Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Curative effect of the probiotic strain Lactobacillus fermentum L23 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 L23 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 rdoRıo Cuarto (UNRC), 5800 Rıo Cuarto, Co ba, Argentina. E-mail: [email protected] 2013/1793: received 2 September 2013, revised 18 February 2014 and accepted 12 March 2014 doi:10.1111/lam.12249
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 L23 after vaginal administration in female BALB/c mice infected with Gardnerella vaginalis. One dose of Lact. fermentum L23 containing 109 CFU ml1 was administered locally in a murine vaginal model. L23 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 1 9 106 CFU ml1 suspension. Infection with the pathogen was observed in the vaginal tract for 4 days. At 144 h after inoculation, levels of 4 log10 CFU ml1 were observed. The curative effect of L23 was evaluated with one administration at 1 9 109 CFU ml1 72 h after the inoculation with G. vaginalis. Lactobacillus fermentum L23 inhibited the growth of G. vaginalis. The results of suppression of G. vaginalis using different concentrations of L23 were favourable due that these concentrations are normally used in commercial formulas. The obtained results indicate that Lact. fermentum L23 inhibited the growth of G. vaginalis. Therefore, L23 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. 2010). 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. 2006). Thus, BV is characterized by changes in the vaginal microbiota, from the predominant biota of lactobacilli to an increase in the Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
populations of Gram variable coccobacilli such as Gardnerella vaginalis, among other species (Catlin 1992; Ngugi et al. 2011). Bacterial vaginosis is characterized by a thin, greyishwhite, malodorous discharge, a vaginal pH > 45, and presence of clue cells (Amsel criteria) (Catlin 1992). 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 2008). It has also been suggested that the presence of BV increases the risk of infection by the human immunodeficiency virus (Guedou et al. 2012). 1
Probiotic bacterium in vaginosis
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. 2008b, 2010; Daniele et al. 2011). In this work, their efficacy against major BV is studied. 2
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 CO2 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 L23. In contrast, others authors have reported on the presence of lactobacilli as members of the vaginal microbiota of experimental animals (Zarate et al. 2007). Lactobacillus fermentum L23 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 L23 levels in the vaginal tract remained fairly high for 72 h, ranging from 46 to 38 log10 CFU ml1. At 96 h after inoculation, the level was of 34 log10 CFU ml1. At the end of the study, counts of strain L23 were of 25 log10 CFU ml1 (Fig. 1). The infection by G. vaginalis in a murine vaginal tract model was induced by vaginal administration of a 1 9 106 CFU ml1 suspension of this pathogenic microorganism. Infection with the pathogen was observed in the vaginal tract for 4 days with values of 5 log10 CFU ml1. At 144 h after inoculation, levels of 4 log10 CFU ml1 were observed. At the end of the study,
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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. 2011). 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 patient0 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. 2011). 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 2010). Lactobacilli probiotics may displace G. vaginalis biofilms and could potentially re-establish protective biofilms in the female urogenital tract (Saunders et al. 2007; Turovskiy et al. 2009). Probiotics are defined as living microorganisms which, when administered in adequate amounts, confer a health benefit on the host (Pascual et al. 2010). 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 2002). 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. 2007; Homayouni et al. 2013). The objective of this work was to evaluate the colonization ability and curative effect of Lact. fermentum strain L23 after vaginal administration in female BALB/c mice infected with G. vaginalis.
