25% and 35%, between 50% and 90%, between 50% and 95%, between 50% and 100%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 55% and 75%, between 55% and 80%, between 55% and 85%, between 55% and 90%, between 55% and 95%, between 55% and 100%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 60% and 80%, between 60% and 85%, between 60% and 90%, between 60% and 95%, between 60% and 100%, between 65% and 70%, between 65% and 75%, between 65%and 80%, between 65% and 85%, between 65% and 90%, between 65% and 95%, between 65% and 100%, between 70% and 75%, between 70% and 80%, between 70% and 85%, between 70% and 90%, between 70% and 95%, between 70% and 100%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 75% and 95%, between 75% and 100%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 80% and 100%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between 90% and 100%, between 95% and 100% of total lactate. In some embodiments, L-lactate represents 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of total lactate. In some embodiments, L-lactate represents between 0% - 5%, between 0% - 10%, between 0% - 15%, between 0% - 20%, between 0% - 25%, between 0%- 30%, between 0% - 35%, between 0% - 40%, between 0% - 45%, between 0% - 50%, between 5% - 10%, between 5% - 15%, between 5% - 20%, between 5% - 25%, between 5%- 30%, between 5% - 35%, between 5% - 40%, between 5% - 45%, between 5% - 50%, between 10% - 15%, between 10% - 20%, between 10% - 25%, between 10% - 30%, between 10% - 35%, between 10% - 40%, between 10% - 45%, between 10% - 50%, between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15% - 35%, between 15% - 40%, between 15% - 45%, between 15% - 50%, between 20% - 25%, between 20% - 30%, between 20% - 35%, between 20% - 40%, between 20% - 45%, between 20% - 50%, between 25% - 30%, between 25% - 35%, between 25% - 40%, between 25% - 45%, between 25% - 50%, between 30% - 35%, between 30% - 40%, between 30% - 45%, between 30% - 50%, between 35% - 40%, between 35% - 45%, between 35% - 50%, between 40% - 45%, between 40% - 50%, or between 45% - 50% of total lactate.

[0195] The term “acidification rate,” as used herein, refers to the rate at which bacteria (e.g., lactobacilli) lower the pH (acidity) of their surrounding environment, e.g., through the production of lactic acid. The acidification rate can be monitored by measuring the change in pH over time (e.g., between 0-8 hours, between 8-24 hours). In some embodiments, the natural log of the acidification rate is about -0.020 hour'1, about -0.015 hour'1, about -0.010 hour'1, or about -0.005 hour'1. In some embodiments, the acidification rate is between -0.020 and -0.015 hour'1, between -0.020 and -0.010 hour'1, between -0.020 and -0.005 hour'1, between -0.020 and 0.000 hour'1, between -0.015 and -0.010 hour'1, between -0.015 and - 0.005 hour'1, between -0.015 and 0.000 hour'1, between -0.010 and -0.005 hour'1, between - 0.010 and 0.000 hour'1, or between -0.005 and 0.000 hour'1.

[0196] The terms “time to engraftment” and “successful engraftment at a predetermined time period,” as used herein, refer to the success and efficiency with which the transplanted bacteria (e.g., lactobacilli) become established (e.g., colonize and propagate) within a subject (e.g., a subject’s genitourinary tract, e.g., the cervix, vagina, uterus, and / or fallopian tubes of a subject). The “female genitourinary tract,” as used herein, refers to the female reproductive and urinary systems including ovaries, fallopian tubes, uterus, cervix vagina, external genitalia, and the urinary tract. Engraftment can be influenced by numerous factors, including the health of the subject’s vaginal environment, the quality and diversity of the transplanted bacteria (e.g., lactobacilli), and the specific method used in the transplantation procedure. The time to engraftment can be evaluated by assessing over time the subject’s clinical symptoms, vaginal pH, microbiome composition and diversity (e.g., using sequencing or 16S rRNA profiling), and / or abundance of lactobacilli (e.g., using qPCR).

[0197] In some embodiments, a Lactobacillus isolate of the present disclosure has a time to engraftment of about 6, 12, 18, 24, 36, 48, or 72 hours. In other words, a Lactobacillus isolate may, in some embodiments, engraft in the genitourinary tract of a subject within 6, 12, 18, 24, 36, 48, or 72 hours following administration of the microbial consortium to the subject. In some embodiments, a Lactobacillus isolate of the present disclosure has a time to engraftment of about 1-6, 5-10, 6-12, 10-15, 12-18, 15-20, 18-24, 20-30, 24-36, 30-40, 36-48, or 36-72 hours.

[0198] In some embodiments, at least one Lactobacillus isolate of a microbial consortium of the present disclosure will have a successful engraftment at a pre-determined time period in the genitourinary tract of a subject administration of the microbial consortium. In some embodiments, a pre-determined time period is about 6, 12, 18, 24, 36, 48, or 72 hours. In some embodiments, a pre-determined time period is about 1-6, 5-10, 6-12, 10-15, 12-18, 15-20, 18-24, 20-30, 24-36, 30-40, 36-48, or 36-72 hours.

[0199] In some embodiments, a microbial consortium of the present disclosure is comprised of at least two lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates) that differ in each of the functional characteristics (e.g., growth rate, capability of utilizing a carbon substrate (e.g., glycogen), rate of lactic acid production, acidification rate, and time to engraftment in the female genitourinary tract). In some embodiments, a microbial consortium of the present disclosure is comprised of at least two L. crispatus isolates that differ in each of the functional characteristics (e.g., growth rate, capability of utilizing a carbon substrate (e.g.,glycogen), rate of lactic acid production, acidification rate, and time to engraftment in the female genitourinary tract).

[0200] In some embodiments, a microbial consortium of the present disclosure is comprised of at least two lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates) that differ in their growth rates. In some embodiments, a microbial consortium of the present disclosure is comprised of L. jensenii and at least one L. crispatus that differ in their growth rates. In some embodiments, a microbial consortium of the present disclosure is comprised of at least two L. crispatus that differ in their growth rates.

[0201] In some embodiments, at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) of the present disclosure differ in their acidification rates. In some embodiments, a first lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) has an acidification rates that is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 35%, at least 50%, or at least 75% higher than the acidification rate of a second lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate). In some embodiments, a first lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) has an acidification rates that is between 1-2%, between 1-3%, between 1- 4%, between 1-5%, between 1-6%, between 1-7%, between 1-8%, between 1-9%, between 1- 10%, between 1-11%, between 1-12%, between 1-13%, between 1-14%, between 1-15%, between 4-10%, between 4-11%, between 4-12%, between 4-13%, between 4-14%, between 4-15%, between 5-6%, between 5-7%, between 5-8%, between 5-9%, between 5-10%, between 5-11%, between 5-12%, between 5-13%, between 5-14%, between 5-15%, between 7-12%, between 7-13%, between 7-14%, between 7-15%, between 8-9%, between 8-10%, between 8-11%, between 8-12%, between 8-13%, between 8-14%, between 8-15%, between 9-15%, between 10-11%, between 10-12%, between 10-13%, between 10-14%, between 10- 15%, between 11-12%, between 11-13%, between 11-14%, between 11-15%, between 12- 13%, between 12-14%, between 12-15%, between 13-14%, between 13-15%, or between 10- 40% higher than the acidification rate of a second lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate).

[0202] In some embodiments, the acidification rates are acidification rates in vitro (e.g., in culture) and / or in vivo.

[0203] In some embodiments, at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) of the present disclosure differ in their capability of utilizing glycogen. Insome embodiments, the at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) differ in their capability of utilizing glycogen by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%. In some embodiments, the at least two lactobacilli (e.g., L. jensenii isolate, L. crispatus isolate) differ in their capability of utilizing glycogen by between 1-2%, between 1-3%, between 1-4%, between 1-5%, between 1-6%, between 1-7%, between 1-8%, between 1-9%, between 1-10%, between 1-11%, between 1-12%, between 1-13%, between 1-14%, between 1-15%, between 2-3%, between 2-4%, between 2-5%, between 2- 6%, between 2-7%, between 2-8%, between 7-15%, between 8-9%, between 8-10%, between 8-11%, between 8-12%, between 8-13%, between 8-14%, between 8-15%, between 9-10%, between 9-11%, between 9-12%, between 9-13%, between 9-14%, between 9-15%, between 10-11%, between 10-12%, between 10-13%, between 10-14%, between 10-15%, between 11- 12%, between 11-13%, between 11-14%, between 11-15%, between 12-13%, between 12- 14%, between 12-15%, between 13-14%, between 13-15%, or between 10-40%.

[0204] In some embodiments, at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) of the present disclosure have different carbon utilization profiles. In some embodiments, the carbon utilization profile is selected from the group consisting of: a glycogen utilization profile; a glucose utilization profile; a fructose utilization profile; a mannose utilization profile; a maltodextrin utilization profile; and a mannitol utilization profile.

[0205] In some embodiments, the capability of utilizing glycogen is a capability of utilizing glycogen in vitro (e.g., in culture) and / or in vivo.

[0206] In some embodiments, the capability of utilizing mannitol and glucose is a capability of utilizing mannitol and glucose in vitro (e.g., in culture) and / or in vivo.

[0207] In some embodiments, the capability of utilizing glycogen, glucose, fructose, mannose, maltodextrin, or mannitol is a capability of utilizing glycogen, glucose, fructose, mannose, maltodextrin, or mannitol in vitro (e.g., in culture) and / or in vivo.

[0208] In some embodiments, at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) of the present disclosure differ in their lactic acid production rates. In some embodiments, the at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) differ in their lactic acid production rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%. In some embodiments, the at least two lactobacilli (e.g., L. crispatusisolate, L. jensenii isolate) differ in their lactic acid production rates by between 1-2%, between 1-3%, between 1-4%, between 1-5%, between 1-6%, between 1-7%, between 1-8%, between 1-9%, between 1-10%, between 1-11%, between 1-12%, between 1-13%, between 1-14%, between 1-15%, between 2-3%, between 2-4%, between 2-5%, between 2-6%, between 2-7%, between 2-8%, between 2-9%, between 2-10%, between 2-11%, between 2- 12%, between 2-13%, between 2-14%, between 2-15%, between 3-4%, between 3-5%, between 3-6%, between 7-10%, between 7-11%, between 7-12%, between 7-13%, between 7-14%, between 7-15%, between 8-9%, between 8-10%, between 8-11%, between 8-12%, between 8-13%, between 8-14%, between 8-15%, between 9-10%, between 9-11%, between 9-12%, between 9-13%, between 9-14%, between 9-15%, between 10-11%, between 10- 12%, between 10-13%, between 10-14%, between 10-15%, between 11-12%, between 11- 13%, between 11-14%, between 11-15%, between 12-13%, between 12-14%, between 12- 15%, between 13-14%, between 13-15%, or between 14-15%.

[0209] In some embodiments, at least two L. crispatus of the present disclosure differ in their lactic acid production rate. In some embodiments, the at least two L. crispatus differ in their lactic acid production rate by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%. In some embodiments, the at least two L. crispatus differ in their lactic acid production rate by between 1-2%, between 1-3%, between 1-4%, between 1-5%, between 1- 6%, between 1-7%, between 1-8%, between 1-9%, between 1-10%, between 1-11%, between 1-12%, between 1-13%, between 1-14%, between 1-15%, between 2-3%, between 2-4%, between 2-5%, between 2-6%, between 2-7%, between 2-8%, between 2-9%, between 2- 10%, between 2-11%, between 2-12%, between 6-13%, between 6-14%, between 6-15%, between 7-8%, between 7-9%, between 7-10%, between 7-11%, between 7-12%, between 7- 13%, between 7-14%, between 7-15%, between 8-9%, between 8-10%, between 8-11%, between 8-12%, between 8-13%, between 8-14%, between 8-15%, between 9-10%, between 9-11%, between 9-12%, between 9-13%, between 9-14%, between 9-15%, between 10-11%, between 10-12%, between 10-13%, between 10-14%, between 10-15%, between 11-12%, between 11-13%, between 11-14%, between 11-15%, between 12-13%, between 12-14%, between 12-15%, between 13-14%, between 13-15%, or between 14-15%.

[0210] In some embodiments, the lactic acid production rate is a lactic acid production rate in vitro (e.g., in culture) and / or in vivo.

[0211] In some embodiments, a rate of lactic acid production (e.g., rate of D-lactic acid, rate of L-lactic acid) is an in vitro rate that is calculated after 24 hours of bacterial growth in culture. In some embodiments, a rate of lactic acid production is determined for a particular bacteria or consortia after an inoculation of MRS broth at 37 °C with 5 million CFU / mL.Rates of D-lactic acid production were determined according to the following equation: Rate of D-lactic acid production equals natural logarithm (Ln) of (final concentration of D-lactic acid divided by initial concentration of D-lactic acid) divided by (final time minus initial time). Rates of L-lactic acid production were determined according to the following equation: Rate of L-lactic acid production equals natural logarithm (Ln) of (final concentration of L- lactic acid divided by initial concentration of L-lactic acid) divided by (final time minus initial time).

[0212] In some embodiments, at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) of the present disclosure differ in their time to engraftment in the female genitourinary tract. In some embodiments, the at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) differ in their time to engraftment in the female genitourinary tract by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In some embodiments, the at least two lactobacilli (e.g., L. crispatus isolate, L. jensenii isolate) differ in their time to engraftment in the female genitourinary tract by between 1-6, 5-10, 6-12, 10-15, 12-18, 15- 20, 18-24, 20-30, or 24-36 hours.

[0213] In some embodiments, a L. jensenii and at least one L. crispatus of the present disclosure differ in their time to engraftment in the female genitourinary tract. In some embodiments, the L. jensenii and the at least one L. crispatus differ in their time to engraftment in the female genitourinary tract by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In some embodiments, the L. jensenii and the at least one L. crispatus differ in their time to engraftment in the female genitourinary tract by between 1-6, 5-10, 6-12, 10- 15, 12-18, 15-20, 18-24, 20-30, or 24-36 hours.

[0214] In some embodiments, at least two L. crispatus of the present disclosure differ in their time to engraftment in the female genitourinary tract. In some embodiments, the at least two L. crispatus differ in their time to engraftment in the female genitourinary tract by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In some embodiments, the at least two L. crispatus differ in their time to engraftment in the female genitourinary tract by 1-6, 5-10, 6-12, 10-15, 12-18, 15-20, 18-24, 20-30, or 24-36 hours.

[0215] In some embodiments, the time to engraftment in the female genitourinary tract is determined in vivo.

[0216] In some embodiments, each Lactobacillus isolate of the consortium is obtained from a healthy human donor. The lactobacilli comprised in the defined population may be obtained from at least two different healthy human donors (e.g., 2, 3, 4, 5, or more healthy human donors). In some embodiments, the lactobacilli comprised in the defined population are obtained from three different healthy human donors.

[0217] A Lactobacillus isolate of a microbial consortium may be obtained from the vaginal environment of a healthy human donor, e.g., a human donor, e.g., a female donor, with a eubiotic vaginal microbiome, e.g., a LactoZ?acz7ZM5-dominated microbiome. A Lactobacillus isolate of a microbial consortium may be obtained using the steps as provided in FIG. 1. For example, in some embodiments, a Lactobacillus isolate of a microbial consortia is obtained by (1) administering a vaginal microbiome transplant (VMT) (comprising a substantially complete vaginal microbiome preparation (SCVMP) derived from a single healthy female donor) to the female genitourinary tract of a recipient subject, (2) collecting a sample from the genitourinary tract of the recipient subject and culturing the sample, and (3) collecting an isolate from the cultured sample. In some embodiments, a VMT is as described in DeLong, K. et al., Front Cell Infect Microbiol. 2019 Aug 28;9:306, the content of which is incorporated herein by reference. In some embodiments, a VMT is as described in Lev-Sagie, A. et al., Nat Med. 2019 0ct;25(10):1500-1504, the content of which is incorporated herein by reference. In some embodiments, a VMT is as described in Bosma, E. F. et al. Antibiotic-free vaginal microbiota transplantation (VMT) changes vaginal microbiota and immune profile in women with asymptomatic dysbiosis: reporting of a randomized, placebo-controlled trial. medRxiv (Cold Spring Harbor Laboratory) (2024), the content of which is incorporated herein by reference.

[0218] In some embodiments, a Lactobacillus isolate of a microbial consortium is obtained by (1) administering a substantially complete vaginal microbiome preparation derived from a single healthy female donor to the female genitourinary tract of a recipient subject, (2) collecting a sample from the genitourinary tract of the recipient subject and culturing the sample, and (3) collecting an isolate from the cultured sample.Microbial consortia

[0219] Aspects of this disclosure provide, in part, microbial consortia comprised of a defined population of isolated lactobacilli (e.g., L. crispatus, L. jensenii) configured to promote engraftment in a female genitourinary tract of a subject. In some embodiments, the defined population of isolated lactobacilli (e.g., L. crispatus, L. jensenii) configured to promote engraftment in a female genitourinary tract of a subject comprises an L. jensenii bacteria, an L. crispatus bacteria that thrives in the presence of the L. jensenii bacteria, and at least one additional L. crispatus bacteria that thrives independent of the presence of the L. jensenii bacteria. In some embodiments, the at least one L. crispatus bacteria that thrives independent of the presence of the L. jensenii bacteria is capable of engrafting the female genitourinary tract of the subject for at least a pre-determined of time.

