Probiotic strain lactobacillus gasseri for vaginal infections

Abstract

A probiotic composition is provided comprising a new Lactobacillus gasseri strain deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) under accession number CECT 30648, or a bacterial strain derived thereof. Also provided are uses and methods thereof for the treatment of urogenital infections, in particular for bacterial vaginosis, aerobic vaginitis, urinary tract infections, vulvovaginal candidiasis, vaginal dysbiosis and unfavorable pregnancy outcomes. The probiotic composition is also useful for the treatment of other urogenital diseases or conditions, particularly, hyperoxaluria and oxalate-related health conditions.

Description

[0001] PROBIOTIC STRAIN LACTOBACILLUS GASSERI FOR VAGINAL INFECTIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the fields of medicine and microbiology and particularly, to a novel strain of Lactobacillus gasseri to be used as a probiotic to benefit human health, in particular vaginal health.

[0004] BACKGROUND ART

[0005] Vaginal microbiota is a critical determinant of women’s health, predominantly composed by single or multiple species of Lactobacillus which are considered a hallmark of vaginal health as they play a key role in maintaining vaginal homeostasis. Specifically, microbiotas dominated with L. gasseri, L. crispatus or L. jensenii are associated with health as they offer a greater degree of protection to the host compared to L. iners or microbiotas dominated by other anaerobic bacteria.

[0006] The protective role of beneficial lactobacilli species is related with the production of both lactic acid and other antimicrobial molecules that that lowerthe pH of the microenvironment inhibiting the growth of pathogens and directly impair pathogens growth, respectively. Lactobacilli can also adhere to the vaginal epithelium, preventing pathogen colonization and thus establishing a defence against infections and contributing to a healthy vaginal microbiota.

[0007] However, disruptions to this natural flora, often due to systemic antibiotic treatments, lead to vaginal dysbiosis characterized by a loss of lactobacilli dominance and an increase in microbiota diversity.

[0008] Vaginal dysbiosis is associated with an increased risk of suffering gynaecological conditions including infections (vaginitis) such as bacterial vaginosis (BV), aerobic vaginitis (AV), urinary tract infections (UTIs), vulvovaginal candidiasis (VVC), and desquamative inflammatory vaginitis (DIV). BV is caused by harmful anaerobes such as Gardnerella, Prevotella or Fusobacterium; AV is associated with Escherichia coll, Staphylococcus aureus, Staphylococcus epidermidis, Group B Streptococcus, Enterococcus faecalis and Klebsiella pneumoniae; UTI is frequently caused by E. coll (uropatho- genic, UPEC); and VVC is caused by Candida spp.

[0009] During pregnancy, the dominance of Lactobacilli in the vaginal microbiota increases, which reinforces and extends their protective role to the foetus. Thus, vaginal dysbiosis has also been associated with adverse reproductive outcomes, since increases the risk of spontaneous abortion with miscarriage being linked with Group B Streptococcus, Prevotella, Gardnerella and Fusobacterium, as well as sexually transmitted infections, or the risk of preterm birth caused by E. coll, E. faecalis and S. aureus. Moreover, S. aureus and E. coll are also frequently found in the vaginal fluid of pregnant women from different populations suffering preterm premature rupture of membranes (PPROM) which is a complication associated with preterm labour and neonatal disease. E. coli is also related to stillbirth.

[0010] Moreover, some of these pathogens can provoke neonatal sepsis.

[0011] Current treatments for these infections primarily involve antibiotics and antimycotics that, while effective in treating bacterial and fungal infections, respectively, have several significant drawbacks. One major issue is the high recurrence rate of infections, particularly with bacterial vaginosis, where recurrence rates can reach up to 70% within the first year.

[0012] Additionally, these treatments can disrupt the natural balance of the vaginal microbiota, often leading to secondary infections such as vulvovaginal candidiasis due to the depletion of protective lactobacilli.

[0013] Another critical concern is the growing prevalence of antibiotic-resistant pathogens, making it increasingly difficult to treat infections effectively. It is noteworthy that strains of Candida glabrata tend to display a high resistance against antimycotics, while up to 20 % of the strains of Candida albicans isolated in clinics present resistance to fluconazole, one of the most typical treatments, stressing out the importance of finding new therapeutic tools for the management of vulvovaginal candidiasis.

[0014] These drawbacks underscore the need for alternative treatments that can effectively manage vaginal dysbiosis and associated conditions without the limitations of the conventional therapies.

[0015] Probiotic treatment present a promising solution, provided they meet specific criteria related to safety, viability, adhesion, and beneficial effects. As known by the skilled in the art, the properties of each bacterial strain are unique and cannot be extrapolated to other strains of the same species. Therefore, it is important to find those strains that have a better performance in all probiotic requirements.

[0016] Some strains of Lactobacillus which are useful to treat complications of vaginal health have been identified in the art, thus demonstrating the concept of probiotic for vaginal health. Still, traditional probiotics for vaginal health usually target Candida yeasts as well as anaerobic bacteria such as Gamderella and Prevotella, but do not aim to reduce specific pathogens such as E. coli, S. aureus or Fusobacterium. Since these pathogens are involved in several complications (e.g., AV, DIV, UTI, preterm birth, miscarriage), there is the need of probiotics with specific activity against them, while are compatible with the usual antibiotic pharmaceutical treatments and capable of restoring and maintaining vaginal homeostasis, thereby reducing the incidence of recurrent infections and improving overall vaginal health.

[0017] On the other hand, oxalate is an antinutrient that is present in many vegetable foods. High levels of oxalate may lead to hyperoxaluria and calcium stone formation (which are related with other diseases and conditions e.g. intestinal bowel disease, Chron's disease or vulvodynia). The treatment of hyperoxaluria varies depending on the type (primary or secondary) and severity of the condition. Primary hyperoxaluria (PH) is a rare inherited genetic defect of oxalate metabolism while secondary hyperoxaluria (SH) is seen in states of increased ingestion of oxalate, its precursors or altered gut microbiota. Dietary measures do not play a major role in PH as the excess oxalate in this condition is endogenous, more severe and may lead to renal failure. Therefore, treatment for PH is much more aggressive involving dietary interventions but also use of drugs such as the recently FDA-approved Lumasiran and Nedosiran. On the other hand, SH is more common, and the treatment is typically conservative involving mainly dietary interventions (low fat, low oxalate food), limit excess vitamin C and vitamin D, pyridoxine (vitamin B6), calcium and potassium citrate supplementation, etc.

[0018] Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. In this context, bacteriotherapy is now being considered as an alternative or complementary therapy for the management of hyperoxaluria.

[0019] The most commonly described intestinal bacteria known to degrade oxalate are categorized into two groups: (i) the “generalist oxalotrophs”, including some strains of Bifidobacterium and Lactobacillus, that degrade alternative carbon sources in addition to oxalate; and (ii) the “specialist oxalotrophs”, such as Oxalobacter formigenes, which is a commensal anaerobe that uses only oxalate as its sole carbon source. Gut microbes use two different microbial oxalate degradation pathways. Type I enzymes degrade oxalate in one single step, involving oxalate oxidase (OXDD; EC.1.2.3.4) and oxalate decarboxylase (OXDD; EC.4.1.1.2), while type II enzymes include formyl-CoA transferase (FRC) and, in a second step, oxalyl-CoA decarboxylase (OXC) that metabolizes oxalyl-CoA into CO2 and formyl-CoA.

[0020] While there are several research groups and companies developing Oxalobacter formigenes-based solutions, this bacterium is a gram- negative, obligate anaerobe, pH susceptible, non-QPS, which must be registered as a live biotherapeutics, thus hampering the route to the market. For these reasons, the use of a Lactobacillus spp. strain with the ability to degrade oxalate might be a more efficient strategy.

[0021] SUMMARY OF THE INVENTION

[0022] The present document provides a new strain of Lactobacillus gasseri, L. gasseri CECT 30648 (also named as KABP-064), which has been found to be suitable as a probiotic for the management of vaginal infections and oxalate-related health conditions. The strain of Lactobacillus gasseri was deposited on 17-May-2022 in the Spanish Type Culture Collection (Coleccion Espanola de Cultivos Tipo, CECT, Edificio 3 CUE, Parc Cientlfic, Universitat de Valencia, Catedratico Agustin Escardino, 9, 46980-Paterna, Valencia, Spain), by the depositor AB-Biotics, S.A., sited at Avda. De la Torre Blanca 57-Desp. 3B-11 , 08172-Sant Cugat del Valles (Barcelona, Spain). The strain of L gasseri received the accession number CECT 30648 after the International Authority of Deposit declared the strain as viable.

[0023] The working examples provided herein show that the L. gasseri CECT 30648 probiotic strain has a great adhesion capacity to the vaginal epithelium, a mechanism that adapts the strain to the vaginal environment and prevents pathogen colonization. Probiotics with good adhesion capacity compete with pathogenic microorganisms for union sites thus reducing their infectivity. In addition, the in vivo colonization is a warranty of the probiotic strain to act at the target location.

[0024] Furthermore, it has been demonstrated that the strain is able to survive the gastrointestinal tract passage and colonize the vagina after oral administration, which is a challenge for strains of vaginal origin such as those belonging to L. gasseri species due to the niche-specific adaptation of the strains (Pan et al., 2020). Although L. gasseri strains can be found in the gut, intestinal strains are less interested due to their poor adaptability to the vagina. Tolerance to the gastrointestinal tract passage is a requirement for oral administered probiotics and even a more essential feature for probiotics aimed to reach the vaginal mucosa. Moreover, while vaginal route warranties probiotics act at the target site, oral administration offers advantages such us user-friendliness, improvement of patient compliance and exerting potential systemic benefits beyond the vaginal tract.

[0025] Of note, the examples also demonstrate that L. gasseri CECT 30648 probiotic strain has a high antimicrobial activity against the pathogenic bacteria causative of urogenital infections (e.g., AV, UTI, DIV and preterm labor), such as E. coli, S. aureus and Fusobacterium sp. The strain showed the highest inhibitory activity against E. coli compared with the other probiotic strains of the field. Importantly, L. gasseri CECT 30648 also showed the highest inhibitory activity against S. aureus.

[0026] L. gasseri CECT 30648 strain has also shown inhibitory potential against a wide range of microorganism involved in vaginal infections and unfavorable pregnancy outcomes including Candida spp strains, other Gram-positive and Gram-negative bacteria.

[0027] Furthermore, inventors have proved that the strain is highly compatible for co-administration with antibiotics used in clinical practice, e.g., those used in vaginitis and urinary tract infections (e.g. metronidazole, clindamycin, ciprofloxacin, and fosfomycin).

[0028] Overall, the new strain L. gasseri CECT 30648 shows the greatest capacity for the treatment and prevention of gynecological conditions associated with vaginal dysbiosis, specifically infections caused by bacteria (vaginitis), more specifically bacterial infections of the group of aerobic vaginitis (AV), urinary tract infections (UTI), and bacterial vaginosis (BV), and more specifically bacterial infections with presence of or caused by E. coli UPEC, S. aureus or Fusobacterium; and the support of a healthy pregnancy and prevention of negative reproductive outcomes, specifically prevention of miscarriage and preterm birth, and more specifically prevention of miscarriage and preterm birth caused by E. coli UPEC, S. aureus or Fusobacterium.

[0029] Further, EXAMPLE 8 demonstrates that the new strain L. gasseri CECT 30648 is the most rapid and efficient oxalate degrader, positioning it as the best candidate for the management of oxalate-related health conditions.

[0030] Accordingly, an aspect of the present invention relates to a probiotic composition comprising Lactobacillus gasseri strain deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) under accession number CECT 30648 (also named as KABP-064), or a bacterial strain derived thereof, wherein the derived bacterial strain has a genome with at least 99% average nucleotide identity (ANI) to the genome of the correspondent deposited strain, and / or has a genome fingerprint of the correspondent deposited strain shown in the pulsed field gel electrophoresis (PFGE) of FIG. 4.

[0031] Another aspect relates to a strain of Lactobacillus gasseri deposited in the Spanish Type Culture Collection under the accession number CECT 30648.

[0032] Other aspects of the invention relates to a method to obtain a strain derived from the Lactobacillus gasseri CECT 30648 strain, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant has a genome at least 99% ANI to the genome of the correspondent deposited strain; or to a method to obtain a strain derived from the Lactobacillus gasseri strain CECT 30648, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant retains or enhances the antimycotic and / or antibacterial activities and / or the capacity to colonize the vaginal tract of the parent deposited strain.

[0033] Another aspect of the invention relates to a probiotic composition described herein for use as a medicament.

