Abstract:
The present invention relates to a probiotic bacterial strain belonging to the genus  Lactobacillus  having the ability to colonize the human vagina, or a variant thereof. More specifically the probiotic bacterial strain belongs to a species chosen from the group comprising  Lactobacillus plantarum, Lactobacillus crispatus , and  Lactobacillus gasseri . Further it relates to its use as a medicament, a composition comprising the strain, the composition, e.g., being a food product or a pharmaceutical composition, a hygiene product, a biological pure culture of the strain, and a novel food.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a probiotic bacterial strain belonging to the genus  Lactobacillus , or a variant thereof, having the ability to colonize the human vagina. More specifically the probiotic bacterial strain belongs to a species chosen from the group comprising  Lactobacillus plantarum, Lactobacillus crispatus  and  Lactobacillus gasseri . Further it relates to its use as a medicament, a composition comprising said strain, the composition e g being a food product or a pharmaceutical composition, a hygiene product, a biological pure culture of said strain and a novel food. 
       BACKGROUND OF THE INVENTION 
       [0002]    The healthy vagina is maintained by the interaction of the vaginal epithelium and the microbial flora, where lactobacilli play a crucial role.  Lactobacillus  species maintain the pH acidity in the vagina by the glucose metabolism; moreover, together with the hydrogen peroxide and bacteriocin-like production they suppress the growth of pathogens and other unwanted microorganisms. This contributes to a successful protection against uropathogens causing urinary tract infection (UTI), the disorder bacterial vaginosis and yeast vaginitis by  Candida albicans  (Reid G, Bruce A W. Urogenital infections in women: can probiotics help? Postgrad Med J 2003; 79: 428-432.). 
         [0003]    The  Lactobacillus  species dominating in the vagina has for long been accepted to be  Lactobacillus acidophilus , but the use of genotypic identification methods have demonstrated that the most common lactobacilli species in the healthy vagina are actually  Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners  and  Lactobacillus jensenii . However, separate studies demonstrate differences between the lactobacilli recovered from the vagina showing species such as  Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus plantarum  and  Lactobacillus acidophilus  as the dominating lactobacilli, this is probably due to differences in the handling of samples, vaginal status or the methods preferred for the isolation (Vasquez A, Jakobsson T, Ahrné S, Forsum U, Molin G. Vaginal  Lactobacillus  flora of healthy Swedish women. J. Clin. Mirobiol. 2002; 40: 2746-9). 
         [0004]    The World Health Organization defined probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The development of antibiotic resistance and failures in vaginal infections treatment have risen an increased interest in probiotics as an alternative tool. The need for a vaginal probiotic is clear in terms of the high number of incidence of vaginal infections recurrence (Reid G, Bruce A W, Fraser N, Heinemann C, Owen J, Henning B. Oral probiotics can resolve urogenital infections. FEMS Immunol Med Microbiol 2001; 30: 49-52; Famularo G, Pieluigi M, Coccia R, Mastroiacovo P, De Simone C. Microecology, bacterial vaginosis and probiotics: perspectives for bacteriotherapy. Med Hypotheses 2001; 56: 421-30.). A change in the vaginal flora characterized by the decrease of lactobacilli appears to be the major factor causing the syndrome bacterial vaginosis. Regular administration of a  Lactobacillus  strain with ability to colonize vagina can be an alternative solution for this problem. 
         [0005]    Yoghurt treatment as a household remedy has been used for years for prevention or relief of vaginal disorders. However,  Lactobacillus delbreukii  var.  bulgaricus , the lactobacilli found in yogurt is not an optimal candidate for vaginal restoration by probiotics because it&#39;s not normally found in that environment and does not adhere well to the vaginal epithelial cells for successful colonization (Famularo G, Pieluigi M, Coccia R, Mastroiacovo P, De Simone C. Microecology, bacterial vaginosis and probiotics: perspectives for bacteriotherapy. Med Hypotheses 2001; 56: 421-430). The optimal candidates for such a vaginal probiotic are species tnat are normally found in the vagina and possess qualities to suppress pathogens. 
         [0006]    The normal vaginal flora ascends from the rectal mucosa (Reid G, Bruce A W. Urogenital infections in women: can probiotics help? Postgrad Med J 2003; 79: 428-432), which means that orally administrated microorganisms that survive the gastrointestinal passage will appear in the vagina after a certain time. This fact raises the possibility of a vaginal probiotic that can be given orally to the host which simplify long term administration with the intention to prevent vaginal problems. 
         [0007]    Women given  L. acidophilus  during 6 moths resulting in a decrease in both candidal colonization and infection (Hilton E, Isenberg H D, Alperstein P, France K, Borenstein M T. Ingestion of yogurt containing  Lactobacillus acidophilus  as prophylaxis for candidal vaginitis. Ann Internal Med 1992; 116: 353-357). 
       SUMMARY OF THE INVENTION 
       [0008]    It has now surprisingly been found that specific probiotic bacterial strains belonging to the genus  Lactobacillus , and more specifically to a species chosen from the group comprising  Lactobacillus plantarum, Lactobacillus crispatus  and  Lactobacillus gasseri , have the ability to colonize the human vagina and preserve or improve the vaginal health when administered e g orally, rectally or vaginally. 
         [0009]    In one aspect the present invention relates to a probiotic bacterial strain belonging to the genus  Lactobacillus , or a variant thereof, having the ability to colonize the human vagina with the proviso that the bacterial strain is not  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum  HEAL 19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316,  Lactobacillus plantarum  299, deposition number DSM 6595, or  Lactobacillus plantarum  299v, deposition number DSM 9843. 
         [0010]    In one embodiment said bacterial strain belongs to a species chosen from the group comprising  Lactobacillus plantarum, Lactobacillus crispatus  and  Lactobacillus gasseri , or a variant thereof, and in another embodiment said strain has a mannose-specific adhesin or other adhesive mechanisms that allow the bacteria to bind to the inner surface of the vagina, the gastro intestinal tract, urinary bladder including the urethra. 
         [0011]    In one embodiment of the invention the probiotic bacterial strain is  Lactobacillus  crispatus VPC5, deposition number DSM 16735, or a variant thereof. 
         [0012]    In another embodiment of the invention the probiotic bacterial strain is  Lactobacillus  crispatus VPC40, deposition number DSM 16736, or a variant thereof. 
         [0013]    In still another embodiment of the invention the probiotic bacterial strain is  Lactobacillus  crispatus VPC70, deposition number DSM 16738, or a variant thereof. 
         [0014]    In a yet further embodiment the probiotic bacterial strain is  Lactobacillus  crispatus VPC71, deposition number DSM 16739, or a variant thereof. 
         [0015]    In a yet further embodiment the probiotic bacterial strain is  Lactobacillus  crispatus VPC77, deposition number DSM 16740, or a variant thereof. 
         [0016]    In another embodiment the probiotic bacterial strain is  Lactobacillus  crispatus VPC111, deposition number DSM 16741, or a variant thereof. 
         [0017]    In yet another embodiment the probiotic bacterial strain is  Lactobacillus  crispatus VPC130, deposition number DSM 16742, or a variant thereof. 
         [0018]    In yet another embodiment the probiotic bacterial strain is  Lactobacillus  crispatus VPC177, deposition number DSM 16743, or a variant thereof. 
         [0019]    In a further embodiment the probiotic bacterial strain is  Lactobacillus gasseri  VPG44, deposition number DSM 16737, or a variant thereof. 
         [0020]    In yet another embodiment the bacterial strains described above are used as a medicament. 
