Mixture of lactobacillus crispatus strains

Abstract

The present invention relates to an isolated mixture of strains of Lactobacillus crispatus, comprising strains that have different expression of surface-associated polysaccharides, including a first strain of Lactobacillus crispatus having expression of one or more of glycosyltransferase (GT) 1, GT2, and GT3; and a second strain of Lactobacillus crispatus having a different expression of GT1, GT2, and GT3 as compared to the first strain. The isolated mixture can be used in the treatment or prevention of bacterial vaginosis, preferably after treatment with antibiotic.

Description

[0001] P36697PC00 / MJO Mixture of Lactobacillus crispatus strains Technical fieldThe present invention relates to a formulation comprising Lactobacillus crispatus for use inthe treatment of bacterial vaginosis. Background of the inventionVaginal microbiota compositions that are dominated by Lactobacillus crispatus are associatedwith vaginal health, whereas a vaginal microbiota composition consisting of diverseanaerobes—commonly referred to as vaginal dysbiosis—has been shown to increase awoman’s odds for developing bacterial vaginosis (BV), acquiring sexually transmittedinfection, including HIV, and having adverse pregnancy outcomes.The application of human vaginal L. crispatus isolates as therapeutic agents to treat dysbiosismay have much potential, but currently, there are still many gaps in our knowledgeconcerning the importance of specific physiological properties of L. crispatus for a sustaineddomination on the mucosal surface of the vagina.Currently, it is unclear whether traits pertaining to in vivo dominance are shared by allstrains (i.e. traits encoded on the core genome) or only by a subset of strains (i.e. traitsencoded on the accessory genome) of L. crispatus. For example, both women without andwith (a short or long-term state of) vaginal dysbiosis can be colonized withL. crispatus, and we do not yet fully understand why in some women L. crispatus dominatesand in others it does not.The following bacterial traits have previously been considered to be of importance forL. crispatus to successfully dominate the vaginal mucosa:(1) the formation of an extracellular matrix (biofilm) on the vaginal mucosal surface,(2) the production of antimicrobials such as lactic acid, bacteriocins, and H2O2 that inhibit thegrowth and / or adhesion of urogenital pathogens,(3) efficient utilization of available nutrients—particularly glycogen, as this is the main carbonsource in the vaginal lumen, and(4) the modulation of host-immunogenic responses.In addition, a L. crispatus-dominated vaginal microbiome is associated with an anti-inflammatory vaginal cytokine profile and immune evasion is likely a crucial (but poorlystudied) factor that allows L. crispatus to dominate the vaginal niche. A proposed underlyingmechanism is that L. crispatus produces immunomodulatory molecules, but L. crispatus mayalso accomplish immune modulation by alternating its cell surface glycosylation, as has beensuggested for gut commensals.Taken together, there is a clear need to unravel the properties of more human (clinical)L. crispatus isolates to fully appreciate the diversity within this species. It is an objective of thepresent invention to provide for an L. crispatus formulation that can be successfully used in the prevention or treatment of bacterial vaginosis, in particular an L. crispatus formulationwhich has a high response rate across different patients with bacterial vaginosis.Summary of the invention The present disclosure meets one or more of the above-mentioned objectives by providinga multi-strain Lactobacillus crispatus formulation. The strains comprised in the formulationpreferably have a variation of surface-associated polysaccharides mediating bacteriophageresistance and host immune system evasion.The surface of most bacteria is coated with polysaccharides of variable structures, including cell-wall polysaccharides and lipopolysaccharides, which are crucial for specific interactionsof bacteria with their environment. A high degree of structural diversity is typically observed inthese polysaccharides among bacterial strains of the same species, which is considered to bea consequence of environmental and biological pressures and which results from coevolutionof bacteria with their host and their infecting bacteriophages. The chemical diversity of bacterial polysaccharides is mirrored by the genetic diversity of the loci on the genome encoding the components of their biosynthesis.In agreement with this notion, the present inventors found in a comparative genomics studyon a new collection of over 30 human vaginal Lactobacillus crispatus isolates, a high inter- strain diversity in a gene cluster encoding a cell-wall anchored polysaccharide pellicle (PSP). The cluster may include a set of at least two conserved glycosyltransferases (GTA, GTB) anda variable glycosyltransferase comprising three fragments (GT1, GT2, GT3), all presumablyinvolved in the glycosylation of the cell wall. The inventors identified naturally occurring strains with various combinations of these glycosyltransferase fragments, including (i) none,(ii) GT1, GT2, (iii) GT1, GT3 or (iv) GT1, GT2, GT3 fragments. Importantly, the inventors havefound that strains with all three glycosyltransferase fragments are significantly more presentin Lactobacillus crispatus strains isolated from a dysbiotic vaginal microbiota, which containsrelatively low levels of lactobacilli. The dysbiotic vaginal microbiota is known to be associated with a less protective environment and adverse reproductive health outcomes. The inventorsconsider that a gene cluster including the two conserved and variable fragmentedglycosyltransferases is involved in the biosynthesis of a cell-wall anchored polysaccharide pellicle (PSP) and governs bacteriophage sensitivity, as the extracellular polysaccharides of the PSP are known to act as bacteriophage receptors. Various surface-associated glycoconjugates have been identified in bacteria that promote survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. However, the presence of these glycoconjugates (PSP) also leads to bacteriophage sensitivity. The present inventors considered that a mixture of L. crispatus strains with natural variation in the presence and polysaccharide composition of the PSPwould facilitate bacteriophage resistance and expand the number of niches enhancing theefficacy of the probiotic formulation considering the inter and intra individual (temporal) variation in the conditions of the vagina. It should be noted that the dysbiotic environment is in many ways different form the eubiotic or Lactobacillus-dominated environment, most probably demanding different surface-associated properties (in particular the PSP) of the colonizing Lactobacillus crispatus. These differences include an (i) activated immune system (inflamed state of the host), (ii) relatively high shedding rate of host epithelial cells, (iii) higher pH ~ 4.5, and a highly competitive microbial environment (high bacterial load and high diversity). Single-strain probiotic formulations of bacterial species show limited efficacy becausebacterial strains have evolved in a specific environment in the host – with particular referenceto the variation in the bacterial cell wall surface mediating bacteriophage resistance and the colonization of multiple niches. Although multi-strain probiotics are very common (i.e. a formulation containing multiple species represented by the same number of strains) , multistrain probiotics of a single species remain underexplored. For example, the recent study onlactin-V (N Engl J Med.2020 May 14;382(20):1906-191) was carried out with a formulation ofa single strain of L. crispatus containing a gene cluster with all GT1, GT2, GT3 fragments,making this strain highly sensitive to bacteriophage attack. Efficacy of probiotics may improveand the number of non-responders may reduce by the use of multi-strain formulation, inparticular in case of Lactobacillus crispatus with natural variation of surface-associatedpolysaccharides mediating bacteriophage resistance (and occupation of multiple niches by multiple strains). Detailed description of the disclosureThe present disclosure relates to an (isolated) mixture of strains of Lactobacillus crispatus,wherein the mixture comprises at least two strains of Lactobacillus crispatus that differ fromeach other with respect to the presence or absence of glycosyltransferase (GT) 1, GT2, and / or GT3. In particular, the mixture according to the disclosure may comprise strains that have differentexpression of surface-associated polysaccharides. The expression of surface-associatedpolysaccharides can be assessed by determining if the strains have a different cell envelopethickness, e.g. by transmission electron microscopy (TEM, see protocol in the Experimentalsection). Cell envelope thickness is indicative for the presence of a polysaccharide pellicle(cell envelope thickness ≥ 65 nm) or absence of a polysaccharide pellicle (cell envelopethickness < 65 nm). The strains thus preferable comprise at least one strain having a cellenvelope thickness ≥ 65 and at least one strain having a cell envelope thickness < 65 nm.The cell envelope can be regarded as a combination of the cell membrane, cell wall, andouter membrane, if present. Usually, this envelope is a characteristic of prokaryotes likebacteria. The cell envelope provides structural integrity to the cell.The inventors envisage that such a mixture can treat a condition of vaginal dysbiosis withhigh response rates amongst patients and leads to recovery of the local microbiota balance. Lactobacillus crispatus as referred to herein is a well-known bacterial species, and preferably has a 16S rRNA gene sequence with at least 95, 96, 97, 98, 99, or 100% sequence identity toSEQ ID NO:8 or the 16S rRNA gene sequence of the type strain of Lactobacillus crispatus,deposited under DSM 20584. In a particularly preferred embodiment, the (isolated) mixture may comprise- a first strain of Lactobacillus crispatus having expression of (or comprising) one or more ofglycosyltransferase (GT) 1, GT2, and GT3; and- a second strain of Lactobacillus crispatus having a different expression of GT1, GT2, andGT3 as compared to the first strain. Preferably the first strain does not have expression ofGT1 while optionally (or also not) expressing GT2 and / or GT3; and / or the second strain has expression of GT1 and GT2, GT1 and GT3, or GT1, GT2, and GT3. In addition or alternatively, the (isolated) mixture comprises- a first strain of Lactobacillus crispatus not comprising (not having expression of) of GT1while optionally comprising (having expression of) GT2 and / or GT3; and- a second strain of Lactobacillus crispatus comprising (having expression of) at least GT1and optionally comprising (having expression of) GT2 and / or GT3.In addition or alternatively, the (isolated) mixture comprises- a first strain of Lactobacillus crispatus comprising (having expression of) at