Curative effect of the probiotic strain Lactobacillus fermentum L23 in a murine model of vaginal infection by Gardnerella vaginalis

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1 Letters in Applied Microbiology ISSN 66-8 ORIGINAL ARTICLE Curative effect of the probiotic strain Lactobacillus fermentum L in a murine model of vaginal infection by Gardnerella vaginalis M. Daniele, L. Pascual and L. Barberis Departamento de Microbiologıa e Inmunologıa, Universidad Nacional de Rıo Cuarto (UNRC), Rıo Cuarto, Argentina Significance and Impact of the Study: The use of the probiotic strain Lactobacillus fermentum L as a biotherapeutic agent can be expected to prevent and treat genital infections, particularly recurrent bacterial vaginosis, with similar concentrations to those normally used in commercial formulas. It is likely that the use of this probiotic strain for the treatment of bacterial vaginosis will provide a natural and nontoxic treatment modality. Keywords bacterial vaginosis, curative effect, Gardnerella vaginalis, lactobacilli, probiotic. Correspondence Liliana Pascual, Departamento de Microbiologıa e Inmunologıa, Universidad Nacional de Rıo Cuarto (UNRC), 8 Rıo Cuarto, Cordoba, Argentina. lpascual@exa.unrc.edu.ar /79: received September, revised 8 February and accepted March doi:./lam.9 Abstract Bacterial vaginosis is a common vaginal infection characterized by changes in the vaginal microbiota. The objective of this work was to evaluate the colonization ability and curative effect of Lactobacillus fermentum L after vaginal administration in female BALB/c mice infected with Gardnerella vaginalis. One dose of Lact. fermentum L containing 9 CFU ml was administered locally in a murine vaginal model. L colonized the vaginal tract of BALB-c mice after one inoculation. The infection by G. vaginalis in a murine model was induced by vaginal administration of a 9 6 CFU ml suspension. Infection with the pathogen was observed in the vaginal tract for days. At h after inoculation, levels of log CFU ml were observed. The curative effect of L was evaluated with one administration at 9 9 CFU ml 7 h after the inoculation with G. vaginalis. Lactobacillus fermentum L inhibited the growth of G. vaginalis. The results of suppression of G. vaginalis using different concentrations of L were favourable due that these concentrations are normally used in commercial formulas. The obtained results indicate that Lact. fermentum L inhibited the growth of G. vaginalis. Therefore, L might be used as a potential biotherapeutic agent for the elimination of this bacterium. Introduction The healthy vaginal environment of women in the childbearing age is characterized by a complex ecology of micro-organisms where lactobacilli are the predominant species (Ehrstr om et al. ). Bacterial vaginosis (BV) is a common vaginal infection causing significant gynecological and obstetric morbidity. BV is a polymicrobial syndrome where the lactobacilli-dominated vaginal microbiota is replaced by a mix of other, mainly anaerobic bacterial species (Anukam et al. 6). Thus, BV is characterized by changes in the vaginal microbiota, from the predominant biota of lactobacilli to an increase in the populations of Gram variable coccobacilli such as Gardnerella vaginalis, among other species (Catlin 99; Ngugi et al. ). Bacterial vaginosis is characterized by a thin, greyishwhite, malodorous discharge, a vaginal ph >, and presence of clue cells (Amsel criteria) (Catlin 99). In addition to cause irritating symptoms, BV has been associated with pelvic inflammatory disease, infections following gynecological surgery, and preterm birth (Modares Nejad and Shafaie 8). It has also been suggested that the presence of BV increases the risk of infection by the human immunodeficiency virus (Guedou et al. ). Letters in Applied Microbiology 9, The Society for Applied Microbiology 9