[0034] Another aspect relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of a urogenital infection; or alternatively, to a method of preventing, ameliorating or treating a urogenital infection, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0035] Other aspects relate to a probiotic composition described herein for use in the prevention, amelioration or treatment of a vaginal infection; or for use in the prevention, amelioration or treatment of atrophic vaginitis; or for use in the prevention, amelioration or treatment of desquamative inflammatory vaginitis; or for use in the prevention, amelioration or treatment of a urinary tract infection; or for use in reducing or preventing the risk of a unfavorable pregnancy outcome (or an adverse reproductive outcome). Alternatively, these aspects can be formulated as methods of preventing, ameliorating or treating a vaginal infection; or preventing, ameliorating or treating atrophic vaginitis; or preventing, ameliorating or treating desquamative inflammatory vaginitis; or preventing, ameliorating or treating a urinary tract infection; or reducing or preventing the risk of an unfavorable pregnancy outcome, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0036] Another aspect of the invention relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of hyperoxaluria and oxalate-related health conditions.

[0037] Another aspect of the invention relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of an urogenital disease or condition, including as explained herein e.g. urogenital infections, hyperoxaluria and oxalate-related health conditions.

[0038] Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes, and without intending to be limiting to the present invention.

[0039] DESCRIPTION OF DRAWINGS

[0040] FIG. 1 shows the growth inhibition (%) of probiotic strains against pathogenic bacteria investigated by microplate growth inhibition assay using neutralized supernatants from probiotics monocultures: in (A) the pathogenic bacteria investigated is E. co / / UPEC DSM 10650; in (B) the pathogenic bacteria investigated is S. aureus CIP 107860.

[0041] FIG. 2 shows the growth inhibition (%) of probiotic strains against F. necrophorum DSM 20698 bacteria investigated by microplate growth inhibition assay using and neutralized supernatants from probiotic + pathogen cocultures.

[0042] FIG. 3 shows the percentage of subjects with L. gasseri CECT 30648 detected in vaginal samples in probiotic ( / _. gasseri) and placebo group. Statistical analysis was performed by Chi-square test.

[0043] FIG. 4 shows the pulsed field gel electrophoresis (PFGE) pattern of CECT 30648 strain after digestion with Smal enzyme. Order: Marker 1 , L. gasseri CECT 30648, Marker 2, L. crispatus KABP 39_01 , Marker 2. FIG. 5 shows (A) the percentage of oxalate degradation (10 mM, 72 h of incubation) of all strains included in the study, and (B) the percentage of oxalate degradation of the three most active strains after 24 h of incubation in 10 mM oxalate concentration. The results are expressed as a percentage of oxalic acid consumed.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is directed to a probiotic composition and their uses and methods, particularly for the treatment of urogenital infections. Also provided are products comprising the probiotic composition, and procedures and doses of the probiotic compositions. It is understood that the term "probiotic composition" in this description means any probiotic composition as defined herein, e.g., comprising Lactobacillus gasseri CECT 30648 or a derived strain, and any other excipient / carrier, as well as in any product form (e.g., pharmaceutical composition, etc).

[0046] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this description, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present description. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0047] The headings provided herein are not limitations of the various aspects of the description, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present description will be limited only by the appended claims.

[0048] Accordingly, the terms defined immediately below are more fully defined by reference to the description in its entirety.

[0049] Definitions

[0050] Probiotic: As used herein, the term “probiotic” refers to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and / or subtypes of non-pathogenic bacteria are currently recognized as probiotic. The probiotic can be a variant or a mutant strain of bacterium. Probiotic bacteria can be naturally mutated or genetically engineered modified to retain, enhance or improve desired biological properties, e.g., survivability to provide probiotic properties or to retain, enhance or improve probiotic properties such the ones described herein. Derived from: The terms "derived from," "derivative", "variant", "mutant" (e.g., "mutant strain"), or any grammatical variant thereof, as used herein, refer to a component that is isolated from or made using a specified molecule / substance (e.g., a strain of the present invention). For example, a bacterial strain that is derived from a first bacterial strain (e.g., a deposited strain) can be a strain that is identical or substantially similar to the first strain. In the case of bacterial strains, the derived strain can be obtained by, e.g., naturally occurring mutagenesis, artificially directed mutagenesis, artificially random mutagenesis or other genetic engineering techniques, and it retains, enhances or improves at least one ability of the deposited strain.

[0051] Excipient / Carrier: The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a e.g., pharmaceutical composition, to further facilitate administration of a compound, e.g., a bacterial strain of the present invention. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, e.g., polysorbate. The terms “physiologically acceptable excipient / carrier” and “pharmaceutically acceptable excipient / carrier” which may be used interchangeably, refer to a substance or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these terms.

[0052] Composition: As used herein, the term “composition” refers to a mixture of at least one compound useful within the present invention with an excipient / carrier. For example, "pharmaceutical composition" refers to a preparation of the bacteria of the present invention with other components such as a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition facilitates the administration of the compound to a patient or subject.

[0053] Identity: As used herein, the term "identity" refers to the overall conservation of the monomeric sequence between polymeric molecules, e.g., between DNA molecules and / or RNA molecules. The term "identical" without any additional qualifiers, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical," is equivalent to describing them as having, e.g., "70% sequence identity.”

[0054] Calculation of the percent identity of two polymeric molecules, e.g., polynucleotide sequences, can be performed, e.g., by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second polynucleotide sequences for optimal alignment). In certain embodiments, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the length of the reference sequence. The bases at corresponding base positions, in the case of polynucleotides, are then compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, considering the number of gaps, and the length of each gap, which can be determined using a mathematical algorithm. Suitable software programs are available for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, which performs a comparison between two sequences using either the BLASTN (used to compare nucleic acid sequences) or BLASTP (used to compare amino acid sequences) algorithm. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs. Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, MAUVE, MUMMER, RAST, etc.

[0055] In certain embodiments, the percentage identity (% ID) of a first sequence to a second sequence is calculated as %ID = 100 x where Y is the number of amino acid residues or nucleobases scored as identical matches in the alignment of the first and second sequences (e.g., as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. When comparing complete or near complete genomic nucleobase sequences, % ID is sometimes referred to as ANI (Average Nucleotide Identity). Calculating ANI usually involves the fragmentation of genome sequences, followed by nucleotide sequence search, alignment, and identity calculation.

[0056] Subject: The terms "subject", "patient", "individual", and "host", and variants thereof are used interchangeably herein and refer to any mammalian subject, particularly humans, but also including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) forwhom diagnosis, treatment, ortherapy is desired. The methods described herein are applicable to both human therapy and veterinary applications.

[0057] Subject in need thereof: As used herein, "subject in need thereof includes subjects, such as mammalian subjects, that would benefit from administration of the probiotic compositions of the present invention.

[0058] Therapeutically effective amount: The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to the amount of the probiotic composition of the present invention that is sufficient to a produce a desired therapeutic effect, pharmacologic and / or physiologic effect on a subject in need thereof. Particularly, the terms refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition. A therapeutically effective amount can, e.g., be sufficient to treat, prevent, reduce the severity, delay the onset, and / or reduce the risk of occurrence of one or more symptoms of a disease or condition. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below. Treatment: The terms "treat," "treatment," "therapy," as used herein describes the management and care of a subject for the purpose of combating the disease, condition, or disorder. Treating includes the administration of a probiotic composition as described herein to reduce the severity of a disease or condition disclosed herein; the mitigation / amelioration or elimination of one or more symptoms, complication, or sequelae associated with a disease disclosed herein; to prevent the onset of the symptoms or complications; to eliminate the disease, condition, or disorder; or the provision of beneficial effects to a subject with a condition / disease disclosed herein, without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or symptoms, complications, or sequelae thereof.

[0059] The term refers to a clinical or nutritional intervention to prevent the disease or condition; cure the disease or condition; delay onset of the disease or condition; delay onset of a symptom, complication or sequela; reduce the seriousness of the disease or condition; reduce the seriousness of a symptom, complication, or sequela; improve one or more symptoms; improve one or more complications; improve one or more sequelae; prevent one or more symptoms; prevent one or more complications; prevent one or more sequelae; delay one or more symptoms; delay one or more symptoms; delay one or more complications; delay one or more sequelae; mitigate / ameliorate one or more symptoms; mitigate / ameliorate one or more complications; mitigate / ameliorate one or more sequelae; shorten the duration one or more symptoms; shorten the duration one or more complications; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more complications; reduce the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more complications; reduce the severity of one or more sequelae; improve the quality of life; increase survival; prevent a recurrence of the disease or condition; delay a recurrence of the disease or condition; or any combination thereof, e.g., with respect to what is expected in the absence of the treatment with the probiotic composition of the present invention.

[0060] Prevent: The terms "prevent", "preventing", "prophylaxis" and variants thereof as used herein, refer, e.g., to partially or completely delaying onset of a disease, disorder and / or condition disclosed herein; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations, complications, or sequelae of a particular disease, disorder, and / or condition disclosed herein; partially or completely delaying onset of one or more symptoms, features, or manifestations, complications, or sequelae of a particular disease, disorder, and / or condition disclosed herein; partially or completely delaying progression from a particular disease, disorder and / or condition disclosed herein; and / or decreasing the risk of developing pathology associated with the disease, disorder, and / or condition disclosed herein.

[0061] Symptom: As used herein, the term "symptom" refers to subjective or physical sign, indication, or evidence of disease or physical disturbance observed by the subject. In general, the term refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. In some embodiments, a symptom can be a mild symptom, a moderate symptom, or severe symptom. As used herein, the term "mild symptom" refers to a symptom that is not life threatening and does not require, e.g., intensive care treatment. As used herein, the term "moderate symptom" refers to a symptom that requires monitoring because it may become life threatening and may require, e.g., hospitalization. As used herein, the term "severe symptom" refers to a symptom that is life threatening and requires, e.g., intensive care treatment.

[0062] Complication: As used herein, the term "complication" refers to a pathological process or event occurring during a disease or condition that is not an essential part of the disease or condition; where it may result from the disease / condition or from independent causes. In some embodiments, a complication can be chronic or permanent. As used herein, the term "sequela" refers to a long term, chronic, or permanent complication. In a particular embodiment, the complication is a pregnancy complication.

[0063] Probiotic composition

[0064] As previously discussed, an aspect of the present invention relates to a probiotic composition comprising Lactobacillus gasseri strain deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) under accession number CECT 30648, or a bacterial strain derived thereof, wherein the derived bacterial strain has a genome with at least 99% average nucleotide identity (ANI) to the genome of the correspondent deposited strain, and / or has a genome fingerprint of the correspondent deposited strain shown in the pulsed field gel electrophoresis (PFGE) of FIG. 4.

[0065] In an embodiment, the derived bacterial strain has a genome with at least 99% average nucleotide identity (ANI) to the genome of the correspondent deposited strain. The genome of Lactobacillus gasseri CECT 30648 corresponds to SEQ ID NO: 1. In some embodiments, the derived bacterial strain further retains the antimicrobial ability (e.g., antimycotic and / or antibacterial) of the corresponding deposited strain and / or the capacity to colonize the vaginal tract of the corresponding deposited strain.

[0066] In an embodiment, the invention relates to a probiotic composition comprising the Lactobacillus gasseri strain deposited under accession number CECT 30648.

[0067] Another aspect of the invention relates to a strain of Lactobacillus gasseri deposited in the Spanish Type Culture Collection under the accession number CECT 30648. The strain was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in the Spanish Type Culture Collection (Universitat de Valencia, Campus de Burjassot, Edif. de Investigation, 46100 Burjassot, Valencia, Spain) on May 24, 2022 (24.05.2022) with accession number CECT 30648. The deposited strain is viable and keeps all their features related to their deposit. The strain was deposited by the same applicant.

[0068] As understood by the skilled person in the present context, a bacterial strain has been isolated from its natural environment, i.e., it is free from other organisms and substances present in the natural environment. Particularly, the Lactobacillus gasseri CECT 30648 was isolated from the vagina of a healthy woman.

[0069] It is clear that by using the deposited strain as starting material, the skilled person in the art can routinely, by conventional mutagenesis or re-isolation techniques, obtain further variants or mutants thereof that retain, enhance or improve the herein described relevant features and advantages of the strain forming the probiotic composition of the present invention. Thus, the invention also relates to variants / mutants of the strain disclosed herein.

[0070] In a particular embodiment, the bacterial strain derived from the deposited strain has a genome with at least 99% average nucleotide identity (ANI) to the genome of the correspondent deposited strain; more particularly, % of identity is 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%. Particularly the % of ANI is at least 99.5%. More particularly, % of ANI is 99.50%, 99.51 %, 99.52%, 99.53%, 99.54%, 99.55%, 99.56%, 99.57%, 99.58%, 99.59%, 99.60%, 99.61 %, 99.62%,

[0071] 99.63%, 99.64%, 99.65%, 99.66%, 99.67%, 99.68%, 99.69%, 99.70%, 99.71 %, 99.72%, 99.73%,

[0072] 99.74%, 99.75%, 99.76%, 99.77%, 99.78%, 99.79%, 99.80%, 99.81 %, 99.82%, 99.83%, 99.84%,

[0073] 99.85%, 99.86%, 99.87%, 99.88%, 99.89%, 99.90%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%,

[0074] 99.96%, 99.97%, 99.98% or 99.99%. In another embodiment, the % of ANI is at least 99.9%; particularly, % of ANI is 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%.