[0220] A microbial consortium of the present disclosure is designed, in some embodiments, to include specific Lactobacillus isolates having a diverse set of functional and genomic characteristics. A microbial consortium as described herein and having a diverse set of functional and genomic characteristics can engraft in the female genitourinary tract of a diverse set of subjects, each of whom have different vaginal environments. For example, a microbial consortium of the disclosure may include two or more Lactobacillus isolates, wherein each isolate has different functional characteristics (e.g., different growth rates, different acidification rates, different carbohydrate utilization profiles, different rates of engraftment). A microbial consortium of the disclosure may include two or more Lactobacillus isolates, wherein each isolate has different genomic characteristics. For example, in determining a set of two or more Lactobacillus isolates to be included in a microbial consortium, in silico analysis can be used to identify a species-level pangenome comprising genes that are linked to engraftment (and beneficial effect on a subject, e.g., reduction of (local) inflammation, e.g., in the genitourinary tract). In some embodiments, in silico analysis can be used to identify a Lactobacillus crispatus pangenome comprising genes that are linked to engraftment (and beneficial effect on a subject, e.g., reduction of (local) inflammation, e.g., in the genitourinary tract). Isolates can be selected for inclusion in the microbial consortium such that a combination of isolates provides maximal coverage of a preselected portion (or all) of the pangenome (e.g., the genes of the pangenome that are linked to engraftment). In other words, a combination of isolates can be combined into a microbial consortium such that the consortium encompasses a preselected portion (or all) ofthe pangenome (e.g., the genes of the pangenome that are linked to engraftment) even though any single isolate might not encompass the preselected portion by itself. In some embodiments, a microbial consortium is generated to encompass a maximal portion of the pangenome with the minimum number of necessary Lactobacillus isolates. Maximal coverage of a pangenome (e.g., a Lactobacillus crispatus engrafting pangenome) within a microbial consortium allows for the consortium to be capable of successfully engrafting a diverse set of vaginal environments (e.g., belonging to diverse set of subjects).

[0221] In some embodiments, an exemplary microbial consortium of the disclosure is comprised of a Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770; a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771; a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772; and a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

[0222] In some embodiments, an exemplary microbial consortium of the disclosure is comprised of:(a) Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%);(b) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%);(c) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%); and

[0223] (d) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value (e.g., a threshold value of 99.81%, 99.90%, or 99.96%).

[0224] In some embodiments, the microbial consortium is an n-mcmbcr consortium comprising n bacterial isolates, in which n is an integer reflecting the number of different bacterial isolates in the consortium. In some embodiments, n is at least 2, at least 3 or at least 4. In some embodiments, n is in the range of 2 to 10, 2 to 6, or 2 to 4. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the microbial consortium is a 2- member consortium comprising two bacterial isolates. In some embodiments, the microbialconsortium is a 3-member consortium comprising three bacterial isolates. In some embodiments, the microbial consortium is a 4-member consortium comprising four bacterial isolates.

[0225] In some embodiments, the defined population of isolated lactobacilli is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and an additional L. crispatus that thrives independent of the presence of the L. jensenii, wherein the relative abundance of the L. jensenii is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells, and the additional L. crispatus that thrives independent of the presence of the L. jensenii is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of the total viable cells. In some embodiments, the defined population of isolated lactobacilli is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and an additional L. crispatus that thrives independent of the presence of the L. jensenii, wherein the relative abundance of the L. jensenii is between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15% - 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, or between 30% - 35% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15% - 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, or between 30% - 35% of the total viable cells, and the additional L. crispatus that thrives independent of the presence of the L. jensenii is between 30% - 35%, between 30% - 40%, between 30% - 45%, between 30% - 50%, between 30% - 55%, between 30% - 60%, between 30% - 65%, between 30% - 70%, between 35% - 40%, between 35% - 45%, between 35% - 50%, between 35% - 55%, between 35% - 60%, between 35% - 65%, between 35% - 70%, between 40% - 45%, between 40% - 50%, between 40% - 55%, between 40% - 60%, between 40% - 65%, between 40% - 70%, between 45% - 50%, between 45% - 55%, between 45% - 60%, between 45% - 65%, between 45% - 70%, between 50% - 55%, between 50% - 60%, between 50% - 65%, between 50% - 70%, between 55% - 60%, between 55% - 65%, between 55% - 70%, between 60% - 65%, between 60% - 70%, or between 65% - 75% of the total viable cells.

[0226] In some embodiments, the defined population of isolated lactobacilli is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, andan additional L. crispatus that thrives independent of the presence of the L. jensenii, wherein the relative abundance of the L. jensenii is about 25% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii about 25% of the total viable cells, and the additional L. crispatus that thrives independent of the presence of the L. jensenii is about 50% of the total viable cells.

[0227] Relative abundance of Lactobacillus isolates within the defined population can be determined using an absorbance measurement (e.g. OD600 measurement of isolates in culture), a viable cell counting methodology, or by weight (e.g., weight of lyophilized isolates).

[0228] In some embodiments, the defined population of isolated lactobacilli is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and an additional L. crispatus that thrives independent of the presence of the L. jensenii, the L. jensenii comprises a plurality of L. jensenii, the L. crispatus that thrives in the presence of the L. jensenii comprises a plurality of L. crispatus that thrives in the presence of the L. jensenii, and the at least one additional L. crispatus that thrives independent of the presence of the L. jensenii comprises a plurality of L. crispatus that thrives independent of the presence of the L. jensenii.

[0229] In some aspects, this disclosure provides a microbial consortium comprising a defined population of isolated lactobacilli configured to promote engraftment in a female genitourinary tract of a subject. In some embodiments, the defined population comprises an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two L. crispatus have different growth rates (e.g., different growth rates in vitro).

[0230] In some embodiments, at least two of the L. crispatus differ in their growth rates, capability of utilizing glycogen, rate of producing lactic acid, acidification rate, and / or time to engraftment in the female urinary tract.

[0231] In some embodiments, the defined population comprises an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two L. crispatus have different growth rates, wherein the relative abundance of the L. jensenii is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells, and the relative abundance of the at least two additional L.crispatus is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells. In some embodiments, the defined population comprises an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two L. crispatus have different growth rates, wherein the relative abundance of the L. jensenii is between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15%- 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, between 25% - 30%, between 25% - 35%, or between 30% - 35% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15%- 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, between 25% - 30%, between 25% - 35%, or between 30% - 35% of the total viable cells, and the relative abundance of the at least two additional L. crispatus is between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15%- 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, between 25% - 30%, between 25% - 35%, or between 30% - 35% of the total viable cells. In some embodiments, the defined population comprises an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two L. crispatus have different growth rates, wherein the relative abundance of the L. jensenii is about 25% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is about 25% of the total viable cells, and the relative abundance of the at least two additional L. crispatus is about 50% of the total viable cells.

[0232] In some embodiments, the defined population comprises an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two L. crispatus have different growth rates, the L. jensenii comprises a plurality of L. jensenii, the L. crispatus that thrives in the presence of the L. jensenii comprises a plurality of L. crispatus that thrives in the presence of the L. jensenii, and each of the at least two additional L. crispatus comprises a plurality of L. crispatus.

[0233] Aspects of this disclosure provide, in part, a microbial consortium comprising a defined population of isolated lactobacilli (e.g., L. jensenii, L. crispatus) configured to promote engraftment in a female genitourinary tract (e.g., cervix, vagina, uterus, and / or fallopian tubes). In some embodiments, the defined population of isolated lactobacilli (e.g., L. jensenii, L. crispatus) configured to promote engraftment in a female genitourinary tract is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two additional L. crispatus havedifferent acidification rates. In some embodiments, the difference in acidification rates between the at least two additional L. crispatus is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%. In some embodiments, the difference in acidification rates between the at least two additional L. crispatus is between 1-10%, between 1-15%, between 1-20%, between 5-10%, between 5-15%, between 5-20%, between 10-20%, between 10- 30%, between 10-40%, or between 10-50%.

[0234] In some embodiments, the defined population is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two additional L. crispatus have different acidification rates, at least two L. crispatus differ in their growth rates, capability of utilizing glycogen, rate of lactic acid production, and / or time to engraftment in the female genitourinary tract.

[0235] In some embodiments, the defined population is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two additional L. crispatus have different acidification rates, the relative abundance of L. jensenii is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells, the relative abundance of L. crispatus that thrives in the presence of the L. jensenii is about 15%, about 20%, about 25%, about 30%, or about 35% of the total viable cells, and the relative abundance of the at least two additional L. crispatus bacteria is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of the total viable cells. In some embodiments, the defined population is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two additional L. crispatus have different acidification rates, the relative abundance of L. jensenii is between 15% - 20%, between 15% - 25%%, between 15% - 30%, between 15% - 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, between 25% - 30%, between 25% - 35%, or between 30% - 35% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15% - 35%, between 20% - 25%, between 20% - 30%, between 20% - 35%, between 25% - 30%, between 25% - 35%, or between 30% - 35% of the total viable cells, and the relative abundance of the at least two additional L. crispatus is between 30% - 35%, between 30% - 40%, between 30% - 45%, between 30% - 50%, between 30% - 55%, between 30% - 60%, between 30% - 65%, between 30% - 70%, between 35% - 40%,between 35% - 45%, between 35% - 50%, between 35% - 55%, between 35% - 60%, between 35% - 65%, between 35% - 70%, between 40% - 45%, between 40% - 50%, between 40% - 55%, between 40% - 60%, between 40% - 65%, between 40% - 70%, between 45% - 50%, between 45% - 55%, between 45% - 60%, between 45% - 65%, between 45% - 70%, between 50% - 55%, between 50% - 60%, between 50% - 65%, between 50% - 70%, between 55% - 60%, between 55% - 65%, between 55% - 70%, between 60% - 65%, between 60% - 70%, or between 65% -70% of the total viable cells. In some embodiments, the defined population is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two additional L. crispatus have different acidification rates, the relative abundance of the L. jensenii is about 25% of the total viable cells, the relative abundance of the L. crispatus that thrives in the presence of the L. jensenii is about 25% of the total viable cells, and the relative abundance of the at least two L. crispatus is about 50% of the total viable cells.

[0236] In some embodiments, the defined population is comprised of an L. jensenii, an L. crispatus that thrives in the presence of the L. jensenii, and at least two additional L. crispatus, wherein the at least two additional L. crispatus have different acidification rates, the L. jensenii comprises a plurality of L. jensenii, the L. crispatus that thrives in the presence of the L. jensenii comprises a plurality of L. crispatus that thrives in the presence of the L. jensenii, and each of the at least two additional L. crispatus comprises a plurality of L. crispatus.

[0237] In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their growth rates. In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their growth rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0238] In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their growth rates. In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their growth rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0239] In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their growth rates. In some embodiments, at least two L. crispatusof a microbial consortium described herein differ in their growth rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0240] In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their acidification rates. In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their acidification rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0241] In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their acidification rates. In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their acidification rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0242] In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their acidification rates. In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their acidification rates by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0243] In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their capability of utilizing glycogen. In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their capability of utilizing glycogen by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0244] In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their capability of utilizing glycogen. In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their capability of utilizing glycogen by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0245] In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their capability of utilizing glycogen. In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their capability of utilizing glycogen by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0246] In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their rate of lactic acid production. In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their rate of lactic acid production by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0247] In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their rate of lactic acid production. In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their rate of lactic acid production by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0248] In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their rate of lactic acid production. In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their capability of rate of lactic acid production by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25%.

[0249] In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their time to engraftment in the female genitourinary tract. In some embodiments, at least two lactobacilli (e.g., L. crispatus, L. jensenii) of a microbial consortium described herein differ in their time to engraftment in the female genitourinary tract by about 1-6, 5-10, 6-12, 10-15, 12-18, 15-20, 18-24, 20-30, or 24- 36 hours.

[0250] In some embodiments, an L. jensenii and at least one L. crispatus of a microbial consortium described herein differ in their time to engraftment in the female genitourinary tract. In some embodiments, an L. jensenii and at least one L. crispatus of a microbialconsortium described herein differ in their time to engraftment in the female genitourinary tract by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

[0251] In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their time to engraftment in the female genitourinary tract. In some embodiments, at least two L. crispatus of a microbial consortium described herein differ in their time to engraftment in the female genitourinary tract by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

[0252] In some embodiments, the isolates of the microbial consortium engraft equally in the female genitourinary tract of the subject. In some embodiments, each of the isolates of the microbial consortium engraft in the female genitourinary tract of the subject. In some embodiments, one, two, three, or four isolates of the microbial consortium engraft in the female genitourinary tract of the subject. In some embodiments, the K jensenii of the microbial consortium is the first bacteria of the consortium to engraft in the female genitourinary tract of the subject.

[0253] In some embodiments, at least one of the lactobacilli bacteria of the microbial consortium is capable of engrafting the female genitourinary tract of the subject within 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 24, 36, or 48 hours after administration of the consortium to the female genitourinary tract of the subject. In some embodiments, at least two or at least three of the lactobacilli bacteria of the microbial consortium is capable of engrafting the female genitourinary tract of the subject within 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 24, 36, or 48 hours after administration of the consortium to the female genitourinary tract of the subject.

[0254] Engraftment of the female genitourinary tract can be quantified, e.g., by counting the number of colony forming units (CFU) / gram and / or performing nucleic acid sequencing of microbes comprised in one or more samples of the female genitourinary tract. In some embodiments, engraftment of the female genitourinary tract is successful if at least one (at least two, or at least three) of the lactobacilli bacteria is detected within the female genitourinary tract after a certain period of time (e.g., 6, 12, 24, 36, 48, or 72 hours). In some embodiments, engraftment of the female genitourinary tract is successful if the relative abundance of at least one of the lactobacilli bacteria is at least 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% of the total microbial abundance in the genitourinary tract. In some embodiments, the integration of transplanted bacteria (e.g., a microbial consortium or individual lactobacilli isolates) into a subject’s existing microbialcommunity represents engraftment. The success of engraftment can be assessed through microbiome analysis (e.g., 16S rRNA sequencing, metagenomic sequencing, or qPCR) or laboratory testing (e.g., culture).

[0255] In some embodiments, at least one of the lactobacilli bacteria of the exemplary microbial consortium described herein is capable of persisting within the female genitourinary tract of the subject for at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, 25, 30, or 35 days after administration to the female genitourinary tract of the subject. In some embodiments, at least one of the lactobacilli bacteria of the exemplary microbial consortium described herein is capable of persisting within the female genitourinary tract of the subject for at least 1, 2, 3, 4, 5, 6, or 8 weeks after administration to the female genitourinary tract of the subject.

[0256] In some embodiments, 1, 2, 3, or 4 of the lactobacilli bacteria of the exemplary microbial consortium described herein is capable of persisting within the female genitourinary tract of the subject for at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, 25, 30, or 35 days after administration to the female genitourinary tract of the subject. In some embodiments, 1, 2, 3, or 4 of the lactobacilli bacteria of the exemplary microbial consortium described herein is capable of persisting within the female genitourinary tract of the subject for at least 1, 2, 3, 4, 5, 6, or 8 weeks after administration to the female genitourinary tract of the subject.

[0257] In some embodiments, the lactobacilli of the consortium are capable of competing with the resident microbiota (e.g., resident lactobacilli and resident nonlactobacilli) within the female genitourinary tract of the subject, and, in embodiments, successfully engraft. In some embodiments, the lactobacilli of the consortium are capable of competing with the resident microbiota (e.g., resident lactobacilli and resident nonlactobacilli) within the female genitourinary tract of the subject for nutrients (e.g., carbohydrates, vitamins), space in the genitourinary tract, and / or growth, and, in embodiments, successfully engraft.

[0258] In some embodiments, the resident microbiota (e.g., resident lactobacilli and resident non-lactobacilli) within the female genitourinary tract of the subject is at least partially removed by a local antiseptic or antibiotic treatment, e.g., for 1, 2, 3, 4, 5, or 6 days, or 1, 2, or 3 weeks prior to administration of the microbial consortium, e.g., to provide space within the genitourinary tract for engraftment. In some embodiments, an antiseptic is an antiseptic solution. An antiseptic solution may comprise chlorhexidine and / or povidone-iodine. In some embodiments, an antiseptic solution comprises about 10% or 5-10% povidone-iodine. In some embodiments, an antiseptic solution comprises about 0.5% chlorhexidine. In some embodiments, an antibiotic is metronidazole or clindamycin.

[0259] The microbial consortium may include at least 1 million colony forming units (CFUs), at least 5 million CFU, at least 6 million CFU, at least 7 million CFU, at least 8 million CFU, or at least 10 million CFU of total bacteria. The microbial consortium may include at least 1 million CFU / mL, at least 5 million CFU / mL, at least 6 million CFU / mL, at least 7 million CFU / mL, at least 8 million CFU / mL, at least 9 million CFU / mL, or at least 10 million CFU / mL of total bacteria. In some embodiments, the microbial consortium includes 1-100 million colony forming units (CFUs), 20-100 million CFU, 50-100 million CFU, or 50- 200 million CFU. In some embodiments, the microbial consortium comprises at least 107, 108, 109, 1010, 1011or 1012CFU of total bacteria. In some embodiments, the microbial consortium comprises at least 107, 108, 109, 1010, 1011or 1012CFU / mL of total bacteria.