         [0021]    In a second aspect the present invention relates to the use of a bacterial strain as described above for the manufacturing of a medicament for treatment and/or prophylaxis of bacterial vaginosis, viral vaginosis, yeast vaginitis, infections in the vagina, sexually transmitted diseases, such as HIV and chlamydia infection, infections endangering the foetus in pregnant women, preterm labour and urinary tract infection. 
         [0022]    In a third aspect the present invention relates to the use of a bacterial strain chosen from the group comprising  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum  HEAL 19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316,  Lactobacillus plantarum  299, deposition number DSM 6595, or  Lactobacillus plantarum  299v, deposition number DSM 9843, or a variant thereof, for the manufacturing of a medicament for treatment and/or prophylaxis of bacterial vaginosis, viral vaginosis, yeast vaginitis, infections in the vagina, sexually transmitted diseases, such as HIV and chlamydia infection, infections endangering the foetus in pregnant women, preterm labour and urinary tract infection with the proviso  Lactobacillus plantarum  299v, deposition number DSM 9843 for urinary tract infection. 
         [0023]    In a fourth aspect the present invention relates to a composition comprising at least one probiotic bacterial strain as described above, and/or  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum HEAL  19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316 , Lactobacillus plantarum  299, deposition number DSM 6595, or  Lactobacillus plantarum  299v, deposition number DSM 9843, or a variant thereof, and/or a fragment or fraction thereof. In one embodiment the composition comprises a carrier material. 
         [0024]    In another embodiment the composition is a food product, preferably comprising a carrier material chosen from the group comprising oat meal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins. In still another embodiment said food product is chosen from the group comprising bread, cheese, yogurt, juice, health bars, spreads, biscuits and cereals, and in yet another one said at least one probiotic bacterial strain is encapsulated or coated, and preferably present in an amount giving, when consumed, an effective daily dose of 107 to 10 12  CFU, preferably from 109 to 10 10  CFU. 
         [0025]    In a further embodiment the composition is a food supplement, preferably comprising a carrier material chosen from the group comprising oat meal gruel, lactic acid fermented foods, resistant starch, dietary fibers, inulines, carbohydrates, proteins and glycosylated proteins. 
         [0026]    In still another embodiment the composition is a pharmaceutical composition for treatment and/or prophylaxis of bacterial vaginosis, viral vaginosis, yeast vaginitis, infections in the vagina, sexually transmitted diseases, such as HIV and chlamydia infection, infections endangering the foetus in pregnant women, preterm labour and urinary tract infection, with the proviso  Lactobacillus plantarum  299v, deposition number DSM 9843 for urinary tract infection. 
         [0027]    In one embodiment said pharmaceutical composition is administered orally, vaginally or rectally, or instilled into the urinary bladder, and preferably said carrier material is at least one pharmaceutically acceptable carrier. 
         [0028]    In a further embodiment said pharmaceutical composition is administered in a combined administration treatment of orally and vaginally, orally and rectally, or orally, vaginally and rectally. 
         [0029]    In another embodiment said pharmaceutical composition is administered in the form of tablets, sucking tablets, sweets, chewing gum, capsules, enterocoated tablets and capsules, suppositories, micro-enemas, vaginal tablets, vaginal gelatin capsules, vaginal troches, cream, gel, ointment, lotion, tampons, napkins, pads, melting strips, condoms, pessaries, sprays and clinical nutrition product. In still another embodiment of the composition, when administered orally, said at least one probiotic bacterial strain is present in an amount giving an effective daily dose of from 10 7  to 10 12  CFU, preferably from 10 9  to 10 10  CFU, and when administered vaginally or rectally, said at least one probiotic bacterial strain is present in an amount giving an effective daily dose of from 10 3  to 10 12  CFU, preferably from 10 5  to 10 9  CFU. 
         [0030]    In one embodiment of the pharmaceutical composition said at least one probiotic bacterial strain is encapsulated or coated, and in another embodiment the composition comprises additives chosen from the group comprising vitamins, minerals, and prebiotics. 
         [0031]    In a fifth aspect the present invention relates to a hygiene product comprising a composition as described above. In one embodiment said hygiene product is chosen from the group comprising tampons, sanitary napkins, sanitary pads, diapers, soaps, shampoos, gels, ointments, creams, sprays and lotions. 
         [0032]    In a sixth aspect the present invention relates to a biological culture of at least one probiotic bacterial strain as described above, and/or  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum  HEAL 19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316 , Lactobacillus plantarum  299, deposition number DSM 6595 , Lactobacillus  plantarum 299v, deposition number DSM 9843, or a variant thereof, and/or a fragment or fraction thereof. 
         [0033]    In a seventh aspect the present invention relates to a novel food comprising at least one probiotic bacterial strain as described above, and/or  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum  HEAL 19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316 , Lactobacillus plantarum  299, deposition number DSM 6595 , Lactobacillus plantarum  299v, deposition number DSM 9843, or a variant thereof, and/or a fragment or fraction thereof. 
         [0034]    In an eighth aspect the present invention relates to a food supplement comprising at least one probiotic bacterial strain as described above and/or  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum  HEAL 19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316,  Lactobacillus plantarum  299, deposition number DSM 6595,  Lactobacillus plantarum  299v, deposition number DSM 9843, or a variant thereof, and/or a fragment or fraction thereof. 
         [0035]    In one embodiment said probiotic bacterial strain is viable. In another embodiment it is inactivated or suppressed. In still another embodiment it is genetically modified, and in still another one it is killed. 
         [0036]    In an eighth aspect of the present invention relates to a fragment or fraction of a probiotic bacterial strain as described above. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0037]      FIG. 1  shows a dendrogram based on the RAPD-analyses of some of the most preferred probiotic bacterial strains. 
           [0038]      FIG. 2  A-I shows the RAPD patterns for some of the most preferred probiotic bacterial strains on which the dendrogram of  FIG. 1  is based. 
           [0039]      FIG. 3A  shows log cfu/g faeces after intake of the four different experimental products of example 3. 
           [0040]      FIG. 3B  shows log cfu/swab in vaginal fluid after intake of the four different experimental products of example 3. 
           [0041]      FIG. 4  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. crispatus  VPC 177 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
           [0042]      FIG. 5  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. crispatus  VPC 5 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
           [0043]      FIG. 6  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. crispatus  VPC 40 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
           [0044]      FIG. 7  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. crispatus  VPC 71 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
           [0045]      FIG. 8  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. crispatus  VPC 111 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
           [0046]      FIG. 9  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. crispatus  VPC 130 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
           [0047]      FIG. 10  shows separated DNA fragments obtained by cleaving chromosomal DNA of the strain  L. gasseri  VPG 44 with the restriction enzymes Hind III (lane 2), Cla I (lane 3), Eco RI (lane 4). Molecular Weight DNA Markers were used as standards (lanes 1 and 5). 
       