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Figure 1 Vaginal colonization of Lactobacillus fermentum L23 in BALB/c mice.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Probiotic bacterium in vaginosis
by Zarate et al. (2007), who needed two doses of 109 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 6 5
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counts were of 35 log10 CFU ml1 (Fig. 2). 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. 2001; Pascual et al. 2010). The curative effect of probiotic L23 was evaluated with one administration of this strain at 1 9 109 CFU ml1 72 h after the inoculation with G. vaginalis. Lactobacillus fermentum L23 inhibited the growth of G. vaginalis (Fig. 3). Inoculation with L23 into the vagina after a G. vaginalis infection resulted in a complete cure. Indeed, L23 exhibited a significant antimicrobial activity in the postinfection treatment. These results agree with those reported in previous experiments by Pascual et al. (2010) on 100 clinical Escherichia coli strains. These findings would reaffirm the antimicrobial activity of L23. The assay of curative effect with different concentrations (104, 106, 108 and 109) of viable L23 cells ml1 was studied in the vaginal tract of mice. Concentrations of 109 and 108 CFU ml1 of Lact. fermentum L23 suppressed G. vaginalis after 72 h, while the inoculation of 106 CFU ml1 suppressed G. vaginalis after 120 h of inoculation (Fig. 4). On the other hand, the inoculation of L23 at 104 CFU ml1 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 L23 (108– 9 1 10 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 106 CFU ml1 of Lact. fermentum L23 can successfully eradicate the pathogen from the vaginal niche of mice. Our results differed from a previous report
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Time (h) Figure 3 Vaginal curative effect of Lactobacillus fermentum in BALB/c mice infected with Gardnerella vaginalis. (–■–) Lact. fermentum and (–♢–) G. vaginalis.
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Figure 2 Gardnerella vaginalis infection in vagina of BALB/c mice.
Figure 4 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 109 CFU ml1; (--♦--) G. v treated with Lact. fermentum 108 CFU ml1; and (–▼–) G v treated with Lact. fermentum 105 CFU ml1.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Probiotic bacterium in vaginosis
et al. 2011). 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. 2011; Sungkar et al. 2012). 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. (2010). 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 119 Oxoid, Basingstoke, UK) containing gentamicin sulphate, nalidixic acid, amphotericin B and human blood (5%), under optimal conditions of incubation at 37°C for 24– 48 h. It was identified as G. vaginalis by Gram staining followed by standard biochemical tests (Bergey 1994). Lactobacillus strain Lactobacillus fermentum L23 was originally isolated from vaginal smears of a healthy woman and was previously characterized by its probiotic properties and technological potential. Lact. fermentum L23 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. 2008a,b). 4
M. Daniele et al.
The strain was grown overnight at 37°C in MRS medium (Biokar Diagnostics, Beauvais, France) under limited aerobic conditions. The strain was identified by standard biochemical tests (Bergey 1994) and an API 50 CH biochemical identification system (bioMerieux, Craponne, France) (Nigatu et al. 2000). The 16S rRNA gene from Lact. fermentum L23 was sequenced with an Applied Biosystems model 3730XL automated DNA sequencing system by Macrogen Laboratories (Korea). The obtained sequence was subjected to a BLAST search (www.ncbi.nlm. 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 24°C, with a cycle of 12-h fluorescent light/12-h darkness. The microbiota of the mice was studied from vaginal samples taken with swabs. Swabs were sown at 37°C on plates of MRS agar under partial CO2 tension and 5% blood agar. Strains grown on MRS and blood agar plates were identified by standard diagnostic procedures (Bergey 1994). In this study were included four mice per group. The first group was used to study microbial colonization by the Lact. fermentum strain L23, 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 L23 Lactobacillus fermentum strain L23 was cultured in MRS broth at 37°C for 24 h, resuspended in PBS (pH 67) and adjusted to an approximate concentration of 1 9 109 CFU ml1. Mice were intravaginally inoculated with 20-ll inoculum of the lactobacillus suspension using an Eppendorf pipette (Pascual 2004). At 4 h postinoculation with L23, the first sample was taken from the vagina, serial dilutions were performed, and 20-ll aliquots were inoculated onto MRS agar plates (CFU ml1 at T0). The microbial colonization of the mice was monitored on days 1–7 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 37°C for 24 h under partial CO2 tension (CO2 incubator). The number of CFU was expressed as log10 CFU ml1 of vaginal fluid (Pascual 2004). Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
