Composition for vaginal therapy comprising lactobacillus species

Abstract

Compositions and methods for treating patients are disclosed. Example compositions may include vaginal microbiota compositions. Vaginal microbiota compositions may include bacterial mixtures. Bacterial mixtures may include *Lactobacillus curvatureii*, *Lactobacillus gasseri*, *Lactobacillus janniae* / *Lactobacillus mutans*, and *Lactobacillus rhamnosus*.

Description

Technical Field

[0001] This disclosure relates to compositions and methods for treating patients. Background Technology

[0002] Various compositions and methods have been developed for treating diseases and / or conditions, such as those of the reproductive system, digestive tract, liver, immune system, etc. Each known composition and method has its own advantages and disadvantages. There remains a persistent need for alternative compositions and methods to treat diseases and / or conditions. Summary of the Invention

[0003] In a first aspect, a composition is provided for use in a therapy, the composition comprising (or consisting of): The composition comprises at least one or more Lactobacillus species, and / or supernatant derived from cultures of the Lactobacillus species, including Lactobacillus curvatureii, Lactobacillus gasseri, Lactobacillus janniae / Lactobacillus muscaria, and / or Lactobacillus rhamnosus, and optionally includes one or more pharmaceutically acceptable adjuvants and / or excipients.

[0004] Further aspects and teachings are provided below and in accordance with the appended claims.

[0005] The composition may contain one, two, three, or all four of the aforementioned *Lactobacillus* species. In one embodiment, the composition contains at least *Lactobacillus rhamnosus*, which may be present in an amount between 10% and 100%, typically between 25% and 95%, for example between 40% and 90% (the percentage age of the total *Lactobacillus* species present in the composition). In one embodiment, the composition comprises a mixture of all four aforementioned *Lactobacillus* species, wherein each species is present in an independent amount between 10% and 50%, for example between 15% and 30%. Conveniently, each species may be provided in approximately equal percentages. Typically, this may be between approximately 20% and 25%.

[0006] This disclosure provides for the design, materials, manufacturing methods, and alternative uses of compositions and methods for treating patients. In one instruction, vaginal microbiota compositions are disclosed. As used herein, the terms “vaginal” or “vagina” should be understood to refer not only to the vaginal region but also to any urogenital region, and therefore should be interpreted broadly unless the context otherwise requires. In addition to the aforementioned *Lactobacillus* species, the vaginal microbiota composition may also contain one or more of the following bacteria: *Lactobacillus inertes*, *Lactobacillus reuteri*, *Lactobacillus acidophilus*, *Lactobacillus vaginalis*, *Lactobacillus johnsonii*, *Lactobacillus helveticus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus salivarius*, and *Lactobacillus delbrueckii*. When any of these additional *Lactobacillus* species are present in the composition, they may be present in any suitable amount. Typically, they may be present independently in amounts of 0.5%–10%, such as 1%–5%. In some cases, the compositions disclosed herein may not include bacterial species other than those of the *Lactobacillus* genus. Therefore, in some cases, the composition may contain bacteria consisting of or substantially consisting of Lactobacillus species.

[0007] According to this disclosure, the compositions described herein can be useful in methods for restoring a beneficial vaginal microbiota and / or increasing resistance to vaginal diseases, including bacterial vaginosis (BV) and / or sexually transmitted diseases. The usefulness of the compositions can be enhanced by applying an acidifier (such as lactic acid) before, during, and / or after application. Conveniently, a mixture of D- and L-form lactic acid can be added. Typically, the ratio of D-lactic acid to L-lactic acid can be between 1:8 and 1:2, for example, 1:6 to 1:3 (w / w).

[0008] The composition can also be filtered to achieve a sterile state and remove particles, aggregates and cells, thereby being applied in filtrate form, and optionally mixed with isolated and cultured Lactobacillus bacteria, or spray-dried or lyophilized (optionally with one or more cryoprotectants), and optionally packaged into a single-dose unit applicator.

[0009] Alternatively or additionally for any of the above embodiments, the composition comprises or includes one or more lyophilized bacterial strains.

[0010] Alternatively or additionally, for any of the above embodiments, the composition is placed in a capsule.

[0011] Alternatively or additionally, for any of the above embodiments, the composition is placed in a suppository.

[0012] Alternatively or additionally for any of the above embodiments, the composition is placed in a soluble shell.

[0013] A composition for use in a method of treating an infection is disclosed. The method includes administering the composition, such as any vaginal composition described herein, to an infected patient. The composition can be used to modulate, for example, attenuate, the immune response of epithelial cells (e.g., vaginal epithelial cells) against pathological microbial species present in the vagina (e.g., in biofilms). The immune response may be associated with the expression of one or more inflammatory markers, and the composition described herein can be used to reduce the expression and / or function of said markers. Accordingly, in another embodiment, a composition is provided for use in a method of treating inflammation, particularly inflammation caused by an infection, especially a vaginal disease (including bacterial vaginosis (BV) and / or sexually transmitted diseases). In one embodiment, the inflammatory marker may be selected from one or more of LDH (lactate dehydrogenase), IL-6, and CXCL8.

[0014] In another aspect, a method for treating an infection is provided, wherein the method comprises administering a composition, such as the vaginal composition of any of the embodiments described herein, to an infected patient. The composition can be used to modulate, for example, attenuate, the immune response of epithelial cells (e.g., vaginal epithelial cells) against pathological microbial species present in the vagina (e.g., in biofilms). The immune response may be associated with the expression of one or more inflammatory markers, and the composition described herein can be used to reduce the expression and / or function of said markers. Accordingly, in another embodiment, a method for treating inflammation, particularly inflammation caused by an infection, especially a vaginal disease (including bacterial vaginosis (BV) and / or sexually transmitted diseases), is provided. In one embodiment, the inflammatory marker may be selected from one or more of LDH (lactate dehydrogenase), IL-6, and CXCL8.

[0015] In another aspect, the use of compositions, such as the vaginal compositions of any of the embodiments described herein, in the preparation of medicaments for treating infections is provided. The compositions can be used to modulate, for example, weaken, the immune response of epithelial cells (e.g., vaginal epithelial cells) against pathological microbial species present in the vagina (e.g., in biofilms). Immune responses can be associated with the expression of one or more inflammatory markers, and the compositions described herein can be used to reduce the expression and / or function of said markers. Accordingly, in another embodiment, the use of compositions in the preparation of medicaments for treating inflammation, particularly inflammation caused by infection, especially vaginal diseases (including bacterial vaginosis (BV) and / or sexually transmitted diseases). In one embodiment, the inflammatory marker may be selected from one or more of LDH (lactate dehydrogenase), IL-6, and CXCL8.

[0016] Alternatively or additionally for any of the above embodiments, the infection may include one or more of bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, and gynecological cancer.

[0017] A method for treating a patient is disclosed. The method includes administering the composition, such as any one of the embodiments described herein, to the patient.

[0018] Alternatively or additionally with respect to any of the embodiments described herein, administration of the vaginal microbiota composition to a patient may include one or more of the following: prevention of preterm birth, prevention of miscarriage, treatment of infertility, treatment of interstitial cystitis, and treatment of polycystic ovary syndrome.

[0019] The above overview of some embodiments is not intended to describe every disclosed embodiment or implementation. The following detailed description illustrates these embodiments in more particular terms. Detailed Implementation

[0020] For the purposes of the following definitions, unless otherwise provided in the claims or elsewhere in this specification, these definitions shall apply.

[0021] All numerical values ​​in this document (whether explicitly stated or not) are assumed to be modified by the term "about". The term "about" generally refers to a range of numbers that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result). In many cases, the term "about" may include numbers rounded to the nearest significant figure.

[0022] A description of a numerical range using endpoints includes all the numbers in that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0023] As used in this specification and the appended claims, the singular forms “a / an” and “the” include plural references unless the context clearly indicates otherwise. As used in this specification and the appended claims, the term “or” is generally used in its meaning as including “and / or” unless the content expressly specifies otherwise.

[0024] It should be noted that references to "one embodiment," "some embodiments," "other embodiments," etc., in the specification indicate that the described embodiments may include one or more specific features, structures, and / or characteristics. However, such descriptions do not necessarily imply that all embodiments include the specific features, structures, and / or characteristics. Furthermore, when a specific feature, structure, and / or characteristic is described in conjunction with one embodiment, it should be understood that regardless of whether such a feature, structure, and / or characteristic is explicitly described (unless clearly stated otherwise), such a feature, structure, and / or characteristic may also be used in conjunction with other embodiments.

[0025] As used herein, the term "supernatant" refers to the liquid phase of a culture that has undergone some form of separation procedure. For example, bacterial cultures subjected to gravity-based separation techniques (such as centrifugation) (e.g., cultures containing any of the bacterial species described herein) produce a precipitate containing the cultured bacteria and a liquid phase referred to as the supernatant. The supernatant will contain chemicals, metabolites, and other compounds produced by the bacteria during culture. Thus, therapeutic benefits attributable to the bacteria themselves can be obtained by using one or more supernatants derived from the various bacteria described herein, which contain therapeutic compounds produced by the cultured bacteria.