    
    
     DESCRIPTION OF THE INVENTION 
       [0048]    The present invention refers inter alia to a composition comprising one or more strains of  Lactobacillus plantarum  or closely related  Lactobacillus  spp. with ability to colonize vagina via oral administration and intestinal establishment. Said composition will e g pre-serve vaginal health, attenuate vaginosis, yeast vaginitis, urinary tract infection, sexually transmitted diseases and infections that might endanger the foetus in pregnant women. 
         [0049]    A healthy uro-genital micro-riora comprises &gt;50 species of micro-organisms. Healthy uro-genital cells are covered by bacterial bio films where lactobacilli predominate. The healthy vagina is maintained by the interaction of the vaginal epithelium and the microbial flora. Pathogens in the faecal flora are the origin of urinary tract infection and bacterial vaginosis. One of the factors in pathogenesis is the ability of the pathogens to attach to epithelial cells. Adhesins and receptor sites are involved in this attachment process. For example the pathogen  E. coli  have a mannose-specific adhesion on epithelial surfaces. Some Lactobacilli have previously been shown to have a mannose-specific adhesion as well. By utilizing this mechanism some probiotic lactobacilli may also prevent pathogens from adhering to mucosal surfaces such as vaginal and urethral mucosa and thus prevent infection. Other factors are maintaining the low pH in the vagina by the glucose metabolism, production of hydrogen peroxide and bacteriocin-like production to suppress growth of pathogens and other unwanted microorganisms. 
         [0050]    The invention especially refers to the strains  Lactobacillus crispatus  VPC5, deposition number DSM 16735,  Lactobacillus  crispatus VPC40, deposition number DSM 16736,  Lactobacillus  crispatus VPC70, deposition number DSM 16738,  Lactobacillus  crispatus VPC71, deposition number DSM 16739,  Lactobacillus  crispatus VPC77, deposition number DSM 16740,  Lactobacillus  crispatus VPClll, deposition number DSM 16741,  Lactobacillus  crispatus VPC130, deposition number DSM 16742 , Lactobacillus crispatus  VPC177, deposition number DSM 16743, and  Lactobacillus gasseri  VPG44, deposition number DSM 16737, which were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on Sep. 17, 2004 and were then given the accession numbers referred to above, or variants thereof. Said strains having the ability to colonize the vagina. 
         [0051]    The invention also refers to the use or the bacterial strains  Lactobacillus plantarum  HEAL 9, deposition number DSM 15312,  Lactobacillus plantarum  HEAL 19, deposition number DSM 15313,  Lactobacillus plantarum  HEAL 99, deposition number DSM 15316, which were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on Nov. 27, 2002 and were then given the accession numbers referred to above, or variants thereof. 
         [0052]    The invention also refers to the use of the bacterial strains  Lactobacillus plantarum  299, deposition number DSM 6595, which were deposited on 2 Jul. 1991, and  Lactobacillus plantarum  299v, deposition number DSM 9843, which were deposited on 16 Mar. 1995 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and were then given the accession numbers referred to above, or variants thereof. 
         [0053]    The phrase “variant thereof” as used herein in reference to the bacterial strains of the invention, especially those which have been deposited, is defined as a bacteria
       belonging to the cluster having a RAPD similarity of at least 80% to said probiotic bacterial strain, by using the Pearson product moment correlation coefficient and the unweighted pair algorithm with arithmetic averages (UPGMA; BioNumerics 2.5, Applied Maths, Kortrijk, Belgium); and/or   belonging to a cluster having a restriction endonuclease analysis similarity of preferably at least 75% to said probiotic bacterial strain, by using the Pearson product moment correlation coefficient and the unweighted pair algorithm with arithmetic averages (UPGMA; BioNumerics 2.5, Applied Maths, Kortrijk, Belgium); and/or   having the ability to colonize the human vagina; and/or   having a mannose-specific adhesin or other adhesive mechanisms that allow the bacteria to bind to the inner surface of the vagina, the gastro intestinal tract, urinary bladder including the urethra.       
 