[0260] In some embodiments, at least a subset of the lactobacilli comprised in the microbial consortia or related compositions described herein is capable of engrafting in a human vagina. In some embodiments, at least a subset of the individual lactobacilli isolates (e.g., L. crispatus isolates, L. jensenii isolates) described herein is capable of engrafting in a human vagina. In some embodiments, engraftment occurs even during menstrual discharge. In some embodiments, engraftment is substantially maintained (stable) after one or more menstrual discharge. The lactobacilli comprised in the microbial consortia, the individual lactobacilli isolates (e.g., L. crispatus isolates, L. jensenii isolates) described herein, or related compositions may continue to reside in the vagina after administration over several menstruation cycles. In some embodiments, the lactobacilli engraft in the vaginal microbial niche over one or more than one (e.g., two, three, four, five or six) menstruation cycle upon vaginal administration. Residence time can be determined, e.g., using nucleic acid sequencing, e.g., for specific lactobacilli, e.g., comparing sequencing results prior to and post administration of the microbial consortia, individual lactobacilli isolates (e.g., L. crispatus isolates, L. jensenii isolates), or related compositions described herein, e.g., to determine the identity of newly added members (e.g., one or more lactobacilli) to the subject’s microbial community, and then at predetermined time intervals (e.g., prior or post a menstruation cycle, optionally, over a number of menstruation cycles) determine (e.g., through nucleic acid sequencing) that all or a certain subset of the newly added members are still substantially present at the specific time interval. In some embodiments, the residence time of the subject's microbiota may be compared to the donor's microbiota. The residence time may vary depending on various factors including hormone levels (e.g., estrogen), diet, sexual activity, acidity status of the vagina, and the presence or absence of genital infections and other microbial perturbations, e.g., treatment with antibiotics.

[0261] Successful engraftment of the lactobacilli comprised in the microbial consortia or related compositions, or of the individual lactobacilli isolates (e.g., L. crispatus isolates, L. jensenii isolates) described herein, may be indicated by one or more of: decreased pH, increased lactic acid content, lower abundance of antibiotic resistance genes, decreased amount of fungal DNA, decreased toxin content, decreased pathogenicity factors, decreased inflammatory cytokines and chemokines, decreased immune cell infiltrates, decreased total bacterial DNA load, decreased total pathogenic DNA load, increased viscoelasticity, increased sialoglycan content, decreased relative or absolute abundance of pathobionts or pathogens, or any combination thereof in the vaginal cavity and the vaginal microbial niche (e.g., when compared to baseline of the same subject (e.g., prior to administration and engraftment) or a non-treated control subject).

[0262] In some embodiments, pathogens are not detectable in a microbial consortium of the present disclosure. A pathogen is any microorganism (e.g., virus, bacteria, fungus, protozoa) that has the capability of causing disease or illness in a host. Non-limiting examples of pathogens include Gardnerella vaginalis, Chlamydiua trachomatis, Herpes Simplex Virus, Human Papillomavirus, Candida species, and Trichomonas vaginalis. In some embodiments, other non-lactobacilli are not detectable. As used herein, the term “other non-lactobacilli” refers to microorganisms that may or may not be pathogenic (e.g., may be commensal organisms or environmental contaminants) and are not part of the lactobacilli genus. Non-limiting examples of other non-lactobacilli include Atopobium species, Prevotella species, and Streptococcus species.Lactobacilli in the vaginal niche

[0263] Lactobacilli are the predominant microorganisms in the vaginal microbial community, and they play a major role in maintaining a healthy urogenital tract, including the vaginal and / or endometrial niche. Lactobacilli are capable of preventing adhesion and growth of pathogenic microorganisms and / or overgrowth of pathobionts through mechanisms that appear to involve secretion of anti-adhesion factors, hydrogen peroxide, bacteriocins and fermenting the glycogen to lactic acid, thereby creating an acidic environment hostile topathogens and pathobionts. The genus Lactobacillus comprises a phenotypically heterogenous group of Gram-positive, aerotolerant, lactic acid producing bacteria. Other typical characteristics include being catalase-negative and rod-shaped, and generally possess DNA with a low content of guanine (G) and cytosine (I), less than about 50%. They are members of the phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillaceae. One skilled in the art will be able to identify Lactobacillus species using standard techniques.

[0264] Species of lactobacilli can be identified phenotypically, as well as genetically, e.g., on the basis of 16S rRNA (ribosomal RNA) sequence (or the DNA encoding the 16S rRNA, generally referred to as 16S rDNA). Genetic analysis can be performed using standard techniques, for example whole genome sequencing analysis as well as widely used typing approaches based on nucleotide variation in several hundred DNA sequences and a few gene fragments: Multi-locus Sequence Typing (MLST), Multi-locus Variable number of tandem repeats Analysis (MLVA), rMLST and cgMLST) discussed, e.g., in Marcos Perez-Losada, M. et al., “Microbial sequence typing in the genomic era”, Infection, Genetics and Evolution, Vol. 63, Sep. 2018, p. 346-359.

[0265] Other identification techniques include: Vaginal pH, Nugent Score, Whiff Test, gas liquid chromatographic analysis of glucose fermentation products, total anaerobe concentrations, total aerobe concentrations, enzymatic activity (e.g., lipase, phospholipase A2 and phospholipase C, hydrogen peroxide production).Stability

[0266] The lactobacilli comprised in the microbial consortia or related compositions comprising the same, are generally viable for up to one month if stored in the refrigerator at 4° C and up to several months (e.g., 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months, or longer) if stored frozen at about -18° C, or preferably at about -80° C in solution or on agar plates. Individual lactobacilli isolates described herein (e.g., L. jensenii isolates, L. crispatus isolates) are generally viable for up to one month if stored in the refrigerator at 4° C and up to several months (e.g., 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months, or longer) if stored frozen at about -18° C, or preferably at about -80° C in solution or on agar plates.

[0267] The lactobacilli comprised in the microbial consortia or related compositions comprising the same, are generally viable for up to several months (e.g., 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months, or longer) or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years) if stored ina lyophilized or freeze-dried format. Individual lactobacilli isolates described herein (e.g., L. jensenii isolates, L. crispatus isolates) are generally viable for up to several months (e.g., 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months, or longer) or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years) if stored in a lyophilized or freeze-dried format. Viability decreases over time. The microbial consortia or related compositions are suitable for administration if they retain at least 30%, 40%, 50%, 60%, 70%, or at least 80% viable bacteria prior to use. Individual lactobacilli isolates described herein (e.g., L. jensenii isolates, L. crispatus isolates) are suitable for administration if they retain at least 30%, 40%, 50%, 60%, 70%, or at least 80% viable bacteria prior to use. Viable cells are generally able to colonize and engraft. Percent viability refers to the percentage of viable bacteria in a population. In some embodiments, the microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or other related composition are further processed by adding a diluent, such as, e.g., saline, e.g., to formulate a pharmaceutical composition. In some embodiments, the microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), one or more lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates) identified by the method of the invention, or compositions thereof described herein are further processed to be lyophilized (e.g., by freeze-drying, spray drying or other technologies known in the art) , e.g., for easy storage, packaging, formulated in, for instance, compositions, and transport, and can be rehydrated before administration.Acidifying Agents

[0268] The microbial consortia, individual lactobacilli isolates (e.g., L. crispatus isolates, L. jensenii isolates) described herein, one or more lactobacilli isolates identified by the method of the invention, or related compositions, may optionally comprise one or more acidifying agents, such as, e.g., organic acids or salts thereof. In some embodiments, acidifying agents may be used to reduce the pH of the compositions, e.g., to below pH 5.5 or 5.0, e.g., to between pH 3.5 to 4.5, or pH 3.0 to 4.5. In decreasing pH such acidifiers may act as anti-microbial agents (e.g., to inhibit Candida or pathogenic bacteria). Acidifying agents comprise, e.g., lactic, acetic, ascorbic, citric, folic sorbic, or boric acid. In other embodiments, the acidifying agent is administered separate from the compositions comprising one or more lactobacilli isolates identified by the method of the invention, a microbial consortia, individual lactobacilli isolates described herein (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions; e.g. prior to, concurrent with, or after administration of thecomposition (e.g., in a different dosage form, such as, e.g. a suppository, cream, gel, powder, douche or similar), e.g., for one to seven days, or one to ten days. In these embodiments, the acidifying agent may be used to promote the survival and engraftment of the one or more lactobacilli isolates identified by the method of the invention, the Lactobacilli comprised in the microbial consortia, individual lactobacilli isolates described herein (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions. Acidifying agents may also be useful as spermicides, e.g., to kill or inactivate residual sperm that may be present in the preparation. In one embodiment, the spermicidal activity is contributed by adding lactic acid. In one embodiment, lactic acid may be provided as a racemic mixture of D- and L-isomers, or at different suitable ratio, including, e.g., only D-lactate or only L-lactate.

[0269] In some embodiments, microbial consortia, compositions comprising individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions are acidified by adding 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 5% of acidifier (e.g., lactic acid). In preferred embodiments, the acidifier is added at a concentration of 0.5% to 1.5%, or 0.2% to 2% (w / w), or a similar concentration that matches a healthy vagina.Viscosity

[0270] In some embodiments, microbial consortia, compositions comprising individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions described herein may comprise one or more pharmaceutically acceptable buffer or diluent or other excipients. In embodiments, one or more excipients is used to formulate the composition in different dosage forms, such as, e.g., suppositories, creams or dissolving films or tablets.

[0271] In some embodiments, the dosage form is liquid, solid or semi-solid. The solid dosage form preferably comprises a tablet, capsule, or a film. The semi-solid dosage form preferably comprises a suppository, ointment, gel, cream or rigid foam. In a preferred embodiment, the dosage form is a gel.Prebiotics

[0272] A “prebiotic” as used herein is a growth substrate, which increases growth of bacteria (such as lactobacilli) comprised in a microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates) described herein, in a composition comprising one or more (isolated) bacterial isolates identified according to the method of the invention, or related compositions as could be measured, e.g., in vitro. If desired, though it isnot generally necessary, a prebiotic may be added to the compositions described herein, e.g., to create a symbiotic mixture, e.g., to increase growth of the bacteria upon administration to the genitourinary tract of a female subject. This may, under certain circumstances, increase successful colonization and engraftment. In other embodiments, prebiotics may be used for the maintenance of an engrafted preparation and / or general vaginal health.

[0273] If a prebiotic is desired, it should be carefully chosen to not be greatly metabolizable by any yeast (e.g., Candida species), pathobionts or pathogens (e.g., E. coli and other Gram- negative bacteria) that may reside in the female subject’s genitourinary tract (e.g., to avoid promoting their growth). Prebiotics include, e.g., lactitol, lactulose, and in some instances also other oligosaccharides and soluble fibers, e.g., fructooligosaccharides (FOS), glucooligosaccharides (GOS), and inulin.Other Active Agents

[0274] If desired, other active agents may be added to microbial consortia, e.g., to address bacterial or fungal infections and / or sexually transmitted diseases in the female subject, for example antimicrobial agents, antifungal agents, antibacterial agents, antiviral agents, antibiotics, antiparasitic agents (e.g., with activities against Trichomonas vaginalis), anti-inflammatory agents, and the like. Care must be taken when formulating these agents into the pharmaceutical composition so as to not substantially interfere with the activity and efficacy of the lactobacilli comprised in the preparations and compositions described herein.

[0275] In some embodiments, the microbial consortia comprise a form of estrogen. Adequate levels of estrogen play a role in the trophism of vaginal mucosa, and estrogens increase the cellular content of glycogen.

[0276] In some embodiments, the microbial consortia comprise thiosulfate, e.g., to potentiate the anti-pathogenic effect of lactobacilli.

[0277] If desired, a treatment regimen could utilize an antibiotic administered to a subject prior to administration of the microbial consortia. An antibiotic may include metronidazole, or one or more antibiotics of the following classes: a macrolide (e.g., azithromycin, clarithromycin and erythromycin), a tetracycline (e.g., doxycycline, tigecycline), a fluoroquinolone (e.g., levofloxacin, ciprofloxacin and mocifloxacin), a cephalosporin (e.g., ceftriaxone, defotaxime, ceftazidime, cefepime), a penicillin (e.g., amoxicillin, amoxicillin with clavulanate, ampicillin, piperacillin, and ticarcillin) optionally with a beta-lactamase inhibitor (e.g., sulbactam, tazobactam and clavulanic acid), such asampicillin-sulbactam, piperacillin-tazobactam and ticarcillin with clavulanate, an aminoglycoside (e.g., amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, and apramycin), a penem or carbapenem (e.g. doripenem, ertapenem, imipenem and meropenem), a monobactam (e.g., aztreonam), an oxazolidinone (e.g., linezolid), vancomycin, glycopeptide antibiotics (e.g. telavancin), and the like.

[0278] In some embodiments, the pharmaceutical compositions can further contain an antimicrobial (an antibiotic or antifungal) selected from metronidazole, tinidazole, secnidazole, clindamycin, nystatin, azithromycin, erythromycin, ofloxacin, doxycycline, levofloxacin, amoxicillin, and fluconazole.

[0279] If desired, the microbial consortia can contain an agent for treating infections with mycobacteria. Suitable agents for treating infections with mycobacteria include an aminoglycoside (e.g., capreomycin, kanamycin, streptomycin), a fluoroquinolone (e.g. ciprofloxacin, levofloxacin, moxifloxacin), isozianid and isozianid analogs (e.g. ethionamide), aminosalicylate, cycloserine, diarylquinoline, ethambutol, pyrazinamide, protionamide, rifampin, and the like.

[0280] If desired, the microbial consortia can contain a suitable antiviral agent, such as remdesivir, foscavir, Cytogam® (cytomegalovirus immune globulin), pleconaril, rupintrivir, palivizumab, motavizumab, cytarabine, docosanol, denotivir, cidofovir, and acyclovir.

[0281] If desired, the microbial consortia can contain a suitable antifungal agent, such as polyene (e.g., nystatin and natamycin) and imidazole antifungals (e.g., flucanozole and clotrimazole).

[0282] If desired, the donor sample or compositions comprising one or more bacterial isolates identified by the inventive method, can contain one or more suitable steroids. For example, the donor sample or composition may include androgens / anabolic steroids, estrogen, progestagens, corticosteroids, neurosteroids, estradiol, estropipate, drospirenone, noresthisterone, levonorgestrel, testosterone, fluoxymesterone, methylesterosterone, oxandrolone, and oxymetholone.Dosage Forms and Formulations

[0283] The microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions may be formulated into isolate preparations and (pharmaceutical) compositions and various dosage forms. In the following, the preferreddosage forms are provided, wherein all dosage forms are equally suitable for the microbial consortia or individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates).

[0284] Multiple dosage forms are contemplated herein, and include a suspension, spray, gel, cream, ointment, powder, (gelatin or vegetable cellulose) capsule, solution for lavages or douches, foams, films, ovules, a vaginal insert (e.g. tampon), tablets, disk, wafer (e.g., drying on film, by vaporization), or a microencapsulated product employing excipients and formulation techniques known to those skilled in the art. Particularly preferred dosage forms include formed gels, lyophilized gels, tablets, frozen formulations and films. In one embodiments, the dosage form is a capsule.

[0285] A number of suitable excipients can be used to formulate the microbial consortia, compositions comprising individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions such as bulking agents, polymers, carbon substrates, mucoadhesive agents, or pH modifiers and / or buffers.

[0286] The carbon substrate excipients may act as a carbon substrate for the microbiota contained in the microbial consortia, compositions comprising individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions. Such carbon courses comprise mannitol, and Guar gum. Some excipients may further serve as mucoadhesive agents or as viscosity agents. Bulking agents may comprise one or more of mannitol, microcrystalline cellulose, guar gum, inulin, or alginic acid (e.g., sodium alginate). Polymers may comprise structural polymers. In some embodiments, polymers comprise one or more of mucin, hyaluronic acid, polyvinyl alcohol, sodium CMC, polyvinylpyrrolidone, hydroxypropyl methylcellulose (e.g., Carbopol 934), and poloxamer (e.g., poloxamer 407). Mucoadhesive agents comprise, e.g., alginic acid (sodium alginate) and sodium CMC. Viscosity agents comprise, e.g., to Guar gum and Carbopol 934.

[0287] Some excipients serve as pH modifiers and / or buffers, such as lactic acid and acetate buffer.

[0288] Suitable formulations show little to no flow on suitable vertical surfaces and maintain high bacterial viability (e.g., CFU count) both upon formulation and during (longterm) storage.

[0289] Desired formulation selection parameters include, for example, water activity / moisture content (e.g., of dried formulations), e.g., between 0.5 - 3% water (e.g., for longer term dried formulation stability); microbial diversity, e.g., absence of microorganisms suchas, Pseudomonas aeruginosa, Candida albicans, Staphylococcus aureus, Ph Eur criteria 5.1.4, 2.6.12 & 2.6.13).