M. Daniele et al.
Vaginal infection A suspension of G. vaginalis adjusted to 1 9 106 CFU ml1 was prepared in PBS. A tip, containing 20 ll of the suspension, was inserted into the vagina. Infection levels on days 1–7 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 5% sheep blood (bioMerieux) and grown at 37°C for 24 h and 48 h. Results were expressed as log10 CFU ml1. Colonies were identified by standard methods using morphological characteristics, Gram stain and standard biochemical tests suggested by Catlin B W (Catlin 1992). Curative effect A G. vaginalis suspension (1 9 106 CFU ml1) was vaginally inoculated. After 72 h, a suspension of Lact. fermentum L23 (1 9 109 CFU ml1) 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 L23 was incubated under limited aerobic conditions for 24 h. Aerobic conditions were required for G. vaginalis. Rating of the effective dose Doses of 104, 106, 108 and 109 viable L23 cells ml1 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. 2.0, for Windows NT and 3.1; SPSS, Chicago, IL) was used for differences in numbers of viable micro-organisms from different treatment groups. A P value of 005 was considered statistically significant. Acknowledgements This study was supported by the Secretarıa de Ciencia y Tecnica de la Universidad Nacional de Rıo Cuarto, C ordoba, Argentina. Conflict of Interest The authors declare that they have no conflict of interest.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Probiotic bacterium in vaginosis
References Anukam, K.C., Osazuwa, E.O., Ahonkhai, I. and Gregor, R. (2006) Lactobacillus vaginal microbiota of women attending a reproductive health care service in Benin City, Nigeria. Sex Transm Dis 33, 59–62. Asahara, T., Nomoto, K., Watanuki, M. and Yokokura, T. (2001) Antimicrobial activity of intraurethrally administered probiotic Lactobacillus casei in a murine model of Escherichia coli urinary tract infection. Antimicrob Agents Chemother 45, 1751–1760. Bergey (1994) in Manual of Determinative Bacteriology 9th edn, pp. 71–174, 175-289, 527-558.Edited by Holt, J.G. Baltimore: Williams & Wilkins. Catlin, B. (1992) Gardnerella vaginalis: Characteristics, clinical consideration and controversies. Clin Microbiol Rev 5, 213–237. Daniele, M., Ruiz, F., Pascual, L. and Barberis, L. (2011) Ureaplasma urealyticum and Mycoplasma hominis sensitivity to bacteriocins produced by two Lactobacilli strains. Curr Microbiol 63, 360–365. Ehrstr€ om, S., Daroczy, K., Rylander, E., Samuelsson, C., Johannesson, U., Anzen, B. and Pahlson, C. (2010) Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis. Microbes Infect 12, 691–699. Falagas, M., Betsi, G.I. and Athanasiou, S. (2007) Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infect 13, 657–664. Guedou, F.A., Van Damme, L., Mirembe, F., Solomon, S., Becker, M., Deese, J., Crucitti, T. and Alary, M. (2012) 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, 545–551. Homayouni, A., Bastani, P., Ziyadi, S., Mohammad-AlizadehCharandabi, S., Ghalibaf, M., Mortazavian, A.M. and Mehrabany, E.V. (2013) Effects of Probiotics on the recurrence of bacterial vaginosis: a review. J Low Genit Tract Dis 18, 79–86. Kumar, N., Behera, B., Sagiri, S., Pal, K., Ray, S. and Roy, S. (2011) Bacterial vaginosis: etiology and modalities of treatment - A brief note. J Pharm Bioallied Sci 3, 496–503. Modares Nejad, V. and Shafaie, S. (2008) The association of bacterial vaginosis and preterm labor. J Pak Med Assoc 58, 104–106. Ngugi, B.M., Hemmerling, A., Bukusi, E.A., Kikuvi, G., Gikunju, J., Shiboski, S., Fredricks, D.N. and Cohen, C.R. (2011) Effects of bacterial vaginosis-associated bacteria and sexual intercourse on vaginal colonization with the probiotic Lactobacillus crispatus CTV-05. Sex Transm Dis 38, 1020–1027.