most one ofglycosyltransferase (GT) 1, GT2, and GT3, preferably none of GT1, GT2, and GT3; and- a second strain of Lactobacillus crispatus comprising (having expression of) at least two ofGT1, GT2, and GT3, preferably at least GT1 and one of GT2 and GT3, more preferably all of GT1, GT2, and GT3. The term “isolated” means that the mixture is not in its natural environment (e.g. notcomprised in a vagina and / or not comprised in (natural) vaginal microbiota) and / or that themixture is be produced by an ex vivo process). It is preferred that at least one and preferablyall of the strains in the mixture are viable and / or able to survive and / or grow in the (human)vagina. Ther term “expression” preferably refers to presence (yes or no) of the recitedelement, e.g. GT1, GT2, and / or GT3. “Different” expression thus may refer to difference in the presence( / absence) of the respective recited element, e.g. GT1, GT2 and / or GT3. For example, the (isolated) mixture comprises- a first strain of Lactobacillus crispatus comprising (having expression of) none ofglycosyltransferase (GT) 1, GT2, and GT3;- a second strain of Lactobacillus crispatus comprising (having expression of) GT1 and GT2while not comprising (having expression of) GT3;- a third strain of Lactobacillus crispatus comprising (having expression of) GT1 and GT3while not comprising (having expression of) GT2; and / or- a fourth strain of Lactobacillus crispatus comprising (having expression of) all of GT1, GT2,and GT3.Similarly, provided is for an (isolated) mixture of strains of Lactobacillus crispatus, wherein- the first strain of Lactobacillus crispatus comprises (has expression of) none ofglycosyltransferase (GT) 1, GT2, and GT3;- the second strain of Lactobacillus crispatus comprises (has expression of) all of GT1, GT2,and GT3; and / or- a third strain of Lactobacillus crispatus is comprised which comprises (has expression of)GT1 and GT2 while not comprising GT3. In any of the strains of the mixture, GT1, GT2 and / or GT3 (if present) are preferably comprised in a gene cluster (encoding a cell-wall anchored polysaccharide pellicle (PSP))which may further preferably comprise glycosyltranferase A (GTA) and / or GTB. The clusterpreferably further comprises flippase, and / or uridine diphospho (UDP)-galactopyranose mutase. In addition (or alternatively), the cluster may comprise oligosaccharide repeat unit polymerase Wzy (Domain: Transmembrane & Transmembrane helix), DUF4422 domain-containing protein (Molecular functions: Glycosyltransferase & Transferase), putative sugar transferase EpsL(Molecular function: Transferase, Biological process: Exopolysaccharide synthesis), Tyrosine-protein phosphatase (Molecular function: Hydrolase & Protein phosphatase), Tyrosine-protein kinase CpsD (Molecular function: Kinase, Transferase & Tyrosine-protein kinase, Biological process: Capsule biogenesis / degradation & Exopolysaccharide synthesis), and / or Capsular polysaccharide biosynthesis protein CpsC (Biological process: Capsule biogenesis / degradation & Exopolysaccharide synthesis).The gene cluster may encode a (cell-wall anchored) polysaccharide pellicle (PSP).Hence, strains comprising (or having expression of) any one of GT1, GT2, and / or GT3 (andGTA and / or GTB and / or flippase and / or UDP-gal) may have a (cell-wall anchored)polysaccharide pellicle (PSP) and / or may have a cell envelope thickness of more than 61, 62,63, 64, 65, 66, 67, 68, 69 or 70 nm. Conversely, strains not comprising (or having expressionof) any one of GT1, GT2, and / or GT3 (and GTA and / or GTB and / or flippase and / or UDP-gal)may not have a (cell-wall anchored) polysaccharide pellicle (PSP) and / or may have a cellenvelope thickness of less than 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nm. Accordingly, also provided is for an (isolated) mixture comprising- a first strain of Lactobacillus crispatus having a (cell-wall anchored) polysaccharide pellicle(PSP) and / or having a cell envelope thickness of more than 61, 62, 63, 64, 65, 66, 67, 68, 69or 70 nm;- a second strain of Lactobacillus crispatus not having a (cell-wall anchored) polysaccharidepellicle (PSP) and / or having a cell envelope thickness of less than 61, 62, 63, 64, 65, 66, 67,68, 69 or 70 nm. Cell envelope thickness can be measured by transmission electron microscopy (TEM), e.g. with the protocol as disclosed in the experimental section. In addition, strains comprising (having expression of) any one of GT1, GT2, and / or GT3 (andGTA and / or GTB and / or flippase and / or UDP-gal) may have better immune evasioncapabilities as compared to a strains not comprising any one of GT1, GT2, and / or GT3 (andGTA and / or GTB and / or flippase and / or UDP-gal). In particular, strains comprising any one ofGT1, GT2, and / or GT3 (and GTA and / or GTB and / or flippase and / or UDP-gal) may be lessprone to be killed by CD4+ T cells isolated from vaginal swap as compared to a strains notcomprising any one of GT1, GT2, and / or GT3 (and GTA and / or GTB and / or flippase and / orUDP-gal).In addition or alternatively, strains not comprising (having expression of) any one of GT1,GT2, and / or GT3 (and GTA and / or GTB and / or flippase and / or UDP-gal) may be moreresistant to infection by bacteriophages as compared to a strains comprising any one of GT1,GT2, and / or GT3 (and GTA and / or GTB and / or flippase and / or UDP-gal).Preferably, the (isolated) mixture according to the disclosure comprises 3, 4, 5, 6, 7, 8 ormore strains of Lactobacillus crispatus, wherein at least 2, 3, 4 or even each of the strainshas different expression of surface-associated polysaccharides (e.g. different expressionprofile for GT1, GT2, and / or GT3). In order to be effective it is preferred to have more thantwo strains in the mixture and particularly good results have been obtained when using four strains or more.In a particularly preferred embodiment, the (isolated) mixture according to the disclosurecomprises:- a first strain of Lactobacillus crispatus comprising (having expression of) none of GT1 andGT2, and none of GTA and GTB and none of flippase, UDP-gal and GT3, and none of oligosaccharide repeat unit polymerase Wzy, DUF4422 domain-containing protein, putative sugar transferase EpsL, Tyrosine-protein phosphatase, Tyrosine-protein kinase CpsD, and Capsular polysaccharide biosynthesis protein CpsC;- a second strain of Lactobacillus crispatus comprising (having expression of) GT1 and GT2,and GTA and GTB, and oligosaccharide repeat unit polymerase Wzy, DUF4422 domain- containing protein, putative sugar transferase EpsL, Tyrosine-protein phosphatase, Tyrosine-protein kinase CpsD, and Capsular polysaccharide biosynthesis protein CpsC; and / or- a third strain of Lactobacillus crispatus comprising (having expression of) GT1 and GT2,GTA and GTB, and oligosaccharide repeat unit polymerase Wzy, DUF4422 domain- containing protein, putative sugar transferase EpsL, Tyrosine-protein phosphatase, Tyrosine- protein kinase CpsD, and Capsular polysaccharide biosynthesis protein CpsC, and flippase, UDP-gal and GT3. GT1 is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:1. GT2 is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:2. GT3 is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:3. Flippase is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:4. UDP-galactopyranose mutase is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:5. GTA is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:6. GTB is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:7. In addition or alternatively, GT1 preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:9. In addition or alternatively, GT2 preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:10. In addition or alternatively, GT3 preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:11. In addition or alternatively, flippase preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:12. In addition or alternatively, UDP-galactopyranose mutase preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:13.In addition or alternatively, GTA preferably comprises an amino acid sequence having at least50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:14.In addition or alternatively, GTB preferably comprises an amino acid sequence having at least50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:15. In addition or alternatively, Oligosaccharide repeat unit polymerase Wzy is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:16. In addition or alternatively, DUF4422 domain-containing protein is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:17. In addition or alternatively, Putative sugar transferase EpsL is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:18. In addition or alternatively, Tyrosine-protein phosphatase is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:19. In addition or alternatively, Tyrosine-protein kinase CpsD is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:20. In addition or alternatively, Capsular polysaccharide biosynthesis protein CpsC is preferably encoded by a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:21. In addition or alternatively, Oligosaccharide repeat unit polymerase Wzy preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:22. In addition or alternatively, DUF4422 domain-containing protein preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:23. In addition or alternatively, putative sugar transferase EpsL preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:24. In addition or alternatively, tyrosine-protein phosphatase preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:25. In addition or alternatively, tyrosine-protein kinase CpsD preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:26. In addition or alternatively, capsular polysaccharide biosynthesis protein CpsC preferably comprises an amino acid sequence having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:27. In a preferred embodiment, at least one or all of the strains in the mixture according to the disclosure are capable of growth on glycogen and / or comprise a pullulanase type I gene. The inventors observed that strains from Dysbiotic Vaginal Microbiota (DVM) were more likely to carry the three gene fragments of a single glycosyltransferase (GT) than strains isolated from Lactobacillus-dominated Vaginal Microbiota (LVM). GTs are enzymes that are involvedin the transfer of a sugar moiety to a substrate and are thus important for synthesis ofglycoconjugates like exopolysaccharides, glycoproteins, and glycosylated teichoic acids. The first fragment (GT1) of typically 510 bp may contain the true GT fold domain and is thus likely involved in the catalytic activity of the GT. The second (GT2) and third (GT3) fragments aregenerally considerably shorter, respectively typically 228 and typically 328 bp. The presentinventors hypothesize that these latter two fragments play a role in steering the specificactivity of the GT (e.g., towards donor or substrate specificity). This might point towards L.crispatus harnessing its genetic potential to