[0026] The human microbiome (or human microbiota) is an aggregate of microorganisms residing on the surface and deep layers of the skin, in saliva and oral mucosa, in the conjunctiva, and in the human gastrointestinal tract, urogenital tract, and / or vagina, including microorganisms related to reproductive health, such as the placental microbiome. The human microbiome is composed of bacteria, but may also include fungi, bacteriophages, viruses, archaea, and others. Some of these organisms perform tasks useful to the human host, but the functions of most of the organisms that make up the human microbiome remain to be studied. Under normal circumstances, these microorganisms do not cause disease in the human host but rather participate in maintaining health.

[0027] The vaginal microbiota may play a role in many different health conditions. Some of these conditions may include infections such as bacterial vaginosis, candidiasis, human papillomavirus (HPV) infection, urinary tract infections, and / or sexually transmitted infections. Other conditions may include gynecological cancers (e.g., cervical cancer), premature birth, miscarriage, infertility, interstitial cystitis, polycystic ovary syndrome (PCOS), etc. Restoring, standardizing, and / or otherwise altering the composition of the vaginal microbiota can help alleviate these and other conditions. This article discloses compositions and methods for treating patients. At least some of the compositions include vaginal microbiota compositions. At least some of the compositions are used in treatment methods, including methods for treating bacterial vaginosis, treating candidiasis, treating HPV infection, treating urinary tract infections, treating sexually transmitted infections, treating gynecological cancers (e.g., cervical cancer), preventing premature birth, preventing miscarriage, treating infertility, treating interstitial cystitis, treating PCOS, etc.

[0028] As used herein, the term "urogenital region" refers to the region of the anus and genitals. In some embodiments, the female urogenital region includes the cervix, vagina, vulva, clitoris, urethral orifice, vulvar vestibule, perineum, and / or anus. In one teaching, the term "urogenital" refers to the distal urinary tract region and the genital region. In some embodiments, the female urogenital region includes the cervix, vagina, vulva, clitoris, vaginal orifice, urethral orifice, urethral fold, vulvar vestibule, and / or perineum.

[0029] As used herein, the term "vaginal microbiota" refers to the collective microorganisms that are normally colonized in the vulva, clitoris, vestibule, and vagina. As used herein, the term "vaginal microbiota" refers to the collective microorganisms that are normally colonized in the reproductive area and are non-pathogenic. The vaginal microbiota can refer to the vaginal microbiota of a female subject (e.g., the vaginal mucosal microbiota), the cervical microbiota of a female subject, the vulvar microbiota of a female subject, or any combination thereof.

[0030] In healthy women of reproductive age, bacteria of the genus *Lactobacillus* tend to dominate the vaginal microbiome. Four species were consistently identified in women: *Lactobacillus curvatureii*, *Lactobacillus indolentii*, *Lactobacillus gasseri*, and *Lactobacillus janseri*. These species are listed in order of their frequency of dominance in Caucasian, African, Asian, and Hispanic women. Generally, lactobacillus dominance in the vaginal microbiome is associated with health. This dominance of a single genus or species results in a low-diversity microbiome. Increased vaginal microbiome diversity is associated with poor health and a higher risk of infection. For example, bacteria from the genera *Sneathia*, *Megasphaera*, *Atopobium* (also known as *Fannyhessea*), *Peptoniphilus*, *Dialister*, *Prevotella*, *Mobiluncus*, and / or *Gardnerella* may tend to be associated with an unhealthy vaginal microbiota, as they have been identified in women with other poor vaginal health indicators (high Nugent score and vaginal pH). However, these profiles may vary across different populations.

[0031] The vaginal microbiome can change between pre-pubertal, premenopausal, and postmenopausal women. For example, pre-pubertal women may have relatively low relative abundance of lactobacilli and relatively high microbial diversity (as well as relatively thin vaginal epithelium, a relatively thin mucus layer, and relatively low glycogen levels). Premenopausal women may have relatively high relative abundance of lactobacilli and relatively low microbial diversity (as well as relatively thick vaginal epithelium, a relatively thick mucus layer, relatively high estrogen levels, and relatively low glycogen levels). Postmenopausal women may have moderate relative abundance of lactobacilli and relatively low or low microbial diversity (as well as relatively thin vaginal epithelium, a relatively thin mucus layer, relatively moderate estrogen levels, and relatively moderate glycogen levels). Postmenopausal women with symptoms of vaginal infection (e.g., bacterial vaginosis) may have lower relative abundance of lactobacilli and relatively high microbial diversity. Overall, compositions that tend to include or otherwise increase the relative abundance of lactobacilli and help reduce microbial diversity may be beneficial.

[0032] As used in this article, bacterial vaginosis (“BV”) refers to the overgrowth of one of the several non-lactobacterial bacteria that are normally present in the vagina, disrupting the natural balance of vaginal bacteria.

[0033] In at least some cases, the compositions disclosed herein (e.g., vaginal microbiota compositions) utilize vaginal microbiota collected from vaginal microbiota donors. However, other sources of vaginal microbiota are considered, including bacterial cultures, etc. Potential vaginal microbiota donors can be identified and may undergo numerous screening processes. In some cases, health history questionnaires can be used to screen potential donors. Example questionnaires may be the same as or similar to those used by the Red Cross to screen potential blood donors. In some such cases and in others, potential donors may be screened for common infectious diseases and other conditions. Such screening may include blood tests, stool tests, urine tests, vaginal swab tests, etc. Such tests may include testing for the presence of HIV, hepatitis (hepatitis A, hepatitis B, and / or hepatitis C), syphilis, Clostridium difficile, bacterial pathogens, ovum, and parasites and / or the like. These are merely examples. The health of a donor can be monitored by performing blood tests, analyzing stool samples, analyzing urine samples, analyzing vaginal secretions, periodically updating health history, etc. Vaginal swabs can be collected to assess vaginal health via Gram staining and Nugent score, white blood cell count, and detection of yeast presence. Urine and / or vaginal swabs can be collected to test for sexually transmitted infections.

[0034] Once a potential donor has been determined to be sufficiently healthy to donate, a sample collection kit (e.g., a self-collection kit) is provided to the donor. The sample collection kit may include donor instructions, a vaginal DNA / RNA collection kit for sequencing analysis (e.g., the OMNIgene Vaginal Collection Kit ORM-130, commercially available from DNAgeneotek™), and a sample collection and transport kit (e.g., the eSwab 480C Copan Liquid Amis Elution Swab Collection and Transport System, commercially available from Copan Diagnostics). Informed consent may be required from the donor. Two samples may be collected from each donor. The first sample is used for sequencing analysis. For example, after handwashing, a swab from a vaginal DNA / RNA collection kit can be used to collect a vaginal sample by inserting the swab a few inches into the vagina and wiping the vaginal wall for approximately 20 seconds. The swab can then be inserted into a tube containing a stability buffer, and the tube / sample can be further analyzed. The second sample can be used to culture vaginal microbiota. For example, a swab from a sample collection and transport kit can be used to collect a vaginal sample. The swab is then inserted into a tube containing a liquid acetylene solution (e.g., which can preserve aerobic, anaerobic, and recalcitrant bacteria for up to 48 hours). After collection, the donor places the sample tube and material in a biohazard bag and then places the sample in a donation facility.

[0035] To process samples for sequencing (e.g., shotgun sequencing), first rehydrate the protease (e.g., QIAGEN protease) using sterile, PCR-standard water to produce an 80 mg / mL solution (this may include inverting the sample 10 or more times). The sample (e.g., a vaginal DNA / RNA collection kit sample) can be removed from the refrigerator and vortexed for 30 seconds. The collection tube can be shaken three times to ensure the solution containing the sample reaches the bottom. Five microliters of rehydrated protease can be added to the collection tube containing the vaginal swab sample. The sample can be inverted 10 or more times. The sample is then incubated at 50°C for 2 hours. The sample is then vortexed for 30 seconds. The collection tube can then be shaken three times to ensure the solution containing the sample reaches the bottom. The swab can be removed from the tube (this may include pressing the swab against the side of the tube to recover the sample absorbed into the swab). 500 μL of sample can be aliquoted into two 2 mL cryovials for storage and can be stored at -80°C until ready for sequencing analysis (it can be stored in suitable processing / analysis facilities such as Divergent). TM (The event was conducted in St. Paul, Minnesota).

[0036] To process samples for culture, samples (e.g., sample collection and transport kit samples) can be placed in the airlock of an anaerobic culture chamber and introduced into the chamber. While the swab is still in its original position, the tube can be vigorously swirled for 5 seconds to release the sample from the swab tip. This is undiluted or 10... 0 Diluent. The swab can then be removed. A 10-fold serial dilution can be performed by transferring 100 μL of sample to a tube containing 900 μL of sterile saline (0.9%). The sample can be vigorously vortexed for 5 seconds and / or mixed by inverting. This is 10 -1 Diluent. 100 microliters of 10 -1 Transfer the diluent to a new tube containing 900 μL of sterile physiological saline (0.9%). The sample can be vigorously vortexed for 5 seconds and / or mixed by inverting. This is 10 -2 Diluent. This process can be repeated until the sample has been diluted to 10⁻⁶. -6 Diluent.