[0075] In some embodiments, the mutant is obtained by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. In an embodiment, the bacterial strain derived from the deposited strain is obtained by using recombinant DNA technology. In another embodiment, the mutants are obtained by random mutagenesis. Thus, another aspect of the invention relates to a method to obtain a strain derived from the Lactobacillus gasseri CECT 30648 strain, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant has a genome at least 99% ANI to the genome of the correspondent deposited strain.

[0076] Another aspect relates to a method to obtain a strain derived from the Lactobacillus gasseri strain deposited in the Spanish Type Culture Collection under accession number CECT 30648, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant retains or enhances the antimycotic and / or antibacterial activities and / or the capacity to colonize the vaginal tract of the parent deposited strain.

[0077] In an embodiment, the strain forming part of the probiotic composition is in the form of viable cells. Alternatively, the strain is in the form of non-viable cells. This can include thermally killed microorganisms or microorganisms killed by exposure to altered pH, sonication, radiation or high pressure. Product preparation is simpler with non-viable cells, as cells can be incorporated easily into dietary, pharmaceuticals cr edible products, and storage requirements are much less limited than viable cells. A probiotic composition comprising the strain of the present invention as non-viable cells can comprise products derived from the strain which are in the medium.

[0078] Thus, the probiotic composition can comprise the strain as well as the bioactive compounds released by this strain, the supernatants and the cultures of the strain, the fractions / extracts containing the bioactive compounds, lysates and the formulation of any of these in e.g., food and pharmaceutical formulations.

[0079] For the purposes of the present invention, the term "bioactive compounds or products" derived from the strain of the present invention, are defined as its cell components, and the compounds and molecules that form part of the strain, such as the metabolites and molecules secreted thereof, such as: intracellular components (e.g., DNA, peptides, fatty acids, etc.), or cell-wall components (proteins, peptides, fatty acids, etc.) that confer the desired preventive or therapeutic activity.

[0080] As used herein “lysate” refers to a sample of the probiotic strain which has been subject to lysis. A lysate can contain one or more soluble metabolites of the probiotic strain useful in the methods described herein. Lysis can occur by chemical or physical disruption, e.g., by addition of an osmotic agent or enzyme to the bacteria, or by the application of physical pressure, e.g., through sonic disruption. The lysate can be a mixture comprising the cellular contents of the probiotic strain. For example, one or more of fragments of cell wall or cell membrane, proteins, nucleic acids, carbohydrates, and organelles (disrupted or intact). The lysate can be suspended, e.g., in aqueous medium.

[0081] The present invention thus provides a method of producing a culture supernatant from the strain, the method comprising a step of culturing the isolated strain and separating the supernatant from the culture, thereby producing the culture supernatant.

[0082] The present invention also provides a method of producing a fraction / extract or a metabolite from strain cells (comprising bioactive compounds of the strain), the method comprising a step of lysing the cells of the isolated strain and separating the extract of the lysed cells, thereby producing the fraction / extract or the metabolite. The strain disclosed herein is produced by cultivating (or fermenting) the bacteria in a suitable artificial medium and under suitable conditions. By the expression, “artificial medium” is understood to be a medium containing natural substances, and optionally synthetic chemicals such as the polymer polyvinyl alcohol which can reproduce some of the functions of serums. Common suitable artificial media are nutrient broths that contain the elements including a carbon source (e.g., glucose), a nitrogen source (e.g., amino acids and proteins), water and salts needed for bacterial growth. Growth media can be liquid form or often mixed with agar or another gelling agent to obtain a solid medium. The strain can be cultivated alone to form a pure culture, or as a mixed culture together with other microorganisms, or by cultivating bacteria of different types separately and then combining them in the desired proportions. After cultivation, and depending on the final formulation, the strain can be used as purified bacteria, or alternatively, the bacterial culture or the cell suspension can be used, either as such or after an appropriate post-treatment. In this description, the term “biomass” is understood to be the bacterial strain culture obtained after cultivation (or fermentation as a term synonymous to cultivation).

[0083] In an embodiment, the strain is fermented in an artificial medium and submitted to a post-treatment after fermentation, to obtain bacterial cells, and the resulting bacterial cells are in a liquid medium or in a solid form. Particularly, the post-treatment is selected from the group consisting of drying, freezing, freeze-drying, fluid bed-drying, spray-drying and refrigerating in liquid medium, and more particularly, is freeze-drying.

[0084] By the term “post-treatment” is to be understood in the present context, any processing carried out on the biomass with the aim of obtaining storable bacterial cells. The objective of the post-treatment is decreasing the metabolic activity of the cells in the biomass, and thus, slowing the rate of cellular deleterious reactions. As a result of the post-treatment, the bacterial cells can be in solid or liquid form. In solid form, the stored bacterial cells can be a powder or granules. In any case, both the solid and liquid forms containing the bacterial cells are not present in nature, hence, are not naturally- occurring, since they are the result of artificial post-treatment process(es). The post-treatment processes can in particular embodiments require the use of one or more of so-called post-treatment agents. In the context of the present invention, the expression “post-treatment agent” refers to a compound used to perform the herein described post-treatment processes. Among the post-treatment agents are to be included, without limitation, dehydrating agents, bacteriostatic agents, cryo- protective agents (cryoprotectants), inert fillers (also known as lyoprotectants), carrier material (also known as core material), etc., used either alone or in combination.

[0085] There are two basic approaches to decrease the metabolic activity of the bacterial cells, and thus, two approaches to carry out the post-treatment. The first one is decreasing the rate of all chemical reactions, which can be done by lowering the temperature by refrigerating or freezing using refrigerators, mechanical freezers, and liquid nitrogen freezers. Alternatively, decreasing the rate of all chemical reactions can be achieved by adding substances that inhibit the growth of the bacterial cells, namely a bacteriostatic agent, abbreviated Bstatic.

[0086] The second approach to carry out the post-treatment is to remove water from the biomass, a process which can involve sublimation of water using a lyophilizer. Suitable techniques to remove water from the biomass are drying, freeze-drying, spray-drying or fluid bed-drying. Post-treatments that result in solid form can be drying, freezing, freeze-drying, fluid bed-drying, or spray-drying.

[0087] In an embodiment, the post-treatment is freeze-drying, which involves the removal of water from frozen bacterial suspensions by sublimation under reduced pressure. This process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most water, and secondary drying to remove bound water. Due to objective and expected variability of industrial processes for manufacturing and isolation of lyophilized bacterial cultures, the latter commonly contain a certain amount of inert filler also known as lyoprotectant. Its role is to standardize the content of live probiotic bacteria in the product. The following inert fillers in commercially available lyophilized cultures are used: sucrose, saccharose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharmaceutically acceptable non-hygroscopic fillers. Optionally, other stabilizing or freeze-protecting agents like ascorbic acid, are also used to form a viscous paste, which is submitted to freeze-drying. In any case, the so-obtained material can be grinded to appropriate size, including to a powder.

[0088] Alternatively to having biomass preserved in solid form, biomass can be also preserved in liquid form. This can be done by adding a bacteriostatic agent as described above to stop bacteria growth to the culture medium or with an intermediate step of harvesting cells, re-suspending the pellet in saline solution with a bacteriostatic agent, and optionally refrigerating it.

[0089] Sometimes, as described for instance above in the fluid bed-drying process, the probiotic composition is subjected to an immobilization and / or coating, or encapsulation process in order to improve the shelf life and / or functionalities. Several techniques for immobilization, coating or encapsulation of bacteria are known in the art.

[0090] In other embodiments, the probiotic composition is formulated for sustained-release administration e.g., by means of the encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. The suitable sustained-release forms as well as materials and methods for their preparation are well known in the state of the art. Thus, the orally administrable form of any of the probiotic compositions of the present invention is in a sustained-release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semisynthetic or synthetic polymers, water-insoluble or modified, waxes, fats, fatty alcohols, fatty acids, natural, semisynthetic or synthetic plasticizers or a combination of two or more of the same. Enteric coatings can be applied using conventional processes known to those skilled in the art. The effective amount of colony forming units (cfu) for the strain in the probiotic composition can be determined by the skilled in the art and will depend upon the final formulation. The term "colony forming unit" ("cfu") is defined as the number of bacterial cells as revealed by microbiological counts on agar plates.

[0091] In some embodiments, the strain of the present invention is present in an amount of between 105and 1012cfus in the composition, particularly between 107and 1011cfus, and more particularly in an amount of between 109and 1 O10cfus. In another embodiment, the strain of the present invention is present in an amount of between 1x109and 6x109cfus in the composition, and particularly in an amount of about 2x109cfus.

[0092] As known by the skilled person, the effective amount of colony units can also be measured by the effective amount of active fluorescent units. The term “active fluorescent unit" ("afu") is defined as the number of bacterial cells as revealed by flow cytometry counts in a gate specific for fluorescence characteristics of presumed live cells. Therefore, the skilled person would consider the above-mentioned specific quantities of cfu to be about the same quantity of afu.

[0093] In one embodiment, the probiotic composition is a solid composition. In another embodiment, the probiotic composition is a liquid composition.

[0094] In an embodiment, the probiotic composition comprises a cryoprotectant. Particularly, the probiotic composition comprises at least one cryoprotectant that is an allergen-free cryoprotectant. In some embodiments, the probiotic composition comprises at least one cryoprotectant such as maltose, trehalose, mannitol (particularly, d-mannitol), saccharose, lactose, dextrose, sodium ascorbate, sodium citrate, L-cysteine, maltodextrin, anhydrous dextrose, starch, cellulose and inulin. In a particular embodiment, the cryoprotectant and / or the pharmaceutically acceptable carrier is selected from the group consisting of trehalose, D-mannitol, dextrose, sodium ascorbate, sodium citrate, L-cysteine, maltodextrin, starch, and cellulose. Particularly, the starch is corn, maize starch and / or potato starch.

[0095] In a particular embodiment, the probiotic composition is a solid composition comprising: a freeze- dried bacterial biomass comprising from about 105cfu to about 1012cfu of each strain; and a cryoprotectant.

[0096] More particularly, the probiotic composition further comprises a pharmaceutically acceptable carrier chosen from a tablet, a capsule, an emulsion, a suspension, a gel, a paste, granules, a powder, a suppository (e.g., vaginal suppository), a vaginal ovule, and a gum. Particularly, the carrier is an allergen-free carrier. In some embodiments, the probiotic composition comprises one or more carriers selected from the group consisting of: maltodextrin, cellulose, starches, inulin, lactose, trehalose, dextrose, sodium citrate, sodium ascorbate, D-mannitol, L-cysteine, magnesium sulphate, sodium glutamate, histidine and marie acid.

[0097] In some embodiments, the probiotic composition is in the form of a tablet, a pill, a capsule, a sachet, a suppository (e.g., vaginal suppository), a vaginal ovule, an emulsion, a suspension, a gel, a paste, granules, a powder, or a gum. Particularly, the probiotic composition is in the form of a capsule, a tablet, a pill, a sachet, or a suspension. In a particular embodiment, the probiotic composition is in the form of a tablet. In another particular embodiment, the probiotic composition is in the form of a capsule. In another particular embodiment, the probiotic composition is in the form of a vaginal suppository or vaginal ovule. Particularly, the capsule comprises maltodextrin carrier (e.g., E1400, qs).

[0098] Product forms of the probiotic composition

[0099] Pharmaceutical / nutraceutical forms

[0100] In some embodiments, the probiotic composition described herein is in a pharmaceutical / nutraceutical form, such as a capsule, a powder, a suspension, or a tablet.

[0101] The term “pharmaceutical form" is understood in its widest meaning, including any composition that comprises an active ingredient, in this case, the probiotic composition described herein together with at least a pharmaceutically (also referred as nutraceutically or veterinary) acceptable excipient. The term "pharmaceutical form" is not limited to medicaments but includes e.g., pharmaceutical compositions, nutraceutical compositions or veterinary compositions. A pharmaceutical form can adopt different names depending on the product regulatory approval route and also depending on the country.

[0102] In an embodiment, the probiotic composition is for use as a pharmaceutical form or as a probiotic.