change its surface glycome. Such a process istermed phase variation and allows bacteria to rapidly adapt and diversify their surface glycans, resulting in an evolutionary advantage in the arms race between the immune system and invading bacteria. Modulation of the surface glycome by phase variation of the GT coding sequence is a common immune evasion strategy, which has been extensively studied in pathogenic bacteria like Campylobacter jejuni, but could be utilized by commensals as well. The presentinventors hypothesize that L. crispatus in Dysbiotic Vaginal Microbiota (DVM) exploits thisgenetic variation to allow for (a higher) variation in cell wall glycoconjugates providing amechanism for L. crispatus to persist at relatively low levels in DVM and remain stealth fromthe immune system. The presence of the fragmented GT gene may be a selective advantagefor L. crispatus under dysbiotic conditions.The strains of L. crispatus as proposed herein can easily be obtained from freshly obtainedsamples. In particular, the strains of L. crispatus according to the present disclosure can beisolated from vaginal swab from at least one woman, or preferably from at least one womanwith bacterial vaginosis and at least one woman without bacterial vaginosis. For this, vaginalswabs are plated on e.g. tryptic soy agar supplemented with 5% sheep serum and 0.25%lactic acid and pH set to 5.5 with acetic acid, and incubated under a microaerobic atmosphere(e.g. using an Anoxomat; Mart Microbiology B.V., The Netherlands) at 37 °C for 48–72 h.Candidate Lactobacillus spp. strains can be selected based on colony morphology (white,small, smooth, circular, opaque colonies), and single colonies are subjected to sequencing fordetermining presence of absence of GT1, GT2, and / or GT3 (and / or GTA / GTB).Alternatively, the strains of L. crispatus according to the present disclosure can be obtainedfrom the bacteria collection at Westerdijk Fungal Biodiversity Institute (Uppsalalaan 8; 3584 CT, Utrecht; The Netherlands) under number NCCB 100711, NCCB 100713, NCCB 100714, NCCB 100715, NCCB 100716, NCCB 100717, NCCB 100718, NCCB 100719, NCCB 100720, NCCB 100721, NCCB 100722, NCCB 100723, NCCB 100724, NCCB 100725, NCCB 100726, NCCB 100727, NCCB 100728, NCCB 100729, NCCB 100730, and / or NCCB 100731. For the production of sufficient bacteria to produce the mixture of the present disclosure, cultures of the strains as isolated from one woman with bacterial vaginosis and / or at least onewoman without bacterial vaginosis (or as obtained from deposits) may be produced byculturing the bacteria using methods well known in the art.For example, Lactobacillus - MRS Agar may be used (introduced by De Man, J. et al., J. Appl.Bact.23: 130-135, I960)) (LMRS AGAR) which is an enriched selective medium intended forthe isolation and cultivation of Lactobacillus found in clinical specimens and dairy and foodproducts. The basis of this medium consists of peptones yeast extract and glucose. This medium is supplemented with sorbitan monooleate complex (a source for fatty acids) and magnesium for additional growth requirements. Sodium acetate and ammonium citrate areadded to inhibit normal flora, such as gram- negative bacteria, oral flora and fungi. With theaddition of both of these inhibiting agents, the medium has been shown to selectively improvethe growth of Lactobacillus. The pH is adjusted to 6.3 - 6.7 to favor the growth ofLactobacillus. This medium is prepared, dispensed, stored and packaged under oxygen-free conditions to prevent the formation of oxidized products prior to use.Alternatively, for the culture enrichment of Lactobacilli of the vaginal flora a so-called CDMmedium as described in Geshnizgani A.M., and Onderdonk A.B., (J Clin Microbiol.1992 May;30(5): 1323-6) may be used. In this study, a chemically defined medium that simulatesfemale genital tract secretions was developed for the growth of the vaginal microflora.Qualitative and quantitative studies of the growth of predominant components of the vaginal microflora indicated that all vaginal isolates tested were able to grow in this defined medium.The Lactobacillus crispatus strains to be used in the (isolated) mixture or compositionaccording to the present disclosure are preferably capable of producing lactic acid. In additionor alternatively, the strains are capable of vaginal colonization. In addition or alternatively, thestrains are capable of maintaining a pH of below 4.5 in the vagina.The present disclosure further provides for composition comprising the (isolated) mixtureaccording to the disclosure and glycogen, lactose and / or a carbohydrate; preferably a carbohydrate mixture. In addition or alternatively, the composition may comprise poly- unsaturated fatty acids; preferably the unsaturated fatty acids are selected from the group of omega-3 and omega-6 fatty acids.The present disclosure also provides for a (probiotic) composition comprising a mixtureaccording to the disclosure and optionally lactose. The lactose in this composition may provide a substrate for (at least part of) the bacteria in the mixture. Further, the lactose in such a composition would also have a beneficial effect on the vaginal microbiota as has been demonstrated previously and thus can be used as an additional medicament for the prevention or treatment of bacterial vaginosis. The composition may also contain other carbohydrates that can serve as substrate for the bacteria.In addition or alternatively, the (probiotic) composition according to the present disclosuremay contain glycogen. Although, it has been thought that glycogen cannot be used as a substrate by L. crispatus, because it apparently lacks the enzymatic machinery for glycogen degradation (Ojala, T. et al., BMC Genomics 15: 1070, 2014), the present inventors have shown previously that about 50% of the strains that can be isolated from a healthy vaginal microbiota are able to use glycogen. Also glycogen has been proposed for use in the prevention and treatment of bacterial vaginosis. Accordingly, the glycogen in the composition has similar advantages as lactose. It is further preferred that the (probiotic) composition further comprises additional carbohydrates that can function as substrate for the bacteria. In particular, the (probiotic) composition, which comprises the (isolated) mixture according to according to the present disclosure may comprise lactose, and preferably additionally comprised a carbohydrate mixture comprising two or more carbohydrates selected from the group of glycogen, galactose, glucose, lactulose, mannose, n-acetylglucosamine, cellobiose, maltose, mannose, mannitol, raffinose, trehalose, saccharose, starch, amygdalin, arbutin, salicin and esculin.The term "(probiotic) composition" as used herein refers to a composition comprising one ormore (probiotic) organisms and one or more acceptable excipients suitable for application toa mammal, preferably a human. It will be appreciated that acceptable excipients will be wellknown to the person skilled in the art of (probiotic) composition preparation.Examples of such acceptable excipients for oral administration of the probiotic composition include: sugars such as sucrose, isomerized sugar, glucose, fructose, maltose, mannose, sorbose, rhamnose, arabinose, palatinose, trehalose, lactose, cellobiose, melibiose and xylose; sugar alcohols such as sorbitol, mannitol, xylitol, inositol, erythritol, lactitol, palatinol,reduced glutinous starch syrup and reduced glutinous maltose syrup; polysaccharides asmaltodextrins, inulins, starches like maize starch, rice starch, potato starch and wheat starch, glucosides like amygdalin, erbutin, esculin, salicin, N-acetylglucosamine and the like, emulsifiers such as sucrose esters of fatty acid, glycerin esters of fatty acid and lecithin;thickeners (stabilizers) such as carrageenan, xanthan gum, guar gum, pectin and locust beangum; acidifiers such as citric acid, lactic acid and malic acid; fruit juices such as lemon juice, orange juice and berry juice; vitamins such as vitamin A, vitamin B, vitamin C, vitamin D and vitamin E; and minerals such as calcium, iron, manganese and zinc.For topical administration of a (probiotic) composition the composition can additionally oralternatively comprise polymers, such as natural polymers including proteins such as zein, modified zein, casein, gelatin, gluten, serum albumin, and collagen, polysaccharides such as cellulose, dextrans, and polyhyaluronic acid, or synthetic polymers includingpolyphosphazenes, poly(vinyl alcohols), polyamides, polycarbonates, polyacrylates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and copolymers thereof. Examples of suitablepolyacrylates include poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate) and poly(octadecyl acrylate). Synthetically modified natural polymers include cellulose derivatives such as alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocelluloses. Examples ofsuitable cellulose derivatives include methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate and cellulose sulfate sodium salt.Also usable are degradable polymers, such as polysaccharides such as alginate, dextran, cellulose, collagen, and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), and proteins such as albumin, zein and copolymers and blends thereof, alone or incombination with synthetic polymers. It is also possible that the (probiotic) composition isadministered in the form of a hydrogel. Suitable hydrogels can be formed from synthetic polymers such as polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylates, poly (ethylene terephthalate), polyvinyl acetate), and copolymersand blends thereof, as well as natural polymers such as cellulose and alginate, as described above. In a particularly preferred embodiment, the (isolated) mixture or composition according to thepresent disclosure is for use in therapy. As will be clear, the (isolated) mixture according tothe disclosure or a composition containing said mixture can be used as a medicament in the treatment or prevention of bacterial vaginosis. The prevention or treatment according to the disclosure may involve administering the (isolated) mixture according to the present disclosure to a subject in need thereof, preferably a female human.Bacterial vaginosis may refer to dysbiosis in the vagina which is defined as a state in whichthe microbiota produce harmful effects via (a) qualitative and quantitative changes in the content or amount of the microbiota itself, (b) changes in their metabolic activities; and / or (c) changes in their local distribution. Specifically, dysbiosis in the vagina is defined as an aberration of the healthy state. A healthy state is defined in this case as a condition with a relatively low susceptibility to sexually transmitted diseases. It has been widely accepted thatthe activity of Lactobacillus spp. contributes to maintain this low susceptibility through the protection of the vaginal environment against pathogens by the production of lactic acid,resulting in a low pH. Hence, vaginal dysbiosis