[0037] Triple copies can be prepared from the following dilution tubes on CDC anaerobic 5% sheep blood agar, De Man, Rogosa and Sharpe (MRS) agar, and 50% MRS agar plates: 10 -3 10 -4 10 -5In short, 100 µL can be transferred directly from a suitable tube to the surface of an agar plate, and the inoculum is spread over the entire plate surface using a sterile L-shaped spreader and plate spinner. The plate can be incubated at 35°C under anaerobic conditions for 24–120 hours. The appearance of new colonies can be checked daily. If the plate begins to dry, it can be wrapped with paraffin film. Colonies can be observed, and colony counts and morphology can be recorded (this may include capturing photographs of the plate to aid in recording colony morphology). Individual colonies can be selected for further characterization and streaked onto new plates. If desired, selective media such as Bifidobacterium selective agar (BSA), Enterococcus agar (ECA), cetyltrimethylammonium bromide agar (CA), mannitol agar (MSA), etc., can be used.

[0038] In some cases, Gram staining can be used. When doing so, the sample can be applied to a glass slide. If staining liquid cultures, a sterile inoculating loop can be used to apply the culture directly to a glass slide as a smear. If staining colonies from a plate, a drop of sterile saline can be added to the center of a glass slide using an inoculating loop, and then a small number of single colonies can be collected and added to the drop. If possible, the drop and sample can be gently mixed to produce a thin, homogeneous smear. The drop can be dried. The glass slide can be fixed with methanol by immersing it in anhydrous methanol for 1–2 minutes, followed by rinsing with tap water. The fixed smear can be immersed in a primary stain (e.g., crystal violet) for 1 minute. The primary stain can be removed by gently rinsing with tap water. The fixed smear can be immersed in a secondary stain / mordant (e.g., Gram's iodine) for 1 minute, followed by gentle rinsing with tap water. The slide can be decolorized until the solvent flowing from the slide is colorless (e.g., about 3-60 seconds), and the slide can be gently washed with tap water. Then, the slide can be immersed in a counterstain (e.g., safranin; if the initial counterstaining result is unsatisfactory, basic fuchsin can be substituted) for 1 minute. The slide can be washed with tap water and allowed to dry. Examples of smears can be illustrated under an oil immersion lens, and the results / observations can be recorded.

[0039] Other sample tests can be performed, including a catalase test, which may involve adding hydrogen peroxide to differentiate between staphylococci and streptococci. Another sample test that can be performed may include a coagulase test, which may involve adding plasma to differentiate between staphylococci and streptococci.

[0040] Isolates identified as *Lactobacillus* can be further characterized to determine their antibiotic susceptibility. This may include suspending *Lactobacillus* colonies from an overnight plate in a broth consisting of 90% Iso-sensitest broth and 10% MRS broth until the suspension is equivalent to a McFarland standard of 1. A sterile swab can then be dipped into the inoculum, and the tube wall squeezed to remove excess liquid. The swab can be streaked across the entire agar surface (rotating two or more times to ensure even distribution). After excess moisture has been absorbed, a minimum inhibitory concentration (MIC) test strip can be applied (e.g., this may include applying a test strip with upward-facing graduations and a code to the outside of the location). The test strip can be pressed against the agar surface, ensuring the full length of the antibiotic gradient is in complete contact with the agar surface (repositioning if necessary). The plate can be inverted and incubated at 35°C for 24–48 hours (or longer). These results can be interpreted.

[0041] Isolated bacterial strains can be cryopreserved and stored. This can include inoculating overnight cultures with well-isolated colonies selected from streak plates. If many colonies are to be selected, a 96-well plate can be used. Suitable broth can be used (e.g., MRS might be used for Lactobacillus). Cultures can be incubated overnight in an anaerobic incubator at 35°C. If the culture is not turbid by the next morning, it can be incubated for an additional time. Once the culture becomes visibly turbid, 200-300 μL can be transferred to a new 96-well plate containing a cryoprotectant (e.g., 50-60 μL of 50% glycerol). The final concentration of glycerol can be 10%. The 96-well plate can be sealed and stored in a freezer at -80°C.

[0042] Cryoprotectants are any agents that prevent the formation of ice crystals that can rupture cell membranes, and therefore, cryoprotectants refer to substances used to protect biological cells or tissues from the effects of freezing. Cryoprotectants include extracellular cryoprotectants that do not penetrate bacterial cell walls and intracellular cryoprotectants that do penetrate bacterial cell walls. Examples of cryoprotectants include dextrose, betaine, glycine, sucrose, polyvinyl alcohol, polyethylene glycol (PEG), Pluronic F-127, mannitol, Tween 80, ethylene glycol, 1,3-propanediol, hydroxypropyl cellulose, glycerol, PEG / glycerol mixtures, milk (e.g., skim milk), and propylene glycol. In some embodiments, PEG may be preferred.

[0043] As suggested in this article, a single microbial strain (e.g., a single bacterial strain or species) can be isolated from a swab. This can include one or more processes involving plating on an agar medium selective for the desired strain, followed by “purifying” the strain as a single colony isolate. The single microbial strain can be grown / cultured and can be species identified / characterized through sequencing, biochemical tests, colony morphology, cell morphology, microscopic mortality, and sensitivity to different representative antibiotics. The microbial strain can be altered. In some cases, the microbial strain may include one or more of the following: *Lactobacillus rhamnosus*, *Lactobacillus curvaturei*, *Lactobacillus gasseri*, *Lactobacillus janseri* / *Lactobacillus muhrii*, and optionally one or more of the following: *Lactobacillus reuteri*, *Lactobacillus acidophilus*, *Lactobacillus vaginalis*, *Lactobacillus indolenti*, *Lactobacillus johnsonii*, *Lactobacillus helveticus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus salivarius*, *Lactobacillus d'Erbrücke*, *Gardnerella vaginalis*, non-lactobacterial bacteria: *Atopobium vaginae*, *Prevotella bivia*, other species of the following genera: *Macrococcus*, *Trichophyton*, *Microbacterium*, *Peptospira*, fungi, combinations thereof, etc.

[0044] It should be noted that this article refers to *Lactobacillus janniae* and *Lactobacillus muscaria*. In 2020, *Lactobacillus muscaria* was identified as a new bacterial species, and analysis indicated that many *Lactobacillus janniae* species should be reclassified as *Lactobacillus muscaria*. Therefore, and for the purposes of this disclosure, *Lactobacillus janniae* and *Lactobacillus muscaria* are used together and are considered synonyms.

[0045] In some cases, each component can be lyophilized. The total number of microorganisms in the example composition can be approximately 1 x 10⁻⁶. 5 Up to 1 x 10 15 CFU / ml, or approximately 1 x 10 6 Up to 1 x 10 12 CFU / ml, or approximately 1 x 10 7 Up to 1 x 10 10 CFU / ml, or 1 x 10 8 Up to 1 x 10 9 CFU / ml. Therefore, the number of microorganisms can be understood as being related to the number of microorganisms prior to lyophilization. The composition can be placed in a suitable delivery medium, such as capsules, suppositories, soluble shells, and / or the like. In some cases, the composition can be administered orally. In other cases, the composition can be administered topically (e.g., by inserting capsules, suppositories, soluble shells, etc., into the vagina).

[0046] Example compositions may include strains of *Lactobacillus curvatureii*, *Lactobacillus gasseri* / *Lactobacillus mutans*, *Lactobacillus janniae*, and *Lactobacillus rhamnosus* having the desired properties. For example, strains of each component may be selected to have pH-lowering activity, lactic acid production, inhibitory activity against other microorganisms, other unique activities, combinations thereof, and / or such.

[0047] Example compositions may include lyophilized *Lactobacillus curvatureii*, lyophilized *Lactobacillus gasseri*, lyophilized *Lactobacillus janniae* / *Lactobacillus mutans*, and lyophilized *Lactobacillus rhamnosus*. Each component can be isolated from a donor. For example, a composition may include about 20%-85% *Lactobacillus rhamnosus*, or about 25%-75% *Lactobacillus rhamnosus*, or about 30%-60% *Lactobacillus rhamnosus*, or about 40% *Lactobacillus rhamnosus*. A composition may include about 1%-10% *Lactobacillus gasseri*, or about 2%-8% *Lactobacillus gasseri*, or about 5% *Lactobacillus gasseri*. A composition may include about 1%-10% *Lactobacillus janniae*, or about 2%-8% *Lactobacillus janniae*, or about 5% *Lactobacillus janniae*. A composition may include about 0.1%-5% *Lactobacillus curvatureii*, or about 0.5%-4% *Lactobacillus curvatureii*, or about 1% *Lactobacillus curvatureii*. One or more components may be derived / isolated from a donor sample and / or bacterial culture. Prior to freeze-drying, the total number of microorganisms in the example composition can be approximated to about 1 x 10⁻⁶. 5 Up to 1 x 10 15 CFU / ml, or approximately 1 x 10 6 Up to 1 x 10 12 CFU / ml, or approximately 1 x 10 7 Up to 1 x 10 10 CFU / ml, or 1 x 10 8 Up to 1 x 10 9 CFU / ml. The composition can be placed in a suitable delivery medium, such as capsules, suppositories, soluble shells, and / or the like. In some cases, the composition can be administered orally. In other cases, the composition can be administered topically (e.g., by inserting capsules, suppositories, soluble shells, etc., into the vagina).