[0103] A nutraceutical composition can also be named e.g., as food supplement or dietary supplement. A nutraceutical composition is understood as a preparation or product intended to supplement the diet, made from compounds usually used in foodstuffs, which provide nutrients or beneficial ingredients that are not usually ingested in the normal diet or cannot be consumed in sufficient quantities. Nutraceutical compositions are usually sold “over the counter”, i.e., without prescription.

[0104] In some embodiments, the probiotic composition is formulated as pharmaceutical form in which the strain is the only active agent or is mixed with one or more other active agents and / or are mixed with pharmaceutically / nutraceutically / veterinary acceptable excipients. Particularly, the additional active agent or agents are other probiotic bacteria which are not antagonistic to the strain forming the probiotic composition of the present invention. Depending on the formulation, the strain can be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture which has been post-treated, and alone or together with suitable carriers or other ingredients.

[0105] The term "pharmaceutically / nutraceutical / veterinary acceptable" is art-recognized, and includes excipients, compounds, materials, compositions, carriers, vehicles and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., human or animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical / nutraceutical / veterinary texts.

[0106] Thus, some embodiments of the present invention relate to a pharmaceutical composition, a nutraceutical composition, and / or a veterinary composition comprising the probiotic composition described herein together with at least a pharmaceutically / nutraceutically / veterinary acceptable excipient as described above.

[0107] Alternatively, the probiotic composition is in the form of a pharmaceutical composition, a nutraceutical composition, and / or a veterinary composition, comprising the probiotic composition described herein together with at least one pharmaceutically / nutraceutically / veterinary acceptable excipient.

[0108] In an embodiment, the probiotic composition is in the form of a pharmaceutical product, a nutraceutical product, a veterinary product, a medical food, a food product, an edible product, a food supplement or a personal hygiene product.

[0109] Some non-limiting examples of materials which can serve as pharmaceutically / nutraceutically / veter- inary acceptable excipients or carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; or phosphate buffer solutions.

[0110] Excipients are selected, without limitation, from the group comprising: fillers / diluents / bulking agents, binders, antiadherents, disintegrants, coatings, anti-caking agents, antioxidants, lubricants, sweeteners, flavors, colors, or tensides. Fillers are selected, without limitation, from the group comprising: inulin, oligofructose, pectin, modified pectins, microcrystalline cellulose, lactose, starch, maltodextrin, saccharose, glucose, fructose, mannitol, xylitol, non-crystallizing sorbitol, calcium carbonate, dicalcium phosphate, other inert inorganic and organic pharmacologically acceptable fillers, and mixtures of these substances. At dosage form of oral suspension, fillers or diluents are selected from the group comprising: vegetable oil, oleic acid, oleyl alcohol, liquid polyethylene glycol, other pharmacologically acceptable inert liquids, or mixtures of these substances.

[0111] Binders are used in solid dosage forms, e.g., to hold the ingredients in a tablet together, to ensure that tablets and granules can be formed with required mechanical strength, and to give volume to low active dose tablets. Binders in solid dosage forms like tablets are: lactose, sucrose, corn (maize) starch, modified starches, microcrystalline cellulose, modified cellulose (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxyethylcellulose), other water-soluble cellulose ethers, polyvinylpyrrolidone (PVP) also known as povidone, poly-ethylene glycol, sorbitol, maltitol, xylitol and dibasic calcium phosphate; other suitable pharmacologically acceptable binders, or mixtures of these substances.

[0112] Antiadherents are used to reduce the adhesion between the powder (granules) and the punch faces and thus prevent sticking to tablet punches. They are also used to help protect tablets from sticking. The most commonly used is magnesium stearate.

[0113] As disintegrants and su perdis integrants in solid dosage forms like tablets and capsules, the following substances, without limitation, are used: cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, and formaldehyde-casein, other suitable pharmacologically acceptable disintegrant and superdisintegrant, or their mixtures.

[0114] Coatings in the case of solid dosage forms, such as tablets and granules for capsule filling, protect the ingredients from deterioration by moisture in the air, make large, unpleasant-tasting tablets easier to swallow and / or in the case of enteric coatings ensure intact passage through a strong acidic medium of gastric juice (pH around 1), and which allow release in duodenum or ileum (small intestine). For most coated tablets, a cellulose ether hydroxypropyl methylcellulose (HPMC) film coating is used. Occasionally, other coating materials are used, e.g., synthetic polymers and co-polymers like polyvinylacetate phthalate (PVAP); co-polymers of methyl acrylate-metacrylic acid; co-polymers of methyl metacrylate-metacrylic acid; shellac, corn protein zein or other polysaccharides; waxes or wax-like substances such as beeswax, stearic acid; higher fatty alcohols like cetyl or stearyl alcohol; solid paraffin; glycerol monostearate; glycerol distearate, or their combinations. Capsules are coated with gelatin or hydroxypropyl methylcellulose. Enteric coatings control the rate of drug release and determine where the drug will be released in the digestive tract. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers and their mixtures, also in combination with other above-mentioned coatings.

[0115] An anticaking agent is an additive placed in powdered or granulated materials to prevent the formation of lumps (caking) and for easing packaging, transport, and consumption. As anti-caking agents in solid dosage forms like tablets, capsules, or powders, the following are used: magnesium stearate, colloidal silicon dioxide, talc, other pharmacologically acceptable anticaking agents, or their mixtures.

[0116] Lubricants are used in solid dosage forms, in particular in tablets and capsules, to prevent ingredients from clumping together and from sticking to the tablet punches or capsule filling machine, and also in hard capsules. As lubricants talc or silica, and fats, e.g., vegetable stearin, magnesium stearate or stearic acid, and mixtures thereof, are the most frequently used lubricants in tablets or hard gelatin capsules.

[0117] Sweeteners are added to make the ingredients more palatable, especially in solid dosage forms, e.g., chewable tablets, as well as in liquids dosage forms, like cough syrup. Sweeteners can be selected from artificial, natural or synthetic or semi-synthetic sweeteners; non-limiting examples of sweeteners are aspartame, acesulfame potassium, cyclamate, sucralose, saccharine, sugars or any mixture thereof.

[0118] Flavors can be used to mask unpleasant tasting active ingredients in any dosage form. Flavorings can be natural (e.g., fruit extract) or artificial. For example, to improve: (1) a bitter product, mint, cherry or anise can be used; (2) a salty product, peach or apricot or liquorice can be used; (3) a sour product, raspberry; and (4) an excessively sweet product, vanilla.

[0119] Except auxiliary substances from the class of excipients, the formulation from the present invention can contain other pharmacologically active or nutritive substances including, but not limited, to prebi- otics, vitamins, such as vitamin D (calciferol) in the pharmaceutically acceptable chemical form, salt or derivatives; minerals in the form of pharmaceutically and nutritive acceptable chemical form; and L-amino acids.

[0120] In certain embodiments, the probiotic composition further comprises one or more probiotics. Probiotics resist breakdown in the gastrointestinal tract and increase probiotic functionality by acting as a food source for naturally occurring and added beneficial bacteria. Probiotics promote the growth and proliferation of beneficial bacteria in the digestive system. In such embodiments where one or more probiotics is present, such probiotics are present in a quantity between about 10 to 99.9 percent weight. Particularly, the concentration of probiotics ranges from about 15 to 25 weight percent. Examples of probiotics are fructo-oligosaccharides (e.g., inulin), galacto-oligosaccharides, xylo- oligosaccharides, arabinoxylan-oligosaccharides, pectins, beta-glucans, human milk oligosaccharides (e.g., Lacto-N-tetraose) gum acacia, resistant starch, chicory root extract or partially hydrolyzed guar gum.

[0121] In each case the presentation of the probiotic composition will be adapted to the type of administration used by means known by the person skilled in the art. Thus, the probiotic composition can be presented in the form of solutions or any other form of clinically permissible administration and in a therapeutically effective amount. The probiotic composition can be thus formulated into solid, semisolid or liquid preparations, such as tablets, capsules, powders (such as those derived from lyophilization (freeze-drying) or air-drying), granules, solutions, suppositories, gels or microspheres. In a particular embodiment, the probiotic composition is formulated for administration in liquid form or in solid form.

[0122] Pharmaceutical unit dosage forms of the compounds of this invention are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., bolus injection), topical, or trans- dermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. The pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, e.g., Remington et al., 2000).

[0123] In an embodiment, the probiotic composition is in solid form such as tablets, lozenges, sweets, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles or coated tablets, tablet, pills, troches, g astro- resista nt tablets and capsules, dispersible strips and films. More particularly, the probiotic composition is in form of a capsule, a powder, a tablet, a pill, lozenges, sachets, or granules.

[0124] In an embodiment, the probiotic composition is in form of a powder which is put in contact with an aqueous phase to form a solution. The aqueous phase can comprise fibers such as inulin. The two components (the powder and the aqueous phase) can be in separate compartments / containers and the two components are mixed for in situ reconstitution. In an embodiment where the probiotic formulation is delivered as e.g., a capsule or tablet, the probiotic composition can further comprise one or more excipients to facilitate the manufacturing process by preventing the ingredients from adhering to machines. Moreover, such excipients render the capsule or tablet form easier to swallow and digest through the intestinal tract. The excipients can be a vegetable stearate, magnesium stearate, steric acid, ascorbyl palmitate, retinyl palmitate, or hyproxypropyl methylcellulose. In a particular embodiment, the excipients are present in an amount between about 0 to 30 weight percent.

[0125] In an embodiment, the probiotic composition is in form of gelatin capsules. In a particular embodiment, the probiotic composition is in the form of a vegetable capsule and comprises hydroxypropyl methylcellulose (HPMC).

[0126] In another embodiment, the probiotic composition is in liquid form such as oral solutions, drops, suspensions (e.g., oil), emulsions and syrups. Particularly, the probiotic composition is in form of drops. More particularly, the probiotic composition is in form of oily drops.

[0127] In some embodiments, the probiotic composition is in the form of an oily suspension to be administered alone or mixed with a liquid. The oily suspension comprises at least one edible oil such as olive oil, maize oil, soybean oil, linseed oil, sunflower oil or rice oil. The oil is present in a quantity of at least 70% weight / weight. In a particular embodiment, the oily suspension also comprises at least one excipient which is an emulsifier, stabilizer or anti-caking agent, in an amount of 0.1-15% w / w. Suitable agents are silicon dioxide, silica gel, colloidal silica, precipitated silica, talc, magnesium silicate, lecithin, pectin, starch, modified starches, konjac gum, xanthan gum, gellan gum, carrageenan, sodium alginate, mono- or diglycerides of fatty acids such as glycerol monostearate or glycerol monooleate and citric acid esters of mono- or diglycerides.

[0128] In an embodiment, the e.g., capsule, sachet or stick, tablet or pill have a weight of about 150 mg to about 8000 mg. More particularly, the capsule has a weight of about 200 mg to about 600 mg. More particularly, the sachet or stick has a weight of about 1 .5 g to about 6 g. More particularly, the tablet or pill have a weight of about 400 mg to about 1200 mg.

[0129] In an embodiment, the e.g., spray, oily drops have a volume of about 3 ml to about 50 ml. More particularly, the spray has a volume of about 5 ml to about 50 ml. More particularly, the oil drops have a volume of about 3 ml to about 30 ml.

[0130] Regarding the preparation of the formulations of the present invention, it is within the scope of ordinary person skilled in the art and will depend upon the final dosage formulation. For instance, and without limitation, when the final dosage form is an oral solid one, such as tablets, capsules, powder, granules, oral suspension, etc. the process for preparation of solid dosage forms of the formulation includes homogenization of: (1) the active ingredients), comprising post-treated probiotic bacteria in an effective amount; (2) with one or more excipients to form homogeneous mixture which is, e.g., according to requirements, subjected to lubrication with magnesium stearate or other lubricants yielding final dosage form of powder. Such homogeneous powder is filled into ordinary gelatin capsules or, alternatively, into gastro-resistant capsules. In the case of tablets, they are manufactured by direct compression or granulation. In the first case, a homogeneous mixture of active ingredients and suitable excipients such as anhydrous lactose, non-crystallizing sorbitol, and others is prepared. In the second case, tablets are processed of the mixture in granulated form. Granules are prepared by granulation process of active ingredients of the formulation with suitable fillers, binders, dis integrants, and small amount of purified water. Such prepared granules are sieved and dried until the water content of <1 % w / w.

[0131] Regarding the process for preparation of liquid dosage forms (e.g., oral suspension), it involves homogenization of the active ingredient(s) of the formulation comprising post-treated probiotic bacteria in an effective amount in an inert liquid diluent (filler) such as various vegetable oils like sunflower, soybean or olive oil; oleic acid; oleyl alcohol; liquid polyethylene glycols like PEG 200, PEG 400 or PEG 600; or other inert pharmacologically acceptable liquids. The process further involves treatment of homogeneous mixture with one or more processes selected from the group comprising: (1) stabilization of the formulation, by addition and homogenization of suspension stabilizers like beeswax, colloidal silicon dioxide, etc.; (2) sweetening of the formulation, by addition and homogenization of sweetener; (3) flavoring of the formulation, by addition and homogenization of flavoring.