2 Probiotic bacterium in vaginosis M. Daniele et al. As the treatment for urogenital infections is not always effective, and problems remain due to bacterial and yeast resistance, recurrent infections and side effects, it is not surprising that alternative therapies are of interest to both the patients and their caregivers (Kumar et al. ). It is assumed that recurrences are due to antimicrobials failing to eradicate the pathogens, perhaps because of biofilm resistance, or virulent organisms coming back from their source (the patient s gut, or a sex partner) and attacking a host whose defences are suboptimal. Young girls who suffer from urinary tract infection (UTI) are more likely to have repeated episodes during their adulthood. Indeed, many UTI, BV and yeast vaginitis patients will have a recurrence (Kumar et al. ). Recurrent infections may also be due to the elimination of the commensal organisms from the vagina by antimicrobial compounds, thereby increasing susceptibility to recolonization by pathogens. This is one of the main reasons for considering the use of probiotics, that is, to replenish the commensal microbes as a way to lower the risk of reinfections (Quigley ). Lactobacilli probiotics may displace G. vaginalis biofilms and could potentially re-establish protective biofilms in the female urogenital tract (Saunders et al. 7; Turovskiy et al. 9). Probiotics are defined as living microorganisms which, when administered in adequate amounts, confer a health benefit on the host (Pascual et al. ). Lactobacilli are the most common organisms used as probiotics. The use of lactobacilli isolated from humans as probiotics is promising for restoring and maintaining a healthy genital tract (Reid and Burton ). As the vaginal microbiota of women with BV has been found to contain a reduced number of lactobacilli in comparison with healthy women, lactobacilli administered orally or intravaginally have been tested for their effectiveness in colonizing the vagina and curing women with BV, or at least preventing its recurrence (Falagas et al. 7; Homayouni et al. ). The objective of this work was to evaluate the colonization ability and curative effect of Lact. fermentum strain L after vaginal administration in female BALB/c mice infected with G. vaginalis. Results and discussion Our research group has isolated lactobacilli from human vagina, selecting those with beneficial or probiotic properties. These lactobacilli produce different bacteriocins of similar activity and play an important role in maintaining the vaginal health, which can prevent the overgrowth of micro-organisms in the vaginal environment (Pascual et al. 8b, ; Daniele et al. ). In this work, their efficacy against major BV is studied. By analysing the direct vaginal spreads obtained from each of the mice for the study of the microbiota, we observed the presence of Gram-positive cocci and bipolar bacilli. These observations correlated with the white colonies that grew on blood agar plates under partial CO tension conditions. There was no growth on Man Rogosa Sharpe (MRS) plates or typical colonies of G. vaginalis on blood agar. This indicated an absence of Lactobacillus and G. vaginalis strains. Our results demonstrated that the microbiota of mice would not interfere with the results obtained after the experiences of colonization, infection and curative effect. The presence of lactobacilli in the microbiota of the animal model could be a problem for the evaluation of the biotherapeutic effect of Lact. fermentum strain L. In contrast, others authors have reported on the presence of lactobacilli as members of the vaginal microbiota of experimental animals (Zarate et al. 7). Lactobacillus fermentum L strain colonized the vaginal tract of female BALB-c mice. One inoculation was sufficient for the probiotic strain to establish in the vaginal tract of mice. Lactobacillus fermentum L levels in the vaginal tract remained fairly high for 7 h, ranging from 6 to8 log CFU ml. At 96 h after inoculation, the level was of log CFU ml. At the end of the study, counts of strain L were of log CFU ml (Fig. ). The infection by G. vaginalis in a murine vaginal tract model was induced by vaginal administration of a 9 6 CFU ml suspension of this pathogenic microorganism. Infection with the pathogen was observed in the vaginal tract for days with values of log CFU ml. At h after inoculation, levels of log CFU ml were observed. At the end of the study, Log cfu ml Time (h) Figure Vaginal colonization of Lactobacillus fermentum L in BALB/c mice. 9 Letters in Applied Microbiology 9, The Society for Applied Microbiology