[0048] Example compositions may include strains of *Lactobacillus curvatureii*, *Lactobacillus gasseri*, *Lactobacillus janniae* / *Lactobacillus mutans*, and *Lactobacillus rhamnosus* with the desired properties. For example, strains of each component may be selected to have pH-lowering activity, lactic acid production (typically a combination of D- and L-forms), inhibitory activity against other microorganisms, other unique activities, combinations thereof, and / or such.

[0049] As mentioned above, prior to freeze-drying, the total number of microorganisms in the example composition can be approximated to about 1 x 10⁻⁶. 5 Up to 1 x 10 15CFU / ml, or approximately 1 x 10 6 Up to 1 x 10 12 CFU / ml, or approximately 1 x 10 7 Up to 1 x 10 10 CFU / ml, or 1 x 10 8 Up to 1 x 10 9 CFU / ml. This may correspond to the total number of microorganisms in a single capsule, suppository, soluble shell, and / or such. Alternatively, this may correspond to the total number of microorganisms in a suitable dose of a composition, which may comprise one or more capsules, suppositories, soluble shells, and / or such.

[0050] A single capsule, suppository, soluble shell, and / or similar encapsulated pharmaceutical product or dose may be administered to a patient. This may include administering the dose to the patient using a suitable dosing regimen. This may include administering one or more doses to the patient. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may be administered to the patient. In some such cases and others, the encapsulated pharmaceutical product may be administered to the patient over one or more days. For example, these doses may be administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. In some such cases and others, these doses may be administered to the patient once or more daily. For example, these doses can be administered to patients once, twice, three times, four times, five times, six times or more per day.

[0051] One example dosing regimen may include administering two doses to a patient twice daily for two days. Another example dosing regimen may include administering four doses to a patient twice daily for two days. Another example dosing regimen may include administering four doses to a patient twice daily for four days. Another example dosing regimen may include administering two doses to a patient daily for four days. Another example dosing regimen may include administering one dose to a patient daily for eight days. These are merely examples. In at least some of these examples, each dose may comprise a lyophilized material containing a mixture of *Lactobacillus curlis*, *Lactobacillus gasseri*, *Lactobacillus janniae*, and *Lactobacillus rhamnosus*.

[0052] In at least some cases, after application of the composition as described herein, the vaginal microbiota composition can substantially (e.g., < 1 x 10⁻⁶) be effective. 2 -1 x 10 3The vaginal microbiota composition (CFU / ml) does not contain bacteria from the following genera: Trichophyton, Megacoccus, Miasma, Peptone, Microbe, Prevotella, Mobiluncus, and / or Gardnerella. For example, the vaginal microbiota composition may not contain bacteria from Miasma, Gardnerella, and Prevotella.

[0053] The vaginal microbiota composition disclosed in this article can be used to treat a variety of different health conditions. For example, the vaginal microbiota composition can be used to treat bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, gynecological cancer (e.g., cervical cancer), reduce and / or prevent premature birth, reduce and / or prevent miscarriage, treat infertility, treat interstitial cystitis, and treat polycystic ovary syndrome, etc.

[0054] Also considered vaginal microbiota compositions include combinations of full-spectrum vaginal microbiota with one or more additional microorganisms (e.g., those collected and isolated from a donor). For the purposes of this disclosure, full-spectrum vaginal microbiota can be understood as a collection of microorganisms present in the vagina of a typical woman. No intentional manipulation of the full-spectrum vaginal microbiota is made to alter the presence or absence of any particular microorganism in the sample, but rather it is intended to represent the complete community of organisms in the sample. It is understood that the composition of the vaginal microbiota may vary from person to person. Therefore, full-spectrum vaginal microbiota may vary. In some cases, considered vaginal microbiota compositions include full-spectrum vaginal microbiota combined, mixed, or doped with one or more of the following: *Lactobacillus rhamnosus*, *Lactobacillus curvatureii*, *Lactobacillus gasseri*, *Lactobacillus janseri* / *Lactobacillus mutans*, and optionally one or more of the following: *Lactobacillus reuteri*, *Lactobacillus acidophilus*, *Lactobacillus vaginalis*, *Lactobacillus indolentiary*, *Lactobacillus johnsonii*, *Lactobacillus helveticus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus salivarius*, *Lactobacillus d'Eau*. In at least some cases, the full spectrum of vaginal microbiota and other microbes are derived from vaginal microbiota donors.

[0055] Bacteria / microbes can be placed in suitable containers (e.g., capsules, suppositories, soluble shells, and / or similar) for oral or topical delivery. In some cases, simulated vaginal fluid can be incorporated into the vaginal microbiota composition. In some such cases and others, the vaginal microbiota composition may include glycogen. The total volume contained in the container / suppository can be approximately from about 100 microliters to about 10 milliliters.

[0056] In at least some cases, the bacteria / microbes in the vaginal microbiota composition may include lyophilized bacteria / microbes. This may include a lyophilization process in which the bacteria / microbes are freeze-dried. The bacteria / microbes and / or lyophilized bacteria / microbes may be placed in suitable containers (e.g., capsules, suppositories, soluble shells, and / or the like) for oral or topical delivery.

[0057] Treatment of a patient may include administering a vaginal microbiota composition to the patient. One therapeutic goal may be to normalize the patient's vaginal microbiota by effectively replacing it with the vaginal microbiota composition. This may include direct administration of the vaginal microbiota composition to the patient's vagina via implantation, suppositories, or another suitable route of administration.

[0058] In at least some cases, multiple vaginal microbiota compositions can be stored in or deposited in suitable storage devices / facilities. Storage devices may include temperature-controlled devices such as freezing devices (e.g., 4°C freezing devices), freezers (e.g., -20°C freezers), deep freezers (e.g., -80°C freezers), etc. In some cases, an indexing system may also be linked to or otherwise associated with a vaginal microbiota composition library. The indexing system may include data that can be used to match vaginal microbiota compositions from a given donor, including a suitable patient, such as donor data. Donor data may include age at collection, reproductive status at collection (non-menopausal, menopausal), ethnicity, menstrual stage at collection, history of bacterial vaginosis, history of Candida infection, history of human papillomavirus (HPV), other medical histories (e.g., history of Clostridium difficile infection, cancer diagnosis / treatment history, etc.), combinations thereof, and / or the like. Clinicians can use donor data to tailor treatment plans for specific patients. For example, patients with bacterial vaginosis can be treated with a vaginal microbiota composition derived from a donor with a history of successful remission of bacterial vaginosis.

[0059] In some cases, donor data may include a pregnancy history. For example, some donors may have a high probability of pregnancy when seeking pregnancy or otherwise having an increased actual or perceived fertility. To attempt to increase or enhance a patient's fertility, a vaginal microbiome composition comprising microorganisms from such donors may be administered to patients experiencing fertility challenges. In such cases, the vaginal microbiome composition may be administered as an oral capsule. Alternatively, the vaginal microbiome composition may be administered as an implant and / or otherwise directly inserted into the patient's vagina.

[0060] U.S. Patent No. 9,675,648 is incorporated herein by reference.

[0061] U.S. Patent No. 9,629,881 is incorporated herein by reference.

[0062] U.S. Patent No. 10,226,431 is incorporated herein by reference.

[0063] U.S. Patent Application Publication No. US 2018 / 0289750 is incorporated herein by reference.

[0064] Example

[0065] This disclosure can be further illustrated by referring to the following examples and the accompanying drawings, which show:

[0066] Figure 1 Biofilm-forming ability of Lactobacillus isolates. (A) Crystal violet determination of single-species biofilms at 24 hours. Macroscopic images taken before dye leaching (top), with absorbance readings after ethanol leaching shown (bottom). Graphs show the mean and standard deviation for n = 3. Statistical data were analyzed using one-way ANOVA with multiple comparisons supplemented by Tukey post-hoc tests. p < 0.001 indicates comparison between BPL5 and all other isolates. (B) Live / dead cell fluorescence staining of single-species biofilms at 24 hours using SYTO9 (live) and propidium iodide (PI; dead) dyes. Images taken at 40x objective on an AMG EVOS fl microscope.

[0067] Figure 2 Lactobacillus treatment of bacterial vaginosis biofilms. Experimental overview (A), followed by viable cell levels of Gardnerella vaginalis (B), Fannie Hercetella vaginalis (C), Molecularcurvatus (D), and Prevotella ilex (E). The values ​​for each individual species are summed (F) and the composition of viable cells within the biofilm is calculated (G).