[0290] Suitable testing methods include standard assays, such as, plate count (e.g., MRS agar), e.g., for life bacteria count, dose determination, shelf-life; rheometer, e.g., for mucoadhesion and viscosity, pH meter, Karl Fisher / water activity meter, Ph Eur testing, e.g., for microbial loads.

[0291] The one or more bacterial isolates identified by the methods of the invention, the microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions can be lyophilized and formulated into, e.g., gels and tablets as well as other dosage forms that can be filled with lyophilized products, such as, e.g., capsules. The microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions can also be formulated into gels that can be frozen, as well as into liquid media (e.g., with glycerol) that can be frozen. The composition comprising one or more bacterial isolates identified by the methods of the invention, the microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions can also be formulated into (air-dried) films, that could be, e.g., shaped like disks.Lyophilized Formulations

[0292] The microbial consortia, composition comprising individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions, can be lyophilized. The lyophilized preparation or lyophilized composition is stable and may be stored for extended periods of time at 2-8°C. The lyophilized preparation or lyophilized composition comprises the lyophilized microbial consortia or lyophilized individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates). The lyophilized preparation or lyophilized composition may be supplied in a vial. The lyophilized preparation or lyophilized composition can be reconstituted prior to administration (e.g., in a clinical or home setting) with a reconstitution agent. In some embodiments, the reconstitution agent comprises a gel, a gel forming agent, or a liquid. The liquid may comprise water, saline or another liquid suitable for reconstitution and subsequent administration to a subject.

[0293] Sequencing may be performed to assess the microbial community of the donor microbiota sample and to select suitable donor females.

[0294] Optionally, nucleic acid sequencing is performed to identify the presence of any antimicrobial resistance (AMR) genes in the donor sample, e.g., to determine that a donor sample is substantially free of antimicrobial resistance (AMR) genes. Antimicrobial resistance (AMR) genes include genes that confer resistance to one or more antibiotics, including, e.g., aminoglycosides, beta-lactams, tetracyclines, and sulfonamides (e.g., as described and cataloged in the NCBI National Database of Antibiotic Resistant Organisms (NDARO)). It will be appreciated that due to extensive use of antibiotics the cut-off is not zero. Some reasonable allowance for the presence of AMR genes is made. The precise cut-off can be determined by one of ordinary skill, based on, e.g., the nature of the AMR genes (e.g., degree of health concern) and public health recommendations.Methods of Administration

[0295] Aspects of this disclosure provide, in part, methods for vaginal administration to a human female subject of a microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions described herein. Further aspects of this disclosure provide, in part, a method of increasing the relative abundance of lactobacilli (e.g., L. crispatus, L. jensenii) in the female genitourinary tract of a subject comprising administering a microbial consortium described herein to the genitourinary tract of the subject.

[0296] In some embodiments, the female genitourinary tract is dysbiotic. In some embodiments, the genitourinary tract of the subject exhibits dysbiosis. Further, the invention provides the microbial consortium for use in therapy. In one embodiment, the invention provides the microbial consortium described herein for use in a method of treating dysbiosis of the genitourinary tract of a female subject, said method comprising administering the microbial consortium described herein to the genitourinary tract of the subject.

[0297] In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 90% of the total bacterial population prior to administration of a microbial consortium of the present disclosure. In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, or less than 20% of the total bacterial population prior to administration of a microbial consortium ofthe present disclosure. In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is between 20% - 25%, between 20% - 30%, between 20% - 35%, between 20% - 40%, between 20% - 45%, between 20% - 50%, between 20% - 55%, between 20% - 60%, between 20% - 65%, between 20% - 70%, between 20% - 75%, between 20% - 80%, between 20% - 85%, between 20% - 90-%, between 25% - 30%, between 25% - 35%, between 25% - 40%, between 25% - 45%, between 25% - 50%, between 25% - 55%, between 25% - 60%, between 25% - 65%, between 25% - 70%, between 25% - 75%, between 25% - 80%, between 25% - 85%, between 25% - 90%, between 30% - 35%, between 30% - 40%, between 30% - 45%, between 30% - 50%, between 30% - 55%, between 30% - 60%, between 30% - 65%, between 30% - 70%, between 30% - 75%, between 30% - 80%, between 30% - 85%, between 30% - 90%, between 35% - 40%, between 35% - 45%, between 35% - 50%, between 35% - 55%, between 35% - 60%, between 35% - 65%, between 35% - 70%, between 35% - 75%, between 35% - 80%, between 35% - 85%, between 35% - 90%, between 60% - 65%, between 60% - 70%, between 60% - 75%, between 60% - 80%, between 60% - 85%, between 60% - 90%, between 65% - 70%, between 65% - 75%, between 65% - 80%, between 65% - 85%, between 65% - 90%, between 70% - 75%, between 70% - 80%, between 70% - 85%, between 70% - 90%, between 75% - 80%, between 75% - 85%, between 75% - 90%, between 80% - 85%, between 80% - 90%, or between 85% - 95% of the total bacterial population prior to administration of a microbial consortium of the present disclosure.

[0298] In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 70% of the total bacterial population following administration of a microbial consortium of the present disclosure. In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% of the total bacterial population following administration of a microbial consortium of the present disclosure. In some embodiments, the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is between 70% - 75%, between 70% - 80%, between 70% - 85%, between 70% - 90%, between 70% - 95%, between 70% - 100%, between 75% - 80%, between 75% - 85%, between 75% - 90%, between 75% - 95%, between 75% - 100%, between 80% - 85%, between 80% - 90%,between 80% - 95%, between 80% - 100%, between 85% - 90%, between 85% - 95%, between 85% - 100%, between 90% - 95%, between 90% - 100%, or between 95% - 100% of the total bacterial population following administration of a microbial consortium of the present disclosure.

[0299] In some embodiments, the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 5% following administration of a microbial consortium of the present disclosure. In some embodiments, the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% following administration of a microbial consortium of the present disclosure. In some embodiments, the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by between 5% - 10%, between 5% - 15%, between 5% - 20%, between 5% - 25%, between 5% - 30%, between 5% - 35%, between 5% - 40%, between 5% - 45%, between 5% - 50%, between 5% - 55%, between 5% - 60%, between 10% - 15%, between 10% - 20%, between 10% - 25%, between 10% - 30%, between 10% - 35%, between 10% - 40%, between 10% - 45%, between 10% - 50%, between 10% - 55%, between 10% - 60%, between 15% - 20%, between 15% - 25%, between 15% - 30%, between 15% - 35%, between 15% - 40%, between 15% - 45%, between 15% - 50%, between 15% - 55%, between 15% - 60%, between 20% - 25%, between 20% - 30% , between 20% - 35%, between 20% - 40%, between 20% - 45%, between 20% - 50%, between 20% - 55%, between 20% - 60%, between 35% - 60%, between 40% - 45%, between 40% - 50%, between 40% - 55%, between 40% - 60%, between 45% - 50%, between 45% - 55%, between 45% - 60%, between 50% - 55%, between 50% - 60%, or between 55% - 60% following administration of a microbial consortium of the present disclosure.

[0300] Vaginal dysbiosis refers to an imbalance or disruption in the normal microbial community within the vagina. It is often characterized by a decrease in the population of Lactobacillus species and an overgrowth of other microorganisms, which may include pathogenic bacteria, anaerobic bacteria, or yeasts. This shift in microbial composition can result in an increase in vaginal pH, a decrease in lactic acid production, and alterations in the overall ecosystem of the vagina (Lev-Sagie A, et al. The Vaginal Microbiome: II. Vaginal Dysbiotic Conditions, J Low Genit Tract Dis 26(1): 79-84 (2022); Saraf VS, et al. Vaginalmicrobiome: normalcy vs dysbiosis, Archives of Microbiology 203: 3793-3802 (2021); van de Wijert JHHM, et al. The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization, PLoS One 9(8): el05998 (2014)). Method for treating vaginal dysbiosis are known in the art and may include administration of probiotics (e.g., as oral supplements or applied directly to the vagina using suppositories or creams), administration of antibiotics (e.g., in cases where vaginal dysbiosis is associated with a bacterial infection, such as bacterial vaginosis), administration of antifungal medications (e.g., in cases where yeast, such as Candida, is the cause of dysbiosis), hormone replacement therapy or local estrogen therapy, administration of a vaginal microbiome transplant (e.g., as a suppository or gel), or administration of acidifying agents or gels.

[0301] The methods may include using a device for administration, e.g., these methods would normally be carried out by a healthcare provider (e.g., in a clinic). Alternatively, the methods include using an alternative dosage form described herein, such as, e.g., a suppository, tablet, capsule, film, cream, etc. The methods can, if desired, be carried out by the subject herself, e.g., by self-administration (e.g., at home). The methods may also include other healthcare related activities, such as diagnosing a health issue and providing standard of care in addition to providing a microbial consortia, composition comprising individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related composition described herein. The activities can include one or more combination therapies provided herein. For example, the methods for administration described herein may further comprise administering antimicrobial agents, antifungal agents, antibacterial agents, antiviral agents, antibiotics, antiparasitic agents (e.g., with activities against Trichomonas vaginalis), antiinflammatory agents, and the like.

[0302] In embodiments in which administration is carried out using a device, the device typically includes an open end (e.g., a tip) for insertion into the vaginal cavity, and a dispensing end (e.g., a plunger or piston) to expel the composition through the open end. The administration steps include: a) introducing the open end into a vaginal cavity, b) expelling the composition into the vaginal cavity, c) removing the device from the vaginal cavity (after administering the desired dose). Administration is preferably carried out with the subject being in a lithotomy position, e.g., with the female subject in a lithotomy position. In some embodiments, the subject is to remain in a lithotomy position for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes before returning to an upright position to allow the microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L.crispatus isolates), or related composition sufficient residence time in the vaginal cavity, e.g., sufficient contact time with the mucosal or endometrial surfaces of the vagina. In some embodiments, the administration is carried out targeting areas high in the vaginal cavity, e.g., near the vaginal fomices.

[0303] If desired, the menstrual cycle of the subject female is taken into account when determining the timing of administration. For example, administration may be avoided during menstrual discharge. In some embodiments, a time other than during menstrual discharge is preferred for carrying out the administration, including, e.g., during a time window that includes prior to ovulation and prior to menstrual discharge.

[0304] A microbial consortium may be administered to a subject as necessary. In some embodiments, a microbial consortium is administered to a subject one, two, or three times within 3-21 days. In some embodiments, a microbial consortium is administered to a subject once daily for 3, 5, 10, 15 or 20 days. In some embodiments, a microbial consortium is administered to a subject once daily for 5 days. In some embodiments, a microbial consortium is administered to a subject once daily for 15 days. In some embodiments, a microbial consortium is administered to a subject once every one to four weeks for a period of one to six months. In some embodiments, a microbial consortium is administered to a subject twice weekly, three times weekly or four times weekly for 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0305] In some embodiments, the precise steps, timing, and length of the administration varies between subjects (and is, e.g., determined by a healthcare provider) in order to provide optimal conditions for the bacteria (e.g., lactobacilli) comprised in the microbial consortia or related compositions to colonize and become established (engrafted) in the vagina of the female subject.Health Symptoms and Biomarker Assessment

[0306] In some embodiments, successful colonization and engraftment is associated with changes in (a) the composition of the microbiota of the female subject’s genitourinary tract (e.g., the vaginal and or endometrial tract) and / or (b) one or more disease-associated health symptoms over a predetermined time period. These desired changes may be included as additional selection criteria for the one or more isolates that are identified as being capable of colonization and engraftment. In a preferred embodiment, the one or more isolates identified by the methods described herein are not only capable of colonizing and engrafting in theurogenital tract but also are associated with desired changes in the composition of the microbiota of the female subject’s genitourinary tract and / or (b) one or more disease- associated health symptoms. Such isolates may preferably be selected for isolation, propagation and formulation into preparations and (pharmaceutical) compositions.

[0307] Desirable changes of the composition of the microbiota of the female subject’s genitourinary tract that can be determined include, e.g., a reduction in the relative abundance of pathogen and / or pathobiont residing in the genitourinary tract (e.g., less than 10% relative abundance of pathogen and / or pathobiont), and / or an increase in relative abundance of Lactobacillus above 50% (above 60%, or above 70%).

[0308] Desirable changes in one or more disease-associated health symptoms that can be determined include, e.g., (a) a reduction in one or more pro-inflammatory markers (e.g., local or systemic cytokines and chemokines) and / or decreased immune cell infiltrates; (b) a lowering of vaginal pH (e.g., by at least pH 0.3, 0.5, 1.0, or 1.5) and / or increased lactic acid content of the vaginal tract; and / or (c) a change in grade based on the Ison-Hay scoring system (e.g., to grade 0 or grade I), when compared to the values of (a), (b), and / or (c) determined in the same subject prior to administration of one or more isolates capable of colonization and engraftment.

[0309] Other desirable changes in one or more disease-associated health symptoms that can be determined include, lower abundance of antibiotic resistance genes, decreased amount of fungal DNA, decreased toxin content, and / or decreased pathogenicity and / or virulence factors.

[0310] The vaginal health status of a female subject can be assessed prior to and / or after administration at predetermined intervals. Testing may include using one or more of: Amsel Criteria, Nugent Gram-stain scoring system, and Hay-Ison Criteria.

[0311] Amsel Criteria include the presence of three of the following four symptoms: (a) thin homogeneous malodorous discharge; (b) vaginal pH fluid >4.5; (c) an amine odor from vaginal fluid when 10% KOH is added; and (d) the presence of “clue” cells (vaginal epithelial cells with adherent bacteria that obscure cell margins) (Amsel et al., Am. J. Med. 74:14-22 (1983)).

[0312] The Nugent Gram-stain scoring system involves assessment of a normally prepared Gram stain for relative abundance of three morphotypes of bacteria, and then calculating the so-called Nugent score based on the amounts of large Gram-positive rods (lactobacilli morphotype; decrease in lactobacilli is scored as 0 to 4), small Gram-negativeand variable rods (Bacteroides and Gardnerella morphotype; scored as 0 to 4), and curved gram-variable rods (Mobiluncus spp. morphotype; scored as 0 to 2). The Nugent score can range from 0 to 10, with scores of 0-3 deemed normal (non-BV), 4-6 intermediate, and 7-10 positive for BV.

[0313] Hay-Ison Criteria (alternatively Ison-Hay scoring system) suggests five grades of flora: a) Grade 0, epithelial cells with no bacteria; b) Grade I, normal vaginal flora (Lactobacillus morphotypes alone); c) Grade II, reduced numbers of Lactobacillus morphotypes with a mixed bacterial flora; d) Grade III, mixed bacterial flora only, few or absent Lactobacillus morphotypes; e) Grade IV, Gram positive cocci only.

[0314] Grades 0, 1, and IV are found in women without BV. Grade II is intermediate and not found in women with BV as defined by Amsel criteria. Grade III is consistent with BV as diagnosed by Amsel criteria. Grade III flora are indicative of BV (C.A. Ison and P.E. Hay, Sex Transm. Infect. 2002 Dec;78(6):413-5).

[0315] Further desirable changes in one or more disease-associated health symptoms that can be determined include beneficial immune response, which may be assessed by e.g. proteomics (e.g., Olink) and transcriptomics (e.g., Fluidigm or RNAseq).

[0316] In some embodiments, the desired changes associated with successful colonization and engraftment in the female subject’s genitourinary tract are assessed within one, two, three, four, six, eight, ten, or twelve week(s), or 6 months, 8 months, 10 months, 12 months, 18 months, or within 24 months post administration of the donor sample.

[0317] In some embodiments, the desired changes associated with successful colonization and engraftment in the female subject’s genitourinary tract are assessed within 1, 2, 3, 4, 5, 6 or 7 day(s) post administration of the donor sample.

[0318] In some embodiments, the desired changes associated with successful colonization and engraftment in the female subject’s genitourinary tract are detectable within 1, 2, 3, 4, 5, 6, or 7 day(s) post administration of the donor sample.

[0319] In some embodiments, the desired changes associated with successful colonization and engraftment in the female subject’s genitourinary tract are detectable within one, two, three, four, six, eight, ten, or twelve week(s), or 6 months, 8 months, 10 months, 12 months, 18 months, or within 24 months post administration of the donor sample.Nucleic Acid Sequencing

[0320] In one embodiment, methods include sequencing of nucleic acids in the sample to identify the bacterial taxa present in the sample, e.g., a sample from the female subject’s genitourinary tract prior to and after administration of the donor sample, or a composition comprising one or more bacterial isolates. Genetic information from the sample can be obtained by nucleic acid extraction from the sample. Methods for extracting nucleic acid from a sample are known in the art. For sequencing, nucleic acid is extracted from the bacterial samples collected. Numerous standard DNA extraction protocols exist. Preferably, extraction protocols are used that specifically enrich for microbial DNA (e.g., protocols that reduce the amount of human DNA in the sample). Exemplary commercial kits include MoLysis Completes kit, and Qiagen DNAeasy Blood & Tissue kit. In a preferred embodiment, human DNA is removed prior to sequencing. DNA may be extracted using the Molysis Completes kit (MolZym), which uses a differential lysis method to extract microbial DNA and remove human DNA.