3 M. Daniele et al. Probiotic bacterium in vaginosis counts were of log CFU ml (Fig. ). The clinical G. vaginalis strain used in this experimental infection persisted in the animal model in high numbers for more than 7 days. Increased homogeneous vaginal discharge was observed in a murine vaginal tract. This persistence was achieved without a pretreatment to traumatize the mucosal surface (Asahara et al. ; Pascual et al. ). The curative effect of probiotic L was evaluated with one administration of this strain at 9 9 CFU ml 7 h after the inoculation with G. vaginalis. Lactobacillus fermentum L inhibited the growth of G. vaginalis (Fig. ). Inoculation with L into the vagina after a G. vaginalis infection resulted in a complete cure. Indeed, L exhibited a significant antimicrobial activity in the postinfection treatment. These results agree with those reported in previous experiments by Pascual et al. () on clinical Escherichia coli strains. These findings would reaffirm the antimicrobial activity of L. The assay of curative effect with different concentrations (, 6, 8 and 9 ) of viable L cells ml was studied in the vaginal tract of mice. Concentrations of 9 and 8 CFU ml of Lact. fermentum L suppressed G. vaginalis after 7 h, while the inoculation of 6 CFU ml suppressed G. vaginalis after h of inoculation (Fig. ). On the other hand, the inoculation of L at CFU ml caused a high and constant colonization level by the pathogen (data not shown). The results of suppression of the bacterial pathogen (BV) using different concentrations of L ( 8 9 CFU ml ) were favourable due that these concentrations are normally used in commercial formulas. Furthermore, in this study, we have shown that the use of a concentration of 6 CFU ml of Lact. fermentum L can successfully eradicate the pathogen from the vaginal niche of mice. Our results differed from a previous report by Zarate et al. (7), who needed two doses of 9 lactobacilli to eliminate another pathogen in the same animal model. To our knowledge, there is a lack of works where different concentrations of a Lactobacillus strain are studied to prove the biological control activity of this probiotic micro-organism against G. vaginalis on this animal model. With the advent of increasing cases of antibiotic-resistant pathogenic micro-organisms, the use of probiotics for the treatment of BV is being extensively studied. The use of probiotics for the treatment of BV has provided a ray of hope by natural and nontoxic treatment modality (Kumar Log cfu ml Time (h) Figure Vaginal curative effect of Lactobacillus fermentum in BALB/c mice infected with Gardnerella vaginalis. ( ) Lact. fermentum and ( ) G. vaginalis. 7 6 Log cfu ml Log cfu ml Time (days) Time (h) Figure Gardnerella vaginalis infection in vagina of BALB/c mice. Figure Curative effect found with different concentrations of lactobacilli on the vaginal infection by Gardnerella vaginalis in mice. ( ) G. vaginalis; (-- --) G. v treated with Lactobacillus fermentum 9 CFU ml ; (-- --) G. v treated with Lact. fermentum 8 CFU ml ; and ( ) Gvtreated with Lact. fermentum CFU ml. Letters in Applied Microbiology 9, The Society for Applied Microbiology 9

4 Probiotic bacterium in vaginosis M. Daniele et al. et al. ). The antimicrobial metabolites produced by the probiotics have shown a great potential not only to restrict the growth of antimicrobial resistant strains but also to kill them. Although the probiotics have shown a positive effect on the treatment of BV, their effectiveness has differed from patient to patient and their reaction time against the infection can be slow (Kumar et al. ; Sungkar et al. ). The use of certain Lactobacillus strains such as Lact. fermentum for treatment of genital infection is promising, especially for recurrent BV. This model animal might partly explain differential susceptibility to vaginal infection by G. vaginalis within the human population. Therefore, the consumption of probiotics could be useful to improve public health among