[0132] Food products

[0133] In some embodiments, the probiotic composition is in the form of a food product or an edible composition, such as infant formulas or food, milk-based fermented products (e.g., yogurt, cheese, curd), vegetable-based fermented products, breads, bars (e.g., energetic bars), spreads, biscuits, syrups, beverages, dressings, sauces, fillings, soups, ice creams, oils, dressings or confectionaries.

[0134] The term “food product or edible composition” are used herein in its broadest meaning, including any type of product, in any form of presentation, which can be ingested by an animal, particularly a human, but excluding pharmaceutical, nutraceutical and veterinary products. Such foods products can be in solid or liquid / drinkable form. Further, the food product can contain all customary additives, including but not limited to, proteins, vitamins, minerals, trace elements, and other nutritional ingredients.

[0135] In an embodiment, the probiotic composition is included in an infant formula or food. Particularly, the probiotic composition is included in a beverage.

[0136] Examples of other food products are meat products, chocolate spreads, fillings and frostings, chocolate, confectionery, baked goods, sauces and soups, fruit juices and coffee Whiteners. The food product particularly comprises a carrier material such as oatmeal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins. In a particular embodiment the strain is encapsulated or coated. Particularly, milks can be either of animal or vegetable origin. Administration

[0137] A therapeutically effective dose level will depend on many factors. In addition, it is well-known within the skill of the art to start doses of the active composition at relatively low levels, and increase the dosage until the desired effect is achieved. Efficacy of the methods provided herein can be determined using any appropriate method.

[0138] Clinicians, physicians, and other health care professionals can administer the probiotic composition to a subject in need thereof according to a method provided herein. In some cases, a single administration of the probiotic composition can be sufficient. In other cases, more than one administration of the probiotic composition is performed at various intervals (e.g., once per week, twice per week, daily, monthly) or according to any other appropriate treatment regimen. The duration of treatment can be a single dose or periodic multiple doses for as long as administration of the probiotic composition provided herein is tolerated by the subject.

[0139] In some embodiments, the probiotic composition is administered in a single dose or repeated dose at specific time intervals, e.g., can be administered daily for a specific number of days or according to a specific dosing schedule. In an embodiment, the probiotic composition is administered from 10 days to 90 days. More particularly, it is administered from 10 days to 60 days or from 15 to 45 days, more particularly for 30 days. In another embodiment, the probiotic composition is administered for at least 12 weeks or for at least 90 days.

[0140] In some embodiments, the probiotic composition is administered from once every three days to thrice a day, particularly, once or twice a day.

[0141] In an embodiment, the probiotic composition contains a single (unit) dose of the strain. Suitable doses of the strain (intact or lysed) can be in the range 105to 1012colony forming units (cfu), particularly between 107and 1011cfus, and more particularly in an amount of between 109and 101° cfus. In another embodiment, the strain of the present invention is present in an amount of between 1x109and 6x109cfus in the composition, and particularly in an amount of about 2x109cfus.

[0142] In an embodiment, the probiotic composition comprises the strain in an amount between 104cfu and 1012cfu per gram or milliliter of composition, particularly between 106and 1012cfu / g or cfu / ml or between 107and 1012cfu / g or cfu / ml.

[0143] In an embodiment, the probiotic composition comprises the strain in an amount between 104cfu and 1012cfu per daily dose, and particularly in an amount of about 109cfu.

[0144] In an embodiment, the probiotic composition comprises: a freeze-dried bacterial biomass comprising from about 104cfu to about 1012cfu of the strain; particularly from about 105cfu to about 1012cfu of the strain; more particularly from about 109cfu to about 1012cfu of the strain; more particularly about 1011cfu.

[0145] In some embodiments, the probiotic composition can be administered orally, rectally, parenterally, topically, ocularly, aurally, nasally, vaginally or to the buccal cavity, to give a local and / or a systemic effect. Particularly, the probiotic composition is orally or vaginally administered. More particularly, the probiotic composition is orally administered.

[0146] In an embodiment, the probiotic composition is administered via oral administration. Alternatively, the probiotic composition is an oral composition.

[0147] Medical uses and clinical outcomes

[0148] Inventors have demonstrated with high evidence in the working examples that the administration of the probiotic composition has antimicrobial activity on pathogens present in several urogenital infections, such as bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis, vaginal dysbiosis, and also unfavorable pregnancy outcomes or reproductive problems. It has been shown that L. gasseri CECT 30648 inhibits a wide range of pathogens associated to vaginal dysbiosis including Gram-positive and Gram-negative bacteria and yeast, such as E. coli, S. aureus, Fuso- bacterium, and several strains of Candida. Further, inventors have found that the strain CECT 30648 can be used for the prevention, amelioration and treatment of a hyperoxaluria and oxalate-related health conditions, since this strain is an effective oxalate degrader.

[0149] Accordingly, an aspect of the invention relates to a probiotic composition described herein for use as a medicament.

[0150] Another aspect relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of a urogenital infection. Alternatively, the invention also encompasses a method of preventing, ameliorating or treating a urogenital infection, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0151] A urogenital infection is an infection that affects any part of the urinary and genital systems, which include the kidneys, ureters, bladder, urethra, as well as the reproductive organs such as the vagina, cervix, uterus, fallopian tubes, ovaries in females, and the prostate, testes, and epididymis in males. These infections can be caused by a variety of pathogens, including bacteria, viruses, fungi, and parasites. Common symptoms can vary depending on the specific area affected but often include pain, discomfort, abnormal discharge, itching, and changes in urination such as increased frequency, urgency, or pain during urination. Examples of urogenital infections include urinary tract infections, bacterial vaginosis, aerobic vaginitis, yeast infections, sexually transmitted infections, and prostatitis. Accordingly, in an embodiment, the urogenital infection is selected from the group consisting of a vaginal infection, vaginal dysbiosis, a urinary tract infection (UTI), a genital infection, an infection of the female reproductive tract, an infection of the male reproductive tract, and a sexually transmitted infection (STI).

[0152] In an embodiment, the urogenital infection is a vaginal infection. Vaginal infections are infections that occur within the vagina. Vaginal infections are associated to symptoms such as abnormal discharge, itching, odour, burning, dyspareunia, menstrual toxic shock syndrome, vaginism, mucosal inflammation, yogurt-like discharge, and redness. Vaginal infections also increase the risk of urinary tract infections and sexually transmitted diseases. Vaginal infections also increase the risk of unfavorable pregnancy outcomes, including neonatal sepsis, stillbirth, preterm birth, miscarriage, spontaneous abortion, and endometriosis. Particularly, the vaginal infection can be a bacterial infection, a fungal infection, or a parasitic infection.

[0153] In a particular embodiment, the vaginal infection is a bacterial vaginal infection. Particularly, the bacterial vaginal infection is selected from the group consisting of bacterial vaginosis, aerobic vaginitis, atrophic vaginitis, group B Streptococcus (GBS) infection, Mycoplasma genitalium, Chlamydia, and gonorrhea.

[0154] In an embodiment, the bacterial vaginal infection is bacterial vaginosis (BV, also called bacterial vaginitis). BV is an endogenous infection produced by the overgrowth of harmful anaerobes such as Gardnerella or Prevotella. BV communities are associated with higher vaginal pH, abnormal discharge, itching and odour. Women with BV are more likely to be colonized by Fusobacterium, a pathogen linked with pregnancy complications. The strain of the present invention has shown significant activity against Fusobacterium. Thus, in a particular embodiment, the bacterial vaginosis is caused by a Fusobacterium. In another embodiment, the administration of the probiotic composition of the invention results in an improvement of pregnancy complications, e.g., improvement of neonatal sepsis, stillbirth, preterm birth, miscarriage and spontaneous abortion.

[0155] In another embodiment, the bacterial vaginal infection is aerobic vaginitis (AV). AV is an exogenous infection in which lactobacilli are replaced by aerobic bacteria of intestinal origin. The most frequent microorganisms found in AV are Escherichia coli, Staphylococcus aureus, Staphylococcus epider- midis, Group B Streptococcus, Enterococcus faecalis and Klebsiella pneumoniae. The strain of the present invention has shown significant activity against both E. coli and S. aureus. Thus, in a particular embodiment, the aerobic vaginitis is caused by Escherichia coli and / or Staphylococcus aureus.

[0156] E. co / / has been repeatedly linked to preterm birth and stillbirth. In an embodiment, the administration of the probiotic composition of the invention results in an improvement or prevention of adverse reproductive outcomes, such as preterm birth or stillbirth. E. coli and S. aureus are among most often isolated bacteria from patients with desquamative inflammatory vaginitis (DIV), a severe form of AV with symptoms of copious vaginal discharge, burning and dyspareunia. Thus, in an embodiment, the aerobic vaginitis is desquamative inflammatory vaginitis. In another embodiment, the vaginal infection is desquamative inflammatory vaginitis.

[0157] Another aspect of the present invention relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of desquamative inflammatory vaginitis; or alternatively, to a method of preventing, ameliorating or treating desquamative inflammatory vaginitis, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0158] Of note, women colonized by S. aureus and with AV have been suggested to be more susceptible to menstrual toxic shock syndrome (TSS) which is a life-threatening disease associated with the use of tampons. S. aureus is involved in nearly all cases of menstrual TSS. Therefore, in a particular embodiment, the aerobic vaginitis is menstrual toxic shock syndrome. In another embodiment, the vaginal infection is menstrual toxic shock syndrome.

[0159] In another embodiment, the bacterial vaginal infection is atrophic vaginitis. Atrophic vaginitis is caused by decline of estrogens and local immunity after menopause provoking a reduction of epithelial barrier function that facilitates pathogen colonization. Atrophic vaginitis is characterized by dryness, dyspareunia and abnormal vaginal discharge, and linked to lower levels of lactobacilli and increased bacterial diversity involving Streptococcus and Prevotella, among others. Thus, in a particular embodiment, the atrophic vaginitis is caused by Streptococcus and / or Prevotella.

[0160] Another aspect relates to a probiotic composition for use in the prevention, amelioration or treatment of atrophic vaginitis; or alternatively, to a method of preventing, ameliorating or treating atrophic vaginitis, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0161] In a particular embodiment, the vaginal infection is a fungal vaginal infection. Particularly, the fungal vaginal infection is vulvovaginal candidiasis. Vulvovaginal candidiasis (VVC), commonly known as candidiasis or a yeast infection, is an infection of the vagina and vulva caused by an overgrowth of Candida spp, which manifests with mucosal inflammation, dyspareunia, vaginism, yogurt-like discharge, burning, itching and redness. As said above, strain L. gasseri CECT 30648 displays a significant activity against Candida species, e.g., Candida albicans DSM 1386, Candida glabrata DSM 11226, or Candida tropicalis DSM 5991 . Thus, in a particular embodiment, the vulvovaginal candidiasis is caused by Candida species, more particularly Candida albicans, Candida glabrata, and / or Candida tropicalis.

[0162] In a particular embodiment, the vaginal infection is a parasitic vaginal infection. Particularly, the parasitic vaginal infection is trichomoniasis. In an embodiment, the vaginal infection is selected from the group consisting of bacterial vaginosis, aerobic vaginitis, desquamative inflammatory vaginitis, atrophic vaginitis, menstrual toxic shock syndrome, group B Streptococcus infection, mycoplasma, chlamydia, gonorrhea, vulvovaginal candidiasis and trichomoniasis. In a particular embodiment, the vaginal infection is selected from the group consisting of bacterial vaginosis, aerobic vaginitis, desquamative inflammatory vaginitis, and vulvovaginal candidiasis. Particularly, the vaginal infection is selected from the group consisting of bacterial vaginosis, aerobic vaginitis, and vulvovaginal candidiasis.

[0163] Another aspect of the present invention relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of vaginal infections (e.g., bacterial vaginosis, aerobic vaginitis, or vulvovaginal candidiasis); or alternatively, to a method of preventing, ameliorating or treating a vaginal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0164] In an embodiment, the urogenital infection is vaginal dysbiosis, i.e., alterations of the vaginal microbiota. Vaginal dysbiosis refers to an imbalance or disruption in the normal microbiota present in the vagina. Normally, the vagina is colonized by a diverse array of microorganisms, predominantly by various species of lactobacilli. These bacteria help maintain a slightly acidic environment, which is essential for keeping potentially harmful bacteria, viruses, and fungi in check. However, when there is a disturbance in this delicate balance, characterized by a decrease in beneficial lactobacilli and an overgrowth of other microorganisms, vaginal dysbiosis occurs. This imbalance can lead to various symptoms such as abnormal vaginal discharge, odor, itching, irritation, and increased susceptibility to infections. Vaginal dysbiosis is associated with conditions such as bacterial vaginosis, yeast infections (e.g., vulvovaginal candidiasis), and other forms of vaginitis. Treatment aims to restore the normal balance of vaginal microbiota and alleviate associated symptoms.