[0068] Figure 3 Supernatant treatment. Viable cell count from the BVAB biofilm (left) after application of Lactobacillus supernatant. The table shows the pH values ​​from Lactobacillus soup with and without pH adjustment.

[0069] Figure 4 Lactic acid against Gardnerella vaginalis. Showing D- and L-lactic acid levels produced by probiotic isolates at 24 (A) and 48 (B) hours. pMIC of D-lactic acid (C) and L-lactic acid (D) against Gardnerella vaginalis is shown. Synergistic effects of D- and L-lactic acid (E) and biofilm treatment using lactic acid (F) are also shown.

[0070] Figure 5Mixture treatment. BVAB biofilms were treated with a mixture of Lactobacillus bacteria. Data show the biofilm composition after 4 hours (A) and 24 hours (B). The actual amount of biofilm after 24 hours is also shown (C).

[0071] Figure 6 Lactobacillus treatment attenuates the inflammatory potential of BV biofilms on VK2 cells. (AC): BV bacteria (Gardnerella vaginalis, Fannie H. Negri, Prevotella 2, and Molecularis koraiensis) or lactobacilli (Lactobacillus curvularia, Lactobacillus gasseri, Lactobacillus mirabilis, and Lactobacillus rhamnosus) are used as a floatation mixture at 1 x 10⁻⁶. 7 CFU / mL was applied to the top of the VK2 monolayer for 24 hours. (DF): Individual BV biofilms or 1 x 10⁻⁶ CFU / mL was applied. 7 BV biofilms treated with a CFU / mL Lactobacillus mixture were sonicated into 1 mL of KSFM medium and applied to a VK2 cell monolayer for 24 hours. (G): Live / dead imaging of VK2 cells after the addition of the sonicated biofilm. The staining agents were SYTO9 (green) and propidium iodide (red). Images were taken with a 20x objective lens under an EVOS fluorescence microscope.

[0072] Materials and methods

[0073] Determination of crystal violet biomass

[0074] Single-species biomass was evaluated using a crystal violet assay in 24-well microtiter plates. After 24 hours of incubation, the biofilms were gently washed three times in sterile PBS to remove dead and non-adhering cells. Following washing, the biofilms were dried by incubation at 55°C for 15 minutes before applying 500 μL of 0.05% crystal violet solution (w / v) to each well. The biofilms were incubated at room temperature for 15 minutes and then washed by immersion in a beaker containing tap water. To leach the dye, 500 μL of 100% ethanol was applied and gently mixed eight times by pipetting. From each biofilm, 5 x 75 μL aliquots were transferred to new flat-bottomed 96-well microtiter plates. The absorbance of each well was read at 570 nm using a FLUOstar Omega microplate reader. The final biomass reading for each biofilm was given using the average of the five aliquots per well. All absorbance readings were corrected for a culture medium control.

[0075] Live / dead cell fluorescence microscopy

[0076] Use LIVE / DEAD TM BacLight TMA bacterial viability kit (Invitrogen, Paisley, UK) was used to image single-species Lactobacillus biofilms. The kit contains SYTO9 (3.34 μM) and propidium iodide (20 μM) dyes, which fluoresce in green and red, respectively. Cells with intact membranes stained green, while cells with damaged membranes (dead or dying) stained red. For this assay, single-species biofilms were gently washed once with sterile distilled water after 24 hours. A dye stock solution was prepared by adding SYTO9 and propidium iodide to sterile distilled water at a 1:1 volume ratio. The dye was then applied in 250 μL to the biofilm to cover the bottom of the microtiter plate and incubated in the dark for 15 minutes. After staining, the dye was removed, and the biofilm was washed three more times with sterile distilled water. Fluorescence imaging was performed using EVOS (AMG EVOS fl) at 40x objective magnification.

[0077] Multi-species biofilm formation

[0078] A multi-species BVAB biofilm was grown using a 'Gardnerella vaginalis pre-excitation system,' allowing the organism to colonize for 24 hours before the remaining bacteria were introduced. This is similar to previously developed two-species biofilm models, where Gardnerella vaginalis was first cultured alone before introducing another organism (Machado et al., 2013, Castro et al., 2019, Castro et al., 2022). For this model, Gardnerella vaginalis was normalized to approximately 1 x 10⁻⁶. 8 CFU / mL (OD5500.2), and diluted 1:10 to 1 x 10 in NYC III broth. 7 CFU / mL. Then, 500 μL was added to the wells of a 24-well microtiter plate containing sterile 13 mm Nunc™ Thermanox™ coverslips (Fisher Scientific). The microtiter plate was incubated anaerobically at 37°C for 24 hours. After incubation, the culture medium was removed, and *Fanny Hesse*, *Prevotella ilex*, and *Aerobiculatus koraiensis* were each normalized to approximately 1 x 10⁻⁶ CFU / mL. 8 CFU / mL (OD550 values ​​of 0.15, 0.2, and 0.2, respectively). Each species was then combined to produce a product containing 1 x 10⁻⁶ CFU / mL. 7 A mixture of each bacterium was prepared at a final concentration of CFU / mL. The plate was incubated anaerobically at 37°C for another 24 hours. On day 3, the treatment (Lactobacillus) was administered.

[0079] Probiotic treatment of multi-species biofilms

[0080] For probiotic treatment of biofilms, Lactobacillus isolates were cultured anaerobically at 37°C in NYC III medium for 24 hours. Once turbid growth was observed, each isolate was normalized to approximately 1 x 10⁸ CFU / mL (OD₆₀₀ 0.5) in NYC III. For single-species biofilm treatment, three concentrations of probiotics were applied to the biofilm (approximately 1 x 10⁸ CFU / mL). 7 1 x 10 8 and 1 x 10 9 CFU / mL). Cells were directly administered (1 x 10⁸ CFU / mL) or diluted 1:10 in NYC III (1 x 10⁸ CFU / mL). 7 (CFU / mL) or concentrated to 1 x 10⁻⁶ 9 CFU / mL. For concentration, a solution containing 1 x 10⁻⁶ CFU / mL will be used. 8 Centrifuge CFU / mL Falcon tubes at 3500 RPM for 10 minutes and resuspend in one-tenth of the volume. For example, if centrifuging 25 mL of broth, resuspend it in 2.5 mL of fresh, sterile NYC III medium. Once the final concentration is obtained, gently remove helper pathogens from the biofilm and apply 500 μL of each probiotic to the BVAB biofilm for 4 or 24 hours.

[0081] For mixture treatment, apply the same concentration of total cells (approximately 1 x 10⁻⁶). 7 1 x 10 8 and 1 x 10 9 (CFU / mL). For these experiments, each species was concentrated to 1 x 10⁻⁶. 9 CFU / mL was added in equal volumes to the final mixture of four species. This means that the concentration of each species was approximately 2.5 x 10⁻⁶. 8 CFU / mL, producing approximately 1 x 10⁻⁶ CFU / mL. 9 The final mixture concentration was determined by Miles-Misra colony counts, and then diluted 1:10 and 1:100 to produce a final concentration of approximately 1 x 10⁻⁶ CFU / mL. 8 and 1 x 10 7 A mixture of CFU / mL. Once prepared, the mixture is applied by adding 500 μL of each concentration to the BVAB biofilm for 4 or 24 hours.

[0082] PMAxx TM deal with

[0083] Following multi-species biofilm treatment on day 4, coverslips were gently washed three times by immersion in a culture dish containing sterile PBS and placed in bijoux's microsample tubes containing 1 mL PBS. To remove bacteria from the coverslips, the microsample tubes were sonicated at 35 kHz for 10 minutes, after which the samples were aliquoted into 500 µL aliquots. Using a method modified according to a previously published protocol (Latka et al., 2022), PMAxx was used to analyze the bacteria. TM One of these samples was treated with a dye (Biotium, California, USA). In short, PMAxx was applied at 25 µM. TM And gently mix the mixture in the tube. Then incubate the tube in the dark at 4°C for 15 minutes. Repeat this process a total of three times to obtain the final PMAxx. TM The concentration reached 75 μM. (At PMAxx) TM After treatment, the sample was then exposed to the PMA-lite device (Biotium) for 15 minutes.

[0084] DNA extraction

[0085] DNA was extracted from all samples using the MasterPure Complete DNA and RNA Purification Kit following its 'Cell Samples' protocol (Biosearch Technologies, Hoddesdon, UK). For this protocol, PMAxx TM The treated bacterial cells were precipitated by centrifugation at 10,000 RPM for 5 minutes. The supernatant was discarded, and the precipitate was resuspended in 300 μL of tissue and cell lysis solution supplemented with 1 μL of proteinase K (50 μg / μL). The samples were incubated on a hot plate at 65°C for 15 minutes with pulsed vortexing every 5 minutes. Each sample was then placed in a cold chamber at 4°C for 10 minutes, followed by the addition of 150 µL of MPC protein precipitation reagent. The solution was pulsed vortexed and centrifuged at 15,000 xg for 10 minutes at 4°C. The supernatant was then removed, and the precipitate was transferred to fresh DNase-free Eppendorf tubes containing 500 µL of 100% isopropanol, followed by 30–40 tube inversions. All samples were centrifuged at 15,000 xg for 10 minutes at 4°C, the supernatant was removed, and the precipitate was washed twice with 500 µL of 70% ethanol. The remaining precipitate was then resuspended in 35 μL of TE buffer. The DNA was stored at -20°C for later use.