         [0058]    The phrase “probiotic bacterial strain(s)” as used herein in reference to the invention is meant live microorganisms which when administered in adequate amounts confer a health benefit on the host. In addition, when the bacterial strains are taken orally, the bacterial strains must survive the passage through the gastro-intestinal tract and, when the bacterial strains are taken vaginally or rectally, the bacterial strains must colonize the vagina and rectum, respectively. 
         [0059]    The phrase “colonize the human vagina” as used herein in reference to the bacterial strains of the invention is meant that the viable count of lactobacilli (log cfu/swab) in the vaginal fluid and/or faeces is at least increased after taking the bacterial strain orally, rectally or vaginally compared to the total lactobacilli counts initially, i.e. before taking any of the bacterial strains. 
       DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 
     Genotypic Identification by REA 
       [0060]    The strains (Lactobacillus crispatus VPC5, deposition number DSM 16735,  Lactobacillus crispatus  VPC40, deposition number DSM 16736,  Lactobacillus crispatus  VPC71, deposition number DSM 16739 , Lactobacillus crispatus  VPC111, deposition number DSM 16741,  Lactobacillus crispatus  VPC130, deposition number DSM 16742,  Lactobacillus crispatus  VPC177, deposition number DSM 16743, and  Lactobacillus gasseri  VPG44, deposition number DSM 16737, were examined as to the cleavage pattern of the chromosomal DNA, through restriction-endonuclease analysis—REA—method according to Stahl M, Molin G, Persson A, Ahrne S &amp; Stahl S, International Journal of Systematic Bacteriology, 40:189-193, 1990, and further developed by Johansson, M-L, et al., International Journal of Systematic Bacteriology 45:670-675, 1995. Schematically REA can be described as follows: Chromosomal DNA from the strains involved in the study were prepared and cleaved by restriction endonucleases. 0.75 μg of each DNA was separately digested at 37° C. for 4 h with 10 units of Hind III, Cla I and EcoRI; each endonuclease was used separately. The cleaved DNA fragments are separated as to size by gel electrophoresis using submerged horizontal agarose slab gels. The gels consisted of 150 ml of 0.9% agarose (ultrapure DNA grade; low electro-endo osmosis; BioRad Laboratories, Richmond, USA) and were cast as slab gels (150 by 235 mm). 0.2 μg of the High Molecular Weight DNA marker (Bethesda Research Laboratories, Md., USA) together with 0.5 μg of a DNA molecular weight marker VI (Roche, Germany) were used as standards. Minimal band distortion and maximal sharpness were achieved by applying the sample DNA in Ficoll loading buffer (2 g of Ficoll, 8 ml of water, 0.25% bromphenol). 
         [0061]    Gels were run at a constant voltage of 40V for 18 h at about 6-8° C. The buffer (89 mM Tris, 23 mM H 3 PO 4 , 2 mM sodium EDTA, pH 8.3) was recirculated during the running period. Thereafter, the gels were stained for 20 minutes in ethidium bromide (2 μg/ml) and destained in distilled water, visualized at 302 nm with a UV transilluminator (UVP Inc., San Gabriel, USA) and photographed. This way of running the gel electrophoresis gave well distributed and relatively well-separated band down to a molecular weight of 1.2×10 6 . 
         [0062]    The results of the analysis are presented in the  FIGS. 4-10 . 
       Example 1 
     Volunteers and Strains 
       [0063]    Ten healthy women at different ages with no vaginal infections or any intestinal diseases participated in the study. The study subjects provided information about their age, menstrual cycle and health behavior by a questionnaire. One week before the first samples were taken the volunteers had a washout period in which they avoided all kind of probiotic containing formulas. 
         [0064]    The bacterial strains used in the study are presented in Table 1. They were chosen among a battery of 23  Lactobacillus  after qualities such as Viable Count numbers after culturing in the laboratory, fermentation of oat meal gruel and ability to withstand freezing and thawing. 
         [0065]    Twelve of the most resistant strains were selected for administration to the volunteers; bacterial concentration and incubation time for culture are given in Table 1. The administration was performed during 10 days. The bacterial strains were prepared from a fresh culture in LCM (Lactobacillus carrying medium) (Efthymiou C, Hansen C A. An antigenic analysis of  Lactobacillus acidophilus . J Infect Dis 1962; 110: 258-267). Cells of each culture equivalent a concentration of about 109 bacteria in a total volume of 100 ml were harvested by centrifugation and washed once in Millipore—H 2 O. The pellets were then dissolve in 20 ml oat milk (Beneviva, Finland) and 80 ml blueberry soup (Ekstroms, Sweden), which was the drink administrated to the subjects. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Selected bacterial  Lactobacillus  (L.) strains for the administration. 
               