[0165] During pregnancy, the dominance of Lactobacilli in the vaginal microbiota increases, which reinforces and extends their protective role to the foetus. Vaginal dysbiosis increases the risk of spontaneous abortion with miscarriage being linked with Group B Streptococcus, Prevotella, Gardnerella and Fusobacterium, as well as sexually transmitted infections. Several pathogens have been linked to preterm birth. Most frequently isolated bacteria include E. coll, E. faecalis and S. aureus. Moreover, S. aureus and E. coll are also frequently found in the vaginal fluid of pregnant women from different populations suffering preterm premature rupture of membranes (PPROM) which is a complication associated with preterm labour and neonatal disease. E. coll is also related to stillbirth. Moreover, some of these pathogens can provoke neonatal sepsis.

[0166] Accordingly, another aspect relates to a probiotic composition for use in reducing or preventing the risk of an unfavorable pregnancy outcome (or adverse reproductive outcome); or alternatively, to a method of reducing or preventing the risk of an unfavorable pregnancy outcome, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0167] Unfavourable pregnancy outcomes refer to a spectrum of adverse events that can occur during pregnancy, labour, or shortly after birth, posing significant risks to both the mother and the infant. These outcomes include neonatal sepsis, preterm birth, miscarriage, spontaneous abortion, endometriosis or any other adverse event related to pregnancy and newborns birth episode.

[0168] Thus, in an embodiment, the unfavorable pregnancy outcome is selected from the group consisting of neonatal sepsis, stillbirth, preterm birth, miscarriage, spontaneous abortion, preterm premature rupture of membranes, and endometriosis. In a particular embodiment, the miscarriage, spontaneous abortion or sexually transmitted infection is caused by Group B Streptococcus, Prevotella, Gardnerella and / or Fusobacterium. In another particular embodiment, the preterm birth is caused by E. coll, E. faecalis and / or S. aureus. In another particular embodiment, the preterm premature rupture of membranes is caused by S. aureus and / or E. coll. In another particular embodiment, the stillbirth is caused by E. coll. In a particular embodiment, the probiotic composition is for use in the prevention of spontaneous abortion and preterm delivery.

[0169] In an embodiment, the urogenital infection is a urinary tract infection (UTI). UTIs are also associated with vaginal microbiota imbalances, since vagina (together with gut) serves as a reservoir for pathogens that can transit in the urinary tract. E. coll (uropathogenic, UPEC) is also the most frequent causative agent of UTIs (in both community and pregnant women). S. aureus can also cause UTIs in pregnant women. Importantly, women with UTI during pregnancy are more likely to have preterm delivery, E. co / / UPEC being the most common uropathogen in those patients. UTI symptoms include frequent urination, burning sensation and pain. Studies showed women with a history of UTIs have the same E. coll UPEC strain in the urinary tract and vagina (Lewis and Gilbert, 2020; Navas-Nacher et al., 2001). Accordingly, in an embodiment, the administration of the probiotic composition of the invention results in an improvement of adverse reproductive outcomes, such as preterm delivery. In another embodiment, the adverse reproductive outcome, particularly preterm delivery, is caused by E. coll UPEC. In another embodiment, the urinary tract infection is caused by Escherichia coll and / or Stapylococcus aureus.

[0170] Particularly, the UTI can be a lower UTI or an upper UTI. In a particular embodiment, the UTI is a lower UTI. Particularly, the lower UTI is cystitis (i.e., bladder infection) or urethritis (i.e., urethra infection). In another particular embodiment, the UTI is an upper UTI. Particularly, the upper UTI is pyelonephritis (e.g., kidney infection) or ureteritis (ureter infection).

[0171] In some embodiments, the UTI is selected from the group consisting of cystitis, urethritis, pyelonephritis, ureteritis, prostatitis, epididymitis, orchitis, cystourethritis, interstitial cystitis, and acute bacterial prostatitis. In a particular embodiment, the UTI is selected from the group consisting of cystitis, urethritis, pyelonephritis, and ureteritis.

[0172] Another aspect relates to a probiotic composition for use in the prevention, amelioration or treatment of an urinary tract infection; or alternatively, to a method of preventing, ameliorating or treating an urinary tract infection, comprising administering to a subject in need thereof a therapeutically effective amount of a probiotic composition of the invention.

[0173] In an embodiment, the urogenital infection is a genital infection. Genital infections encompass all infections that occur in the genital area, which includes both internal and external reproductive organs, e.g., the vagina, vulva, cervix, uterus, fallopian tubes, ovaries or external genitalia (labia, clitoris). Genital infections can be bacterial infections, viral infections, fungal infections, or parasitic infections. Non-limiting examples of genital infections are human papillomavirus (HPV), herpes simplex virus (HSV), human immunodeficiency virus (HIV) / AIDS, hepatitis B and C, syphilis, chancroid, Mycoplasma genitalium, candidiasis, pubic lice, or scabies.

[0174] In an embodiment, the urogenital infection is an infection of the female reproductive tract. Non-limiting examples of infections of the female reproductive tract are pelvic inflammatory disease (PID) (i.e, infection of the upper female reproductive organs, including the uterus, fallopian tubes, and ovaries), endometritis (i.e., infection of the lining of the uterus), salpingitis (i.e., infection of the fallopian tubes), and oophoritis (i.e., infection of the ovaries).

[0175] In an embodiment, the urogenital infection is an infection of the male reproductive tract. Non-limiting examples of infections of the male reproductive tract are prostatitis (i.e., infection of the prostate gland), epididymitis (i.e., infection of the epididymis), orchitis (i.e., infection of the testes), and balanitis (i.e., infection of the glans penis).

[0176] In an embodiment, the urogenital infection is a sexually transmitted infection (STI). Examples of STIs are chlamydia, gonorrhea, syphilis, human papillomavirus (HPV), herpes simplex virus (HSV), human immunodeficiency virus (HIV) / AIDS, trichomoniasis, Mycoplasma genitalium, and Ureaplasma urealyticum.

[0177] In some embodiments, the administration of the probiotic composition of the invention results in at least one outcome (i.e., effect) selected, but not limited to, from the group consisting of:

[0178] - reduction of the abundance of non-Lactobacillus species in the vaginal microbiota;

[0179] - reduction of the abundance of non-Lactobacillus species in the urinary tract;

[0180] - improvement or restoration of vaginal microbiota / ecosystem;

[0181] - reduction of itching;

[0182] - reduction of irritation;

[0183] - pH stabilization; - reduction of abnormal discharge (e.g., thick, white, clumpy discharge, or yogurt-like discharge);

[0184] - reduction of odour;

[0185] - reduction of burning sensation or pain;

[0186] - reduction of redness;

[0187] - amelioration or treatment of dyspareunia;

[0188] - amelioration or treatment of menstrual toxic shock syndrome;

[0189] - amelioration or treatment of vaginism;

[0190] - reduction of swelling;

[0191] - prevention of complications;

[0192] - enhanced sexual health;

[0193] - improved reproductive health;

[0194] - restoration of normal urinary function (e.g., frequent urination); and

[0195] - reduced inflammation or reduced mucosal inflammation.

[0196] In a particular embodiment, the administration of the probiotic composition results in at least one outcome selected from the group consisting of:

[0197] - reduction of the abundance of non-Lactobacillus species in the vaginal microbiota;

[0198] - reduction of the abundance of non-Lactobacillus species in the urinary tract; and

[0199] - improvement or restoration of vaginal microbiota / ecosystem.

[0200] Another aspect of the present invention relates to a probiotic composition described herein for use in the prevention, amelioration or treatment of a hyperoxaluria and oxalate-related health conditions.

[0201] While urinary oxalate concentrations are in the millimolar range with an average of 0.25 mmoles / 24 h in healthy controls, hyperoxaluria is defined by a urinary excretion greater than 0.45 mmoles / 24 h.

[0202] Primary hyperoxaluria is a rare inherited genetic defect of oxalate metabolism while secondary hyperoxaluria is seen in states of increased ingestion of oxalate, its precursors or altered gut microbiota. Thus, in one embodiment the hyperoxaluria is primary hyperoxaluria, and in another embodiment, the hyperoxaluria is secondary hyperoxaluria.

[0203] The most common oxalate toxicity is calcium oxalate nephrolithiasis (kidney stones), which accounts for more than 70% of overall nephrolithiasis, affecting 9% of the US population with a 20% 5-year recurrence rate. Thus, in a particular embodiment, the oxalate-related health condition is nephrolithiasis.

[0204] When hyperoxaluria progresses and kidney function declines, oxalate crystals can accumulate in various tissues and organs throughout the body, leading to a condition known as systemic oxalosis. In an embodiment, the oxalate-related health condition is systemic oxalosis. This systemic involvement can result in a range of significant health problems. This systemic involvement can result in a range of significant health problems in the cardiovascular system (oxalate crystal deposits may lead to heart block or other arrhythmias and cardiomyopathy), the skeletal system (bone pain and fractures as well as such as synovitis and chondrocalcinosis), the hematologic system (anemia can develop due to oxalate deposition in the bone marrow), the nervous system (peripheral neuropathy, retinopathy and cerebral infarcts due to oxalate deposition), the skin and vascular system (skin ulcers, gangrene and refractory hypotension). Further, in children, hyperoxaluria can severely impact to growth and development. Therefore, in some embodiments, the systemic oxalosis involves health problems in the cardiovascular system, the skeletal system, the hematologic system, the nervous system, the skin and vascular system, children growth and development. Therefore, in some embodiments, the oxalate-related health conditions are selected from the group consisting of cardiovascular conditions (e.g., arrhythmias and cardiomyopathy), skeletal conditions (e.g., (bone pain, fractures, synovitis and chondrocalcinosis), hematological conditions (e.g., anemia), nervous system conditions (e.g., peripheral neuropathy, retinopathy and cerebral infarcts due to oxalate deposition), skin and vascular conditions (e.g., skin ulcers, gangrene and refractory hypotension) and children growth and development conditions.

[0205] Further, IBD (inflammatory bowel disease) and Chron’s disease patients have increased oxalate levels and prevalence of stones formation. Thus, in a particular embodiment, the oxalate-related health condition is IBD. In another embodiment, the oxalate-related health condition is Chron’s disease.

[0206] Of note, it has been recently reported that dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis. Thus, in a particular embodiment, the oxalate-related health condition is atherosclerosis.

[0207] In women, high oxalate levels have been also linked to vulvodynia, which is defined as a type of long-term pain or discomfort around the outer part of the female genitals (the vulva), that lasts at least three months and has no clear cause. In a particular embodiment, the oxalate-related health condition is vulvodynia.

[0208] Therefore, an aspect of the invention relates to a probiotic composition described herein for use in the prevention, amelioration ortreatment of an urogenital disease or condition, including as explained herein e.g. urogenital infections, hyperoxaluria and oxalate-related health conditions.

[0209] Combination with an antimicrobial agent / other probiotic strains

[0210] Remarkably, EXAMPLE 6 of the working examples demonstrates that the probiotic composition of the present invention can be combined with antibiotics used in the clinical practice. Results confirmed that L. gasseri CECT 30648 is highly compatible, i.e., no susceptible at the maximum concentration tested, with several antibiotics, particularly with those that are commonly used in vaginitis and urinary tract infections, indicating that it can be administered together with these treatments.

[0211] Accordingly, the invention is also directed to combinations comprising the probiotic composition described herein, i.e., which comprises Lactobacillus gasseri CECT 30648, together with one or more antimicrobial agent. The probiotic composition and the other antimicrobial agent can be formulated for a separate, sequential, concomitant administration, or in a mixture in a single pharmaceuti- cal / nutraceutical / veterinary composition.

[0212] Thus, in some embodiments, the probiotic composition of the present invention is combined with one or more antimicrobial agent. Particular embodiments of antimicrobial agents are described hereinafter.

[0213] The term “antimicrobial” has in the present invention the normal meaning in the field, namely, an agent that kills microorganisms or inhibits their growth. They can be further grouped according to the microorganisms they act primarily against, e.g. against bacteria (antibacterials) and against fungi (antifungals).

[0214] In an embodiment, the antimicrobial agent is selected from the group consisting of an antibiotic, an antiseptic, an antifungal, an antiviral and an antiparasitic.