[0086] Quantitative PCR (qPCR)

[0087] All qPCR reactions were performed using 20 µL of reaction volume on a ViiA 7 real-time PCR system (Applied Biosystems). Each reaction mixture consisted of 10 μL of 2x PowerUp SYBR greenmastermix (Feisher Technologies), 7 μL of Hyclone molecular-grade water, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), and 1 μL of DNA. This resulted in a final primer concentration of 0.5 μM for each reaction. After preparation, plates were incubated on a ViiA 7 analyzer under the following cycling conditions: 40 cycles of 50°C for 2 min, 95°C for 2 min, 95°C for 3 s, followed by 60°C for 30 s (with melting curve). Primer sequences targeting each species (or genus) used throughout the study are highlighted below (Table 1). For each organism, primers were prepared from known concentrations of target bacteria (1 x 10⁻⁶). 3 -1 x 10 8 A standard curve was prepared from DNA extracted at CFU / mL. This standard curve was used to quantify the bacterial levels in each sample. A template-free control (NTC) was included in each reaction, in which 1 μL of Hyclone molecular-grade water was used to replace the DNA.

[0088] Table 1: Primer sequences used in this study to determine the composition of biofilm samples using qPCR. Original references are highlighted.

[0089] Conditioned culture medium

[0090] Each isolate (LC01, LG01, LJ01, BPL5, or as a mixture) was first standardized to 1 x 10⁻⁶ in NYC III broth. 7 Conditioned culture media were prepared using CFU / mL. For each culture, 15 mL was added to a volume of 75 cm³. 3 Cells were cultured in Corning Costar flasks and incubated anaerobically at 37°C for 24 hours to promote biofilm formation. The next day, the culture medium was removed, the biofilm was washed three times with sterile PBS, and then 15 mL of fresh culture medium was applied for an additional 24 hours. After incubation, the biofilm supernatant was harvested as follows: the supernatant was transferred to a sterile 50 mL tube and centrifuged at 3500 RPM. The remaining supernatant was transferred to a fresh 50 mL tube and sterilely filtered using a syringe attached to a 0.22 µM filter.

[0091] The supernatant was used fresh for processing. This involved preparing 5 x NYC III broth solutions containing 1.25 g HEPES (Sigma-Aldrich, Gillingham, UK), 7.5 g protease-peptone (Sigma-Aldrich), 1.875 g yeast extract (Fisher Chemical, Loughborough, UK), and 2.5 g sodium chloride (NaCl; Fisher Chemical), added to 40 mL of distilled water. The solution was autoclaved at 121°C, and 10 mL of glucose (v / v, 25 g / 100 mL in H2O, equivalent to 2.5 g / 100 mL) and 50 mL of heat-inactivated horse serum (Gibco™, Fisher Chemical, Renfrew, UK) were added. The final solution was 100 mL, containing 5 times the amount of each NYC III component.

[0092] The treatment used an 80% conditioned medium consisting of 80% Lactobacillus supernatant or water as a control, plus 20% 5x NYC III broth. Therefore, all variables contained equal nutrient proportions. For subsequent experiments, the pH of this conditioned medium was adjusted by adding 3000 mg / L sodium bicarbonate and 50 mM HEPES or a suitable aqueous solvent control. This pH adjustment has previously been used to neutralize Lactobacillus broth in vaginal epithelial cell co-culture experiments (Anton et al., 2022).

[0093] Culture medium supplemented with citric acid and lactic acid

[0094] Adjust the pH of the culture medium using citric acid or lactic acid. For the citric acid assay, add 20 mM sodium citrate (Sigma-Aldrich) to a 50 mL aliquot of NYC III (w / v = 0.258 g in 50 mL). Then slowly add citric acid (w / v, Sigma-Aldrich) while continuously measuring the pH. At specific pH values ​​(untreated, pH 5, pH 4.5, pH 4), aliquot 10 mL into fresh 50 mL Falcon tubes and filter sterilize (0.22 µM). Apply this pH-adjusted medium to treat the pre-formed BVAB biofilm. For lactic acid, supplement the medium to the determined concentration using D-lactic acid (Cambridge Bioscience, catalog 4003254.0250) or L-lactic acid (Sigma-Aldrich, catalog L1750-10G). It was then applied to a biofilm to evaluate the minimum inhibitory concentration (defined as the concentration that inhibits ≥ 90% growth) against Gardnerella vaginalis, synergistic effects, and biofilm efficacy.

[0095] Planktonic and biofilm inhibition

[0096] Minimum inhibitory concentration (pMIC) for the airborne transport of D- and L-lactic acid 90 In NYC III, the microdilution method using CLSI M11-A8 broth was employed, as previously reported for BVAB and Lactobacillus (Landlinger et al., 2021, Arroyo-Moreno et al., 2022). In short, Gardnerella vaginalis was first normalized to 1 x 10⁻⁶ in NYC III. 8 CFU / mL, then further diluted to 1 x 10⁶ CFU / mL in sterile medium (1:100). D- and L-lactic acid were purchased as powders and prepared into stock concentrations of 100 g / L (D-lactic acid) and 1000 g / L (L-lactic acid) in sterile distilled water. Working concentrations were prepared by dilution in NYC III medium and serial dilutions in sterile medium at a 1:2 ratio. First, 100 μL of D- and L-lactic acid was added to a 96-well round-bottom microtiter plate at twice the desired concentration, followed by 100 μL of 1 x 10⁶ CFU / mL. 6CFU / mL Gardnerella vaginalis. Therefore, the final bacterial concentration in the wells was 5 x 10⁵ CFU / mL, as recommended for pMIC assays (Wiegand et al., 2008). The plates were incubated at 37°C under anaerobic conditions for 24 hours. pMIC was determined by visual observation of growth inhibition and confirmed by absorbance at 550 nm on a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

[0097] Colony count

[0098] Colony-forming units (CFU / mL) analysis was performed on BVAB biofilms exposed to conditioned medium and supplemented with citrate and lactate in 24-well microtiter plates. For this purpose, the treatment was removed and the biofilm was gently washed twice with sterile PBS. After washing, 1 mL of PBS was applied to the wells, and the biofilm was scraped off. Colony counting was performed using the Miles & Misra method (1938), in which serially diluted biofilm was applied three times (20 μL) to Columbia blood agar plates. The plates were anaerobically incubated for 48 hours, and CFU / mL was estimated using the average of the three replicates.

[0099] Lactic acid measurement

[0100] Lactate measurements were performed by Rebiotix staff. In short, probiotic lactobacillus was incubated in MRS broth for 24 or 48 hours. Lactate was then measured on the clarified supernatant. All measurements were performed using the Neogen Megazyme D- / L-lactic acid (D- / L-lactate) (rapid) assay kit.

[0101] Synergistic effect assessment

[0102] The synergistic effect between D-lactic acid and L-lactic acid was assessed using a checkerboard assay. On Gardnerella vaginalis, 5 x 10⁻⁶ samples were used. 5The assay was performed at CFU / mL (same as pMIC), and the plate layout is shown below. Experiments were conducted using a protocol developed by Emery Pharma©, available at: (https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing / ). In short, plates containing a standard curve were prepared, consisting of D- or L-lactic acid alone, and combinations of both at different concentrations. The inhibitory amounts of each acid, both individually and in combinations, were used to determine the fractional inhibitory concentration index (FICi) calculated as follows. Where A and B are the MICs of each acid in the combination (in a single well). 90 Furthermore, MIC(a) and MIC(b) are the individual MICs for each drug. 90 A value < 0.5 indicates a synergistic effect, while a value > 4 indicates an antagonistic effect.

[0103] Culture VK2 cells

[0104] The VK2 / E6E7 cell line (referred to as VK2 in this paper) was established in 1996 from normal vaginal mucosal tissue obtained from premenopausal women who underwent anterior-posterior vaginal repair surgery. These cells were used in all experiments described in this report. The original cell line was immortalized at passage 3 (p3), and all experiments were performed using cells with p < 10. For routine culture, VK2 cells were grown in KSFM medium supplemented with 0.1 ng / ml recombinant human EGF, 0.05 mg / mL bovine pituitary extract, and an additional 44.1 mg / L calcium chloride (final concentration 0.4 mM). Cells were cultured at 75 cm⁻¹. 3 Grow in a flask (T75) until 80%-90% confluence.