[0321] Nucleic acid sequencing may be conducted by any method known in the art, including whole genome sequencing or other standard nucleic acid techniques, such as Southern blot, sequence analysis, electrophoresis, and PCR (including quantitative PCR (qPCR)). Sequences can be determined, e.g., by Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing, solid-phase sequencing, sequencing with mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), and sequencing by hybridization. Examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis.

[0322] Other exemplary sequencing methods include: dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, shotgun sequencing, polymerase chain reaction (PCR), real-time polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), multiplex PCR, ligase chain reaction, pyrosequencing, sequencing by synthesis, sequencing by ligation, massively parallel signature sequencing, polony sequencing, SOLiD sequencing, DNA nanoball sequencing, mass spectrometry sequencing, microfluidic sequencing, high-throughput sequencing, Illumina sequencing, HiSeq sequencing, MiSeq sequencing, 16S ribosome sequencing,sequencing by chain termination and gel separation, as described by Sanger et al., PNAS, 74(12): 5463 67 (1977); chemical degradation of nucleic acid fragments. See, Maxam et al., PNAS, 74: 560 564 (1977); sequencing by hybridization. See, e.g., Harris et al., (U.S. patent application number 2009 / 0156412); Illumina (MiSeq and HiSeq), Helicos True Single Molecule Sequencing (tSMS) (Helicos Biosciences) See Harris T. D. et al. (2008) Science 320:106-109; see also, e.g., Lapidus et al. (U.S. Pat. No. 7,169,560), Lapidus et al. (U.S. patent application number 2009 / 0191565), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake et al. (U.S. patent application number 2002 / 0164629), and Braslaysky, et al., PNAS, 100: 3960-3964 (2003); 454 sequencing, e.g., Roche 454 GS FLX (Roche) (Margulies, M et al. 2005, Nature, 437, 376-380); SOLiD technology, e.g., SOLiD 5500 series (Applied Biosystems); Ion Torrent / IonProton / Ion Proton sequencing, e.g., PGM, Proton, S5 Series, ; single molecule, real-time (SMRT) technology (Pacific Biosciences); nanopore sequencing (Oxford Nanopore Technologies) (Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001); Qiagen Gene Reader; chemical-sensitive field effect transistor (chemFET) arrays (See e.g., US Patent Application Publication No. 2009 / 0026082); and use of an electron microscope (Moudrianakis E. N. and Beer M. PNAS USA. 1965 March; 53:564-71), or combinations thereof, incorporated by reference herein. Exemplary next generation sequencing methods are described, e.g., in Muneer Ahmad Malla et al., “Exploring the Human Microbiome: The Potential Future Role of Next-Generation Sequencing in Disease Diagnosis and Treatment”, Front. Immunol., 07 January 2019, incorporated by reference herein). If desired, the extracted nucleic acids can be amplified. Suitable amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, Immuno-amplification, nucleic acid sequence -based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). For example, the PCR can be real-time PCR. In some embodiments, the PCR is quantitative real-time PCR (QRT- PCR).

[0323] If desired, nucleic acids synthesized as the result of gene transcription and / or metagenomic molecules can be detected. For example, in the case of the 16S rRNA gene, genomic DNA corresponding, in whole or part, to regions of the 16S rRNA gene, messenger RNA (mRNA) transcripts, in whole or part, of the 16S rRNA gene, and / or functional 16SrRNA may be detected and used to enumerate the abundance of a microbial taxon, e.g., characterized by sequence homology of a particular 16S rRNA gene sequence.

[0324] Genetic analysis can be performed using standard techniques, for example whole genome sequencing analysis as well as widely used typing approaches based on nucleotide variation in several hundred DNA sequences and a few gene fragments: Multi-locus Sequence Typing (MLST), Multi-locus Variable number of tandem repeats Analysis (MLVA), rMLST and cgMLST) (discussed, e.g., in Marcos Perez-Losada, M. et al., “Microbial sequence typing in the genomic era”, Infection, Genetics and Evolution, Vol. 63, Sep. 2018, p. 346-359).

[0325] Sequencing by any of the methods described above and known in the art produces sequence reads. Sequence reads can be analyzed according to any number of methods known in the art to identify microbes (including bacterial taxa) in a sample, e.g., a sample of vaginal fluid from a healthy donor.

[0326] Identification of microorganisms and sorting of them into taxa may also be achieved by other means such as analyzing proteomes, transcriptomes, metabolomes, or combinations thereof. For example, microbial RNA transcripts, proteins, non-16S genes, etc. may be profiled.

[0327] Other methods that may be employed to identify microbes include microarrays or other oligonucleotide probe-based technology (DNA or RNA-based) or antibody-based detection methods, including enzyme-linked immunosorbent assay (ELISA), western blot, immunohistochemistry, immunocytochemistry, flow cytometry and fluorescence-activated cell sorting (FACS), immunoprecipitation, and enzyme linked immunospot (ELISPOT).Bacterial Isolate Isolation and Propagation

[0328] The methods for identifying bacterial isolates capable of colonizing and engrafting the female genitourinary tract optionally further comprise one or more steps to isolate, test, propagate and formulate (e.g., dosage forms, preparations, compositions, such as, e.g., pharmaceutical compositions) the one or more isolates that are identified using the methods described herein. As described herein, one or more isolates capable of colonizing and engrafting the female genitourinary tract can be identified by comparing the constituents (bacterial taxa) of the donor female with those of the female subject (e.g., by nucleic acid sequencing), after a predetermined amount of time and / or after determining a desired outcome (e.g., changes in dysbiosis or reduction in one or more disease-associatedsymptoms). Isolates identified from the sample that match one or more isolates from the donor sample by a predetermined degree of sequence identity are thought to be capable of colonization and engraftment, and, in some embodiments, associated with resolution of dysbiosis and / or disease-associated health symptoms, or another desired outcome. Upon obtaining the identification of the isolates that are successful in colonizing and engrafting the microbial niche in vivo, the isolates so identified are then isolated from the healthy donor sample comprising the healthy donor microbial community.

[0329] Isolation and propagation of bacterial isolates are known in the art and a variety of methods exist to accomplish bacterial isolation and propagation in vitro.

[0330] For example, the lactic acid producing bacteria (e.g., from the donor sample comprising the donor (vaginal) microbial community) or the sample comprising the subject (vaginal) microbial community) are isolated, e.g., from human vaginal fluid / cervicovaginal secretions, selected and cultured in vitro in a suitable nutrient medium providing at least one isolated bacterial isolate. The at least one isolated isolate is then optionally tested for its ability to colonize and engraft by administering the isolated isolate to a female subject. The at least one isolate is then tested for residence time in the microbial niche of the urogenital tract of the subject and / or its associating with one or more health-related outcomes, e.g., resolution of dysbiosis or reduction of one or more disease-associated symptoms exhibited by the female subject prior to administration of the one or more isolated isolates.

[0331] Alternatively, or in addition, the isolated isolates are tested for the presence of antimicrobial resistance (AMR) genes. Preferably, the isolated isolates comprise a low abundance (or are substantially free) of AMR genes. Antimicrobial resistance (AMR) genes include genes that confer resistance to one or more antibiotics, including, e.g., aminoglycosides, beta-lactams, tetracyclines, and sulfonamides (e.g., as described and cataloged in the NCBI National Database of Antibiotic Resistant Organisms (NDARO)). It will be appreciated that due to extensive use of antibiotics the cut-off is not zero. Some reasonable allowance for the presence of AMR genes is made. The precise cut-off can be determined by one of ordinary skill, based on, e.g., the nature of the AMR genes (e.g., degree of health concern) and public health recommendations.

[0332] Alternatively, or in addition, the isolated isolates are tested for the presence of one or more pathogenicity factors / bacterial virulence factors, and / or colonization factors.

[0333] Further, if desired, though not generally necessary for the methods described herein, the one or more isolated isolates can undergo one or more in vitro functionality tests(either prior to, concurrent with or after administration to a female subject), e.g., to supplement or complete the data obtained from in vivo testing, including a) growth in a culture medium, b) antagonism of pathogens (bacterial, viral, and / or fungal) that inhabit the vaginal mucosa, c) high viability during fermentation, d) persistent colonization of mucosal surfaces (e.g., as judged by in vitro adherence assays, e.g., VEC), e) high lactic acid production, f) genetic stability upon repeated cultivation and in vivo upon vaginal administration, and / or g) high viability after final processing, e.g., preserved in a diluent, in lyophilized (desiccated) or (micro)encapsulated from, and storage. Each of the distinguishing characteristics described herein can be detected according to standard methods known in the art.

[0334] For example, antagonism to pathogens may be determined by measuring the production of hydrogen peroxide by the isolated isolate in vitro, e.g., by colorimetric assay (e.g., using tetramethylbenzidine-containing medium or using commercially available hydrogen peroxide detection strips). Alternatively, or in addition antagonism to pathogens may be determined by measuring plating assays with the isolated isolate(s) and one or more pathogens and determining the size (e.g., diameter) and number of the inhibition zones around the colonies of the isolated isolate(s). For example, rapid growth can be measured, e.g., by determining the growth rate (population doubling) into late log phase or stationary phase, e.g., in comparison to a comparator isolate or known growth rates of other members of the same species. For example, a high rate of viability and percent viability (e.g., after culturing or desiccation) can be measured, e.g., by determining the growth rate and / or number of dead / life cells, after exposure to stress, e.g., variations in culturing temperature, pH (e.g., low pH, high pH) richness of the growth medium, concentration of buffering agents, etc., and exposure to desiccation (e.g., freeze-drying, lyophilization) and storage (and measuring the percent viable cells at predetermined intervals). Eife / dead cell counts can be performed visually, e.g., with a microscope, e.g., with a dark field condenser and a Petroff- Hausser counting chamber to obtain a total cell count, or by colony forming units (CFU) on appropriate media plates, and the Eive / Dead ratio calculated as CFU per ml / total cells per ml. For example, lactic acid levels can be determined, e.g., in culture media, using a commercially available lactate assay or using common chemical separation techniques such as capillary electrophoresis and chromatography (e.g., HPEC). For example, the ability of the isolated isolate(s) to adhere to the vaginal mucosa in vivo may be assessed in vitro using vaginal epithelial cells (VEC), cultured Caco-2 cells or other cell lines (e.g., HeEa) bydetermining an adherence value, e.g., by calculating the percent vaginal epithelial cell (VEC) cohesion value, defined as the percentage of VECs, where at least one bacterial cell is adhered to in the total number of VECs. Alternatively, the adherence is determined by calculating the percent vaginal epithelial cell (VEC) cohesion value, defined as the percentage of VECs, where at least one bacterial cell is adhered in the total number of VECs. Another measure of in vitro adherence is to count the average number of microbial cells adhered to a pre-defined number of epithelial cells. For example, the stability of the bacterial genetic profile of the isolated isolate(s) upon repeated cultivation in large-scale production, and during manufacturing processes of, for instance, (pharmaceutical) compositions may be tested. In one embodiment, the isolated isolate(s) preserve their genetic profile upon at least 10 repeated cultivations.

[0335] The isolate(s) are preferably evaluated and selected as to genetic stability in vivo upon vaginal administration, e.g., they are genetically stable in vivo for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, ten months, or for at least 12 months.

[0336] The isolated isolate(s) are preferably evaluated and selected as to their viability upon cryo-preservation (e.g., freezing) and storage, as well as freeze-drying / lyophilization. The isolated isolate(s) are preferably viable for up to several months (e.g., 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months, or longer) if stored at 4° C, frozen at about -18° C, or at about -80° C. Viability decreases over time. The isolated isolate(s) are suitable for use if they retain at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or at least 80% viable bacteria prior to use. In some embodiments, the isolated isolate(s) retain at least 30%, 40%, 50%, 60%, 70%, or at least 80% viable bacteria prior to use. Viable cells are generally able to colonize and engraft. Percent viability refers to the percentage of viable bacteria in a population. If desired, the one or more isolated isolates are further processed to be freeze-dried (lyophilized) and / or pelleted, e.g., for easy storage, packaging, formulated in, for instance, compositions, and transport, and can be rehydrated before administration.

[0337] The bacterial isolates capable of colonizing and engrafting the female genitourinary tract, e.g., isolated by the methods described herein can be propagated by any means know in the art. For example, lactic acid producing isolates (including, e.g., lactobacilli isolates) can be grown in a number of different types of media, in either liquid, semisolid (e.g., agar), or solid form. The culturing conditions can be similar to the natural environment inhabited by the bacteria.

[0338] Standard bacterial media generally include salts, a source of carbohydrate, and a pH buffer. For Lactobacillus, pH is generally maintained in the range of about 4.5 to about 7.0, preferably between pH 5, 5-6, 5 with the addition of sodium phosphate, arginine, ammonium hydroxide, sodium hydroxide, potassium hydroxide, etc. Vitamins and growth agents, including amino acid formulations, can also be added. Bacterial media are known and commercially available (e.g., from Sigma- Aldrich), including, e.g., MRS (including Lactobacillus-MRS Agar (De Man, J. et al., J. Appl. Bact. 23:130-135, I960)), Rogosa, NZM, YM, M17, Thayer-Martin, Trypticase Soy, CDM (Geshnizgani A.M.,and Onderdonk A.B., (J Clin Microbiol. 1992 May;30(5): 1323-6)), and Brain-Heart infusion (BHI) broth. Carbohydrates useful for growing the present isolate include D-galactose, D-glucose, D- fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, sucrose, D-trehalose, amidon, and glycogen. Some of the media can be made selective for lactic acid producing bacteria, such as lactobacilli, e.g., by using a low pH, and addition of certain additives, e.g., sodium acetate and ammonium citrate, e.g., to inhibit other flora and fungi. In some embodiments, the medium comprises Brucella blood agar with hemin and vitamin K, which enables growth of a wide range of vaginal bacteria.

[0339] Lactobacilli are aerotolerant organisms highly directed towards fermentation. Lactobacilli naturally colonize mammalian mucosal surfaces, and generally grow optimally at about body temperature, e.g., 37° C, though they also grow at lower (e.g., 30° C) and higher temperatures (e.g., 40° C). The growth rate can be controlled by changing the temperature.

[0340] After reaching the desired bacterial cell density, the bacterial cells can be harvested using any suitable method to remove the cells from the culture medium. Nonlimiting exemplary methods for harvesting the cultured cells include filtration, centrifugation, and sedimentation. In some embodiments, the cell biomass is washed at least once using a physiologically buffered solution. In some embodiments, the wash solution may contain additional components, such as, e.g., glucose. In some embodiments, the wash solution comprises a buffer or one or more buffering agents. Exemplary buffering agents that may be used or added to the wash solution include phosphate salts, (e.g., Na2HPO4, NaH2PO4, NaHCOa, and arginine).Formulation into Preparations and Pharmaceutical Compositions

[0341] The one or more isolates capable of colonizing and engrafting identified by the methods described herein can be formulated into isolate preparations and (pharmaceutical)compositions and various dosage forms, e.g., using one or more carriers, excipients, diluents and / or buffers, that can optionally, be pharmaceutically acceptable. In some embodiments, the dosage form can be an applicator or dispenser, e.g., those commonly used in medicine and science. In some embodiments, the dosage form can be a vaginal suppository that will remain in the vaginal cavity until it is dissolved.

[0342] Other dosage forms suitable for formulation include a suspension, spray, gel, cream, powder, (gelatin or vegetable cellulose) capsule, solution for lavages, douches, ovules, a vaginal insert, tablets, disk, wafer (e.g., drying on film, by vaporization), or a microencapsulated product employing excipients and formulation techniques known to those skilled in the art. Suitable dosage forms are formulated to readily dissolve and / or disperse inside the vaginal cavity.

[0343] Other dosage forms suitable for formulation include absorbent products comprising the bacterial composition, such as, e.g., a feminine hygiene diaper, panty liners, sanitary napkin, tampon, panty guard or an incontinence guard.

[0344] The one or more isolates capable of colonizing and engrafting identified by the methods described herein can be formulated in dry form, e.g., lyophilized or spray dried and then optionally formulated into a dosage form described herein, or reconstituted, e.g., with sterile water, a weak acidic solution, gel, or buffer prior to use. If the composition is not reconstituted, depending on the precise formulation of the dosage form, rehydration may also be achieved inside the vaginal cavity, e.g., aided by resident vaginal fluid. Methods of preserving viable bacteria by lyophilization can promote long-term preservation of the microorganism. One skilled in the art will be able to lyophilize bacteria using standard techniques.

[0345] In all of these embodiments, dyes, perfumes, pH buffering agents, drying or resuspending agents, or other standard materials for drug formulation can be incorporated into the compositions and dosage forms.

[0346] In all of these embodiments, devices and compositions described herein may be packed in a suitable packaging, for example in a bottle, flacon, blister pack, cartridge, applicator or dispenser.Assembly of a Microbial Consortium

[0347] The inventors have recognized that assembling a microbial consortium for therapeutic use may require monitoring the genetic sequence of bacteria for use in theconsortium over time. For example, genetic drift may cause the bacteria to no longer exhibit desirable therapeutic properties if the bacteria’s genetic sequence is impacted by too many mutations (e.g., SNVs, SNPs, or other changes to the genetic sequence). Accordingly, the inventors have developed techniques for computationally identifying whether two bacterial isolates are substantially similar to be considered a same bacteria. FIG. 13 is a flowchart of an illustrative process 1300 for assembling a microbial consortium including two or more bacteria belonging to one or more bacterial species.