             
          
           
               
                   
                   
                   
                 Adminis- 
                   
               
               
                   
                   
                   
                 tration 
                 Incubation 
               
               
                 Bacterial 
                 Strain 
                 Source 
                 conc. 
                 time 
               
               
                 species 
                 number 
                 (human) 
                 (×10 9 ) 
                 (d) 
               
               
                   
               
               
                 
                   L. crispatus 
                 
                 VPC77 
                 vagina 
                 1.1 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC111 
                 vagina 
                 1.0 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC130 
                 vagina 
                 2.7 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC5 
                 vagina 
                 1.2 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC40 
                 vagina 
                 1.3 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC70 
                 vagina 
                 1.6 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC71 
                 vagina 
                 1.1 
                 2 
               
               
                 
                   L. crispatus 
                 
                 VPC177 
                 vagina 
                 1.6 
                 2 
               
               
                 
                   L. gasseri 
                 
                 VPG44 
                 vagina 
                 1.1 
                 1 
               
               
                 
                   L. plantarum 
                 
                 HEAL19 
                 intestinal 
                 1.3 
                 1 
               
               
                   
                   
                 mucosa 
               
               
                 
                   L. plantarum 
                 
                 HEAL9 
                 intestinal 
                 2.2 
                 1 
               
               
                   
                   
                 mucosa 
               
               
                 
                   L. plantarum 
                 
                 HEAL99 
                 intestinal 
                 1.2 
                 1 
               
               
                   
                   
                 mucosa 
               
               
                   
               
             
          
         
       
     
       Sample Collection 
       [0066]    The volunteers delivered faecal and vaginal fluid samples that were cultivated on agar plates within 3 hours after the delivery to the laboratory. The sampling was at the beginning of the treatment (T 0 ), after 10 days of administration (T 1 ) and 7 days after termination of the administration (T 2 ). 
         [0067]    Vaginal samples were taken by inserting a vaginal swab (Copan amies agar gel swabs; Copan innovation, Italy) trough a cap to prevent bacterial contamination. The vaginal swabs were agitated in 9 ml sterile phosphatebuffered saline (PBS) pH 7.2 and serially diluted. One gram faeces were agitated in 9 ml PBS and serially diluted. Aliquots of each dilution for the vaginal samples and the last three dilutions for the faecal samples were plated on Rogosa agar (Oxoid AB, Sollentuna, Sweden) and anaerobically incubated during 3 days in a BBL Gas Pack system at 37° C. 
         [0068]    Six colonies from both faecal and vaginal samples were randomly picked after the incubation. Moreover, colonies with typical morphological appearance were searched for and additionally picked when found. 
       Strain Typing 
       [0069]    Totally 338 isolates were grouped using Randomly Amplified Polymorphic DNA (RAPD) together with the administrated strains. They were cultivated again in Rogosa agar (Oxoid) to verify that they were free from contamination. 
         [0070]    The primer used in the PCR-amplification was a 9-mere with the sequence 3′-ACG CGC CCT-5′ (Scandinavian Gene Synthesis AB, Koping, Sweden). The following PCRamplification and agarose gel electrophoresis was performed, as previously described by Quednau M, Ahrne S, Pettersson A C, Molin G. Identification of clinically important species of  Enterococcus  with in 1 day with Random Amplified Polymorphic DNA (RAPD) Curr. Microbiol. 1998; 36: 332-6. One μl of PCR-templates were used in a total reaction volume of 50 μl containing PCR-buffer with 1.5 mM MgCl 2  (Roche Diagnostics GmbH, Mannheim, Germany), 0.2 mM of each nucleotide (Roche) and 2.5 units of Taq DNA polymerase (Roche). The PCR amplification was according to the following temperature profile: 94° C. for 45 s, 30° C. for 120 s, 72° C. for 60 s, for four cycles followed by 94° C. for 5 s, 36° C. for 30 s (with extension of 1 s for each cycle), 72° C. for 30 s, for 26 cycles. The PCR session concluded with 72° C. for 10 minutes, followed by cooling to 4° C. The products were visualized by agarose gel electrophoresis and photo-negatives of RAPD-gels were scanned by a Hewlett Packard ScanJet 5300C into a computer at a resolution of 300 dpi. The gel images were then analyzed and grouped by GelCompar 4.2 (Applied Maths, Kortrijk, Belgium) with Pearson product moment correlation coefficient (r) and the unweighted pair group method by arithmetic averages (UPGMA). 
       Strain Identification 
     REA-Restriction Endonuclease Analysis 
       [0071]    Thirty-six isolates together with the administrated strains were selected from the RAPD-grouping and further identified by REA (Restriction Endonuclease Analysis) to assure the presence of the exogenous strains in the faecal and vaginal samples of the subjects. 
         [0072]    Preparation of Chromosomal DNA was Carried Out as a procedure described previously (Ulrich R L, Hughes T A. A rapid procedure for isolating chromosomal DNA from  Lactobacillus  species and other gram-positive bacteria. Lett Appl Microbiol 2001; 32: 52-56). Gel electrophoresis was according to an earlier protocol by Johansson et al. (Johansson M L, Quednau M, Ahrne S, Molin, G. Classification of  Lactobacillus plantarum  by restriction endonuclease analysis of total chromosomal DNA using conventional agarose gel electrophoresis. Int J Syst Bacteriol 1995; 45: 670-5). 
         [0073]    DNA (0.75 μg) was separately digested at 37° C. for 4 h with 10 units of HindIII and EcoRI (Roche). Submerged horizontal 0.9% agarose (High Strength Analytical Grade Agarose: low electro-endo osmosis, Bio-Rad Laboratories, Calif., USA) slab gels in size of 150 by 235 mm were used. Amounts of 0.2 μg of a high molecular weight DNA marker (Gibco, Invitrogene Corporation, Sweden) together with 0.5 μg of a DNA molecular weight marker VI (Roche) were used as standards. Gels were run at a constant voltage of 40 V for 18 h at 5° C. Thereafter, the bands were visualized at 302 nm with a UV transilluminator (UVP Inc., San Gabriel, Calif., USA) and photographed. 
       Results 
       [0074]    Five of the administrated strains were recovered from 8 of the 10 subjects, both after the 10 days treatment (T 1 ) and 1 week after that the administration ended (T 2 ). The re-isolated strains at T 1  were  L. crispatus  VPC177,  L. gasseri  VPG44 and  L. plantarum  HEAL 19; whereas  L. plantarum  HEAL 99 and  L. plantarum  HEAL 9 were recovered both at T 1  and T 2  (Table 2). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Re-isolated strains found in vaginal and faecal samples of subjects 
               
               
                 (S). “d” within parenthesis indicates that the strain was 
               
               
                 re-isolated from a randomly picked colony, i.e. the strain represented 
               
               
                 a dominating part of the total  lactobacilli  flora. 
               