[0105] Cell division was initiated by gently rinsing the cells with 2 mL of 0.25% trypsin / 0.03% EDTA solution after removing the culture medium from the T75 flask. This 2 mL solution was removed and replaced with 3 mL of fresh trypsin-EDTA solution, and the flask was incubated at 37°C for 5 minutes at 5% CO2. After incubation, the trypsin was neutralized by adding 7 mL of DMEM:F12 containing 10% fetal bovine serum. The cells were then transferred to 15 mL tubes and centrifuged at 1000 RPM for 10 minutes. The supernatant was discarded, and the cells were resuspended in KSFM with 100 U / mL penicillin (for passage culture) or without (for experiments) and 100 μg / mL streptomycin. For experiments, cells were diced at 1 x 10⁶ cells / mL using antibiotic-free KSFM before adding bacteria. 5 Cells / well were seeded in 24-well plates for 24 hours. Similar cell concentrations have been used in previous host-bacterial co-culture studies in the vagina (Anton et al., 2022) and oral cavity (Brown et al., 2019).

[0106] Supernatant lactate dehydrogenase

[0107] After exposing VK2 cells to bacteria, the supernatant was harvested and clarified by centrifugation at 10,000 RPM for 10 minutes. Following clarification, lactate dehydrogenase (LDH) levels were measured using the CyQUANT LDH Cytotoxicity Kit (Thermo Fisher, catalog number C20301). This assay measures LDH present in the cell culture supernatant as a marker of cytotoxicity. LDH is a cytoplasmic enzyme released when cell death or near-death leads to impaired membrane integrity. For this assay, 50 μL of clarified supernatant was mixed with 50 μL of assay buffer and incubated at room temperature for 30 minutes. After incubation, the reaction was terminated by adding 50 µL of stop solution and measured at 490 °C using a FLUOstar Omega microplate reader (BMG Labtech, Ottenberg, Germany). nm The absorbance was read at 680. nm Correction is performed at this point.

[0108] ELISA of supernatant

[0109] IL-6 and CXCL8 were also measured in the clarified supernatant using sandwich enzyme-linked immunosorbent assays (ELISA). These assays were purchased as pre-made kits from PEPROTECH; human IL-6 TMB EDK (900-T16) and human IL-8 TMB EDK (900-T18, also known as CXCL8). The experiments were performed exactly as described by the manufacturer. Cell culture supernatant was diluted 1:2 or 1:4 with PEPROTECH assay buffer to ensure that each sample fell within the range of the standard curve. Concentrations were determined by interpolation of the absorbance values ​​of the samples into a 5PL curve fitted to the standards.

[0110] Propidium iodide imaging

[0111] Imaging of dead VK2 cells was performed using propidium iodide (PI), a membrane-impermeable fluorescent dye. Therefore, PI cannot pass through the intact cell membrane of living cells and thus selectively fluoresces in dead or dying cells with impaired membrane integrity. For VK2 cell imaging, cells were washed twice with sterile PBS, and then PI was added at 1 μg / mL. Cells were incubated in the dark at room temperature for 15 minutes, after which the dye was removed, and the cells were washed twice more with sterile PBS. Imaging was performed using EVOS (AMG EVOS fl) at 10x objective magnification. For some experiments, PI was combined with SYTO9 at the same concentration, which fluorescently labels living cells green.

[0112] Adding bacteria to VK2 cells

[0113] For the planktonic assay, bacterial isolates were normalized to 1 x 10⁷ CFU / mL in KSFM medium and applied directly to a VK2 monolayer for 24 hours. For the biofilm sonication co-culture assay, BVAB biofilms were grown anaerobically at 37°C on 13 mm Nunc™ Thermanox™ coverslips (Feisel Technologies) and treated with a Lactobacillus mixture. After treatment, the coverslips were gently washed three times by immersion in a culture dish containing sterile PBS and placed in a microsample tube containing 500 µL of KSFM. To remove bacteria from the coverslips, the microsample tubes were sonicated at 35 kHz for 10 minutes, followed by applying 500 µL of sample directly onto the pre-adhered VK2 cells and incubating at 37°C with 5% CO₂ for 24 hours. The next day, the supernatant was harvested, and LDH, CXCL8, and IL-6 were measured as described above. Live / dead cell fluorescence imaging was also performed on VK2 cells.

[0114] Example section

[0115] Bacterial vaginosis (BV) is characterized by an imbalance in the vaginal microbiome, associated with a reduction in symbiotic lactobacilli and the formation of a multimicrobial anaerobic biofilm. Currently, BV is managed with antibiotics (metronidazole and clindamycin), which exhibit unacceptably high relapse rates. Rebuilding a healthy microbiome using probiotics is an attractive treatment option, especially after antibiotic failure. Therefore, this study aimed to investigate the efficacy of four vaginal-derived lactobacilli in an in vitro multimicrobial biofilm model representative of BV.

[0116] Example 1: Lactobacillus monospecies biofilm formation

[0117] The biofilm formation ability of four Lactobacillus strains (Lactobacillus curvatureii-LC01, Lactobacillus gasseri-LG01, Lactobacillus janniae-LJ01, and Lactobacillus rhamnosus-BPL5) was studied. Figure 1 It can be seen that the probiotic Lactobacillus has different biofilm-forming abilities, but Lactobacillus rhamnosus is particularly effective in forming biofilms.

[0118] Example 2: Biofilm treatment of bacterial vaginosis (BV) using a probiotic model A BV model was developed based on the biofilm of the primary pathogen Gardnerella vaginalis, with the introduction of three helper BV pathogens: Prevotella ileiflora, Mobiluncus curtisii, and Vaginal dysbiosis, representing a clinical scenario of dysbiosis. Four lactobacilli (Lactobacillus curvatureii, Lactobacillus gasseri, Lactobacillus mutansii, and Lactobacillus rhamnosus) were isolated from healthy volunteers. Antibiofilm activity was assessed using live / dead qPCR. Additionally, host responses to treated biofilms were investigated using VK2 vaginal epithelial cells. Figure 2 A).

[0119] This model can be used to study direct therapeutic interventions that can be implemented after biofilm formation. Live / dead qPCR can be used for viability-based compositional analysis to demonstrate the therapeutic efficacy of probiotic-based Lactobacillus interventions—both single-species and mixture-based treatments. Figure 2 B- Figure 2 G). As can be seen, this demonstrates that the four probiotic lactobacilli reduced the number of viable bacteria—vaginal disease-associated bacteria—within a pre-formed BV model species biofilm at certain dosage ranges.

[0120] Example 3: pH dependence and lactic acid form of antibacterial activity

[0121] Four Lactobacillus strains, as used in Example 1, were used to consider whether antibacterial activity was pH-dependent. Figure 3It can be seen that the antibacterial effect is pH (acidity) dependent, as the use of buffer solution to maintain the pH of the model BV biofilm resulted in the complete loss of antibacterial activity. The pH of the untreated biofilm was approximately pH 6 after 4 hours and pH 4.3–4.6 after 24 hours of incubation with Lactobacillus. Furthermore, this demonstrates that the supernatant from Lactobacillus induces contact-independent killing of the BVAB biofilm, and that this effect is pH dependent.

[0122] The effects of lactic acid on Gardnerella vaginalis were further investigated. Figure 4 A and Figure 4 B shows the results over 24 hours ( Figure 4 A) and 48 hours ( Figure 4 B) The relative amounts of D- and L-lactic acid produced by the latter four Lactobacillus strains. From Figure 4 C and Figure 4 As can be seen from D, different inhibitory concentration levels of Gardnerella vaginalis were observed between D- and L-lactic acid. Figure 4 E and Figure 4 F showed that the combined use of D- and L-lactic acid had a synergistic inhibitory effect on Gardnerella vaginalis. Figure 4 F shows that the combination of D- and L-lactic acid is optimal in providing antibacterial activity. This is based on the relative levels of D- and L-lactic acid produced by the four strains (see [reference needed]). Figure 4 A and Figure 4 B) and the biofilm-forming ability of Lactobacillus species (see B) Figure 1 The combination of *Lactobacillus rhamnosus* and at least one other tested *Lactobacillus* strain is likely the most effective in providing a suitable combination of D- and L-lactic acid and antibacterial activity.

[0123] Example 4: Biofilm composition of a BV model before and after Lactobacillus treatment

[0124] like Figure 5 As shown, the Lactobacillus applied as a mixture can effectively kill pre-formed BV biofilms within 24 hours and within 4 hours. Figure 5 A, 24 hours is Figure 5 B. Although Lactobacillus species colonize to varying degrees, *Lactobacillus rhamnosus* (BPL5) is the dominant *Lactobacillus* species at both 4 and 24 hours (see [link to article]). Figure 5 A and Figure 5 B). Figure 5 C and Figure 5 D displays the same data as presented in section B; however, this is the absolute number of living cells present, not... Figure 5 The absolute number of living cells in B, expressed as a percentage.

[0125] Example 5: The role of lactobacillus treatment in attenuating the inflammatory potential of BV biofilm on vaginal mucosal cells in a model.