[0348] Various (e.g., some or all) acts of process 1300 may be implemented using any suitable computing device(s). For example, in some embodiments, one or more acts of the illustrative process 1300 may be implemented in a clinical or laboratory setting. For example, one or more acts of the process 1300 may be implemented on a computing device that is located within the clinical or laboratory setting. In some embodiments, the computing device may directly obtain genetic sequencing data from a sequencing apparatus located within the clinical or laboratory setting. For example, a computing device included in the sequencing apparatus may directly obtain the genetic sequencing data from the sequencing apparatus. In some embodiments, the computing device may indirectly obtain genetic sequencing data from a sequencing apparatus that is located within or external to the clinical or laboratory setting. For example, a computing device that is located within the clinical or laboratory setting may obtain genetic sequencing data via a communication network, such as the Internet, a cloud computing environment, a local area network (LAN), wireless local area network (WLAN), wide area network (WAN), or any other suitable network, as aspects of the technology described herein are not limited to any particular communication network.

[0349] Additionally or alternatively, one or more acts of the illustrative process 1300 may be implemented in a setting that is remote from a clinical or laboratory setting. For example, the one or more acts of process 1300 may be implemented on a computing device that is located externally from a clinical or laboratory setting. In this case, the computing device may indirectly obtain genetic sequencing data that is generated using a sequencing apparatus located within or external to a clinical or laboratory setting. For example, the genetic sequencing data may be provided to computing device via a communication network, such as the Internet, a cloud computing environment, a WAN, or any other suitable network.

[0350] It should be appreciated that, in some embodiments, not all acts of process 1300, as illustrated in FIG. 13, may be implemented using one or more computing devices. Forexample, the act 1312 of assembling the microbial consortium may be implemented manually (e.g., by a clinician or manufacturer).

[0351] In some embodiments, the process 1300 may be used to determine whether a purported sample of a bacteria is substantially identical to a reference sample of the bacteria. In determining that the purported sample is substantially identical to the reference sample, it may further be determined that the purported sample and the reference sample can be considered a same bacteria such that the purported sample may be considered suitable for use in the microbial consortium.

[0352] Process 1300 begins at act 1302, where genetic sequencing data obtained from the purported sample of a bacteria for use in the microbial consortium may be obtained. Process 1300 also includes act 1304, which may be performed before, simultaneously with, or after act 1302, and includes obtaining genetic sequencing data from the reference sample of the bacteria for use in the microbial consortium. In some embodiments, the purported sample and / or the reference sample of the bacteria may be a sample of lactobacilli. In some embodiments, the purported sample and / or the reference sample of the bacteria may be a sample of Lactobacilli crispatus.

[0353] In some embodiments, the genetic sequencing data may be obtained by sequencing a biological sample (e.g., an isolate of the purported sample and / or an isolate of the reference sample) obtained using any suitable sequencing technique. The genetic sequencing data may include sequencing data of any suitable type, from any suitable source, and be in any suitable format. In some embodiments, the sequencing apparatus used to sequence the biological sample may be selected from any suitable sequencing apparatus known in the art including, but not limited to, Illumina™, SOLid™, Ion Torrent™, PacBio™, a nanopore -based sequencing apparatus, a Sanger sequencing apparatus, or a 454TM sequencing apparatus. In some embodiments, sequencing apparatus used to sequence the biological sample is an Illumina sequencing (e.g., NovaSeq™, NextSeq™, HiSeq™, MiSeq™, or MiniSeq™) apparatus.

[0354] As one illustrative example, in some embodiments, the genetic sequencing data may comprise bulk sequencing data. The bulk sequencing data may comprise at least 1 million reads, at least 5 million reads, at least 10 million reads, at least 20 million reads, at least 50 million reads, or at least 100 million reads. In some embodiments, the sequencing data comprises bulk RNA sequencing (RNA-seq) data, single cell RNA sequencing (scRNA-seq) data, or next generation sequencing (NGS) data. In some embodiments, the sequencing data comprises microarray data.

[0355] In some embodiments, the genetic sequencing data may be obtained by accessing and / or receiving the genetic sequencing data after it had been obtained by sequencing a biological sample. For example, the genetic sequencing data may be obtained by accessing genetic sequencing data stored in computer-readable memory (e.g., locally stored and / or remotely stored genetic sequencing data). As another example, the genetic sequencing data may be obtained by receiving the genetic sequencing data in a transmission from a remote computer (e.g., via a network transmission over a network including but not limited to the Internet, a cloud computing environment, and / or a local or wide area network).

[0356] In some embodiments, after obtaining the genetic sequencing data of the purported and reference samples, process 1300 may proceed to act 1306, where the genetic sequencing data from the two samples may be aligned. Any suitable method of pairwise alignment may be used to align the obtained genetic sequencing data. For example, the NUCmer command with the -maxmatch flag, as implemented in MUMmer v3.23 or later, may be used to perform pairwise alignment of the obtained genetic sequencing data. Additional aspects related to the MUMmer package are described in S. Kurtz, et al., Versatile and. open software for comparing large genomes, Genome Biology 5:R12 (2004).

[0357] After aligning the obtained genetic sequencing data, process 1300 may proceed to act 1308, where a percent identity value may be calculated using the aligned genetic sequencing data. The percent identity value describes a percent of the aligned genetic sequencing data that is identical between the two sets of genetic sequencing data, and may be written as:where Naiign is the total number of identical basepairs and NSNV is the number of SNVs in the genetic sequencing data of the purported sample relative to the genetic sequencing data of the reference sample. Any suitable technique may be used to determine the values of Naiign and NSNV. For example, the dnadiff command, as implemented in MUMmer v3.23 or later, as described above, may be used to determine the values of Naiign and NSNV.

[0358] After determining the percent identity, process 1300 may proceed to act 1310, where it may be determined that the purported sample and the reference sample are samples of a same bacteria. This determination may be made by determining that the calculatedpercent identity value is greater than or equal to a threshold value. The threshold value may define a distinction between different bacteria of a bacterial species. For example, a calculated percent identity that is smaller than the threshold value may indicate that the purported sample includes a large number of SNVs relative to the reference sample such that the purported sample and the reference sample may not be considered the same bacteria. A calculated percent identity that is greater than or equal to the threshold value may therefore indicate that the purported sample includes an acceptable number of SNVs relative to the reference sample such that the purported sample and the reference sample may be considered the same bacteria.

[0359] In some embodiments, the process 1300 may optionally include determining the threshold value. For example, the threshold value may be determined using genetic sequencing data obtained from two or more samples of a known same bacteria. Percent identities may then be calculated for pairs of the two or more samples using the obtained genetic sequencing data, as described herein (e.g., by aligning the pairs of genetic sequencing data, determining values of Naiign and NSNV, and calculating the percent identities for each pair). The threshold value may then be determined using a largest value of the calculated percent identities, as this largest value indicates a smallest difference between genetic sequences of the same bacteria (e.g., due to genetic drift or other factors that may introduce SNVs).

[0360] In some embodiments, the threshold value may be 99.81%. In some embodiments, the threshold value may be 99.90%. In some embodiments, the threshold value may be 99.96%. Additional aspects of the determination of the threshold value are described herein in connection with FIG. 3.

[0361] After determining that the purported sample and the reference sample are samples of the same bacteria, the process 1300 may proceed to act 1312, in which the microbial consortium may be assembled using the purported sample. The microbial consortium may be assembled by combining multiple lactobacilli bacteria together, including the purported sample. Identification of lactobacilli bacteria for inclusion in the microbial consortium can be determined using a number of different in vitro, in vivo, and in silico characteristics of potential bacteria, such as engraftment capability, growth and acidification rates in vitro and coverage across a species-level pangenome (see, e.g., Examples 2-5).

[0362] The assemblies of the purported samples can be aligned against one or more of the publicly available L. crispatus RefSeq assemblies known to a person of ordinary skill,including GCF_000091765, GCF_000160515, GCF_000161915, GCF_000162255,GCF_000162315, GCF_000165885, GCF-000176975, GCF-000301115, GCF_000301135,GCF_000466885, GCF_OO 1434005, GCF-001541515, GCF_OO 1546025, GCF-001563615,GCF_002088015, GCF-002218645, GCF-002218695, GCF_002218815, GCF_002218845,GCF_002218855, GCF-002218885, GCF_002218895, GCF-002218945, GCF-002218965,GCF_002218975, GCF-002219005, GCF_002219015, GCF_002219045, GCF-002219055,GCF_002219085, GCF-002811165, GCF-002861765, GCF-002861775, GCF_002861815,GCF_002863245, GCF_002863485, GCF_002863505, GCF-003795065, GCF-003971565,GCF_004361125, GCF_004361175, GCF_004361185, GCF_004361195, GCF-004361245,GCF_004361295, GCF-004361345, GCF-004361375, GCF-004361445, GCF-004361465,GCF_004361515, GCF-004361555, GCF-004361565, GCF-004361575, GCF-008694745,GCF_009730275, GCF-009769205, GCF-009933525, GCF_013456995, GCF_013487905,GCF_013778545, GCF-015669875, GCF-016767795, GCF_018885325, GCF-019537355,GCF_020042005, GCF-020042225, GCF_020887095, GCF-021278925, GCF-021278945,GCF_022453955, GCF-022453975, GCF-022454015, GCF-022454025, GCF-022454055,GCF_022454075, GCF_022454115, GCF-022454135, GCF-022454155, GCF-022454175,GCF_022454205, GCF-022454275, GCF-022454315, GCF-022454335, GCF-022454355,GCF_022454375, GCF-022454385, GCF-022454395, GCF-022454435, GCF-022454495,GCF_022454535, GCF-022454555, GCF-022454565, GCF-022454615, GCF-022454695,GCF_022454705, GCF-022454735, GCF-022454755, GCF-022454765, GCF-022454795,GCF_022454815, GCF-022454845, GCF_022454895, GCF-022454915, GCF-022454935,GCF_022454955, GCF-022454965, GCF-022455005, GCF-022455035, GCF-022455065,GCF_022455155, GCF-022455175, GCF_022455185, GCF-022455205, GCF-022455255,GCF_022455295, GCF-022455315, GCF-022455325, GCF-022455375, GCF-022455395,GCF_022455455, GCF-022455475, GCF_022455485, GCF-022455515, GCF-022455535,GCF_022455555, GCF-022455575, GCF-022455595, GCF-022455615, GCF-022455635,GCF_022455655, GCF-022455675, GCF-022455695, GCF-022455705, GCF-022455755,GCF_022455855, GCF-022455875, GCF-022455915, GCF-022455955, GCF-022455975,GCF_022455985, GCF-022456015, GCF-022456035, GCF-022456175, GCF-022456215,GCF_022456295, GCF-022456575, GCF-022456595, GCF-022456605, GCF-022456655,GCF_022456695, GCF-022456705, GCF-022456755, GCF-022456795, GCF-022456805,GCF_022456835, GCF-022456855, GCF_022456875, GCF-022456895, GCF-022456935,GCF 022456975, GCF 022456995, GCF 022457275, GCF 022457405, GCF 022457425,GCF_022457445, GCF_022457465, GCF_022457495, GCF_022457545, GCF_022458295, GCF_022458335, GCF_022458395, GCF_022458415, GCF_022458425, GCF_025194045, GCF_026740115, GCF_027680585, GCF_027682305, GCF_027695305, GCF_029011155, GCF-029011475, GCF_029011495, GCF_029011595, GCF_029011765, and GCF-904382375.

[0363] FIG. 14 is a diagram of an illustrative computer system on which embodiments described herein (e.g., such as the method of FIG. 13) may be implemented. The computer system 1400 may include one or more processors 1410 and one or more articles of manufacture that comprise tangible (e.g., non-transitory) computer-readable storage media (e.g., memory 1420 and one or more non-volatile storage media 1430). The processor 1410 may control writing data to and reading data from the memory 1420 and the non-volatile storage device 1430 in any suitable manner, as aspects of the technology described herein are not limited to any particular techniques for writing or reading data. To perform any of the functionality described herein, the processor 1410 may execute one or more processorexecutable instructions stored in one or more non-transitory computer-readable storage media (e.g., the memory 1420), which may serve as non-transitory computer-readable storage media storing processor-executable instructions for execution by the processor 1410.

[0364] Computing device 1400 may also include a network input / output (VO) interface 1440 via which the computing device may communicate with other computing devices (e.g., over a network), and may also include one or more user VO interfaces 1450, via which the computing device may provide output to and receive input from a user. The user VO interfaces may include devices such as a keyboard, a mouse, a microphone, a display device (e.g., a monitor or touch screen), speakers, a camera, and / or various other types of VO devices.

[0365] The above-described embodiments can be implemented in any of numerous ways. One or more aspects and embodiments of the present disclosure involving the performance of processes or methods may utilize program instructions executable by a device (e.g., a computer, a processor, or other device) to perform, or control performance of, the processes or methods. In this respect, various inventive concepts may be embodied as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one ormore programs that, when executed on one or more computers or other processors, perform methods that implement one or more of the various embodiments described above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various ones of the aspects described above. In some embodiments, computer readable media may be tangible (e.g., non-transitory) computer readable media. In some embodiments, the computer readable media may comprise a persistent memory.

[0366] In this respect, it should be appreciated that one implementation of the embodiments described herein comprises at least one computer-readable storage medium (e.g., RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or other tangible, non-transitory computer-readable storage medium) encoded with a computer program (i.e., a plurality of executable instructions) that, when executed on one or more processors, performs the abovediscussed functions of one or more embodiments. The computer-readable medium may be transportable such that the program stored thereon can be loaded onto any computing device to implement aspects of the techniques discussed herein. In addition, it should be appreciated that the reference to a computer program which, when executed, performs any of the abovediscussed functions, is not limited to an application program running on a host computer.Rather, the terms “computer program” and “software” are used herein in a generic sense to reference any type of computer code (e.g., application software, firmware, microcode, or any other form of computer instruction) that can be employed to program one or more processors to implement aspects of the techniques discussed herein.

[0367] It will be apparent that example aspects, as described above, may be implemented in many different forms of software, firmware, and hardware in the implementations illustrated in the figures. Further, certain portions of the implementations may be implemented as a “module” that performs one or more functions. This module may include hardware, such as a processor, an application- specific integrated circuit (ASIC), or a field-programmable gate array (FPGA), or a combination of hardware and software.

[0368] The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects as described above. Additionally, it should be appreciated that according to one aspect, one or more computerprograms that when executed perform methods of the present disclosure need not reside on a single computer or processor but may be distributed in a modular fashion among a number of different computers or processors to implement various aspects of the present disclosure.

[0369] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0370] When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.

[0371] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, a tablet computer, a quantum computer, or a photonic computer as non-limiting examples. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smartphone, or any other suitable portable or fixed electronic device.

[0372] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible formats.

[0373] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

[0374] Also, as described, some aspects may be embodied as one or more methods. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.DEFINITIONS

[0375] The definitions hereinafter apply to all aspects and embodiments disclosed herein. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is further to be understood that methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0376] In a preferred embodiment, the term “about” shall allow a deviation of + 10%, and even more preferably of + 5% from an indicated numerical value. Unless otherwise indicated, all numbers expressing quantities of components, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods as known to those of ordinary skill in the art.

[0377] The terms “contacting”, “administering” or “subjecting” and more specifically, “vaginal application” or “vaginal administration”, are used interchangeably herein and relate to a subject (e.g., a female subject) or a specific organ or other physiological site (e.g., the urogenital tract or vaginal cavity or a subpart thereof, e.g., to a site on a vaginal wall (mucosal or endometrial surfaces) or vaginal fornices) and a dosage form, preparation or (pharmaceutical) composition that is provided or given to the subject or site, e.g., for the purpose of colonizing and engrafting a desired bacterial community. In one embodiment, administering is performed locally. In one embodiment, administering is performed topically (e.g., vaginally).

[0378] The terms “bacterial isolate” or “isolate” refers to isolated bacteria of the same species that share defined functional and / or genotypic characteristics. In some embodiments,a bacterial isolate is a Lactobacillus crispatus isolate. In some embodiments, a bacterial isolate is a Lactobacillus jensenii isolate. In some embodiments, a bacterial isolate has been separated from other material (e.g., from a vaginal bacterial community, e.g., derived from a sample of cervicovaginal secretions or vaginal fluid). In some embodiments, a bacterial isolate may be propagated in vitro. A bacterial isolate is, in some embodiments, substantially free of contaminants or components that accompany the material it was derived from in its native state (e.g., such as, vaginal mucus and epithelial cells). In some embodiments, a bacterial isolate is characterized by its genomic haplotypes. In some embodiments, two different bacterial isolates do not share a sequence identity of greater than a threshold value (e.g., a threshold value of 99.81%, 99.90% or 99.96%) across their pairwise aligned genomes. In some embodiments, functional characteristics include growth rates, acidification rates, and rates of carbohydrate utilization, among others. In some embodiments, two or more different bacterial isolates (e.g., obtained from different sources, e.g., obtained from different donors) may be combined to generate a microbial consortium.

[0379] As used herein the term “bacterial sample” or “microbial sample” means a sample comprising bacteria. The sample can be, e.g., vaginal fluid / discharge.