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Nigatu, A., Ahrne, S. and Molin, G. (2000) Temperaturedependent variation in API 50 CH fermentation profiles of Lactobacillus species. Curr Microbiol 41, 21–26. Pascual, L.M. (2004). Bacteriocinogenia en el genero Lactobacillus: caracterısticas beneficas de lactobacilos de vagina humana. Doctoral thesis. Universidad Nacional Rıo Cuarto, C ordoba, Argentina. Pascual, L.M., Daniele, M.B., Giordano, W., Pajaro, M.C. and Barberis, I.L. (2008a) Purification and partial characterization of novel bacteriocin L23 produced by Lactobacillus fermentum L23. Curr Microbiol 56, 397–402. Pascual, L.M., Daniele, M.B., Ruiz, F., Giordano, W., Pajaro, C. and Barberis, L. (2008b) Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina. J Gen Appl Microbiol 54, 141–148. Pascual, L., Ruiz, F., Giordano, W. and Barberis, L. (2010) Vaginal colonization and activity of the probiotic bacterium Lactobacillus fermentum L23 in a murine model of vaginal tract infection. J Med Microbiol 59, 360–364. Quigley, E. (2010) Prebiotics and probiotics; modifying and mining the microbiota. Pharmacol Res 61, 213–218. Reid, G. and Burton, J. (2002) Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4, 319–324.
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Saunders, S., Bocking, A., Challis, J. and Reid, G. (2007) Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms. Colloids Surf B Biointerfaces 55, 138–142. Stecher, B., Chaffron, S., Kp€apeli, R., Hapfelmeier, S., Freedrich, S., Weber, T., Kirundi, J., Suar, M. et al. (2010) Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog 6, 1–15. Sungkar, A., Purwosunu, Y., Aziz, M.F., Pratomo, H., Sutrisna, B. and Sekizawa, A. (2012) Influence of early self-diagnosis and treatment of bacterial vaginosis on preterm birth rate. Int J Gynecol Obstet 117, 264–267. Turovskiy, Y., Ludescher, R., Aroutcheva, A., Faro, S. and Chikindas, M. (2009) Lactocin 160, a bacteriocin produced by vaginal Lactobacillus rhamnosus, targets cytoplasmic membranes of the vaginal pathogen Gardnerella vaginalis. Probiotics Antimicrob Proteins 1, 67–74. Zarate, G., Santos, V. and Nader-Macias, M.E. (2007) Protective effect of vaginal Lactobacillus paracasei CRL 1289 against urogenital infection produced by Staphylococcus aureus in a mouse animal model. Infect Dis Obstet Gynecol 2007, Article ID 48358.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
ORIGINAL ARTICLE
Curative effect of the probiotic strain Lactobacillus fermentum L23 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 L23 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 rdoRıo Cuarto (UNRC), 5800 Rıo Cuarto, Co ba, Argentina. E-mail: [email protected] 2013/1793: received 2 September 2013, revised 18 February 2014 and accepted 12 March 2014 doi:10.1111/lam.12249
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 L23 after vaginal administration in female BALB/c mice infected with Gardnerella vaginalis. One dose of Lact. fermentum L23 containing 109 CFU ml1 was administered locally in a murine vaginal model. L23 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 1 9 106 CFU ml1 suspension. Infection with the pathogen was observed in the vaginal tract for 4 days. At 144 h after inoculation, levels of 4 log10 CFU ml1 were observed. The curative effect of L23 was evaluated with one administration at 1 9 109 CFU ml1 72 h after the inoculation with G. vaginalis. Lactobacillus fermentum L23 inhibited the growth of G. vaginalis. The results of suppression of G. vaginalis using different concentrations of L23 were favourable due that these concentrations are normally used in commercial formulas. The obtained results indicate that Lact. fermentum L23 inhibited the growth of G. vaginalis. Therefore, L23 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. 2010). 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. 2006). Thus, BV is characterized by changes in the vaginal microbiota, from the predominant biota of lactobacilli to an increase in the Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
populations of Gram variable coccobacilli such as Gardnerella vaginalis, among other species (Catlin 1992; Ngugi et al. 2011). Bacterial vaginosis is characterized by a thin, greyishwhite, malodorous discharge, a vaginal pH > 45, and presence of clue cells (Amsel criteria) (Catlin 1992). 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 2008). It has also been suggested that the presence of BV increases the risk of infection by the human immunodeficiency virus (Guedou et al. 2012). 1