[0126] from Figure 5 It can be seen that planktonic and biofilm-forming BV model bacteria lead to the production of inflammatory markers (lactase dehydrogenase) and inflammatory cytokines (IL-6 and CXCL8). However, the addition of a mixture of lactobacilli (Lactobacillus curvatureii, Lactobacillus gasseri, Lactobacillus mirabilis, and Lactobacillus rhamnosus) resulted in a significant reduction in all markers when planktonic bacteria were added, while LDH and CXCL8 were significantly reduced when the BV mixture was added.

[0127] in conclusion

[0128] All Lactobacilli induced antibacterial activity against pre-formed BV biofilms and performed well compared to antibiotic treatment in the same model (3); each isolate was able to colonize BV biofilms when applied as a mixture, with Lactobacillus rhamnosus being the most abundant species after 24 hours; conditioned media from each species significantly reduced the bioburden of BV biofilms, which was reversed after pH neutralization with HEPES and sodium bicarbonate; and treatment with Lactobacilli significantly reduced the inflammatory potential of BV biofilms against VK2 cells. References: Machado, A., Jefferson, KK & Cerca, N. 2013. Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation. Int J Mol Sci, 14,12004-12. Castro, J., Machado, D. & Cerca, N. 2019. Unveiling the role ofGardnerella vaginalis in polymicrobial Bacterial Vaginosis biofilms: the impact of other vaginal pathogens living as neighbors. ISME J, 13, 1306-1317. Castro, J., Sousa, L. G. V., França, Â., Podpera Tisakova, L.,Corsini, L. & Cerca, N. 2022. Exploiting the Anti-Biofilm Effect of theEngineered Phage Endolysin PM-477 to Disrupt In Vitro Single- and Dual-Species Biofilms of Vaginal Pathogens Associated with Bacterial Vaginosis.Antibiotics (Basel), 11. Latka, A., Van Simaey, L., Reynders, M., Cools, P., Rogier, T.,Lebbe, B., Corsini, L., Landlinger, C. & Vaneechoutte, M. 2022. Optimizationof Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by theGardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Womenwith Bacterial Vaginosis. Antibiotics (Basel), 11. Zozaya-Hinchliffe, M., Lillis, R., Martin, D. H. & Ferris, M. J.2010. Quantitative PCR assessments of bacterial species in women with andwithout bacterial vaginosis. J Clin Microbiol, 48, 1812-9. Pacha-Herrera, D., Vasco, G., Cruz-Betancourt, C., Galarza, J. M.,Barragán, V. & Machado, A. 2020. Vaginal Microbiota Evaluation andLactobacilli Quantification by qPCR in Pregnant and Non-pregnant Women: APilot Study. Front Cell Infect Microbiol, 10, 303. Anton L, Ferguson B, Friedman ES, Gerson KD, Brown AG, Elovitz MA.Gardnerella vaginalis alters cervicovaginal epithelial cell function throughmicrobe-specific immune responses. Microbiome. 2022; 10(1):119. Published2022 Aug 4. doi:10.1186 / s40168-022-01317-9 Landlinger, C., Tisakova, L., Oberbauer, V., Schwebs, T., Muhammad,A., Latka, A., Van Simaey, L., Vaneechoutte, M., Guschin, A., Resch, G.,Swidsinski, S., Swidsinski, A. & Corsini, L. 2021. Engineered Phage EndolysinEliminates Gardnerella Biofilm without Damaging Beneficial Bacteria inBacterial Vaginosis Ex Vivo. Pathogens, 10. Arroyo-Moreno, S., Cummings, M., Corcoran, D. B., Coffey, A. &Mccarthy, R. R. 2022. Identification and characterization of novel endolysinstargeting Gardnerella vaginalis biofilms to treat bacterial vaginosis. NPJBiofilms Microbiomes, 8, 29. Wiegand, I., Hilpert, K. & Hancock, R. E. 2008. Agar and brothdilution methods to determine the minimal inhibitory concentration (MIC) ofantimicrobial substances. Nat Protoc, 3, 163-75. Brown, J. L., Johnston, W., Delaney, C., Rajendran, R., Butcher, J.,Khan, S., Bradshaw, D., Ramage, G., & Culshaw, S. (2019). Biofilm-stimulatedepithelium modulates the inflammatory responses in co-cultured immune cells.Scientific reports, 9(1), 15779. https: / / doi.org / 10.1038 / s41598-019-52115-7。

Claims

1. A composition for use in a therapy, the composition comprising: The composition comprises at least one or more Lactobacillus species, and / or supernatant derived from cultures of the Lactobacillus species, including Lactobacillus curvatureii, Lactobacillus gasseri, Lactobacillus janniae / Lactobacillus muscaria, and / or Lactobacillus rhamnosus, and optionally includes one or more pharmaceutically acceptable adjuvants and / or excipients.

2. The composition for use according to claim 1, wherein the composition comprises a mixture of Lactobacillus species, wherein the mixture comprises supernatant of cultures of Lactobacillus rhamnosus and / or Lactobacillus rhamnosus and at least one of the other identified Lactobacillus species and / or supernatant of cultures of such other Lactobacillus species.

3. The composition for use according to claim 1 or 2, wherein the composition comprises a mixture of all four identified species, wherein *Lactobacillus rhamnosus* is present in a greater amount than each of these other identified species.

4. The composition for use according to any of the preceding claims, wherein the composition comprises about 20%-85% Lactobacillus rhamnosus, preferably about 25%-75% Lactobacillus rhamnosus, more preferably about 30%-60% Lactobacillus rhamnosus, and even more preferably about 40% Lactobacillus rhamnosus.

5. The composition according to claim 1 for use in a therapeutic manner, wherein the composition is used to reduce microbial species, such as bacteria, yeast, fungi and / or viruses, associated with vaginal infections / conditions.

6. The composition according to claim 5 for use in therapy, wherein the bacterial, yeast, fungal, and / or viral species are present in the vagina as a biofilm.

7. The composition for use in a therapy according to any one of claims 5 or 6, wherein the bacterial species associated with the vaginal infection / condition includes one or more of Gardnerella vaginalis, Fannie Hercetella vaginalis, Prevotella micrantha, and / or Moscurvy.

8. The composition for use in therapy according to any one of claims 5-7, wherein the reduction of the microbial species is consistent with the recolonization of one or more of the applied Lactobacillus species.

9. The composition according to any of the preceding claims for use in a therapy, wherein the composition is used in the treatment of biofilms and / or planktonic microbial species.

10. The composition for use according to any of the preceding claims, wherein the composition lowers the pH at the application site.

11. The composition for use according to claim 10, wherein the decrease in pH is at least in part due to the production of D- and / or L-lactic acid by the Lactobacillus species.

12. The composition for use according to claim 11, wherein the decrease in pH is at least in part due to the production of a combination of D- and L-lactic acid by the Lactobacillus species.

13. The composition for use according to claim 12, wherein the ratio (w / w) of D-lactic acid to L-lactic acid is 1:8 to 1:2, for example 1:6 to 1:

3.

14. The composition for use according to any of the preceding claims, wherein the supernatant of the at least one or more Lactobacillus species and / or cultures derived from the Lactobacillus species modulates (e.g., weakens) the immune response of epithelial vaginal cells against pathological microbial species present in the vagina.

15. The composition for use according to claim 14, wherein the regulation of the immune response comprises reducing one or more inflammatory markers and / or cytokines.

16. The composition for use according to claim 15, wherein the regulation of the immune response comprises reducing one, two, or all three of lactate dehydrogenase, interleukin-6 (IL-6), and / or interleukin-8 (CXCL8).

17. The composition according to any preceding claim for use in a therapy, wherein the composition is used to treat a vaginal infection or condition, wherein the infection / condition includes one or more of bacterial vaginosis (BV), developmental abnormalities, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, or gynecological cancer.

18. The composition according to any of the preceding claims for use in a therapy, wherein the supernatant of the at least one or more Lactobacillus species and / or a culture derived from the Lactobacillus species is placed in a capsule.

19. The composition according to any of the preceding claims for use in therapy, wherein the supernatant of the at least one or more Lactobacillus species and / or a culture derived from the Lactobacillus species is placed in the suppository.

20. The composition according to any preceding claim for use in a therapy, wherein the supernatant of the at least one or more Lactobacillus species and / or a culture derived from the Lactobacillus species is placed in a soluble shell.

21. The composition according to any preceding claim for use in a therapeutic application, wherein the composition comprises 1 x 10 5 Up to 1 x 10 15 CFU / ml, 1 x 10 6 Up to 1 x 10 12 CFU / ml, 1 x 10 7 Up to 1 x 10 10 CFU / ml, or 1 x 10 8 Up to 1 x 10 9 CFU / ml of at least one or more of the aforementioned Lactobacillus species.

22. The composition according to any of the preceding claims, wherein the composition comprises a mixture of lyophilized bacteria.

23. A method for treating an infection, the method comprising: Administering the composition according to any of the preceding claims to an infected patient, wherein the infection includes one or more of bacterial vaginosis, candidiasis, human papillomavirus infection, urinary tract infection, sexually transmitted infection, and gynecological cancer.

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