is, amongst others, characterized by thepresence of a relative high pH, e.g. a pH of more than 4, 4.5, 5 (which can be measured by holding a piece of pH paper against the wall of the vagina for a 2-5 seconds, or by any other means available in the art). A special form of medical use according to the present disclosure is the use in combination or after treatment with an antibiotic. The current most abundant treatment for bacterial vaginosis is the treatment with antibiotics. However, recurrence of bacterial vaginosis after completionof such a treatment is a common problem: symptomatic bacterial vaginosis persists or recursat 3 months in up to 50% to 70% of patients, with long-term recurrence approaching 85%. The application of the present mixture or composition together with or after antibiotictreatment will greatly minimize the recurrence of the symptoms of bacterial vaginosis and willlead to an improved treatment and / or prevention of bacterial vaginosis.Accordingly, the present disclosure also provides for a method for the treatment or preventionof bacterial vaginosis, preferably to decrease recurrence of bacterial vaginosis after treatmentwith antibiotics, comprising (topical) administration of a mixture according to the disclosure.The prevention or treatment of bacterial vaginosis may comprise first treatment with an antibiotic compound, followed by administration of a mixture according to the disclosure.Hence, use of a mixture or composition according to the disclosure is foreseen for themanufacture of a medicament for topical treatment of bacterial vaginosis, preferably to decrease recurrence of bacterial vaginosis after treatment with antibiotics. The type of antibiotic treatment will not actually influence the effectivity of the application ofthe mixture, although - of course - when the mixture is provided together with the antibiotictreatment it will take longer for the Lactobacillus strains in the mixture or composition tocolonize the vagina. The antibiotic used may be the antibiotics that are used as the standard treatment for bacterial vaginosis, such as metronidazole, clindamycine, amoxicillin or imidazole, but other antibiotics may also be used. The antibiotic treatment generally is provided as an oral administration, although metronidazole may also be given as a topically applicable gel.Also the (isolated) mixture according to the present disclosure may be used topically. In fact,the (isolated) mixture of the present disclosure or the composition comprising said mixture ispreferably given through topical application. For this, it may be in the form of a gel or creme,but preferably it will be provided in the form of a vaginal capsule or vaginal tablet. The skilledperson will know how to formulate these topical application formulations. Preferably, the oral or topical formulation comprises a stabilizer, such as maltodextrin; more preferably, the formulation comprises at least 50, 60, 70, 80, 90 wt.% of a stabilizer, wt.% calculated on thetotal weight of the formulation. The disclosure further comprises the use of the mixture orcomposition according to the disclosure for the prevention or treatment or the prevention ofrecurrence of bacterial vaginosis as indicated above. Also provided in the present disclosureis the use of the mixture or composition according to the disclosure for the preparation of amedicament for the prevention or treatment or the prevention of recurrence of bacterial vaginosis. Compositions for topical administration may be applied in the form of cremes or ointments. As such they will ideally comprise an oily substance originating from vegetable, marine or animal sources. Suitable liquid oil includes saturated, unsaturated or polyunsaturated oils. By way of example, the unsaturated oil may be olive oil, corn oil, soybean oil, canola oil, cottonseed oil,coconut oil, sesame oil, sunflower oil, borage seed oil, syzigium aromaticum oil, hempseedoil, herring oil, cod-liver oil, salmon oil, flaxseed oil, wheat germ oil, evening primrose oils or mixtures thereof, in any proportion. These cremes or ointments may further comprise poly- unsaturated fatty acids. In one or more embodiments, said unsaturated fatty acids are selected from the group of omega-3 and omega-6 fatty acids. Examples of such polyunsaturated fatty acids are linoleic and linolenic acid, gamma-linoleic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Such unsaturated fatty acids are known for their skin-conditioning effect, which contribute to the therapeutic benefit of the composition. Thus, the composition can include at least 6% of an oil selected from omega-3 oil, omega-6 oil, and mixtures thereof. Also usable are the essential oils, which are also considered therapeutically active oils, which contain active biologically occurring molecules and, upon topical application, exert a therapeutic effect, which is conceivably synergistic to the beneficial effect of the probiotic mixture in the composition. Another class of therapeutically active oils includes liquid hydrophobic plant-derived oils, which are known to possess therapeutic benefits when applied topically. Silicone oils also may be used and are desirable due to their known skin protective and occlusive properties. Suitable silicone oils include non-volatile silicones, such as polyalkyl siloxanes, polyaryl siloxanes, polyalkylaryl siloxanes and polyether siloxane copolymers, polydimethylsiloxanes (dimethicones) and poly(dimethylsiloxane)-(diphenyl-siloxane) copolymers. These are chosen from cyclic or linear polydimethylsiloxanes containing from about 3 to about 9, preferably from about 4 to about 5, silicon atoms. Volatile silicones such as cyclomethicones can also be used. Silicone oils are also considered therapeutically active oils, due to their barrier retaining and protective properties.The present disclosure also provides for a (vaginal) capsule or (vaginal) tablet comprising amixture according to the disclosure. For vaginal application the (probiotic) composition maybe in the form of a vaginal capsule or vaginal tablet. The capsule preferably is a (hard) shellpharmaceutical capsule. The capsule may comprise a body and cap and may comprise a fillformulation containing the (probiotic) composition. Capsules suitable for use according to thedisclosure include, without limitation NPcapsCR' available from Capsugel which containpullulan, carageenan and potassium chloride, as well as capsules described in US Patent No. 8, 105,625 and US Patent Application Publication No.2005 / 0249676. In one aspect, capsulesfor use according to the disclosure comprise pullulan with a molecular weight between about50 to 500 kDa, between 100 to 400 kDa, between about 150 to 300 kDa and preferably between about 180 and 250 kDa. In another aspect, capsules for use according to thedisclosure comprise pullulan from about 50% to about 100% by weight (unfilled capsule). Inother aspects, the capsules comprise about 60 to 90 or 70 to 90, or 80 to 90 wt % pullulan.Preferably the capsules comprise about 85 to 90 wt % pullulan. Capsules for use according tothe disclosure may further comprise (in addition to pullulan) one or more gelling agents (e.g.hydrocolloids or polysaccharides such as alginates, agar gum, guar gum, carob,carrageenan, tara gum, gum arabic, pectin, xanthan and the like); salts comprising cations such as K , Li , Na , NH4 , Ca , Mg ; and / or surfactants such as sodium lauryl sulphate, dioctyl sodium sulfosuccinate, benzalkonium chloride, benzethonium chloride, cetrimide, fatty acid sugar esters, glycerl monooleate, polyoxyethylene sorbitan fatty acid esters, polyvinylalcohol, dimethylpolysiloxan, sorbitan esters or lecithin, as e.g. described in US Patent Application Publication No.2005 / 0249676.Capsules for use according to the disclosure may further comprise one or more plasticizingagents (e.g. glycerol, propylene glycol, polyvinyl alcohol, sorbitol, maltitol and the like); dissolution enhancing agents (e.g. maltose, lactose, sorbitol, mannitol, xylitol, maltitol and the like); strengthening agents (e.g. polydextrose, cellulose, maltodextrin, gelatin, gums and the like); colorants, and / or opacifiers as e.g. described in US Patent No.8, 105,625. In a preferred embodiment, the capsule comprises pullulan in an amount of 85% to 90% by weight, potassium chloride in an amount of 1.0% to 1.5% by weight, carrageenan in an amount of 0.1 % to 0.4% by weight, one or more surfactants in an amount of 0.1% to 0.2% byweight and water in an amount of 10% to 15% by weight. In a particularly preferredembodiment, the capsule comprises pullulan in an amount of 86.3% by weight, potassium chloride in an amount of 1.32% by weight, carrageenan in an amount of 0.27% by weight, surfactants selected from sugar esters, sorbitan monolaurate and combinations thereof in an amount of 0.15% by weight and water in an amount of 12% by weight. General definitions As used herein, the term “identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest ordermatch is obtained. "Identity" per se has an art-recognized meaning and can be calculatedusing published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. For example NCBI Nucletide Blast with standard settings (blastn, https: / / blast.ncbi.nlm.nih.gov / ). Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403). As an illustration, by a nucleotide sequence or amino acid sequence having at least, for example, 95% "identity" to a reference sequence, it is intended that the nucleotide sequence or amino acid sequence is identical to the reference sequence except that there may be up to five point mutations per each 100 nucleotides or amino acids of the reference sequence. In other words, to obtain a nucleotide sequence or amino acid sequence being at least 95% identical to a reference sequence, up to 5% of the nucleotides or amino acids in the reference sequence may be deleted and / or substituted with another nucleotide or amino acid, and / or a number of nucleotides or amino acids up to 5% of the total nucleotides or amino acids in the reference sequence may be inserted into the reference sequence. Preferably, the sequence identity refers to the sequence identity over the entire length of the sequence. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids or nucleotides) are referred to. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". Sequences referred to: Nucleic acid sequencesGT1 - SEQ ID NO:1:ATGAAATCAGACATAACCGTAATTGTTCCAATGTATAATGTTGAAAAATACGTAAAAAAATGTTTTG AGTCATTAGAAAATCAGACATACAGTAATTTTGAAGTTTTGGCTATTAATGATGGATCGCCAGATAA TTCTAGTAAGTATGCGAAAGAATGTGCTGCGAAAGATAATAGAATTAAAGTAATTGATAAAGAAAAT GGTGGCTATGGTTCAGCGTTAGAGTTAGCTATAAATAGTATTAATTCTGATTACTTTTTGGTGTGTG ATCCTGATGATTGGTTGGAAGAAGATTGTTTAGAAGTACTTTATACATTTGCCAAAAATAATGACAC TGATATTGTAGTAGGGGATCGATTTGATGTGTATGCTGATGATGGAAGTGCTCATAAATGTTCTGT GAAACCAGGATATTTAGAAAGTATTGAACCTAAAAAAGTATATTCAGAAACACAGGATATACAGCT GTTTTTCTACCCAAAGTCAAGTATTATCAATGATTTAAGAAAATAAGT2 - SEQ ID NO:2:TTTCTTTTTTTCAAGTTTCACCGCATGCAAAACTTTATAAAACCCAATTGCTAAAAAATGTTAGTTTC CCTAAACACGTGAGTTATACGGATTTTTTGTTATATTTGGTTGCGTTGTCTAGAGCGAAGAAAGTAT CATATTATGATAAAGCCCTCGCTTTTTATTTACAAGATAGACCAGGAAATACAGCAACTGATATAAG AAAGTCTATAATTAATGATTATTTAAGT3 - SEQ ID NO:3:TTATTTAACCGTGTGGAAAAAAACTTTTGAAATAATCAACAATAATAATGTCGATAATAATGTCGATA ATAATGTCGATTTTTTAATGTATAGACTTTATATGCAACTACGACTTATATTGGCAGAATATAAGAG GGTGTCTATTAATGGTTTTAATAATAAGTATTGGAGTAATATAATGTCAGCAGTTTACGAATTACAA GATATAGAGATAAATACAGTCCCTTATTTTGAAAATAGCATGATAAAAAAAACGTTTTTTAAATTCTT TATGAATAGAAAAACAGCTAACTTTACTGCAAAAATGTATGTTACTCTAAAAAAATAGFLIPPASE – SEQ ID NO:4TTACTTTCCCAACTTCTCCCGAATCAACTTCTTAGCCTCATCCACAATAGGCGCTCTTAGCAGCATCACC ATAACACCGACAACACCAACAATAACTTCGAGTCCCATCATTAGCCAAGAATCTTTCAAGTGTGTATTCA TCCAAAAAACTGGCAAAAACATTACTACACCAGAGACCAAATACTTCCATAGGCTGTTAAATAGAGTCCT ATAGCTCAACAAACCACGAACCGCAATCAGTTGATACAAAGTAACACTAATTTCGGACAAAACAGTTGAC CACATGGCACCATTCAAACCATAAAAGTGAATCAACGGGAAATTTAAAATTATGTTTACTACGGCACCCA CTGTAACAGAAATTGTAAATTGCTTCTGATGGTGAATTGGTAATAGGTACTGCACGCCAAGTGCGTTACT CCAAGCAATCATCAAAATCACGATAGACTCAATCATCATTGCTGGACCTACTGGACCATAGCCGGGACCA TAATATTTTGGAGCTAATGTTAAGGAGATTGCTGCCAAGCCAAACATCATTGGTACAGCCATAGCCGATA CAAAATCAAAAGACTTATACAACATCTGATTTACCTTCTTCATGTCACCATGTGATACCGCATTGGCTAC ATGTGGCAACATGACTGTACCAGTTGCTGTCACTAAAGCCAAAATCAACTTCACTAAGCTATCAGAATAT TGATAGTAACCAGATGCTGTTTCATTAACCATGACGCCCAACATCGTACGATTCAATTGTACATAAACTT GAGTAGCTATTTGCGGTATGAAAAGCTCAACCATTGGTAAAAAGTGAATCCAAGGATTTAAACTCCTAAT CTTAACCTTAGGCAAATCTCGATAAATATCCGGCCACAAAGTTAAGTTACCTAGTAGAGTTGACAATGCC AATACTACAATATATAAAGTTACATCATAGGGTCCCTTGATAAAAACGAAGATCGCTATCATTGATACAA TCTTTACCAGTGAGTTCTTTAAAACCGTTACTTTAAAGTTTTCAACACCTTCATAAAACCAAGAAATGTC AAAAGCCACAGCAATTAGATTAAGCGACTGAGCCAACATATAATCAAACTGTCGAGTATAAAAAATAAGG AAGATTTCAAAAGCAATAATTGATACAAGAGTCATTATTGTTTTAACTATTTGAATCTCCCAAAAAGTTT TGGCCATTTTAGTTCGATTATCTCGAACATAGGCTATTTGCCGATTCCCATAATAACCAATTCCCATACT AGCAAATAAGATAAAGTATTGAATAATGGAATTTGTAAAAGCATTAGCACCTACACCTTCAGGTCGTAAT ACACGACTGACATATGCCGATGTAATAAGCGGAACTATTATTGCTAATACTTGATAACCTACATTATAGA GATAATTTCGAATGACCTTCATUDP-GALACTOPYRANOSE MUTASE – SEQ ID NO:5CTATTCACCAAATTCATTATTTACCGCATCGAGTGCAGCATCAATTACCTGATCCATATTGTAATACTTG TATTGACCAAGACGACCACCAAAGATTACCTTGTCTTGTTTCTTTGCTAATTCAGCATATTTAGCGTACA AACTATTATTTCTATCATTATTCACTGGGTAATATGGCTCGTCTCCACGCTTCCAATCCGCTGGATATTC CCGGGTAATAATCGTCTTATCTGGATCACCCTTACCGAATTCAAAATGCTTGTGTTCAATTATACGAGTG TAGGGGATTTCAGCTTCAGTATAGTTGATCACTGCGTTGCCTTGATAGTTGCCAACATTCTTTTCTTCAG TTTCAAAACGAAGTGAACGATATTCAAGTTCACCTAATTTATAGTCAAAGAACTTATCAATCATACCGGT GTAGACAACTTTATCATAGTTATTAAGATACTCTTCTTTATCATCAAAAAAATCAGTATTTAATTTAACT TCAATATTTGGATGATCAAGCATCTTTTCCACCATTTTTGTGTAACCACCAATAGGAATACCTTGGTAATCATCATTGAAGTAATTGTTATCGTAAATCAAACGCACTGGAAGACGCTTGATAATAAAAGCAGGCAATTCAGTACATGGTCTGCCCCACTGCTTTTCTGTATAACCCTTGATCAACTTTTCATAGATGTCACGTCCAATT AAAGAAATAGCTTGTTCCTCAAGGTTTTGTGGTTCCTTGCCTGCCATTTCTTGTCGTTGTTCATTGATCT TAGCCATAGCTTCTTGCGGAGTCCCTACTCCCCACATCTCGCTAAAAGTATTCATATTAAATGGCAGATT ATACATACGTCCTTTATAGTTGGCAACAGGACTATTAGTATAACGATTAAATTCAGCAAATTGTTGTACA TAATTCCAAACTTCTTTGTTTGAAGTATGAAAAATGTGAGCACCATATTGATGAACCTGAATCCCATCTA CTTCTTTAGTATAAATATTACCTGCAATATGATCTCTTTTTTCAATTACTGTAACTTTGTTTCCACGTTT TGCTACTTCATGAGCAAATACTGCTCCAAAAAGACCAGAACCAATTATTAGATAATTATTTTCCATGTA– SEQ ID NO:6ATGAGGAAACAGCAAGTTGCCGCTATTGTAGTAACATATAATAGAAAAAAGTTACTAAAAGAATGTATTA AAAGCTTATTGGAACAAACCGCAAAGCCAGATATATATATTATTGATAATAATAGTACAGATGGTACAGT TAATTATATTGCAAAATATATTGATGATAACAGTATAAACTATGTTAATACGGGGAAAAATCTTGGTGGT GCTGGTGGATTTCAGTATGGTATAAAGATGGTAGCTCAAAAGGATTATGATTTTGTTTGGATTATGGATG ATGATTGCATACCAACTAAAAATGCTTTGAATGAGTTTCTTAAGGCAGATATAAAACTAAATCATGATTA TGGTTTTTTGTCTAGTAAAACTTTGTGGACCGATAATACCTTGTGTACGATGAATATTCAGAGAAGGACA TTAACAAAAAATGTGTCTGATTTTTCATCTGAATTGATACCTGTTACTTTAGCTTCTTTTGTTTCCTTAT TTGTTCCAGTTTCAGTTATCAAAAAAGTAGGGTTACCAATTAAGGAATTCTTTATTTGGACTGATGATTG GGAATATACAAGACGAATTTCCTCAAAATATGAATGTTATGTTGTAACTAATAGTATTGTTGTACATAAA ACAAAAACTAATGTTGGTGCAAACATTGCAACAGATGTCCCAGAAAGAATAGAAAGATATAGATATTTGT ATAGGAATGATTTTTTCTTTTATAGAAAGGAAGGGCTAAAAGGATATGGATATGAATTTTTACGTTTAAA TGATCACATGATAAGGGTTTTGTTCAAAGCTAAAAATAATAAACGTAAAAGGTTAAAGATGATATTTAAA GGAACAATTGAGGGATTACGTTTTAATCCGTCTATAGAGTATATAAAATAAGTB– SEQ ID NO:7ATGAAAAAGATTTTAGAGGCGTTTGGTGAGCCAATTTTGTATGGCGGGCAAGAGGCTTTTGTATTTAGAA CAATTGAAAATATGAACAAAAAAGGTCTAAAATTCGATTTTTTAACACCATATTATGCTGATAATCCCGA CTATATTAGCTTTATTAAATCACTAAATTCTACGCTATACTCTTTTAATTTACCTTTTAATGTTGGAAAG AATAGATTTAATGTTGTAAATGCATATAAAAAAATTCTGCTAGATAATCAATATGATGTTGTGCATATAA ATAGTGGAAGTATATCTATACTTGCATTATTTACCTTGTATGCTAAAAAAGCAGGTGTGAAAAAAGTTATTGTGCATTCGCACATGTCAGGAAAAAATAAAAATGTAAAGCATGAAGTAATTAAAAAGATTTATGCACCAATCTTTAGTAAATATGCTGACGTTTTGGTTGCTCCAACAAAAAAAGCTGCATATTGGCAATTTTCAAAAA AAATTTTTAACAAAAAAGGAAGAATTTTGAAAAATGGAATTAACATACAGCAGTATGCATATAATATTGG GACTAGAGAACATTATCGTAACAAATTGAATATTTCAGATCATGAAATACTAATTGGGCATGTTGGAAGG CTATCACCTGAGAAAAATCAAATATTTTTAATAAAATTATTAAGTTATTTTATTAAAAATAATACTAAGG CTAAGCTGTTACTTATAGGAGCTGGTCCACAGGAAAAATCAATACGAATGAGTATTAAGCAAAATCGATT AGAGAAATATGTTCAATTAATAGGAAATGTAGATAATGTTGAAGATTATCTACAAGCAATGGATTTATTT ATTTTTCCTTCTGAATATGAAGGACTAGGAATTGCAAGTATTGAGGCTCAGGATGCTGGGCTTCCGGTAT TAGCTTCCACTAATGTTCCTCTAGATATTAAGGTTACTGATAACGTGATGTTTCTTGACTTAAATTTATC GCTTAAAGAGTGGTATAAGCAAAGTTTAAAGTTACTATCCAGGAAGTATGATCGAGCAGAAAATACAAAT TTTTTAAAAAAACAAGGATACGATATTAGAAATACTGCTGAAAAGTTGAAGAAAATTTATTTGGGGTAA16S rRNA - SEQ ID NO:8 (partial 16S rRNA gene sequence of Lactobacillus crispatus (typestrain), deposited under DSM 20584: gccggcgtgc ctaatacatg caagtcgagc gagcggaact aacagattta cttcggtaat 60 gacgttagga aagcgagcgg cggatgggtg agtaacacgt ggggaacctg ccccatagtc 120tgggatacca cttggaaaca ggtgctaata ccggataaga aagcagatcg catgatcagc 180 ttttaaaagg cggcgtaagc tgtcgctatg ggatggcccc gcggtgcatt agctagttgg 240 taaggtaaag gcttaccaag gcgatgatgc atagccgagt tgagagactg atcggccaca 300 ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat cttccacaat 360 ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa ggttttcgga tcgtaaagct 420ctgttgttgg tgaagaagga tagaggtagt aactggcctt tatttgacgg taatcaacca 480 gaaagtcacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 540 cggatttatt gggcgtaaag cgagcgcagg cggaagaata agtctgatgt gaaagccctc 600 ggcttaaccg aggaactgca tcggaaactg tttttcttga gtgcagaaga ggagagtgga 660 actccatgtg tagcggtgga atgcgtagat atatggaaga acaccagtgg cgaaggcggc 720 tctctggtct gcaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac 780 cctggtagtc catgccgtaa acgatgagtg ctaagtgttg ggaggtttcc gcctctcagt 840gctgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa 900 aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 960 agaaccttac caggtcttga catctagtgc catttgtaga gatacaaagt tcccttcggg 1020 gacgctaaga caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080 tcccgcaacg agcgcaaccc ttgttattag ttgccagcat taagttgggc actctaatga 1140gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcatcat gccccttatg 1200 acctgggcta cacacgtgct acaatgggca gtacaacgag aagcgagcct gcgaaggcaa 1260 gcgaatctct gaaagctgtt ctcagttcgg actgcagtct gcaactcgac tgcacgaagc 1320 tggaatcgct agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccatg ggagtctgca atgcccaaag ccggtggcct aaccttcggg 1440aaggagccgt ctaaggcagg gcagatgact ggggtgaagt cgtaacaagg tagccgtagg 1500 agaacctgcg gttgga 1516 Oligosaccharide repeat unit polymerase Wzy SEQ ID NO:16 ATGTTTAAAGAAATAAATTTAAGGAATCATTCATCCTTACACAAACTATTTTACAGTCTCAGATATGTATCTATTT ATATAGGCGTTTTTATTTTTATGTTTGGGCTACAGTTATATAGGCTAAATAGTTTTTCCTTATCTGGGTACACAA TTAAAGATGCTTTAATTCATAATAGATATTATGCGTATATGTTGTTAACGTTTCCGATGACGGAGGTGCTAGCG ACTGCTAAACGAAGAATGAAATTTATTAATAATATATATTATATGGGGGTTGCTATATTAATATTTAGATTTAGCGCGTGGTTTTTGTACAATAAATTACGTATGAATGTAGCTCCAGGTTATTTTGAAGTAATGGGATTTGCTTGGGCGAGAAATGGAGTAAGTAGATTACCAGGTACTTTTTTAGATAATTACGTATGGGTTATGGCTCTTTTCAAAATCA TCAAAGAAAAAAGTGTTGTAATTAAAACACGTAATATCTCAGTTATAGTATTATGCTTTTTATATGCATATATTGT GTATGACTCACGTTCACAACAAATAGCCTATTTATTAAGTTTAGTGGTATTCATACTTATCGTTAGTGATACTTT TAAAAGTAAAGCTCTAGATGTGGTAGGAGCTATAGCCATTAGTCCAATTATTTTTAGAGGAAATTACTTAAATAA ATTGGTTAATACTTTTTCAACTTCAAATGCAGACTATGGATCTAGTACTCAAATAAGAATGATGACATTAAATAC ATATCAAACTCTTTGGATAGAAAGAGGTGTATGGTGGGGATATGGTATATCAAACGATGGGAATTATTTTAATA TATTGGATACACATATGGTAAACCAATCAGATTTAGGTATATTATCTATGTTATTTCAATATGGAATAATAGGTT TTGCTATTTTTATATCGCCATTTGTAATGGGATTATATGTAAGCTTAAAAAAAGTCAAATTTGTTCAAAATAAGTT TTTGTTTTTACTAACTTGCACTACTGCATTAACTAGTATAATGTCACAAAATATGTATGATCCATTTAGATTTCTA ATAGTACCATTTTTATTAAGTTTTATTTCTGTAGTTAGCTTTATGAATAAAAAGGAGAATTAA DUF4422 domain-containing protein SEQ ID NO:17 TTGAAAATAGAAATTTTGGTTGCTGCGCATAAAGAGTTTCCTATGCCCAAAAAGGAAGGATATTTACCAGTATTAGTAGGCGCAGTAAAGAATTATAAACCAGGTATTTCCTATCAGCGTGATGATGATGGTGAAAATATATCCTCTAAAAATCCTAATTACAATGAACTAACTGCTGTTTATTGGGCTTGGAAGAATTTAAAAGATGTTGACGCTATTGGA TTAGTTCACTATAGAAGATTACTCTTTGAGAAAAGACCATATTCTTTAGATAATGTAATTAGTATTGAGAAAGTA AATCAGCTTTTACAAAAATATGATGTAATTTTACCTAAAAAAAGAAATTATTATATTGAAACTAATTATTCACATT ACATTCATGCACATCATAAAGAGCCTTTGGATGAAGCAAGAAAGGTAATTGCTGAAGTATATCCAAATTATCTT CCTGCTTTTGATAAAGTTATGAAAAAGCGTGGGGCTCATATGTTTAATATGTTTATTATGAAAAGGAATGCATTT GAATCATATTGTAGCTTCATGTTTGATGTTCTAAGTAAGCTAGAAAATAGCATTAATATTTCTGAATATTCTGTG CAAGAAGCGAGAGTATTTGGATATATTTCTGAGTTGTTAATGGATGTTTGGCTTGGTACCAATTCATTTACTTAT GTAGAAGTTCCTTGGGGACAAATTGGTGGAAAGAATACTTTAAAGAAAGGCTTTTCTTTAATTAAACGAAAAAT AGGTATAAAAACGAAAACACATTTTTAA Putative sugar transferase EpsL SEQ ID NO:18 GTGTATCACACGATTAAGGGGATATTTGATATACTAGCTAGCGCACTTGGGTTAATTTTACTATCTCCGTTATT TTTATTTTTGATAATAAAAATAAGACATGAAGATGGTGGTCCTGCATTTTATTCTCAAGAACGAATCGGTAAAGATGAGAAGCCGTTCAAGATGTGGAAGTTCCGTTCAATGGTCGTTAACGCTGATCAAATGCTTGATGAACTAGAAGATCAGAACGAAATTGACGGCGCAATGTTCAAGATCAAGGACGATCCAAGAATTACGAAGATCGGACATACAA TTCGCAAGTATAGTCTTGATGAATTGCCACAACTGTGGAATGTCTTGATCGGCGATATGTCACTAGTCGGTCC ACGGCCCCCTCTACCCTCTGAAGTAGCTGAGTATACTGACTACGATAAGCAACGTTTGCTCGTTATGCCAGGT TGTACTGGCTTGTGGCAGGTAACACGCCGTAGTGAAGCTGACTTTGACGAAATGGTTTGGCTTGATATTGTGT ATATCAACCATTCCGGTTTGTGGGAAGATTTGAAGTTGATTGTTAAGACAATTGGGGTGGTTATTCATCCTAAT GGAGCGTATTGA Tyrosine-protein phosphatase SEQ ID NO:19 ATGGTTTTAGTCGATATTCACTGCCACATCTTGCCTGGCATTGATGATGGCTCAAAAAATTGGGACACTTCAAT TAAGTTAGCCAAGGCTGCGGTCAAAGATGGCGTAACGCATGCAATTTGTACGCCGCATACACTGAATGGTCG TTACACTAACCATAAGGATGATATCGTCTGGCTGACTGATCTCTACCAACAGAAGCTAGATGAGGCCAAGGTG CCGTTGACGGTTTTCCCAGGGCAGGAAGTTCGCTTGTCGGGTGACTTAATTGATGCGCTTGATAACGATGATA TCCTGTTCTGCGATGAAGACGGTACTTATATGCTGTTGGAATTTCCAAGTGAGGACGTGCCAACTTATGCGCA AGACACGATCTTCAAGATCATGCAGCGCGGGGTGACGCCGATTATCGTACACCCAGAGCGCAATAGTCGCAT CTTGAAGGAACCGGAAATTTTGCAAGGGATGCTGGAACAAGGCTGCTTGGTGCAAATTACTGCTAGCTCTTAT ACAGGAATCTTTGGCAAGAAAATCGAAGAGATGTCGCGTAAGTTGATTGCGACGGGGCAGGGATGCACCTTT GCTAGCGACGCACACGACTTGCCAAGACGGCAGTATCAATTGAGTGAAGCTTATAAAAAGATGAGTCAAGAAT TCAGTCAAGATCTAGCTCAGCAGTGGCAAGATAATGCTAGAAGTATCATCAATGGCGACCCAGTTCAGATGGA TTGGCACCCATTGAAGCAGAAGAAAAAGTTCTGGTTATTTTAA Tyrosine-protein kinase CpsD SEQ ID NO:20 ATGGCATTATTTAGAAAAAAGCGTGGTACCGACGATACAATTAAGCATGGTGCCAAATTAATCACAGTTGCTG ATCCTCGTAGTGCGGTATCTGAACAATTCAGAACTATCCGGACTAACATCAACTTTATGGCAGTTGATGAAGA AATTAGTACCTTAGCTTTTACTTCAGCTAACATCAGTGAAGGTAAGTCAACCGTTACGGCCAACGTAGCGATCACCTATGCACAAGCAGGTCGCAAGACCTTGCTAATCGATGCCGACTTGCGTCGTCCAACTTTGCACAGTACGTTCAACGTTAAGAACAATACTGGTTTGACAACTGTTCTTACTTCAGAAGCTGACGCAATCAATTTAAATGATGT GGTTGAAGAAAGTGGAATTGATAACTTGTCAATCTTGACTTCTGGTCCGATTCCACCAAACCCAGCTGAATTG ATTGGTTCACGCAGAATGGAAACCTTCATTGAATTGGTTAAGTCACATTATGATATGGTTATCATTGACTTAGC