Probiotic bacterium in vaginosis
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. 2008b, 2010; Daniele et al. 2011). In this work, their efficacy against major BV is studied. 2
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 CO2 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 L23. In contrast, others authors have reported on the presence of lactobacilli as members of the vaginal microbiota of experimental animals (Zarate et al. 2007). Lactobacillus fermentum L23 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 L23 levels in the vaginal tract remained fairly high for 72 h, ranging from 46 to 38 log10 CFU ml1. At 96 h after inoculation, the level was of 34 log10 CFU ml1. At the end of the study, counts of strain L23 were of 25 log10 CFU ml1 (Fig. 1). The infection by G. vaginalis in a murine vaginal tract model was induced by vaginal administration of a 1 9 106 CFU ml1 suspension of this pathogenic microorganism. Infection with the pathogen was observed in the vaginal tract for 4 days with values of 5 log10 CFU ml1. At 144 h after inoculation, levels of 4 log10 CFU ml1 were observed. At the end of the study,
5
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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. 2011). 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 patient0 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. 2011). 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 2010). Lactobacilli probiotics may displace G. vaginalis biofilms and could potentially re-establish protective biofilms in the female urogenital tract (Saunders et al. 2007; Turovskiy et al. 2009). Probiotics are defined as living microorganisms which, when administered in adequate amounts, confer a health benefit on the host (Pascual et al. 2010). 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 2002). 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. 2007; Homayouni et al. 2013). The objective of this work was to evaluate the colonization ability and curative effect of Lact. fermentum strain L23 after vaginal administration in female BALB/c mice infected with G. vaginalis.
M. Daniele et al.
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Figure 1 Vaginal colonization of Lactobacillus fermentum L23 in BALB/c mice.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
M. Daniele et al.
Probiotic bacterium in vaginosis
by Zarate et al. (2007), who needed two doses of 109 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 6 5
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counts were of 35 log10 CFU ml1 (Fig. 2). 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. 2001; Pascual et al. 2010). The curative effect of probiotic L23 was evaluated with one administration of this strain at 1 9 109 CFU ml1 72 h after the inoculation with G. vaginalis. Lactobacillus fermentum L23 inhibited the growth of G. vaginalis (Fig. 3). Inoculation with L23 into the vagina after a G. vaginalis infection resulted in a complete cure. Indeed, L23 exhibited a significant antimicrobial activity in the postinfection treatment. These results agree with those reported in previous experiments by Pascual et al. (2010) on 100 clinical Escherichia coli strains. These findings would reaffirm the antimicrobial activity of L23. The assay of curative effect with different concentrations (104, 106, 108 and 109) of viable L23 cells ml1 was studied in the vaginal tract of mice. Concentrations of 109 and 108 CFU ml1 of Lact. fermentum L23 suppressed G. vaginalis after 72 h, while the inoculation of 106 CFU ml1 suppressed G. vaginalis after 120 h of inoculation (Fig. 4). On the other hand, the inoculation of L23 at 104 CFU ml1 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 L23 (108– 9 1 10 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 106 CFU ml1 of Lact. fermentum L23 can successfully eradicate the pathogen from the vaginal niche of mice. Our results differed from a previous report
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Figure 4 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 109 CFU ml1; (--♦--) G. v treated with Lact. fermentum 108 CFU ml1; and (–▼–) G v treated with Lact. fermentum 105 CFU ml1.