[0215] In an embodiment, the antimicrobial agent is selected from the group consisting of cefaclor, fosfomy- cin, metronidazole, trimethoprim, sulfapyridine, levofloxacin, ciprofloxacin, and nitrofurantoin. More particularly, the antimicrobial agent is selected from the group consisting of cefaclor, fosfomycin, metronidazole, trimethoprim, and sulfapyridine.

[0216] In a particular embodiment, the antimicrobial agent is an antibiotic used in vaginitis, e.g., metronidazole or clindamycin.

[0217] In another particular embodiment, the antimicrobial agent is an antibiotic used in urinary tract infections, e.g., ciprofloxacin or fosfomycin.

[0218] In another particular embodiment, the antimicrobial agent is an antimycotics used in vulvovaginal candidiasis, e.g., triazole drugs (e.g. fluconazole, clotrimazole, myconazole, itraconazole) or nystatin.

[0219] The invention is also directed to combinations comprising the probiotic composition described herein, i.e., comprising Lactobacillus gasseri CECT 30648, together with one or more additional probiotic strains. The strain CECT 30648 and the other probiotic strain can be formulated for a separate, sequential, concomitant administration, or in a mixture in a single pharmaceutical / nutraceutical / vet- erinary composition. In an embodiment, the strain CECT 30648 is in combination with Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485 (KABP-023) and / or Pediococcus acidilactici CECT 7483 (KABP-021), particularly for use in the treatment of vaginal dysbiosis or for use in reducing or preventing the risk of an unfavorable pregnancy outcome such as endometriosis.

[0220] In another embodiment, the strain CECT 30648 is in combination with Lactiplantibacillus plantarum CECT 7481 (KABP-051), particularly for use in the prevention, amelioration or treatment of hyperoxaluria and oxalate-related health conditions.

[0221] EXAMPLES

[0222] EXAMPLE 1. Origin and adaptability of strain L. gasseri CECT 30648 to the vaginal environment

[0223] L. gasseri CECT 30648 was isolated from the vagina of a healthy woman. To further confirm the adaptability of the strain to the vaginal ecosystem, the resistance to simulated vaginal medium was assessed and compared with reference strains.

[0224] 1.1 Methods. Bacterial strains

[0225] Strains were routinely grown in MRS medium supplemented with cysteine (MRSc) at 37°C in anaerobic conditions. Strains used in each experiment are shown in Table 1 . Strains were inoculated into simulated vaginal media (per L: NaCI 3.51g, KOH 1.40g, Ca(OH)2 0.222g, BSA 0.018g, lactic acid 2g, acetic acid 1 g, glycerol 0.16g, urea 0.4g, glucose 8g, adjusted to pH 4.2) and incubated for 6 h. Proliferative bacteria were quantified by serial dilution and plate counting method.

[0226] Table 1 . Bacterial strains used in the study.

[0227] 1 .2 Results

[0228] L. gasseri CECT 30648 showed a good survival after 6 h of incubation in simulated vaginal medium. These results were in the range of those obtained for most of the reference strains except for L. crispatus 21-01 and CECT 30647, and L. acidophilus LAM whose survivals were worst. This outcome confirms the L. gasseri CECT 30648 is well adapted to the vaginal environment.

[0229] Table 2. Tolerance to vaginal medium. Interpretation under the conditions tested: Good, reduction of less than one logarithm of cfu / mL; Fair, reduction of less than two logarithms; Low, reduction of less than three logarithms; Very low, reduction of more than three logarithms.

[0230] EXAMPLE 2. Resistance to the gastrointestinal tract of strain L. gasseri CECT 30648 Tolerance to the gastrointestinal tract passage is a requirement for oral administered probiotics and particularly for probiotics aimed to reach the vaginal mucosa. Thus, the ability of the strain to survive in a simulated gastric solution and in presence of bile salts was tested.

[0231] 2.1 Methods Strains were inoculated in a gastric solution (per 1 L: NaCI 7.3 g, KCI 0.52 g, NaHCCh 3.78 g and pepsin 3 g) at pH 2.3 for 30 min, and in MRScys with 0.25% bile salts for 180 min. Proliferative bacteria were counted by serial dilution and plate counting method.

[0232] 2.2 Results

[0233] L. gasseri CECT 30648 showed a good resistance to gastric condition at a similar level of other commercial probiotic strains (Table 3). Values of tolerance to bile salts were different between strains with L. gasseri CECT 30648 having a much better resistance than the competitor L. gasseri LN40 and the vaginal strain L. crispatus 21-01 . These outcomes indicate despite L. gasseri CECT 30648 being a vaginal strain it possesses a good tolerance to gastrointestinal conditions.

[0234] Table 3. Tolerance to gastric stress and bile salts. Interpretation under the conditions tested: Good, reduction of less than one logarithm of cfu / mL; Fair, reduction of less than two logarithms; Low, reduction of less than three logarithms; Very low, reduction of more than three logarithms.

[0235] EXAMPLE 3. Adhesion capacity to the vaginal epithelium of strain L. gasseri CECT 30648

[0236] Adhesion to human cells is a key characteristic of probiotic strains. Pathogenic microorganisms start infection processes by adhering to human cells. Probiotics with good adhesion capacity compete with pathogenic microorganisms for union sites thus reducing their infectivity. Here, we investigated the adhesion capacity of L. gasseri CECT 30648 to human vaginal epithelial cells. 3.1 Methods

[0237] Human vaginal epithelial HeLa cells were seeded and incubated until confluence. Washed HeLa cells were co-incubated with bacterial strains for 60 minutes. Medium was removed, and the mono- layer was washed to discard unbonded bacteria. Cells were detached, and the suspension of bacteria and Hela cell debris was serially diluted, plated, and incubated to count bacterial cells.

[0238] 3.2 Results

[0239] L. gasseri CECT 30648 showed an efficient adhesion (74.8%) to the vaginal epithelium (Table 4). This value was within the range of other L. gasseri and L. crispatus strains, and was higher than L. gasseri LN40, L. rhamnosus GR1 , L. rhamnosus Lcr35 and RC14, L. rhamnosus HN001 , L. plantarum P17630, L. acidophilus LA14. These results confirm L. gasseri CECT 30648 possesses a high adhesion capacity to the vaginal epithelium indicating it may compete with pathogens for adhesion sites.

[0240] Table 4. Adhesion of strains to human vaginal epithelial HeLa cells.

[0241] EXAMPLE 4. Inhibition of urogenital pathogens of strain L. gasseri CECT 30648 by production of antimicrobials

[0242] Probiotics may improve vaginal health by inhibiting the growth of opportunistic pathogens associated to vaginal dysbiosis. E. coll, S. aureus and Fusobacterium sp were chosen as target strains for the analysis. E. coll (specifically an UPEC strain) was selected as the main causative agent of UTI and its implication in AV, DIV and preterm birth. S. aureus is an etiological agent of AV, DIV, UTI during pregnancy and preterm labor. Fusobacterium sp. is linked to miscarriage and preterm delivery.

[0243] 4.1 Methods Antimicrobial activity was assessed by microplate growth inhibition. Pathogens (Table 5) were grown in supernatants from probiotics monocultures or probiotic + pathogen cocultures. Monoculture were obtained from probiotic culture in a mixture of pathogen fresh medium and MRScys, and cocultures by mixing probiotic active culture and pathogen active culture. Monocultures and cocultures were further incubated for 24 h. Cultures were centrifuged, and supernatants filtered. An aliquot was adjusted to pH 5 to study inhibition independent of organic acids. Then, pathogen culture was mixed with supernatants and cultured in a microplate. A control condition without supernatants was included. OD was monitored for 24 h in a plate reader and Area Under the Curve was calculated (normalizing with baseline OD). Antimicrobial activity was evaluated as % of inhibition growth in relation to that of the control.

[0244] Table 5. Pathogenic strains used as target strains in microplate growth inhibition assay. Growth medium is indicated.

[0245] Presence of bacteriocins in the genome of L. gasseri CECT 30648 was assessed using BAGEL4 webserver. Whole genome sequence was obtained by Illumina Sequencing By Synthesis and genome was assembled and annotated using standardized methods.

[0246] 4.2 Results

[0247] Analysis of E. coli UPEC DSM 10650 growth in neutralized monocultures of probiotics showed different antagonistic activities ranging from 5 to 80 % inhibition (FIG. 1 (A)). Interestingly, L. gasseri CECT 30648 showed the highest inhibitory activity compared with the other probiotic strains. Similarly, neutralized supernatants from monocultures varied in the capacity to inhibit the growth of S. aureus CIP107860 (from 0 to 38 %) (FIG. 1 (B)). Importantly, L. gasseri CECT 30648 also showed the highest inhibitory activity against this pathogen. On the contrary, the inhibition of the growth of F. necrophorum by neutralized monocultures was generally low (lower than 25 %) with small differences between probiotics.

[0248] Production of antimicrobial compounds is sometimes induced by the presence of pathogenic bacteria. To further evaluate whether the probiotics were able to inhibit F. necrophorum, the pathogen was cultured in neutralized supernatants of probiotic + pathogen cocultures. Importantly L. gasseri CECT 30648 inhibited the grown of the pathogen and the effect was greater than 11 out of 12 competitor strains (FIG. 2). This effect was not observed using monoculture supernatants suggesting the presence of the pathogen activates the production of specific inducible antimicrobial compounds by CECT 30648. In addition, the genome of L. gasseri CECT 30648 was screened for the presence of antimicrobial genes. Genes encoding for bacteriocins Helveticin J and Enterolysin A were identified, confirming the ability of this strain to produce antimicrobial peptides.

[0249] All together these results demonstrate L. gasseri CECT 30648 biosynthesizes antimicrobial compounds that effectively inhibit the growth of E. coli, S. aureus and Fusobacterium having the highest antimicrobial capacity among the tested strains. Of note, the antimicrobial compounds against Fusobacterium seem to be of pathogen-induced type only, as the inhibition is observed when the pathogen is present in the culture but not otherwise.

[0250] EXAMPLE 5. Antimicrobial activity of strain L. gasseri CECT 30648

[0251] The antimicrobial activity of L. gasseri CECT 30648 was assessed against a wide range of microorganism involved in vaginal infections and unfavourable pregnancy outcomes including Candida spp strains and other Gram-positive and Gram-negative bacteria.

[0252] 5.1 Methods

[0253] Antimicrobial activity against pathogens described in Table 6 was assessed by agar plug diffusion methods and liquid cultures in tubes using probiotic supernatants. At least one method was used with each target strain. For the agar plug diffusion method plates were inoculated with pathogens and soft agar disks with probiotic were placed on top. After an incubation for 48 h, the inhibition halos were measured from the outer perimeter of the disks. For liquid cultures in tubes, lactobacilli were grown overnight, and cell free supernatants obtained. Supernatants were mixed with an equal volume of pathogen medium, and pathogen was cultured in the mixture. OD was monitored for 24 h and inhibition was evaluated by comparing a control without probiotic supernatant.

[0254] Table 6. Pathogenic strains used as target strains in antimicrobial activity tests. Growth medium is indicated. 5.2 Results

[0255] L. gasseri CECT 30648 was able to inhibit the growth of the 10 pathogens tested (Table 7), showing a strong inhibition against 7 of them.

[0256] Table 7. Antimicrobial activity of L. gasseri CECT 30648 against target strains evaluated by agar plug diffusion method and / or liquid culture method. absent; +, weak; ++, moderate; +++, strong.

[0257] These results indicate L. gasseri CECT 30648 inhibits a wide range of pathogens associated to vaginal dysbiosis including Gram-positive and Gram-negative bacteria and yeast, and support effect of L. gasseri CECT 30648 to reduce pathogens involved in urogenital infections and reproductive problems.

[0258] EXAMPLE 6. Antibiotic susceptibility and compatibility of strain L. gasseri CECT 30648 with antibiotics used in clinical practice

[0259] 6.1 Methods

[0260] Antibiotic susceptibility to a panel of 8 antibiotics required by EFSA and to a panel of 10 antibiotics used in clinical practice to treat genitourinary infections was tested following the ISO 10932:2010. Susceptibility or resistance was determined by comparing minimum inhibitory concentration (MIC) values with cut-offs defined by EFSA forthe 8 antibiotics required. Presence of possible antimicrobial resistance (AMR) genes was investigated in L. gasseri CECT 30648 genome using the two maintained databases ResFindres and CARD as recommended by EFSA 2018.

[0261] 6.2 Results

[0262] Phenotypic experiments revealed that L. gasseri CECT 30648 is susceptible to all antibiotics included in the EFSA panel except for kanamycin and clindamycin (Table 8). No associated resistance gene was found through genome analysis indicating the resistance is due to intrinsic mechanism and therefore does not pose any risk in relation to the potential transfer of antimicrobial resistance and confirming the safety of the strain.