[0380] The term “carbon substrate utilization profile” or “carbon utilization profile” is an indicator (e.g., a composite measure or set of measurements) reflecting the extent to which a bacterial isolate grows on or consumes different carbon substrates selected from a set of one or more (or two or more) carbon substrates, such as mannitol and glucose. In some embodiments, a carbon substrate utilization profile comprises a measure or measures of growth rate and / or acidification rate during log phase growth on two or more carbon substrates.

[0381] The term “comprising” shall be understood to simultaneously also disclose the term “consisting” as a preferred option. For example, if a composition is said to comprise three components, this also discloses a composition consisting of these three components as preferred embodiment.

[0382] If the term “comprising” is used when referring to (a) pharmaceutically active compound(s), bacterium(a), and the like, this shall be understood to simultaneously also disclose that the pharmaceutically active compound(s), bacterium(a), and the like is / are preferably the sole pharmaceutically active compound(s), bacterium(a), and the like. For example, if a microbial consortium is said to comprise Lactobacillus crispatus andLactobacillus jensenii. this simultaneously and preferably discloses that a donor sample contains Lactobacillus crispatus and Lactobacillus jensenii as the sole bacterial species.

[0383] The term “consortium” or “microbial consortium” is a defined mixture of bacteria that does not exist together naturally, i.e., is non-naturally occurring and / or synthetic, and that has emergent properties not possessed by any of the constituent bacteria individually. (“Non-naturally occurring” consortia).

[0384] “Culture-independent method” means methods not involving isolation and / or in vitro propagation of bacterial isolates, e.g., in cultures.

[0385] The terms “distinct” or “phenotypically distinct” refer to isolates having different phenotypes. In some embodiments, distinct or phenotypically distinct isolates have different growth rates. In some embodiments, distinct or phenotypically distinct isolates have different acidifications rates. In some embodiments, distinct or phenotypically distinct isolates have different carbon substrate utilization profiles. In some embodiments, distinct or phenotypically distinct isolates have different carbon substrate utilization capabilities.

[0386] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration (e.g., daily, one or more times per week, per month, or per 3 months); size and tolerance of the individual; severity of the condition; intended result (e.g., treatment, prophylaxis, modulation or restoration of the microbial community), and the route of administration. A baseline dose can be administered and modified based on the initial response of the subject. For example, a single dose of the compositions comprising the substantially complete vaginal microbiota preparations described herein can be in the range of 104to 1010colony forming units (per dose). In other embodiments, a single dose of the composition can be in the range of 103to 1012, 104to 109, 105to 109, 105to 108, 106to 109, 107to 109, or 107to 1010colony forming units (per dose).

[0387] A “dosage form” refers to a particular physical form of a composition or preparation and depends, e.g., on the desired amount of the material to be administered and on the route of administration, e.g., oral or topical (e.g., vaginal). For example, a dosage form can be in a suppository, a tablet, a capsule, a film, a cream, etc., or a device, such as, e.g., an applicator or dispenser, e.g., for vaginal administration. Dosage forms may be single or multiple-use dosage forms.

[0388] “Dysbiosis” as used herein means a microbial imbalance, i.e. an aberration of the healthy state, where normally predominant species are diminished in abundance and less predominant species become more abundant and / or predominant. Vaginal dysbiosis is a microbial imbalance in the vagina, wherein the vaginal imbalance may, in embodiments, extend to the endometrium. Dysbiosis is generally associated with one or more of: (a) qualitative and quantitative changes in the content or amount of the microbiota itself, (b) changes in their metabolic activities; and / or (c) changes in their local distribution (e.g., inside a niche). A dysbiotic human microbiota (or microbiome) refers to a population of microbes that promotes inflammation of a tissue of the urogenital tract (e.g., the vagina) and / or that contributes to or establishes an environment that permits or promotes the colonization or growth of one or more pathogenic microbes. Dysbiosis also refers to a perturbation of the urogenital (e.g., vaginal) homeostasis. In some embodiments, a dysbiotic vaginal microbiota will generally result in increased pH relative to a healthy microbiota, e.g., a pH above 4.5, e.g., a pH of 5.0, 5.5, 6.0, 6.5, 7.0 or higher. In some embodiments, vaginal dysbiosis is characterized by a reduction of Lactobacillus spp., and an increased diversity of vaginal anaerobic bacteria. In some embodiments, vaginal dysbiosis is associated with infections, inflammation, and increased risk of sexual transmitted diseases. Dysbiosis may be characterized by the relative amount of selected pathogens, such as >20% selected pathogens, and the relative low abundance of vaginal lactobacilli, e.g. <10% vaginal lactobacilli (L. crispatus, L. iners, L. jensenii, L. gasseri).

[0389] “Dysbiotic subjects” as used herein comprise subjects having vaginal symptoms (symptomatic subjects) and subjects not having any vaginal symptoms (asymptomatic subjects), wherein symptoms are characterized by Amsel's criteria, Nugent score and / or Hay / Ison score.

[0390] A “desired change” of dysbiosis may thus comprise prophylaxis and / or treatment of any of the above mentioned symptoms associated with dysbiosis, e.g. reduction of inflammation or infection of the urogenital tract, or the restoration and / or rebalancing of the vaginal microbiota (e.g., to achieve homeostasis). The desired change of dysbiosis may alleviate at least one or more symptoms, delay the development of a symptom, alter the course of a symptom (e.g., slowing the progression of a symptom), or reverse a symptom, wherein the symptom may include an increase of beneficial Lactobacillus spp., a reduction of infections, a reduction of the relative amount of selected pathogens, or a reduction in (local) inflammation.

[0391] As used herein, the term “effective amount” means the amount (e.g., of a microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolate), or related compositions) to be administered to a typical subject (e.g., a female subject) that is sufficient to lead to a desired beneficial or therapeutic effect in the subject. The desired beneficial or therapeutic effect includes prophylaxis and / or treatment, e.g., of dysbiosis, inflammation or an infection or urogenital tract, and also includes the restoration and / or rebalancing of the vaginal microbiota (e.g., to achieve homeostasis), e.g., an antiinflammatory and / or anti-pathogenic state of the urogenital tract and the vaginal microbiota. An effective amount, for example, is the amount sufficient (e.g., at dosages and for periods of time necessary) to alleviate at least one or more symptom, delay the development of a symptom, alter the course of a symptom (e.g., slowing the progression of a symptom), or reverse a symptom. Effective amounts generally cause statistically significant, measurable changes.

[0392] As used herein, the term “engraftment” or “colonization” refers to the colonization of an environment (e.g., a microbial niche or microbial community), e.g., the female genitourinary tract of a subject, by a microbe, e.g., a bacterium (e.g., lactobacilli), such that the viable population of that microbe continues to reside, e.g., in the niche or community, for a certain period of time (e.g., a pre-determined period of time). Engraftment can be transient or stable depending on the period of time that the microbe continues to reside in the niche. Engraftment can be quantified, e.g., by counting the number of colony forming units (CFU) / gram and / or performing nucleic acid sequencing of microbes comprised in one or more vaginal samples that are taken over a certain period of time. In some embodiments, successful engraftment of a microbe refers to colonization of the microbe in an environment such that the microbe can be detected within the environment after a certain period of time. In some embodiments, successful engraftment of a microbe refers to colonization of the microbe in an environment such that the relative abundance of the microbe is at least 5% or at least 10% of the total microbial abundance in the environment (e.g., relative abundance of lactobacilli in the female genitourinary tract of a subject is at least 5% or at least 10% of the total microbial abundance). In some embodiments, a microbial consortium of the present disclosure promotes engraftment of at least a subset of the defined population (e.g., at least one Lactobacillus isolate) in a female genitourinary tract of a subject. In some embodiments, a microbial consortium of the present disclosure promotes engraftment of the lactobacilli of the defined population in a female genitourinary tract of a subject. The defined populationmay comprise a Lactobacillus jensenii isolate, a Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii, and at least one additional Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii.

[0393] In one embodiment, the term “promotes engraftment” refers to the engraftment of Lactobacillus jensenii isolate. In one embodiment, the term “promotes engraftment” refers to the engraftment of Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii. In one embodiment, the term “promotes engraftment” refers to the engraftment of Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii. In one embodiment, the term “promotes engraftment” refers to the engraftment of Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii and Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii. In one embodiment, the term “promotes engraftment” refers to the engraftment of Lactobacillus jensenii isolate and Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii. In one embodiment, the term “promotes engraftment” refers to the engraftment of Lactobacillus jensenii isolate and Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii. In one embodiment, the term “promotes engraftment” refers to the engraftment of the defined population, e.g., Lactobacillus jensenii isolate, Lactobacillus crispatus isolate that thrives in the presence of Lactobacillus jensenii, and Lactobacillus crispatus isolate that thrives independent of the presence of Lactobacillus jensenii.

[0394] As a non-limiting example, L. crispatus bacteria that thrives in the presence of L. jensenii is capable of engrafting the female genitourinary tract of a subject for at least a predetermined period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 days) in the presence of an L. jensenii bacteria.

[0395] The term “engraftment potential” refers to a latent capacity to engraft in a recipient. In some embodiments, engraftment potential refers to a quality of an isolate which has been isolated from a donor SCVMP and which has demonstrated successful engraftment in a recipient subject. In some embodiments, FBS-003 has engraftment potential. In some embodiments, FBS-004 has engraftment potential. In some embodiments, FBS-005 has engraftment potential. In some embodiments, FBS-006 has engraftment potential.

[0396] The terms “eubiosis”, “eubiotic”, and “eubiotic microbiome” refer to a healthy vaginal environment wherein the resident bacteria comprise a high relative abundance of Lactobacillus species. A eubiotic vaginal microbiome may be characterized by the relativehigh abundance (e.g., greater than 70%, greater than 80%, greater than 90%, or greater than 95%) of healthy vaginal Lactobacillus species (L. crispatus, L. iners, L. j ens enii, L. gasseri) and a relatively low abundance of selected pathogens. In some embodiments, eubiosis is characterized by a^^Lactobacillus-dommant,,vaginal microbiome. In some embodiments, a eubiotic microbiome is stable, meaning the microbiome persists in the vaginal environment over time (e.g., for 1 month, for 2 months, for 3 months, for 4 months, for 5 months, for 6 months, or for 12 months). In some embodiments, a eubiotic microbiome is stable, meaning the microbiome persists in the vaginal environment across menstrual cycles (e.g., for 1 menstrual cycle, for 2 menstrual cycles, for 3 menstrual cycles, for 4 menstrual cycles, for 5 menstrual cycles, for 6 menstrual cycles, or for 12 menstrual cycles).

[0397] The phrases “excipient” “pharmaceutically acceptable carrier” “diluent” or “buffer” as used herein mean a non-active, pharmaceutically acceptable material, ingredient, composition or vehicle that is added to form part of the final formulation and / or maintains a drug or other agent in a form for delivery to a subject. Each carrier preferably is compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of an active ingredient or agent upon the treatment, e.g., the carrier is pharmaceutically inert. In some embodiments a pharmaceutically acceptable carrier can be a carrier other than water (including, e.g., a cream, emulsion, gel, liposome, nanoparticle, film, ointment and / or vaginal device). In some embodiments, a pharmaceutical composition is provided comprising the substantially complete vaginal microbiota preparations together with a pharmaceutically acceptable carrier and / or diluent (e.g., saline). These compositions allow the easy administration of the preparations by means known to the person skilled in the art. In some embodiments, a buffering agent is added, e.g., a weak acid or base that maintains the acidity at a chosen level (e.g., between pH 3.5 and 4.5) and prevents a rapid change in acidity.

[0398] The term “haplotype” refers to a set of DNA variations or polymorphisms, e.g. single nucleotide polymorphisms (SNPs) or single nucleotide variants (SNVs), on the same chromosome or in defined genetic regions and that are inheritable. In some embodiments, a haplotype can be used as a defining indicator of a particular bacteria or isolate.

[0399] The terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0400] As used herein, the term “isolated nucleic acids” refers to the purification of nucleic acids from one or more cellular components. Isolated nucleic acids can be prepared from specimens using any acceptable method known in the art. For example, cells can be lysed using known lysis agents, and nucleic acids can be purified or partially purified from other cellular components.

[0401] As used herein the term “lactic acid producing bacteria” means bacteria that produce lactic acid by fermentation.

[0402] The terms “lower”, “reduced”, “reduction” or “decrease”, “down-regulate” or “inhibit” mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level (or levels below the limit of detection) as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0403] A “lyophilized”, “spray-dried” or “freeze-dried” composition refers to a composition from which moisture has been removed, e.g., for easy storage and transport. Such compositions can be rehydrated before use (e.g., administration). The lyophilized, spray-dried, or freeze-dried composition may be further pelleted or packaged for easier storage and transportation.

[0404] The term “microbe” is used synonymously with the term “microorganism” and includes bacteria (Archaea, Eubacteria), yeast, fungi, and viruses. The term “species” is used herein to refer to a taxonomically and / or genetically distinct group of microorganisms. Species may include one or more distinguishable (e.g., by sequencing) isolates.

[0405] The term “microbiota” refers to a community of microorganism localized to a distinct shared environment (a “microbial niche”). For example, “vaginal microbiota” is a community of one or more species of microorganisms that are localized to, or found in, a vagina. The term “microflora” or “flora” is used synonymously with the term “microbiota.” Healthy or normal microbiota denominates the community of commensal microorganisms that colonize (inhabit) a particular microbial niche of the host, such as the vagina. Bacteriaare the most numerous microbial components of the normal flora. “Microbiome” refers to the totality of microbes (bacteria, archaea, yeast, fungi and phages) and their genomes.

[0406] A “microbial profile” is a set of the species and / or isolates of microorganisms present in a sample of microorganisms. To the extent that a sample of microorganisms is obtained from, and corresponds to the species found in, a shared environment, the microbial profile details the species present in a microbial community. The term “profile” as used herein refers to both an individual profile and an aggregate profile over a plurality of individual profiles, depending on the context of its use. In some embodiments, an aggregate profile may include, e.g., a pangenome (sequence), e.g., all the genes (or genomes) of all individual members (e.g., isolates) within a particular grouping (e.g., clade).

[0407] “Mucosa” as used herein indicates a mucous membrane. Mucus is a secretion produced by, and covering, mucous membranes. Mucous fluid is viscous and typically produced from mucous cells (e.g., goblet cells) found in mucous membranes and submucosal glands, and rich in antiseptic enzymes (such as lysozyme), immunoglobulins, inorganic salts, proteins such as lactoferrin, and glycoproteins (mucins). Mucosal surfaces include epithelial linings of the reproductive tract (vagina) and, e.g., lactobacilli are capable of colonizing the vaginal mucosal surfaces.

[0408] In contrast, the term “normal” or “healthy” “vaginal flora” or “vaginal niche” “microbiota” “urogenital tract” or “community state” or similar terms connote that a woman has no vaginal complaints and does not exhibit a vaginal pathology (e.g., no sign or symptom corresponding to or resulting from a pathology of the vagina), and the condition of the vagina is such of a relatively low susceptibility to sexually transmitted diseases and pathogens, and generally of low pH, e.g., less than or equal to pH 4.5, e.g., between 3.2 and 4.5; and generally dominated by lactic acid producing bacteria (e.g., Lactobacillus spp.). Normal vaginal microbiota or normal flora are a community of microorganisms that localize to the vagina in a normal, healthy, that is, a non-pathological, non-pathogenic and / or non- dysbiotic, state.

[0409] “Cervicovaginal secretions” or “vaginal fluid” refers to the mixture of mucus secreted by the cervix, shed epithelial cells, vaginal transudate, and bacteria found in the vagina of a woman.

[0410] “Endometrial fluid” refers to a fluid accumulation within the endometrial cavity that contains bacterial cells, host cells and microbe and host derived proteins, nucleotides and metabolites. Typically, the endometrial fluid is obtained by aspiration for further analysis.

[0411] As used herein, a “nucleic acid” includes DNA and RNA (e.g., mRNA, rRNA, or tRNA) and analogs thereof, including nucleic acids comprising conventional nitrogenous bases (e.g., A, G, C, T, U), base analogs (e.g., inosine), derivatives of purine or pyrimidine bases and any substitutions

[0412] The term “ratiometrically equal” refers to a combination of isolates wherein each isolate is approximately equal in proportions or wherein each isolate is present at about the same relative abundance as each other isolate. The ratiometric proportions of isolates in a consortium can be determined by measuring CFUs, by qPCR, dry weight, or volume. In some embodiments, a consortium comprising 2 isolates will comprise about 50% of each isolate. In some embodiments, a consortium comprising 3 isolates will comprise about 33% of each isolate. In some embodiments, a consortium comprising 4 isolates will comprise about 25% of each isolate.

[0413] As used herein, “sequence identity” or “percent identical” as applied to nucleic acid molecules is the percentage of nucleic acid residues in a candidate nucleic acid molecule sequence that are identical with a subject nucleic acid molecule sequence, after aligning the sequences to achieve the maximum percent identity, and not considering any nucleic acid residue substitutions as part of the sequence identity. Nucleic acid sequence identity can be determined using any method known in the art, for example CLUSTALW, T-COFFEE, BLASTN.