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Probiotic bacterium in vaginosis
et al. 2011). 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. 2011; Sungkar et al. 2012). 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. (2010). 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 119 Oxoid, Basingstoke, UK) containing gentamicin sulphate, nalidixic acid, amphotericin B and human blood (5%), under optimal conditions of incubation at 37°C for 24– 48 h. It was identified as G. vaginalis by Gram staining followed by standard biochemical tests (Bergey 1994). Lactobacillus strain Lactobacillus fermentum L23 was originally isolated from vaginal smears of a healthy woman and was previously characterized by its probiotic properties and technological potential. Lact. fermentum L23 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. 2008a,b). 4
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The strain was grown overnight at 37°C in MRS medium (Biokar Diagnostics, Beauvais, France) under limited aerobic conditions. The strain was identified by standard biochemical tests (Bergey 1994) and an API 50 CH biochemical identification system (bioMerieux, Craponne, France) (Nigatu et al. 2000). The 16S rRNA gene from Lact. fermentum L23 was sequenced with an Applied Biosystems model 3730XL automated DNA sequencing system by Macrogen Laboratories (Korea). The obtained sequence was subjected to a BLAST search (www.ncbi.nlm. 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 24°C, with a cycle of 12-h fluorescent light/12-h darkness. The microbiota of the mice was studied from vaginal samples taken with swabs. Swabs were sown at 37°C on plates of MRS agar under partial CO2 tension and 5% blood agar. Strains grown on MRS and blood agar plates were identified by standard diagnostic procedures (Bergey 1994). In this study were included four mice per group. The first group was used to study microbial colonization by the Lact. fermentum strain L23, 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 L23 Lactobacillus fermentum strain L23 was cultured in MRS broth at 37°C for 24 h, resuspended in PBS (pH 67) and adjusted to an approximate concentration of 1 9 109 CFU ml1. Mice were intravaginally inoculated with 20-ll inoculum of the lactobacillus suspension using an Eppendorf pipette (Pascual 2004). At 4 h postinoculation with L23, the first sample was taken from the vagina, serial dilutions were performed, and 20-ll aliquots were inoculated onto MRS agar plates (CFU ml1 at T0). The microbial colonization of the mice was monitored on days 1–7 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 37°C for 24 h under partial CO2 tension (CO2 incubator). The number of CFU was expressed as log10 CFU ml1 of vaginal fluid (Pascual 2004). Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Vaginal infection A suspension of G. vaginalis adjusted to 1 9 106 CFU ml1 was prepared in PBS. A tip, containing 20 ll of the suspension, was inserted into the vagina. Infection levels on days 1–7 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 5% sheep blood (bioMerieux) and grown at 37°C for 24 h and 48 h. Results were expressed as log10 CFU ml1. Colonies were identified by standard methods using morphological characteristics, Gram stain and standard biochemical tests suggested by Catlin B W (Catlin 1992). Curative effect A G. vaginalis suspension (1 9 106 CFU ml1) was vaginally inoculated. After 72 h, a suspension of Lact. fermentum L23 (1 9 109 CFU ml1) 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 L23 was incubated under limited aerobic conditions for 24 h. Aerobic conditions were required for G. vaginalis. Rating of the effective dose Doses of 104, 106, 108 and 109 viable L23 cells ml1 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. 2.0, for Windows NT and 3.1; SPSS, Chicago, IL) was used for differences in numbers of viable micro-organisms from different treatment groups. A P value of 005 was considered statistically significant. Acknowledgements This study was supported by the Secretarıa de Ciencia y Tecnica de la Universidad Nacional de Rıo Cuarto, C ordoba, Argentina. Conflict of Interest The authors declare that they have no conflict of interest.