TCCTGTTCTTGAAGTATCCGATACGCAAGAGTTGGCTAGTCACCTTGACGGCGTAGTCTTAGTAGTACGTCAA GGCGTTACGCAAAAGGCTGGGATTACTAGAGCAGTGCAAATGTTAAAATTTGCCAAAGCTCGGATTCTAGGCT ACGTCATGAACGACATTAGAGCTGAAAACGGTGGCTATGGTTACGGCTACGGGTATGGCTATGGTTATGGAT ATGGCGCTGAGAAGAAAAAGGGCCTGTTCGGTAGAAAAAAGAGTGACGATCAATAA Capsular polysaccharide biosynthesis protein CpsC SEQ ID NO:21 ATGGAACAAGAGCAAAAGCAAACTGAAAATACAATTGATCTTACTCAATTGCTACAAATTTGCCGTCGCCACAT CTGGGCACTGATTATCTGGAGTGTTGGTTTGGCTTTAGTCGGCTGGGGCGTCGCTAATTTTATTATTTCACCA AAGTATACTTCTAATGCACAGATCTTGGTTAACCAAAAGGCTAACAAGAATGACCCGAATGCGGCATATAATAC TCAACAAGCCAACATGCAAATGGTTACTACCTATAAGGATATTGTAACTAGTCATGTTATTTTGCAAGATGCTT CTAATCGTTTAGCTAACCCAGTTAGAGTGGTTAAAAAGGCTAAACCTGCTAAGTACAAGACTAATGCAGAGGG TCGCCGAGTATTAGTAAGAAAGGCTCAACCAGAAGTAGTTGAACGTAGCGGTAAGAGTTACAGCGTTTCTACT AGCGAATTAGCCAAGAGCGTTTCAGTTAATACTCAACAACAATCACAGGTCTTCTCAATTTCTGCTACTGCAGA TACACCTGAAAAGGCAAAGGTTGAAGCTAACGCTGTAGCGCGTTCATTCAGAGATCAAATTCCTAACATCATG AACATTAACAACGTTACGATCGTTGCGCCAGCAACTGATGGTAATCAATCATCACCTAATGTGAAACTCTTTAC CTTGGCTGGCTTTGTTATTGGCTTGGTTTTAAGTTTCGCAGTAGTCTTAATTCGTGAAATGTCCGACACTACAG TTAAGGATGATGCATTCTTGACTGATAACTTAGGTTTAGTTGACTTAGGTCAAGTTTCACACTTCCATGTGTCA TCATCATTTGTCATTAGAAAAAAGAATAAGAATTCTAATGGCCCTAAGCGTAGAAGCCGTCGAGTATAGAmino acid sequencesGT1 SEQ ID NO:9 MKSDITVIVPMYNVEKYVKKCFESLENQTYSNFEVLAINDGSPDNSSKYAKECAAKDNRIKVIDKENGGYG SALELAINSINSDYFLVCDPDDWLEEDCLEVLYTFAKNNDTDIVVGDRFDVYADDGSAHKCSVKPGYLESI EPKKVYSETQDIQLFFYPKSSIINDLRK GT2 SEQ ID NO:10 SFFQVSPHAKLYKTQLLKNVSFPKHVSYTDFLLYLVALSRAKKVSYYDKALAFYLQDRPGNTATDIRKSIIN DYL GT3 SEQ ID NO:11 YLTVWKKTFEIINNNNVDNNVDNNVDFLMYRLYMQLRLILAEYKRVSINGFNNKYWSNIMSAVYELQDI EINTVPYFENSMIKKTFFKFFMNRKTANFTAKMYVTLKK FLIPPASE SEQ ID NO:12 MKVIRNYLYNVGYQVLAIIVPLITSAYVSRVLRPEGVGANAFTNSIIQYFILFASMGIGYYGNRQIAYVR DNRTKMAKTFWEIQIVKTIMTLVSIIAFEIFLIFYTRQFDYMLAQSLNLIAVAFDISWFYEGVENFKVTV LKNSLVKIVSMIAIFVFIKGPYDVTLYIVVLALSTLLGNLTLWPDIYRDLPKVKIRSLNPWIHFLPMVEL FIPQIATQVYVQLNRTMLGVMVNETASGYYQYSDSLVKLILALVTATGTVMLPHVANAVSHGDMKKVNQM LYKSFDFVSAMAVPMMFGLAAISLTLAPKYYGPGYGPVGPAMMIESIVILMIAWSNALGVQYLLPIHHQK QFTISVTVGAVVNIILNFPLIHFYGLNGAMWSTVLSEISVTLYQLIAVRGLLSYRTLFNSLWKYLVSGVV MFLPVFWMNTHLKDSWLMMGLEVIVGVVGVMVMLLRAPIVDEAKKLIREKLGK UDP-GALACTOPYRANOSE MUTASE SEQ ID NO:13 MENNYLIIGSGLFGAVFAHEVAKRGNKVTVIEKRDHIAGNIYTKEVDGIQVHQYGAHIFHTSNKEVWNYV QQFAEFNRYTNSPVANYKGRMYNLPFNMNTFSEMWGVGTPQEAMAKINEQRQEMAGKEPQNLEEQAISLI GRDIYEKLIKGYTEKQWGRPCTELPAFIIKRLPVRLIYDNNYFNDDYQGIPIGGYTKMVEKMLDHPNIEV KLNTDFFDDKEEYLNNYDKVVYTGMIDKFFDYKLGELEYRSLRFETEEKNVGNYQGNAVINYTEAEIPYT RIIEHKHFEFGKGDPDKTIITREYPADWKRGDEPYYPVNNDRNNSLYAKYAELAKKQDKVIFGGRLGQYK YYNMDQVIDAALDAVNNEFGE GTA SEQ ID NO:14 MRKQQVAAIVVTYNRKKLLKECIKSLLEQTAKPDIYIIDNNSTDGTVNYIAKYIDDNSINYVNTGKNLGG AGGFQYGIKMVAQKDYDFVWIMDDDCIPTKNALNEFLKADIKLNHDYGFLSSKTLWTDNTLCTMNIQRRT LTKNVSDFSSELIPVTLASFVSLFVPVSVIKKVGLPIKEFFIWTDDWEYTRRISSKYECYVVTNSIVVHK TKTNVGANIATDVPERIERYRYLYRNDFFFYRKEGLKGYGYEFLRLNDHMIRVLFKAKNNKRKRLKMIFK GTIEGLRFNPSIEYIK GTB SEQ ID NO:15 MKKILEAFGEPILYGGQEAFVFRTIENMNKKGLKFDFLTPYYADNPDYISFIKSLNSTLYSFNLPFNVGK NRFNVVNAYKKILLDNQYDVVHINSGSISILALFTLYAKKAGVKKVIVHSHMSGKNKNVKHEVIKKIYAP IFSKYADVLVAPTKKAAYWQFSKKIFNKKGRILKNGINIQQYAYNIGTREHYRNKLNISDHEILIGHVGR LSPEKNQIFLIKLLSYFIKNNTKAKLLLIGAGPQEKSIRMSIKQNRLEKYVQLIGNVDNVEDYLQAMDLF IFPSEYEGLGIASIEAQDAGLPVLASTNVPLDIKVTDNVMFLDLNLSLKEWYKQSLKLLSRKYDRAENTN FLKKQGYDIRNTAEKLKKIYLG Oligosaccharide repeat unit polymerase Wzy SEQ ID NO:22 MFKEINLRNHSSLHKLFYSLRYVSIYIGVFIFMFGLQLYRLNSFSLSGYTIKDALIHNRYYAYMLLTFPMTEVLATAKR RMKFINNIYYMGVAILIFRFSAWFLYNKLRMNVAPGYFEVMGFAWARNGVSRLPGTFLDNYVWVMALFKIIKEKSV VIKTRNISVIVLCFLYAYIVYDSRSQQIAYLLSLVVFILIVSDTFKSKALDVVGAIAISPIIFRGNYLNKLVNTFSTSNADY GSSTQIRMMTLNTYQTLWIERGVWWGYGISNDGNYFNILDTHMVNQSDLGILSMLFQYGIIGFAIFISPFVMGLYVS LKKVKFVQNKFLFLLTCTTALTSIMSQNMYDPFRFLIVPFLLSFISVVSFMNKKEN DUF4422 domain-containing protein SEQ ID NO:23 MKIEILVAAHKEFPMPKKEGYLPVLVGAVKNYKPGISYQRDDDGENISSKNPNYNELTAVYWAWKNLKDVDAIGLV HYRRLLFEKRPYSLDNVISIEKVNQLLQKYDVILPKKRNYYIETNYSHYIHAHHKEPLDEARKVIAEVYPNYLPAFDKV MKKRGAHMFNMFIMKRNAFESYCSFMFDVLSKLENSINISEYSVQEARVFGYISELLMDVWLGTNSFTYVEVPWG QIGGKNTLKKGFSLIKRKIGIKTKTHF Putative sugar transferase EpsL SEQ ID NO:24 MYHTIKGIFDILASALGLILLSPLFLFLIIKIRHEDGGPAFYSQERIGKDEKPFKMWKFRSMVVNADQMLDELEDQNEI DGAMFKIKDDPRITKIGHTIRKYSLDELPQLWNVLIGDMSLVGPRPPLPSEVAEYTDYDKQRLLVMPGCTGLWQVT RRSEADFDEMVWLDIVYINHSGLWEDLKLIVKTIGVVIHPNGAY Tyrosine-protein phosphatase SEQ ID NO:25 MVLVDIHCHILPGIDDGSKNWDTSIKLAKAAVKDGVTHAICTPHTLNGRYTNHKDDIVWLTDLYQQKLDEAKVPLTV FPGQEVRLSGDLIDALDNDDILFCDEDGTYMLLEFPSEDVPTYAQDTIFKIMQRGVTPIIVHPERNSRILKEPEILQGM LEQGCLVQITASSYTGIFGKKIEEMSRKLIATGQGCTFASDAHDLPRRQYQLSEAYKKMSQEFSQDLAQQWQDNA RSIINGDPVQMDWHPLKQKKKFWLF Tyrosine-protein kinase CpsD SEQ ID NO:26 MALFRKKRGTDDTIKHGAKLITVADPRSAVSEQFRTIRTNINFMAVDEEISTLAFTSANISEGKSTVTANVAITYAQAG RKTLLIDADLRRPTLHSTFNVKNNTGLTTVLTSEADAINLNDVVEESGIDNLSILTSGPIPPNPAELIGSRRMETFIELV KSHYDMVIIDLAPVLEVSDTQELASHLDGVVLVVRQGVTQKAGITRAVQMLKFAKARILGYVMNDIRAENGGYGYG YGYGYGYGYGAEKKKGLFGRKKSDDQ Capsular polysaccharide biosynthesis protein CpsC SEQ ID NO:27 MEQEQKQTENTIDLTQLLQICRRHIWALIIWSVGLALVGWGVANFIISPKYTSNAQILVNQKANKNDPNAAYNTQQA NMQMVTTYKDIVTSHVILQDASNRLANPVRVVKKAKPAKYKTNAEGRRVLVRKAQPEVVERSGKSYSVSTSELAK SVSVNTQQQSQVFSISATADTPEKAKVEANAVARSFRDQIPNIMNINNVTIVAPATDGNQSSPNVKLFTLAGFVIGLV LSFAVVLIREMSDTTVKDDAFLTDNLGLVDLGQVSHFHVSSSFVIRKKNKNSNGPKRRSRRV In case of any difference(s) between the above sequences and the sequences depicted in the sequence listing, the above sequences may be used. Alternatively, the sequences depicted in the sequence listing mat be used. The following Examples illustrate the different embodiments of the invention.Figure descriptionFigure 1 - Lactobacillus crispatus cell envelope thickness on the basis of TEM (box plot withmedian). NCBB100714 and NCBB100715 have no GT, whereas NCBB100728 and NCBB100730 have GT1-2-3.Figure 2 - Transmission electron micrographs of strains with and without GT1, GT2, GT3EXPERIMENTAL SECTIONIsolation and characterization of Lactobacillus crispatus strainsIsolation of Lactobacillus crispatus strains Nurse-collected vaginal swabs were obtained from the Sexually Transmitted Infections clinic in Amsterdam, The Netherlands, from June to August 2012. These vaginal samples came from women with healthy vagina (Nugent score 0–3) and from women with vaginal bacterial dysbiosis (Nugent score 7–10). For determining Nugent score, Gram-stained vaginal swabs were examined under oil immersion objective (1000x magnification) and graded as per standardized, quantitative, morphological classification developed by Nugent (Journal of Clinical Microbiology.29 (2): 297–301). Composite score was categorized into three categories, scores 0–3 being normal, 4–6 being intermediate, and 7–10 being definite bacterial vaginosis: Table 1. Nugent scoring Lactobacillus Curved Gram variable rods morphotypes —— average per highaverage per highGardnerella / Bacteroides morphotypes —powered (1000× oil powered (1000× oil average per high powered (1000× oil immersion) field. Preferably immersion) field. immersion) field. Preferably average of multiple average of multiple fields. Preferably average of fields. (note that this factor is less multiple fields.important — scores of only0–2 are possible) Score 0 for >30 Score 0 for 0 Score 1 for 5–30 Score 1 for <1 (this is an average, so results Score 2 for 1-4 can be >0, yet <1) Score 0 for 0 Score 3 for < 1 (this is Score 2 for 1–4 Score 1 for 1-4 an average, so results Sc Score 2 for >= 5 can be >0, yet <1) ore 3 for 5–30 Score 4 for 0 Score 4 for > 30 The vaginal swabs were plated on tryptic soy agar supplemented with 5% sheep serum and 0.25% lactic acid and pH set to 5.5 with acetic acid and incubated under a microaerobic atmosphere (using an Anoxomat; Mart Microbiology B.V., The Netherlands) at 37 °C for 48–72 h. Candidate Lactobacillus spp. strains were selected based on colony morphology (white,small, smooth, circular, opaque colonies), and single colonies were subjected to 16S rRNAsequencing. L. crispatus isolates were taken forward for whole genome sequencing.The Table below shows an overview of identified L. crispatus strains, which were deposited atthe NCCB Bacteria database. The presence or absence of glycosyltransferase (GT) genefragments GT1, GT2, and / or GT3 is indicated.Table 2. Overview of identified L. crispatus strainsGenus and species strain DVM / LVMLactobacillus crispatus NCCB 100711 ##02 DVMLactobacillus crispatus NCCB 100713 ##07 DVMLactobacillus crispatus NCCB 100714 ##09 LVMLactobacillus crispatus NCCB 100715 ##10 LVMLactobacillus crispatus NCCB 100716 ##13 DVMLactobacillus crispatus NCCB 100717 ##14 DVMLactobacillus crispatus NCCB 100718 ##15 DVMLactobacillus crispatus NCCB 100719 ##16 LVMLactobacillus crispatus NCCB 100720 ##17 DVMLactobacillus crispatus NCCB 100721 ##19 DVMLactobacillus crispatus NCCB 100722 ##20 DVMLactobacillus crispatus NCCB 100723 ##21 DVMLactobacillus crispatus NCCB 100724 ##23 DVMLactobacillus crispatus NCCB 100725 ##24 DVMLactobacillus crispatus NCCB 100726 ##25 DVMLactobacillus crispatus NCCB 100727 ##27 LVMLactobacillus crispatus NCCB 100728 ##28 DVMLactobacillus crispatus NCCB 100729 ##29 LVMLactobacillus crispatus NCCB 100730 ##30 DVMLactobacillus crispatus NCCB 100731 ##33 DVMLactobacillus crispatus CTV 05 -Also other methods are available to the skilled person to obtain L. crispatus strains accordingto the invention. For example by the following steps.Begin with the swab that is a vial. The vial contains a protective buffer that helps the bacteriastay alive as much as possible, so they can grow when we spread them on a growth mediumlater.- First, wash your hands with water. Use a clean surface. Unscrew the cap of the vial andplace the cap upside down (so, bottom up) on a clean surface.- Take the swab out of its packaging. Assume a comfortable position, like when inserting atampon. You can use the swab while sitting, standing, or lying down.- Use one hand to spread your labia. Gently insert the end with the cotton a few centimetresinto the vagina, until about one-third of the stick is in. This should not cause any pain.- Rotate the swab and count to 20 seconds. This collects fluid from the vagina, containingbacteria and cells from the mucous membrane. Remove the swab from your vaginaafterward. Try not to touch your skin outside the vagina, labia, or thigh with the swab. Do nottouch the swab with your hands.- Place the part with the cotton in the vial and gently rotate it. Press all sides of the swabagainst the side. Count to 60 seconds to ensure as much fluid from the vagina as possibleends up in the vial. Discard the swab after this.- Screw the vial shut, still without touching the opening of the vial or the bottom of the cap.The bacteria can be plated and colonies can be analyzed for presence of the genetic elements as according to the invention. Characterization of Lactobacillus crispatus strains from LVM versus DVM source Distribution of glycosyltransferase (GT) gene fragments in L. crispatus strains from LVM versus DVM source The Table below shows a comparison of the distribution of glycosyltransferase (GT) gene fragments in L. crispatus strains from healthy vagina (LVM) and from vagina with bacterial dysbiosis (DVM).