[0414] As used herein, “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0415] As used herein, the term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least about 95% identity to a given reference sequence. Percent identity can be anywhere from 90% to 100%. Most embodiments include at least: 95%, 96%, 97%, 98%, 99% or 99.9% compared to a reference sequence using the programs described herein (e.g., BLAST, using standard parameters).

[0416] The term “subject” refers to a human (e.g., a human female). In one embodiment, the subject is 18 years or older. In one embodiment, the subject is in puberty. In one embodiment, the subject is pregnant. In one embodiment, the subject is of childbearing age. In one embodiment, the subject is pre-menopausal. In one embodiment, the subject is post-menopausal. In one embodiment, the subject is administered the microbial consortia, individual lactobacilli isolates (e.g., L. jensenii isolates, L. crispatus isolates), or related compositions described herein. In some embodiments, a subject is a human female exhibiting a clinical condition related to a microbial imbalance (e.g., a dysbiosis).

[0417] The term “substantially” means to a great or significant extent. For example in the case of a sample that is “substantially free of’ pathogens, the term means the sample is, for the most part, or essentially, but possibly not completely, void of a pathogen. A sample that is substantially free of selected pathogens can refer to a sample that comprises about 5%, <5%, <4%, <3%, <2%, or <1% of the selected pathogens. In a particular embodiment, the term “substantially free” comprises <5%.

[0418] As used herein the term “suitable nutrient medium” means a medium in which the bacteria might be cultivated.

[0419] As used herein, the terms “treat,” “treatment,” “treating” refer to prophylactic and therapeutic treatments, wherein the object is to prevent, reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition or symptom of a condition associated with a disease or disorder. Treatment includes the improvement of symptoms or markers (of the disease or condition) and the cessation or at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Treatment effectiveness can be measured by monitoring one or more symptoms or clinical markers and is compared, e.g., to the subject’s condition and symptoms before administration or to a control subject not undergoing treatment. In one example, treating a vaginal infection refers to reducing the amount of the infective agent (e.g., number of cells or viral particles), reducing the severity of symptoms, and / or reducing the frequency of symptoms. A treatment that reduces the level of a pathogen to one which is kept in check by the immune system or by the state established by a healthy vaginal microbiota (e.g., the infection is no longer detectable, e.g., by symptoms or general diagnostic techniques) is considered effective as the term is used herein.

[0420] “Genitourinary tract” or “urogenital tract” as used herein as used herein includes the uterus, fallopian tubes, ovaries, vagina, cervix, vulva, and urinary tract. In someinstances, used herein are teachings and exemplifications specifically calling out the “vaginal tract”, “vaginal cavity”, “vaginal lumen”, or “vagina”. One of ordinary skill will appreciate that these exemplifications and teachings are illustrative only and non-limiting and, thus also apply, where appropriate, to other anatomical sites of the genitourinary tract or urogenital tract, not just to the vagina.

[0421] As used herein, the term “viable” refers to a cell (e.g., a bacterial cell) that is able to survive in a given condition (e.g., storage for a certain period of time under particular storage conditions, e.g., including, temperature, humidity) and is generally able to colonize and reproduce (e.g., in the urogenital tract) after exposure to the condition. Percent viability refers to the percentage of viable cells in a population. For example, percent viability can refer to the percentage of lactobaci...

Claims

CLAIMSWhat is claimed is:

1. A microbial consortium comprising a defined population of isolated lactobacilli, the defined population comprising:(a) a Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770;(b) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772;(c) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771, and(d) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

2. A microbial consortium comprising a defined population of isolated lactobacilli, the defined population comprising:(a) a Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value;(b) a Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value;(c) a Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value; and(d) a Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value.

3. The microbial consortium of claim 2, wherein the threshold value for (a), (b), (c), and / or (d) is 99.81%.

4. The microbial consortium of claim 2, wherein the threshold value for (a), (b), (c), and / or (d) is 99.90%.

5. The microbial consortium of claim 2, wherein the threshold value for (a), (b), (c), and / or (d) is 99.96%).

6. The microbial consortium of any one of the preceding claims, wherein the isolate of(a) is derived or propagated from a bacterial sample deposited as DSM 34770.

7. The microbial consortium of any one of the preceding claims, wherein the isolate of(b) is derived or propagated from a bacterial sample deposited as DSM 34771.

8. The microbial consortium of any one of the preceding claims, wherein the isolate of(c) is derived or propagated from a bacterial sample deposited as DSM 34772.

9. The microbial consortium of any one of the preceding claims, wherein the isolate of(d) is derived or propagated from a bacterial sample deposited as DSM 34773.

10. The microbial consortium of any one of the preceding claims, wherein the defined population is configured to promote engraftment in a female genitourinary tract of a subject.

11. The microbial consortium of claim 10, wherein at least one of the lactobacilli are capable of engrafting the female genitourinary tract of the subject for at least a predetermined period of time.

12. The microbial consortium of any one of the preceding claims, wherein the defined population of isolated lactobacilli comprises: a relative abundance of 15% to 35% of the total viable cells are bacteria of the Lactobacillus jensenii isolate of (a); a relative abundance of 15% to 35% of the total viable cells are bacteria of the Lactobacillus crispatus isolate of (b); a relative abundance of 15% to 35% of the total viable cells are bacteria of the Lactobacillus crispatus isolate of (c); anda relative abundance of 15% to 35% of the total viable cells are bacteria of the Lactobacillus crispatus isolate of (d).

13. The microbial consortium of any one of the preceding claims, wherein the defined population of isolated lactobacilli comprises: a relative abundance of about 25% of the total viable cells are bacteria of the Lactobacillus jensenii isolate of (a); a relative abundance of about 25% of the total viable cells are bacteria of the Lactobacillus crispatus isolate of (b); a relative abundance of about 25% of the total viable cells are bacteria of theLactobacillus crispatus isolate of (c); and a relative abundance of about 25% of the total viable cells are bacteria of the Lactobacillus crispatus isolate of (d).

14. The microbial consortium of any one of the preceding claims, wherein: the Lactobacillus jensenii isolate of (a) comprise a plurality of Lactobacillus jensenii bacteria; the Lactobacillus crispatus isolate of (b) comprise a plurality of Lactobacillus crispatus bacteria; the Lactobacillus crispatus isolate of (c) comprise a plurality of Lactobacillus crispatus bacteria; and the Lactobacillus crispatus isolate of (d) comprise a plurality of Lactobacillus crispatus bacteria.

15. The microbial consortium of any one of the preceding claims, wherein the Lactobacillus crispatus isolate of (b) thrive in the presence of Lactobacillus jensenii bacteria.

16. The microbial consortium of any one of the preceding claims, wherein the Lactobacillus crispatus isolate of (c) and / or (d) thrive independent of the presence of Lactobacillus jensenii bacteria.

17. The microbial consortium of any one of the preceding claims, wherein at least two of the lactobacilli differ in their growth rates.

18. The microbial consortium of claim 17, wherein the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their growth rates.

19. The microbial consortium of claim 17 or 18, wherein at least two Lactobacillus crispatus differ in their growth rates.

20. The microbial consortium of any one of claims 17-19, wherein the growth rates are growth rates in vitro (e.g., in culture) and / or in vivo.

21. The microbial consortium of any one of the preceding claims, wherein the growth rate of the Lactobacillus jensenii is 0.32-0.36 hours'1at starting pH of 4.7 or 0.41-0.43 hours'1at starting pH of 5.2.

22. The microbial consortium of any one of the preceding claims, wherein the growth rate of the Lactobacillus crispatus isolate of (b) is 0.35-0.41 hours'1at starting pH of 4.7 or 0.39- 0.41 hours'1at starting pH of 5.2.

23. The microbial consortium of any one of the preceding claims, wherein the growth rate of the Lactobacillus crispatus isolate of (c) is 0.27-0.29 hours'1at a starting pH of 4.7 or 0.35-0.37 hours'1at a starting pH of 5.2.

24. The microbial consortium of any one of the preceding claims, wherein the growth rate of the Lactobacillus crispatus isolate of (d) is 0.24-0.26 hours'1at a starting pH of 4.7 or 0.31-0.33 hours'1at a starting pH of 5.2.

25. The microbial consortium of any one of the preceding claims, wherein at least two of the lactobacilli differ in their acidification rates.

26. The microbial consortium of claim 25, wherein the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their acidification rates.

27. The microbial consortium of claim 25 or 26, wherein at least two Lactobacillus crispatus differ in their acidification rates.

28. The microbial consortium of any one of claims 25-27, wherein the acidification rates are acidification rates in vitro (e.g., in culture) and / or in vivo.

29. The microbial consortium of any one of the preceding claims, wherein the acidification rate of the Lactobacillus jensenii is -0.005 to -0.003 at a starting pH of 4.7 or - 0.009 to -0.007 at a starting pH of 5.2.

30. The microbial consortium of any one of the preceding claims, wherein the acidification rate of the Lactobacillus crispatus isolate of (b) is -0.009 to -0.007 at a starting pH of 4.7 or -0.012 to -0.010 at a starting pH of 5.2.

31. The microbial consortium of any one of the preceding claims, wherein the acidification rate of the Lactobacillus crispatus isolate of (c) is -0.007 to -0.005 hours'1at a starting pH of 4.7 or -0.008 to -0.006 hours'1at a starting pH of 5.2.

32. The microbial consortium of any one of the preceding claims, wherein the acidification rate of the Lactobacillus crispatus isolate of (d) is -0.008 to -0.006 hours'1at a starting pH of 4.7 or -0.012 to -0.010 hours'1at a starting pH of 5.2.

33. The microbial consortium of any one of the preceding claims, wherein at least two of the lactobacilli differ in their capability of degrading glycogen.

34. The microbial consortium of claim 33, wherein the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their capability of degrading glycogen.

35. The microbial consortium of claim 33 or 34, wherein at least two Lactobacillus crispatus differ in their capability of degrading glycogen.

36. The microbial consortium of any one of claims 33-35, wherein the capability of degrading glycogen is a capability of degrading glycogen in vitro {e.g., in culture) and / or in vivo.

37. The microbial consortium of any one of the preceding claims, wherein at least two of the lactobacilli differ in their rate of producing lactate.

38. The microbial consortium of claim 37, wherein the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their rate of producing lactate.

39. The microbial consortium of claim 37 or 38, wherein at least two Lactobacillus crispatus differ in their rate of producing lactate.

40. The microbial consortium of any one of claims 37-39, wherein the rate of producing lactate is a rate of producing lactate in vitro (e.g., in culture) and / or in vivo.

41. The microbial consortium of any one of the preceding claims, wherein at least two of the lactobacilli differ in their rate of engraftment in the female genitourinary tract.

42. The microbial consortium of claim 41, wherein the Lactobacillus jensenii and at least one Lactobacillus crispatus differ in their rate of engraftment in the female genitourinary tract.

43. The microbial consortium of claim 41 or 42, wherein at least two Lactobacillus crispatus differ in their rate of engraftment in the female genitourinary tract.

44. The microbial consortium of any one of claims 41-43, wherein the rate of engraftment in the female genitourinary tract is determined in vivo.

45. The microbial consortium of any one of the preceding claims, wherein at least one of the lactobacilli bacteria is capable of engrafting the female genitourinary tract of the subject within 24 hours after administration to the female genitourinary tract of the subject.

46. The microbial consortium of any one of the preceding claims, wherein at least one of the lactobacilli bacteria is capable of persisting within the female genitourinary tract of the subject for at least 4 days after administration to the female genitourinary tract of the subject.

47. The microbial consortium of claim 46, wherein 2, 3 or 4 of the lactobacilli bacteria is capable of persisting within the female genitourinary tract of the subject for at least 4 days after administration to the female genitourinary tract of the subject.

48. The microbial consortium of any one of the preceding claims, wherein the lactobacilli of the consortium are capable of competing with the resident microbiota within the female genitourinary tract of the subject.

49. The microbial consortium of any one of the preceding claims, wherein each of the lactobacilli of the consortium were obtained from a healthy human donor.

50. The microbial consortium of any one of the preceding claims, wherein the lactobacilli comprised in the defined population were obtained from at least two different healthy human donors.

51. The microbial consortium of any one of the preceding claims, wherein the lactobacilli comprised in the defined population were obtained from three different healthy human donors.

52. The microbial consortium of any one of the preceding claims, wherein the Lactobacillus jensenii isolate and the Lactobacillus crispatus isolate that thrives in the presence of the Lactobacillus jensenii bacteria were obtained from different healthy human donors.

53. The microbial consortium of any one of the preceding claims, wherein the Lactobacillus crispatus isolate of (c) and the Lactobacillus crispatus isolate of (d) were obtained from the same healthy human donor.

54. A method of increasing the relative abundance of lactobacilli in the female genitourinary tract of a subject comprising administering the microbial consortium of any one of claims 1-53 to the genitourinary tract of the subject.

55. The method of claim 54, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 90% (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, or 20%) of the total bacterial population prior to administration of the microbial consortium.

56. The method of claim 54 or 55, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 70% (e.g., greater than 80%, 85%, 90%, 95%, or 98%) of the total bacterial population following administration of the microbial consortium.

57. The method of claim 54 or 55, wherein the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 5 percentage points (e.g., at least 10, 15, 20, 25, 30, 40, 50, or 60 percentage points) following administration of the microbial consortium.

58. The microbial consortium of any one of claims 1-53 for use in therapy.

59. The microbial consortium of any one of claims 1-53 for use in a method of treating dysbiosis of the female genitourinary tract of a subject, said method comprising comprising administering the microbial consortium to the genitourinary tract of the subject, optionally wherein the subject is asymptomatic.

60. The microbial consortium for use of claim 59, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 90% (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, or 20%) of the total bacterial population prior to administration of the microbial consortium.

61. The microbial consortium for use of claim 59 or 60, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 70% (e.g., greater than 80%, 85%, 90%, 95%, or 98%) of the total bacterial population following administration of the microbial consortium.

62. The microbial consortium for use of claim 59 or 60, wherein the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 5 percentage points (e.g., at least 10, 15, 20, 25, 30, 40, 50, or 60 percentage points) following administration of the microbial consortium.

63. A composition comprising Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770.

64. A composition comprising Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772.

65. A composition comprising Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771.

66. A composition comprising Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

67. A composition comprising two or more Lactobacillus bacteria selected from the group consisting of:(a) a Lactobacillus jensenii isolate obtained from a bacterial sample deposited as DSM Deposit Number 34770;(b) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34772;(c) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34771, and(d) a Lactobacillus crispatus isolate obtained from a bacterial sample deposited as DSM Deposit Number 34773.

68. A composition comprising Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value.

69. A composition comprising Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value.

70. A composition comprising Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value.

71. A composition comprising Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value.

72. A composition comprising two or more Lactobacillus bacteria selected from the group consisting of:(a) Lactobacillus jensenii isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34770, has a percent identity greater than or equal to a threshold value;(b) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34771, has a percent identity greater than or equal to a threshold value;(c) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34772, has a percent identity greater than or equal to a threshold value; and(d) Lactobacillus crispatus isolate having a genome that, when aligned with the genome of a bacterial sample deposited as DSM 34773, has a percent identity greater than or equal to a threshold value.

73. The composition of any one of claims 68-72, wherein the threshold value is 99.81%, 99.90%, or 99.96%.

74. A pharmaceutical composition comprising the microbial consortium or composition of any one of claims 1-73 and a pharmaceutically acceptable buffer, diluent or excipients.

65. A method of increasing the relative abundance of lactobacilli in the female genitourinary tract of a subject comprising administering the microbial consortium of any one of claims 1-64 to the genitourinary tract of the subject.

66. The method of claim 65, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 90% (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, or 20%) of the total bacterial population prior to administration of the microbial consortium.

67. The method of claim 65 or 66, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 70% (e.g., greater than 80%, 85%, 90%, 95%, or 98%) of the total bacterial population following administration of the microbial consortium.

68. The method of claim 65 or 66, wherein the relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 5 percentage points (e.g., at least 10, 15, 20, 25, 30, 40, 50, or 60 percentage points) following administration of the microbial consortium.

69. The microbial consortium of any one of claims 1-64 for use in therapy.

70. The microbial consortium of any one of claims 1-64 for use in a method of treating dysbiosis of the female genitourinary tract of a subject, said method comprising administering the microbial consortium to the genitourinary tract of the subject, optionally wherein the subject is asymptomatic.

71. The microbial consortium of any one of claims 1-64 for use in a method of treating inflammation of the female genitourinary tract of a subject, said method comprising administering the microbial consortium to the genitourinary tract of the subject, optionally wherein the subject is asymptomatic.

72. The microbial consortium for use of claim 70 or 71, wherein the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is less than 90% (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, or 20%) of the total bacterial population prior to administration of the microbial consortium.

73. The microbial consortium for use of any one of claims 70-72, wherein: the female genitourinary tract of the subject comprises a bacterial population having a relative abundance of Lactobacillus that is greater than 70% (e.g., greater than 80%, 85%, 90%, 95%, or 98%) of the total bacterial population following administration of the microbial consortium; orthe relative abundance of Lactobacillus in the bacterial population of the female genitourinary tract of the subject is increased by at least 5 percentage points (e.g., at least 10, 15, 20, 25, 30, 40, 50, or 60 percentage points) following administration of the microbial consortium.