             
          
           
               
                   
                   
                 Subject 
                 Sampling time 
                   
               
               
                   
                 Strain 
                 number 
                 T1 
                 T2 
               
               
                   
                   
               
               
                   
                 
                   L. crispatus 
                 
                 S7 
                 faeces 
                 — 
               
               
                   
                 VPC177 
               
               
                   
                 
                   L. gasseri 
                 
                 S2 
                 faeces 
                 — 
               
               
                   
                 VPG44 
                 S10 
                 faeces (d)* 
                 — 
               
               
                   
                 
                   L. plantarum 
                 
                 S3 
                 faeces (d) 
                 — 
               
               
                   
                 HEAL 19 
                 S4 
                 faeces 
                 — 
               
               
                   
                   
                 S5 
                 faeces (d); 
                 — 
               
               
                   
                   
                 S6 
                 vagina 
                 — 
               
               
                   
                   
                 S7 
                 faeces (d) 
                 — 
               
               
                   
                   
                 S8 
                 faeces (d); 
                 — 
               
               
                   
                   
                 S9 
                 vagina (d) 
                 — 
               
               
                   
                   
                 S10 
                 faeces (d) 
                 — 
               
               
                   
                   
                   
                 faeces 
               
               
                   
                   
                   
                 vagina (d) 
               
               
                   
                 
                   L. plantarum 
                 
                 S3 
                 faeces (d) 
                 vagina (d) 
               
               
                   
                 HEAL 99/9 
                 S4 
                 — 
                 vagina (d) 
               
               
                   
                   
                 S7 
                 faeces (d) 
                 — 
               
               
                   
                   
                 S9 
                 faeces (d) 
                 — 
               
               
                   
                   
                 S10 
                 faeces (d) 
                 — 
               
               
                   
                   
               
             
          
         
       
     
       RAPD—Randomly Amplified Polymorphic DNA (RAPD) 
       [0075]    The most interesting bacterial strains were also analyzed with the PCR-based method of Randomly Amplified Polymorphic DNA (RAPD) as described by Johansson et al 1995 Randomly Amplified Polymorphic DNA (RAPD) for rapid typing of  Lactobacillus plantarum  strains. Lett. Appl. Microbiol. 21:155-159, with the following modifications. Crude cell extracts were prepared from 3-5 colonies of a pure culture from MRS agar plates. The cells of pure culture were washed twice in 1 ml sterile Milli-Q water, and disrupted in an Eppendorf tube with glass beads (2 mm in diameter) using an Eppendorf Mixer (5432; Eppendorf, Hamburg Germany) for 10 minutes. The primer used had the sequence 5′-CCGCAGCCAA-3′ and a concentration of 15 μM. Taq mastermix from Qiagen was used in the PCR reaction. The band patterns for the gels were analyzed with Pearson product moment correlation coefficient (r) and the Unweighted Pair Group Method with Arithmetic averages (UPGMA; Romersburg, 1984) by using BioNumerics 2.5 (Applied Maths, Kortrijk, Belgium). The computerized cluster analysis of the RAPD-patterns was combined with a visual comparison. 
         [0076]    By using the RAPD method described above on the eight  L. crispatus  strains and one  L. gasseri  strain of table 1 two clusters containing strains with an 80% similarity could be identified (see the dendrogram of figure 1). Strains within such a cluster are regarded as variants of each other and predicted to have similar properties. One of the clusters contains the strains VPC 70, VPC 71 and VPC 5 and the other contains strains VPC 177, VPC 40 and VPC 111. Strain VPC 177 for example could be found in the vagina after oral administration and it is therefore likely other strains within the same RAPD cluster also have the ability to do so. 
       Conclusion 
       [0077]    The experiment demonstrates an unexpected ability of the chosen  L. plantarum  strains to colonize the vagina via oral administration and intestinal establishment. It should be pointed out that the results is achieved with relatively low doses of probiotics administrated for short times and that the  L. plantarum  strains were compared with strains more typical for the vagina, i.e. more frequently occurring species and strains isolated from vagina. 
       Example 2 
     Yeast Agglutination as a Measure of Mannose Adhesion 
       [0078]    In this experiment the ability of the bacteria to agglutinate yeast cells were determined by eye as visible precipitation of yeast cells and bacteria. Washed bacteria were suspended at a concentration of 2×10 10  cells per ml in PBS (pH 7.2). A solution of 2.5% (wt/vol) baker&#39;s yeast ( Saccharomyces cerevisiae ) suspended in PBS or 0.125% (wt/vol) D-Mannose containing PBS were added to a equal volume of bacterial solution on a microscope slide. The bacteria and the yeast solutions were mixed by gentle rocking of the slide and the number of turns were calculated until a precipitation of agglutinated yeast and bacterial cells appeared. This number of turns was compared between the solution with and without mannose. The yeast agglutination was classified as mannose sensitive if the number of turns until visible precipitation appear differ more than ten times between the samples with or without mannose. 
         [0079]    During the experiment it was shown that  Lactobacillus crispatus  VPC 177 and VPC 70 have the ability to agglutinate yeast in a mannose sensitive manner. Mannose sensitive yeast agglutination has previously been shown to correlate with the ability to adhere to cells of the human colonic carcinoma cell line HT-29. (Adlerberth, I., Ahrné, S., Johansson, M-L., Molin, G., Hanson, L-A., and Wold, A. E. (1996). A mannose-specific adherence mechanism in  Lactobacillus plantarum  conferring binding to the human colonic cell line HT-29 , Appl. Environ. Microbiol.,  62, 2244-2251.), and is thus a factor promoting the colonization and persistence of the bacteria in question in the intestine. Therefore, it is with high probability likely true that this feature of the bacteria also is of importance for colonisation in the vagina and urinary tract, and thereby a mechanism to compete with uropathogenic bacterial strains of  Escherichia coli . This feature, thus a mannose-specific adherence mechanism, has previously also been shown for  Lactobacillus plantarum  299, deposition number DSM 6595, and  Lactobacillus plantarum  299v, deposition number DSM 9843. 
       Example 3 
       [0080]    The aim of the experiment below was to investigate the establishment of 4  Lactobacillus  strains in the intestines and vagina by studying its presence in faeces and vaginal fluid following the intake of the lactic acid bacteria in a freeze dried formula orally. 
         [0081]    It is important to emphasize that the experiment is carried out in vivo in humans, as neither in vitro studies nor animal studies would reflect the degree of survival when administered to humans. The ability of these bacteria to become established in the intestine when administered directly after cultivation is documented in an earlier study (Moreno, Alejandra Vasquez 2004 PhD thesis: Systematics of  Lactobacillus  spp. of probiotic potential Food Technology; Lund University.). In said study fresh bacterial cells from overnight cultivation were used. However, it would be more convenient to use a lyophilized product in a real product. Therefore, a lyophilized formulation has been chosen for that reason in this experiment. 
         [0082]    The primary objective was to investigate the presence in faeces and vaginal fluid of four  Lactobacillus  strains, originating from vagina, following oral ingestion of the strains in an open study. 
         [0083]    Since many diseases in the vagina such as urinary tract infection arise from the rectal mucosa it is beneficial to see an increase in viable count of healthy lacotbacilli in both faeces and vaginal fluid. If the healthy bacterial strains of the invention affect rectum in addition to the vagina, the healthy lactobacilli bacteria of the invention will suppress the non-desirable bacteria in rectum, thereby avoiding diseases in the vagina ascending from rectum. 
       Materials and Methods 
     Experiment Design 
       [0084]    The experiment was an open study and was carried out on one group of subjects. The experiment was divided into eight periods:
       1. A wash-out period of 14 days (day 1-14)   2. An ingestion period of  Lactobacillus  crispatus VPC 111 during 14 days orally (day 15-28).   3. A wash-out period of 14 days (day 29-42)   4. An ingestion period of  Lactobacillus gasseri  VPG 44 during 14 days orally (day 43-56).   5. A wash-out period of 14 days (day 57-70)   6. An ingestion period of  Lactobacillus  crispatus VPC 177 during 14 days orally (day 71-84).   7. A wash-out period of 14 days (day 85-98)   8. An ingestion period of  Lactobacillus plantarum  HEAL 9 during 14 days orally (day 99-112).       
 