[0002] Table 3. Comparison of glycosyltransferase (GT) gene fragments in Lactobacilluscrispatus dominated or dysbiotic vaginal microbiota.Comparison cell envelope thickness of Lactobacillus crispatus strains with and without GT1,GT2, GT3 by transmission electron microscopy (TEM)It was found that the presence of the gene cluster containing GT1, GT2, GT3 leads to a thicker cell wall with a presumed additional polysaccharide pellicle (PSP). Thickness cellenvelope increases in L. crispatus strains from 61 (##09, no gene cluster) and 62 nm (##10,no gene cluster) to 70 nm on average (##30, with gene cluster). See Figure 1, Figure 2 and Table 4 below.

[0003] Table 4. Presence of GT1, GT2, GT3 leads to a thicker cell envelope with a presumedadditional polysaccharide pellicle (PSP). ##9 ##10 ##30The following potassium cyanoferrate TEM protocol was used to determine cell envelopethickness. -Pellet cells and remove supernatant. (14.000 rpm for 5 minutes)- Resuspend pellet in 1 ml of 2.5% glutaraldehyde in 0.1M phosphate / citrate buffer(fixative 1), incubate for 1 hour at RT while keeping the cells resuspended. -Pellet the cells and wash 2 times with 0.1M phosphate / citrate buffer. (washing buffer)- Pellet the cells, remove the supernatant completely and resuspend in 100 µl of 3%gelatin in 0.1M phosphate / citrate buffer. (centrifugation can be done to increase change of finding bacteria)- Incubate for 20 minutes at 4OC. (might take longer, wait until gelatin is solidified)- Pry the solidified gelatin specimen lose and cut specimen into small pieces between1mm3and 3mm3for TEM while keeping them wet in fixative 1.- Add fixative 1 and incubate for at least 1 hour at RT. (may be longer, depended onpenetration of fixative into specimen)- Remove fixative 1 and wash specimen at least 6 times for 10 minutes in washingbuffer.- Add 1% osmium tetroxide + 1.5% potassium cyanoferrate in 0.1M phosphate / citratebuffer. (fixative 2) and incubate for approx.1 hour at RT. (HIGHLY TOXIC, fixation done in fume hood)- Remove fixative 2 and wash specimen at least 3 times for 10 minutes in MilliQ.- Dehydrate the specimen using ethanol (or acetone):o 10% - 10 minuteso 30% - 10 minuteso 50% - 10 minuteso 70% - 10 minutes (possible break and storage step at RT)o 80% - 10 minuteso 90% - 10 minuteso 96% - 10 minuteso 100% - 10 minuteso 100% - 20 minutes- Infiltrate the specimen with resin. (Spurr, LR white or LR gold)o 1:2 resin:ethanol - 30 minuteso 1:1 resin:ethanol - 30 Minuteso 2:1 resin:ethanol - 30 Minuteso 100% resin - 60 minuteso 100% resin – overnight at RTo 100% resin – 60 minutes- Specimen are transferred to BEEM(Spurr) or gelatin(LR white) capsules andcompletely filled with resin.- The capsules containing specimen are polymerized in an oven for 8 hours at 70OC- Polymerized samples are sectioned into 50 thick sections using a Leicaultramicrotome UC7 and attached to the desired grids. (normally 100 mesh carbon grids)- Incubate grids for 10 minutes in 2% uranyl acetate.- Wash 5 times with MilliQ.- Incubate grids for 10 minutes in ready to use lead citrate(EMS)o NOTE: This must be done in a CO2-free environment!!!- Wash 2 times in 0.01 N CO2-free water.- Wash 3 times in MilliQ.TEM specs: JEOL JEM1400, 120kV Comparison bacteriophage susceptibility of Lactobacillus crispatus strains with and without GT1, GT2, GT3 Lactobacillus crispatus strains with and without GT1, GT2, GT3 were subjected to mediumcomprising bacteriophages (isolated from vaginal swap). It was found that Lactobacilluscrispatus strains without GT1, GT2, GT3 are resistant to infection by bacteriophages, and that Lactobacillus crispatus strains with one or two of GT1, GT2, GT3 are sensitive to infection by bacteriophages, and that Lactobacillus crispatus strains with all of GT1, GT2, GT3 are very sensitive to infection by bacteriophages. Comparison immune evasion capability of Lactobacillus crispatus strains with and without GT1, GT2, GT3 Lactobacillus crispatus strains with and without GT1, GT2, GT3 were subjected to medium comprising CD4+ T cells (isolated from vaginal swap). It was found that Lactobacillus crispatus strains without GT1, GT2, GT3 are prone to eradication by the said T cells, and thatLactobacillus crispatus strains with one or two of GT1, GT2, GT3 are less prone toeradication by the said T cells, and that Lactobacillus crispatus strains with all of GT1, GT2, GT3 are not prone to eradication by the said T cells. Probiotic formulations with mixtures Lactobacillus crispatus strainsFrom the isolated Lactobacillus crispatus strains, the following are cultivated and mixed informulations according to Table 5: 1. Lactobacillus crispatus with none of GT1, GT2, GT3 2. Lactobacillus crispatus with only GT1 3. Lactobacillus crispatus with GT1 and GT2 4. Lactobacillus crispatus with GT1 and GT3 5. Lactobacillus crispatus with all of GT1, GT2, and GT3 The above strains are used in a treatment of women with bacterial vaginosis (Nugent score 7-10), according to the treatment arms as depicted in Table 5.Table 5. Response rates for different mixtures of Lactobacillus crispatus strains (with differentdistribution of glycosyltransferase (GT) gene fragments)*. Number and type of Lactobacillus No. of patients in Response ratecrispatus strains in testedtreatment arm (based on % of patients with formulation Nugent score 0-3 after treatment)Strain 1 10 + / -Strain 2 10 + / -Strain 3 10 + / -Strain 5 10 + / -Strain 1 and Strain 3 10 ++Strain 1 and Strain 4 10 ++Strain 3 and Strain 5 10 + / -Strain 1 and Strain 5 10 ++Strain 1, Strain 3, Strain 5 10 +++Strain 1, Strain 3, Strain 4, Strain 5 10 ++++*The putative results in the above Table may be obtained in larger patient cohorts.Notably, the number of non-responders is much lower if mixtures of Lactobacillus crispatusstrains are used which differ in distribution of glycosyltransferase (GT) gene fragments).