[0263] The compatibility of the strain with antibiotics commonly used in clinical practice to treat was investigated. L. gasseri CECT 30648 was highly compatible (i.e no susceptible at the maximum concentration tested) with cefaclor, fosfomycin, metronidazole, trimethoprim and sulfapyridine, and showed intermediate susceptibility to levofloxacin, ciprofloxacin and nitrofurantoin. Of note, L. gasseri CECT 30648 is naturally compatible with antibiotics very commonly used in vaginitis (metronidazole, clindamycin) and urinary tract infections (ciprofloxacin, fosfomycin), indicating it can be administered together with these treatments.

[0264] Table 8. MIC values of L. gasseri CECT 30648 to antibiotics required to be tested by EFSA and antibiotics used in clinical practice. S, susceptibility; R, resistance.

[0265] EXAMPLE 7. Colonization of the vagina after oral administration of strain L. gasseri CECT 30648

[0266] A clinical trial with healthy volunteers was carried out to study the ability of the strain to migrate to the vagina after oral administration. 7.1 Methods

[0267] A prospective, randomized, double-blinded, and placebo-controlled clinical trial with was conducted against placebo to evaluate the safety and capacity of L. gasseri CECT 30648 to migrate to the vagina after oral administration in healthy women. The trial adhered to the ethical principles of the World Medical Association (WMA), Declaration of Helsinki as well as the Good Clinical Practice (GCP) guidelines. The main outcome was the presence of L. gasseri CECT 30648 in the vagina confirmed by qPCR. Adverse events were recorded for safety assessment.

[0268] Forty-eight healthy premenopausal women aged 18-45 years that provided informed consent were recruited voluntarily. Individuals with vaginal infection, relevant medical conditions, chronic gastrointestinal issues, antibiotic, probiotic, or antifungal use a month before study start, pregnancy, lactation, immunomodulator or corticosteroid use, substance abuse, intrauterine device use, recent use of spermicides or vaginal lubricants, irregular menstrual cycles, and other incompatible conditions were excluded. Participants were randomized in a 3:1 ratio to receive L. gasseri CECT 30648 10E9 CFUs / capsule or placebo daily. Probiotic and placebo capsules were indistinguishable and contained maltodextrin as a carrier, magnesium stearate as an anti-adherent, and hypromellose as a capsule coating. The randomization list was generated using a specific software and kept confidential. Participants and personnel involved were blinded through the study period.

[0269] Intervention started the day following the cessation of menstruation and participants consumed one capsule of probiotic or placebo daily between menses for up to 18 days (average inter-menstruation period). Participants collected vaginal swabs every three days. Volunteers were asked to abstain from sexual intercourse and the use of intravaginal products (such as lubricants and spermicides) for 24 hours before sample collection.

[0270] DNA was extracted from vaginal swabs using standardized protocols. L. gasseri CECT 30648 presence was studied by qPCR using strain-specific primers (Forward: tgg gca eta ggt aat aag aac (SEQ ID NO: 2); reverse: ggg tgt cca gat ata tat cca (SEQ ID NO: 3)). Total microbiota was analyzed by massive sequencing of the hypervariable V3-V4 region of the 16S gene using MySeq sequencer, followed by taxonomical identification against Silva v1 .3.8 database. Adverse events (AEs) and medication updates were documented in a diary. Statistical differences were assessed by Chi-square test and paired Student’s T-Test (two-sided p-value <0.05).

[0271] 7.2 Results

[0272] Forty-six out of 48 participants completed the study. Two participants were excluded due to lack of compliance. Probiotic and placebo groups were balanced regarding demographic characteristics, menstrual cycle, and obstetric history at baseline (Table 9). Results showed no adverse event was attributed to the probiotic. Table 9. Baseline demographic and clinical characteristics of the population.

[0273] Strain-specific qPCR analysis was positive for L. gasseri CECT 30648 in the vaginal samples of 18 women (53%) of the probiotic group in at least one time-point (FIG. 3). On the contrary, a positive qPCR result was obtained in only 1 volunteer (from a single time-point sample) of the placebo group (8 %) which can be explained by the natural presence of the strain in the volunteer. The difference between probiotic and placebo group was statistically significant (p= 0.008). This outcome confirms L. gasseri CECT 30648 can tolerate the in vivo conditions of the human gastrointestinal tract and migrate and colonize the vagina.

[0274] Healthy, non-symptomatic women often have non-lactobacillus bacteria in their vaginal microbiota, although not in a predominant manner. In our study, 30-40% of the bacteria in the vaginal microbiota were not of the lactobacillus family on average. Importantly, a statistically significant decrease in abundance of non-lactobacilli was noted in the probiotic group (absolute reduction of 6.0%, p=0.047), in line with the documented antimicrobial activity of strain L. gasseri CECT 30648. Conversely an increase was noted in the placebo group (absolute increase of 5.5%), although not statistically significant.

[0275] EXAMPLE 8. Oxalate degradation by strain L. gasseri CECT 30648

[0276] The aim of this work was to screen AB-BIOTICS collection and competitors for the presence of oxc and frc homologous and to validate and compare oxalate degradation activity of different Lactobacillus spp., strains and species.

[0277] 8.1 Materials and methods

[0278] 8. 1. 1 Strains and growth conditions

[0279] Strains investigated in this study are listed in Table 10. Lactobacilli were routinely grown in MRS medium at 37 °C for 48 h in anaerobiosis.

[0280] Table 10. Strains investigated in this study.

[0281] 8.1.2 In silico analysis

[0282] The proteome of L. gasseri CECT 30648 was screened for the presence of Type I and Type II oxalate degradation pathways (ODPs) (Table 11).

[0283] Table 11 . EC codes for Type I and Type II ODPs investigated.

[0284] DNA sequences of Formyl-CoA:Oxalate CoA-transferase (FRC) and Oxalyl-CoA decarboxylase (OXC) genes were identified in the genome of L. gasseri CECT 30648 retrieved and used as template for the detection of homologous genes in several Lactobacillus spp. through BLAST.

[0285] 8.1.3 Oxalate degradation assay

[0286] To screen for oxalate metabolizing lactobacilli, bacterial strains (Table 10) were precultured in MRS broth at 37 °C in anaerobiosis to an optical density of ~108CFU / ml and 0.1 mL was used to inoculate 10 ml of MRS broth containing 10 mM of sodium oxalate. Bacteria were cultured for 72 h and oxalate was quantified at different timepoints through HPLC as follows. Samples were prepared by mixing 0.4 mL of the cultured growth with 4.6 mL of 4.5 mM sulfuric acid and centrifuging at 5000xg for 15 min at 4 °C (Eppendorf 581 OR centrifuge). The supernatant was filtered through 0.22 pm PVDF membrane filters (Teknokroma, Sant Cugat del Valles, Spain). Extracts (20 pL) were injected in duplicate and eluted with 3 mM sulfuric acid at 65 °C and a flow rate of 0.7 mL min-1 on a 300x7.8 mm HPX-87H Aminex ion-exchange column protected by a Micro-Guard H+ cation cartridge (Bio-Rad Laboratories, Richmond, CA, USA) in a chromatograph, Agilent 1260 Infinity II with a DAD detector at 210 nm for citric, pyruvic, lactic, formic, acetic, propionic, and butyric acids, and oxalic, and at 280 nm for orotic and uric acids. Organic acids were quantified by the external standard method. The results were expressed as a percentage of oxalic acid consumed.

[0287] 8.2 Results

[0288] A large number of Lactobacillus species was screened using BLAST against Nucleotide collection (nr / nt) database filtering by Taxid, and oxc and frc homologous genes were detected only in members of L. acidophilus group of Table 12. Two frc copies were detected in L. crispatus, while the remaining species displayed only one copy of each gene, both organized in an operon.

[0289] Table 12. Presence of oxc and frc genes in Lactobacillus spp.

[0290] Presence of oxc and frc genes seems restricted to L. acidophilus group, which includes dominant members of healthy vaginal microbiota such as L. crispatus and L. gasseri species. Oxalate degradation experiments were in line with in silico results as only L. gasseri, L. crispatus and L. acidophilus strains in our collection showed oxalate degradation, all of them belonging to the L. acidophilus group (FIG. 5(A)). After 72 h, strains L. acidophilus LA-14 and L. gasseri CECT 30648 showed high but comparable oxalate degradation (88.3% and 86.3, respectively, p=0.997) followed by L. gasseri 18_02 (79.7%, p=0.01 vs Lg CECT 30648), motivating analyzing oxalate catabolism by these strains at shorter time point.

[0291] After 24 h (FIG. 5(B)), L. gasseri strains Lg CECT30648 and 18_02 showed comparable oxalate degradation (28.5% and 27.7%, p=0.55), significantly higher (p<0.0001) than L. acidophilus LA-14 (16.9%). Taken together, we conclude that L. gasseri CECT 30648 is the most effective strain in oxalate degradation in terms of percentage and time.

[0292] 8.3 Conclusions

[0293] Only species belonging to L. acidophilus complex harbour oxc and frc genes and display oxalate degradation activity.

[0294] L. gasseri and L. acidophilus species seem to be the most effective oxalate degraders in our collection, although such capacity is also strain-specific.

[0295] Taking all results together, L. gasseri strain CECT 30648 is the most rapid and efficient oxalate degrader in our collection, positioning it as the best candidate for the management of oxalate-related health conditions.

[0296] REFERENCES

[0297] Pan, M., Hidalgo-Cantabrana, C., Goh, Y. J., Sanozky-Dawes, R., and Barrangou, R. (2020). Comparative Analysis of Lactobacillus gasseri and Lactobacillus crispatus Isolated From Human Urogen- ital and Gastrointestinal Tracts. Front. Microbiol. 10, 499288.

[0298] Lewis, A. L., and Gilbert, N. M. (2020). Roles of the vagina and the vaginal microbiota in urinary tract infection: evidence from clinical correlations and experimental models. GMS Infect. Dis. 8, Doc02. Navas-Nacher, E. L., Dardick, F., Venegas, M. F., Anderson, B. E., Schaeffer, A. J., and Duncan, J. L. (2001). Relatedness of Escherichia coli colonizing women longitudinally. Mol. Urol. 5, 31-36.

Claims

CLAIMS1 . A probiotic composition comprising Lactobacillus gasseri strain deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) under accession number CECT 30648, or a bacterial strain derived thereof, wherein the derived bacterial strain has a genome with at least 99% average nucleotide identity to the genome of the correspondent deposited strain.

2. The probiotic composition according to claim 1 , wherein the derived bacterial strain has a genome with at least 99.5% average nucleotide identity to the genome of the correspondent deposited strain.

3. The probiotic composition according to any of claims 1-2, wherein Lactobacillus gasseri strain is the strain deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) under accession number CECT 30648.

4. The probiotic composition according to any of claims 1-3, which is administered via oral administration.

5. The probiotic composition according to any of claims 1-4, wherein the composition is in the form of a pharmaceutical product, a nutraceutical product, a veterinary product, a medical food, a food product, an edible product, a food supplement or a personal hygiene product.

6. A probiotic composition as defined in any of claims 1-5, for use in the prevention, amelioration or treatment of an urogenital disease or condition.

7. The probiotic composition for use according to claim 6, in the prevention, amelioration or treatment of hyperoxaluria and oxalate-related health conditions.

8. The probiotic composition for use according to claim 6, in the prevention, amelioration or treatment of a urogenital infection.

9. The probiotic composition for use according to claim 8, wherein the urogenital infection is selected from the group consisting of a vaginal infection, vaginal dysbiosis, a urinary tract infection, a genital infection, an infection of the female reproductive tract, an infection of the male reproductive tract, and a sexually transmitted infection.

10. The probiotic composition for use according to claim 9, wherein the vaginal infection is selected from the group consisting of bacterial vaginosis, aerobic vaginitis, desquamative inflammatory vaginitis and vulvovaginal candidiasis.11 . The probiotic composition for use according to claim 9, wherein the urinary tract infection is selected from the group consisting of cystitis, urethritis, pyelonephritis, and ureteritis.

12. A probiotic composition as defined in any of claims 1-5, for use in reducing or preventing the risk of an unfavorable pregnancy outcome.

13. The probiotic composition for use according to claim 12, wherein the unfavorable pregnancy outcome is selected from the group consisting of neonatal sepsis, stillbirth, preterm birth, miscarriage, spontaneous abortion, preterm premature rupture of membranes and endometriosis.

14. A strain of Lactobacillus gasseri deposited in the Spanish Type Culture Collection under the accession number CECT 30648.

15. A method to obtain a strain derived from the Lactobacillus gasseri strain deposited in the Spanish Type Culture Collection under accession number CECT 30648, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant has a genome at least 99% ANI to the genome of the correspondent deposited strain.

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