       Subjects and Recruitment 
       [0093]    36 Healthy women aged 18-65 years were recruited by sending out information to the employees at Ideon and Chemical Centre (Lund University) in Lund. Employees of Probi AB, known intolerance or allergy to any ingredient included in the formulations, current treatment for severe gastrointestinal disorders and current treatment for vaginal disorders were excluded. 
         [0094]    The experimental products contained lyophilized  Lactobacillus crispatus  VPC177,  Lactobacillus crispatus  VPC 111,  Lactobacillus plantarum  HEAL9 and  Lactobacillus gasseri  VPG44, respectively. Sucrose, maltodextrine and hydrolysed gelatine were added as cryoprotectants. 
         [0095]    The daily intake was 2×1 g lyophilized lactobacilli (approximately 1×10 9  cfu/day) orally. The doses were taken morning and evening and were to be ingested in association with a meal. The product was supplied in sachets. 
       Dietary Regulations 
       [0096]    From day 1 to day 113, the subjects were not allowed to ingest products containing probiotic bacteria. Each subject was provided with a list of probiotic products not allowed to be consumed during the study period. This was done in order to secure that other probiotic bacteria could not interfere with the experiment. 
       Concomitant Medication 
       [0097]    All concomitant medication was to be noted on the Case Report Form (CRF) and in the diary. 
         [0098]    Exclusion criteria during the study were subjects who had not taken &gt;4 doses (2 days) in succession or have not taken a total of &gt;8 doses (4 days) during each ingestion period of the study, subjects who had not followed the dietary regulations, subjects who had started a course of medical treatment for severe gastrointestinal disease between day 1 and day 112 (on the discretion of the principal investigator), and subjects who wished to discontinue the study. 
         [0099]    Faecal and vaginal samples were handed in on days 15, 29, 43, 57, 71, 85, 99 and 113. The sample on day 15, 43, 71 and 99 was collected before taking the first dose of the periods experiment product. The samples were collected no more than 18 hours before being handed in for analysis, and during this period stored in a refrigerator. The samples were analyzed for lactobacilli. 
         [0100]    Vaginal samples was collected by inserting a vaginal swab (Copan amies agar gel swabs; Copan innovation, Brescia, Italy) through a cap to prevent bacterial contamination. The samples were collected no more than 3 hours before being handed in for analysis, and during this period stored in a refrigerator. 
         [0101]    The samples were analyzed microbiologically (according to Johansson et al., 1998) with regard to the lactobacilli flora. (Rogosa agar, anaerobically, 3 days at 37° C.) The different lactobacilli were identified with Randomly Amplified Polymorphic DNA analysis (RAPD) according to Johansson et al., Lett. Appl. Microbiol. 1995; 21: 155-159. 
         [0102]    The statistical values were calculated according to Wilcoxon signed ranks test, see table 1 and 2. 
       Results 
       [0103]    Of the 36 women included 31 participated in period 1 and 2 (WO+VPC111), 34 in period 3 and 4 (WO+VPG44), 28 in period 5 and 6 (WO+VPC177) and 31 in period 7 and 8 (HEAL 9). 25 of the women participated in all of the study periods. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                   Lactobacilli  (mean (min-max)) in faecal samples before 
               
               
                 and after 2 weeks intake of experimental product. Values 
               
               
                 under detection level are set to 3.0 and values over detection 
               
               
                 level are set to 9.0 to enable calculation. 
               