Claims

Claims 1. An isolated mixture of strains of Lactobacillus crispatus, comprising- a first strain of Lactobacillus crispatus not having expression of glycosyltransferase (GT) 1,while optionally having expression of GT2 and / or GT3; and- a second strain of Lactobacillus crispatus having expression of at least GT1 and optionallyhaving expression of GT2 and / or GT3.

2. Isolated mixture of strains of Lactobacillus crispatus according to claim 1, comprising- a first strain of Lactobacillus crispatus having expression of none of GT1, GT2, and GT3;and- a second strain of Lactobacillus crispatus having expression of at least GT1, preferablyexpression of at least GT1 and GT2, or GT1 and GT3.

3. Isolated mixture of strains of Lactobacillus crispatus according to any of the preceding claims, wherein- the first strain of Lactobacillus crispatus has expression of none of GT1, GT2, and GT3;- the second strain of Lactobacillus crispatus has expression of all of GT1, GT2, and GT3;and / or- a third strain of Lactobacillus crispatus is comprised which has expression of GT1 and GT2while not expressing GT3.

4. Isolated mixture according to any one of the preceding claims, wherein said mixturecomprises 4 or more strains of Lactobacillus crispatus that each have a different expressionof surface-associated polysaccharides.

5. Isolated mixture of strains of Lactobacillus crispatus according to any one of the preceding claims, wherein the strains of Lactobacillus crispatus have been isolated from vaginal swab from at least one woman, preferably from at least one woman with bacterial vaginosis and at least one woman without bacterial vaginosis.

6. Isolated mixture according to any of the preceding claims for use in therapy.

7. Isolated mixture according to any one of the preceding claims for use in the treatment or prevention of bacterial vaginosis, preferably after treatment with antibiotic.

8. Isolated mixture for use according to claim 6 or 7, wherein said use is topical use.

9. Vaginal capsule or vaginal tablet comprising a mixture according to any of claims 1 - 5.

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