women, coinciding with observations by Stecher et al. (). A great deal of scientific evidence supports, by in vivo studies, the effectiveness of probiotics to prevent the attachment or stimulate the removal of vaginal pathogens. Probiotics are increasingly being used to treat and to prevent urogenital infections. However, further research and larger studies with optimal administration procedures are needed. Future controlled randomized assays of use of lactobacilli in bacterial genitourinary infections in women should be adequately developed to detect treatment effects, should use Lactobacillus strains and dosing strategies that have a documented efficacy, and should monitor the integrity of probiotic products over the course of the study. Materials and methods Bacterial strain A strain of G. vaginalis was isolated from a patient with BV. Nugent score and Amsel criteria were used for BV confirmation. The strain was cultured on Columbia agar (SR 9 Oxoid, Basingstoke, UK) containing gentamicin sulphate, nalidixic acid, amphotericin B and human blood (%), under optimal conditions of incubation at 7 C for 8 h. It was identified as G. vaginalis by Gram staining followed by standard biochemical tests (Bergey 99). Lactobacillus strain Lactobacillus fermentum L was originally isolated from vaginal smears of a healthy woman and was previously characterized by its probiotic properties and technological potential. Lact. fermentum L displayed relevant properties such as bacteriocinogenic and colonization abilities, self-aggregation, adherence to vaginal epithelial cells, and co-aggregation with bacterial pathogens (Pascual et al. 8a,b). The strain was grown overnight at 7 C in MRS medium (Biokar Diagnostics, Beauvais, France) under limited aerobic conditions. The strain was identified by standard biochemical tests (Bergey 99) and an API CH biochemical identification system (biomerieux, Craponne, France) (Nigatu et al. ). The 6S rrna gene from Lact. fermentum L was sequenced with an Applied Biosystems model 7XL automated DNA sequencing system by Macrogen Laboratories (Korea). The obtained sequence was subjected to a BLAST search ( nih.gov/blast/) to find identities between sequences. Assay with animal model Mice were obtained from stocks maintained at the National University of Rıo Cuarto. For this assay, 7-weekold female BALB/c mice were used. Mice received a normal diet and water ad libitum, and were kept in isolation cubicles at a constant temperature of C, with a cycle of -h fluorescent light/-h darkness. The microbiota of the mice was studied from vaginal samples taken with swabs. Swabs were sown at 7 C on plates of MRS agar under partial CO tension and % blood agar. Strains grown on MRS and blood agar plates were identified by standard diagnostic procedures (Bergey 99). In this study were included four mice per group. The first group was used to study microbial colonization by the Lact. fermentum strain L, the second one for vaginal infection with G. vaginalis and the other group for determining curative effect. The assay was performed by triplicate. Colonization of vaginal tract by L Lactobacillus fermentum strain L was cultured in MRS broth at 7 C for h, resuspended in PBS (ph 67) and adjusted to an approximate concentration of 9 9 CFU ml. Mice were intravaginally inoculated with -ll inoculum of the lactobacillus suspension using an Eppendorf pipette (Pascual ). At h postinoculation with L, the first sample was taken from the vagina, serial dilutions were performed, and -ll aliquots were inoculated onto MRS agar plates (CFU ml at T ). The microbial colonization of the mice was monitored on days 7 by counting colonies of viable bacteria (CFU) recovered from the vaginal fluids of mice, and serially diluted. To determine the CFU, each dilution was inoculated onto MRS agar plates. The plates were incubated at 7 C for h under partial CO tension (CO incubator). The number of CFU was expressed as log CFU ml of vaginal fluid (Pascual ). 96 Letters in Applied Microbiology 9, The Society for Applied Microbiology