             
          
           
               
                   
                 Faecal counts (cfu/g) 
                   
               
             
          
           
               
                   
                 Strain in exp 
                 Total 
                 Administered 
               
               
                   
                 product 
                 
                   lactobacilli 
                 
                 exp. strain 
               
               
                   
                   
               
               
                   
                 VPC111 
                   
                   
               
               
                   
                 Before intake 
                 4.9 (&lt;3-7.3) 
                 3.0 (&lt;3-4.6) 
               
               
                   
                 After intake 
                 6.2 (&lt;3-&gt;9) 
                 3.0 (&lt;3) 
               
               
                   
                   
                 p = 0.01 
               
               
                   
                 VPG44 
               
               
                   
                 Before intake 
                 5.8 (&lt;3-&gt;9) 
                 3.0 (&lt;3-4.2) 
               
               
                   
                 After intake 
                 6.1 (3.8-8.7) 
                 3.3-(&lt;3-5.9) 
               
               
                   
                 VPC177 
               
               
                   
                 Before intake 
                 5.4 (&lt;3.0-9.0) 
                 3.1 (&lt;3-4.5) 
               
               
                   
                 After intake 
                 7.0 (&lt;3.0-9.8) 
                 3.3 (&lt;3-8.1) 
               
               
                   
                   
                 p = 0.000 
               
               
                   
                 HEAL9 
               
               
                   
                 Before intake 
                 6.9 (&lt;3-&gt;9) 
                 3.0 (&lt;3) 
               
               
                   
                 After intake 
                 7.2 (4.0-9.4) 
                 5.8 (&lt;3-8.0) 
               
               
                   
                   
                   
                 p = 0.000 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   Lactobacilli  (mean (min-max)) in vaginal samples before and after 
               
               
                 2 weeks intake of experimental product. Values under detection level 
               
               
                 are set to 2.0 for vaginal samples to enable calculation. 
               
             
          
           
               
                   
                 counts (log cfu/swab) 
                   
               
             
          
           
               
                   
                 Strain in exp. 
                 Total 
                 Administered 
               
               
                   
                 product 
                 
                   lactobacilli 
                 
                 exp. strain 
               
               
                   
                   
               
               
                   
                 VPC111 
                   
                   
               
               
                   
                 Before intake 
                 5.0 (&lt;2-8.2) 
                 2.0 (&lt;2-4.6) 
               
               
                   
                 After intake 
                 4.9 (&lt;2-8.0) 
                 2.3 (&lt;2-7.3) 
               
               
                   
                 VPG44 
               
               
                   
                 Before intake 
                 5.1 (&lt;2-8.8) 
                 2.0 (&lt;2) 
               
               
                   
                 After intake 
                 5.2 (&lt;2-8.4) 
                 2.0 (&lt;2) 
               
               
                   
                 VPC177 
               
               
                   
                 Before intake 
                 4.7 (&lt;2-8.0) 
                 2.2 (&lt;2-6.6) 
               
               
                   
                 After intake 
                 5.3 (&lt;2-8.2) 
                 2.2 (&lt;2-7.2) 
               
               
                   
                   
                 p = 0.03 
               
               
                   
                 HEAL9 
               
               
                   
                 Before intake 
                 5.4 (&lt;2-8.3) 
                 2.0 (&lt;2) 
               
               
                   
                 After intake 
                 5.3 (&lt;2-8.5) 
                 2.1 (&lt;2-4.2) 
               
               
                   
                   
               
             
          
         
       
     
         [0104]    The two strains of  Lactobacillus  crispatus VPC111 and VPC177 caused significant increases of total lactobacilli in faecal flora. In addition significant increases in total lactobacilli in vaginal fluid were observed after intake of VPC177 orally. Thus, it is very interesting to note that after taking the probiotic bacteria  Lactobacillus  crispatus VPC111 and VPC177 orally a total increase of the lactobacilli in faeces is observed. A significant increase of total Lactobacilli is also observed in vaginal fluid after taking  Lactobacillus crispatus  VPC177 orally. Thus, it is not necessary to achieve beneficial effects in the vagina or rectum in terms of healthy Lactobacilli by taking the strain in a suppository or vaginal insert. It has now been shown that intake of certain Lactobacilli, such as crispatus VPC111 and VPC177, orally leads to a total increase of lactobacilli in faeces as well as in vaginal fluid. 
         [0105]    The lack of effect from  L. plantarum  HEAL9 on both faecal and vaginal flora could be due to the high initial concentration of total lactobacilli. It could be speculated that the wash-out period after intake of VPC177 was not long enough ( FIGS. 1 and 2 ). However, it is worth noting that in 27 women out of 31 , L. plantarum  HEAL 9 could be recovered by Randomly Amplified Polymorphic DNA analysis (RAPD) according to Johansson et al., Lett. Appl. Microbiol. 1995; 21: 155-159. The other experimental strains were also recovered in women tested by (RAPD) after the intake. 
         [0106]    In conclusion, the significant increase of total lactobacilli concentration in faeces and vaginal fluid indicates that the Lactobacilli were active and established. Since no other probiotic material was supplied during each intake period it must be the taken probiotic bacterial strain that achieves the effect. 
         [0107]    Thus, vaginal lactobacilli strains, i.e. strains that have been isolated from the vagina originally, can survive the passage through the gastrointestinal tract via a lyophilised product taken orally and are able to re-colonize the vagina.  Lactobacillus  crispatus VPC177 in particular increases the total concentration of lactobacilli in vagina. This is very surprising since it is known that the stability of isolated vaginal Lactobacilli strains is very low due to a very high sensitivity. To increase the beneficial effects of the bacterial strains of the invention in terms of vaginal health, administration of the bacterial strain both orally and vaginally will help the vagina to stay healthy even more.