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References Anukam, K.C., Osazuwa, E.O., Ahonkhai, I. and Gregor, R. (2006) Lactobacillus vaginal microbiota of women attending a reproductive health care service in Benin City, Nigeria. Sex Transm Dis 33, 59–62. Asahara, T., Nomoto, K., Watanuki, M. and Yokokura, T. (2001) Antimicrobial activity of intraurethrally administered probiotic Lactobacillus casei in a murine model of Escherichia coli urinary tract infection. Antimicrob Agents Chemother 45, 1751–1760. Bergey (1994) in Manual of Determinative Bacteriology 9th edn, pp. 71–174, 175-289, 527-558.Edited by Holt, J.G. Baltimore: Williams & Wilkins. Catlin, B. (1992) Gardnerella vaginalis: Characteristics, clinical consideration and controversies. Clin Microbiol Rev 5, 213–237. Daniele, M., Ruiz, F., Pascual, L. and Barberis, L. (2011) Ureaplasma urealyticum and Mycoplasma hominis sensitivity to bacteriocins produced by two Lactobacilli strains. Curr Microbiol 63, 360–365. Ehrstr€ om, S., Daroczy, K., Rylander, E., Samuelsson, C., Johannesson, U., Anzen, B. and Pahlson, C. (2010) Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis. Microbes Infect 12, 691–699. Falagas, M., Betsi, G.I. and Athanasiou, S. (2007) Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infect 13, 657–664. Guedou, F.A., Van Damme, L., Mirembe, F., Solomon, S., Becker, M., Deese, J., Crucitti, T. and Alary, M. (2012) 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, 545–551. Homayouni, A., Bastani, P., Ziyadi, S., Mohammad-AlizadehCharandabi, S., Ghalibaf, M., Mortazavian, A.M. and Mehrabany, E.V. (2013) Effects of Probiotics on the recurrence of bacterial vaginosis: a review. J Low Genit Tract Dis 18, 79–86. Kumar, N., Behera, B., Sagiri, S., Pal, K., Ray, S. and Roy, S. (2011) Bacterial vaginosis: etiology and modalities of treatment - A brief note. J Pharm Bioallied Sci 3, 496–503. Modares Nejad, V. and Shafaie, S. (2008) The association of bacterial vaginosis and preterm labor. J Pak Med Assoc 58, 104–106. Ngugi, B.M., Hemmerling, A., Bukusi, E.A., Kikuvi, G., Gikunju, J., Shiboski, S., Fredricks, D.N. and Cohen, C.R. (2011) Effects of bacterial vaginosis-associated bacteria and sexual intercourse on vaginal colonization with the probiotic Lactobacillus crispatus CTV-05. Sex Transm Dis 38, 1020–1027.
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Nigatu, A., Ahrne, S. and Molin, G. (2000) Temperaturedependent variation in API 50 CH fermentation profiles of Lactobacillus species. Curr Microbiol 41, 21–26. Pascual, L.M. (2004). Bacteriocinogenia en el genero Lactobacillus: caracterısticas beneficas de lactobacilos de vagina humana. Doctoral thesis. Universidad Nacional Rıo Cuarto, C ordoba, Argentina. Pascual, L.M., Daniele, M.B., Giordano, W., Pajaro, M.C. and Barberis, I.L. (2008a) Purification and partial characterization of novel bacteriocin L23 produced by Lactobacillus fermentum L23. Curr Microbiol 56, 397–402. Pascual, L.M., Daniele, M.B., Ruiz, F., Giordano, W., Pajaro, C. and Barberis, L. (2008b) Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina. J Gen Appl Microbiol 54, 141–148. Pascual, L., Ruiz, F., Giordano, W. and Barberis, L. (2010) Vaginal colonization and activity of the probiotic bacterium Lactobacillus fermentum L23 in a murine model of vaginal tract infection. J Med Microbiol 59, 360–364. Quigley, E. (2010) Prebiotics and probiotics; modifying and mining the microbiota. Pharmacol Res 61, 213–218. Reid, G. and Burton, J. (2002) Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4, 319–324.
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Saunders, S., Bocking, A., Challis, J. and Reid, G. (2007) Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms. Colloids Surf B Biointerfaces 55, 138–142. Stecher, B., Chaffron, S., Kp€apeli, R., Hapfelmeier, S., Freedrich, S., Weber, T., Kirundi, J., Suar, M. et al. (2010) Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog 6, 1–15. Sungkar, A., Purwosunu, Y., Aziz, M.F., Pratomo, H., Sutrisna, B. and Sekizawa, A. (2012) Influence of early self-diagnosis and treatment of bacterial vaginosis on preterm birth rate. Int J Gynecol Obstet 117, 264–267. Turovskiy, Y., Ludescher, R., Aroutcheva, A., Faro, S. and Chikindas, M. (2009) Lactocin 160, a bacteriocin produced by vaginal Lactobacillus rhamnosus, targets cytoplasmic membranes of the vaginal pathogen Gardnerella vaginalis. Probiotics Antimicrob Proteins 1, 67–74. Zarate, G., Santos, V. and Nader-Macias, M.E. (2007) Protective effect of vaginal Lactobacillus paracasei CRL 1289 against urogenital infection produced by Staphylococcus aureus in a mouse animal model. Infect Dis Obstet Gynecol 2007, Article ID 48358.
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