5 M. Daniele et al. Probiotic bacterium in vaginosis Vaginal infection A suspension of G. vaginalis adjusted to 9 6 CFU ml was prepared in PBS. A tip, containing ll of the suspension, was inserted into the vagina. Infection levels on days 7 after inoculation were evaluated by quantifying the number of viable cells. Samples were collected from all of the mice, and serial dilutions were performed. One aliquot was plated with onto Columbia blood agar plates with % sheep blood (biomerieux) and grown at 7 C for h and 8 h. Results were expressed as log CFU ml. Colonies were identified by standard methods using morphological characteristics, Gram stain and standard biochemical tests suggested by Catlin B W (Catlin 99). Curative effect A G. vaginalis suspension ( 9 6 CFU ml ) was vaginally inoculated. After 7 h, a suspension of Lact. fermentum L ( 9 9 CFU ml ) in PBS was inoculated. To evaluate the curative activity, serial dilutions of vaginal swabs were cultured daily for 7 days on blood and MRS agar plates. Lactobacillus fermentum L was incubated under limited aerobic conditions for h. Aerobic conditions were required for G. vaginalis. Rating of the effective dose Doses of, 6, 8 and 9 viable L cells ml were used. We followed the same procedure as in the curative effect assays. Statistical analysis All bacterial counts were expressed as means SD and were log-transformed for each experiment. An analysis of variance (SIGMA STAT STATISTICAL Software ver.., for Windows NT and.; SPSS, Chicago, IL) was used for differences in numbers of viable micro-organisms from different treatment groups. A P value of was considered statistically significant. Acknowledgements This study was supported by the Secretarıa de Ciencia y Tecnica de la Universidad Nacional de Rıo Cuarto, Cordoba, Argentina. Conflict of Interest The authors declare that they have no conflict of interest. References Anukam, K.C., Osazuwa, E.O., Ahonkhai, I. and Gregor, R. (6) Lactobacillus vaginal microbiota of women attending a reproductive health care service in Benin City, Nigeria. Sex Transm Dis, 9 6. Asahara, T., Nomoto, K., Watanuki, M. and Yokokura, T. () Antimicrobial activity of intraurethrally administered probiotic Lactobacillus casei in a murine model of Escherichia coli urinary tract infection. Antimicrob Agents Chemother, Bergey (99) in Manual of Determinative Bacteriology 9th edn, pp. 7 7, 7-89, 7-8.Edited by Holt, J.G. Baltimore: Williams & Wilkins. Catlin, B. (99) Gardnerella vaginalis: Characteristics, clinical consideration and controversies. Clin Microbiol Rev, 7. Daniele, M., Ruiz, F., Pascual, L. and Barberis, L. () Ureaplasma urealyticum and Mycoplasma hominis sensitivity to bacteriocins produced by two Lactobacilli strains. Curr Microbiol 6, 6 6. Ehrstr om, S., Daroczy, K., Rylander, E., Samuelsson, C., Johannesson, U., Anzen, B. and Pahlson, C. () Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis. Microbes Infect, Falagas, M., Betsi, G.I. and Athanasiou, S. (7) Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infect, Guedou, F.A., Van Damme, L., Mirembe, F., Solomon, S., Becker, M., Deese, J., Crucitti, T. and Alary, M. () Intermediate vaginal flora is associated with HIV prevalence as strongly as bacterial vaginosis in a cross-sectional study of participants screened for a randomised controlled trial. Sex Transm Infect 88,. Homayouni, A., Bastani, P., Ziyadi, S., Mohammad-Alizadeh- Charandabi, S., Ghalibaf, M., Mortazavian, A.M. and Mehrabany, E.V. () Effects of Probiotics on the recurrence of bacterial vaginosis: a review. J Low Genit Tract Dis 8, Kumar, N., Behera, B., Sagiri, S., Pal, K., Ray, S. and Roy, S. () Bacterial vaginosis: etiology and modalities of treatment - A brief note. J Pharm Bioallied Sci, 96. Modares Nejad, V. and Shafaie, S. (8) The association of bacterial vaginosis and preterm labor. J Pak Med Assoc 8, 6. Ngugi, B.M., Hemmerling, A., Bukusi, E.A., Kikuvi, G., Gikunju, J., Shiboski, S., Fredricks, D.N. and Cohen, C.R. () Effects of bacterial vaginosis-associated bacteria and sexual intercourse on vaginal colonization with the probiotic Lactobacillus crispatus CTV-. Sex Transm Dis 8, 7. Letters in Applied Microbiology 9, The Society for Applied Microbiology 97

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