PRESENTATION
Effectiveness of the Two Microorganisms Lactobacillus fermentum LF15 and Lactobacillus plantarum LP01, Formulated in Slow-release Vaginal Tablets, in Women Affected by Bacterial Vaginosis A Pilot Study Franco Vicariotto, MD,* Luca Mogna, PhD,w and Mario Del Piano, MDz
Background: Bacterial vaginosis (BV) is the most common reason for abnormal vaginal discharge in reproductive-age women and one of its most important causative agents is the gram-variable bacterium Gardnerella vaginalis. BV is not accompanied by significant local inflammation, whereas the “fishy odor” test is always positive. In contrast, aerobic vaginitis (AV) is predominantly associated with Escherichia coli, but Streptococcus agalactiae and Staphylococcus aureus are also involved. Standard treatment of BV consists of oral or intravaginal antibiotics, although these are unable to spontaneously restore normal flora characterized by a high concentration of lactobacilli. The main limitation is the inability to offer a long-term defensive barrier, thus facilitating relapses and recurrences. This study was undertaken firstly to assess the ability of selected lactobacilli to in vitro antagonize G. vaginalis to determine an association with a strain able to inhibit E. coli, thus identifying a possible use in AV. The second step of the study was to conduct a human pilot trial in women affected by BV using an association of the most promising and active bacteria. Materials and Methods: For this purpose, neutralized supernatants of individual lactobacilli were tested at percentages ranging from 0.5% to 4% to determine their ability to hinder the growth of G. vaginalis American Type Culture Collection 10231. The bacterium that was able to exert the strongest inhibition was subsequently tested with Lactobacillus plantarum LP01 in a human intervention, placebo-controlled, pilot trial involving 34 female subjects (aged between 18 and 50, mean 34.7 ± 8.9, no menopausal women) diagnosed with BV. The 2 microorganisms Lactobacillus fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) were delivered to the vagina by means of slow-release vaginal tablets, also containing 50 mg of tara gum. The amount of each strain was 400 million live cells per dose. The women were instructed to apply a vaginal tablet once a day for 7 consecutive nights, followed by 1 tablet every 3 nights for a further 3-week application (acute phase) and, finally, 1 tablet per week to maintain a long-term vaginal colonization against possible recurrences. A clinical examination was performed and the Nugent score was quantified for each patient at enrollment (d0), after 28 days (d28), and at the end of the second month of relapse prevention (d56). A statistical comparison was made between d28, or d56, and d0, and between d56 and d28 to quantify the efficacy against possible recurrences.
From the *Gynaecology Unit, “San Pio X” Nursing Home, Milan; wBiolab Research Ltd.; and zGastroenterology Unit, Maggiore della Carita` Hospital, Novara, Italy. L.M. is an employee of Biolab Research Ltd. The remaining authors declare that they have nothing to disclose. Reprints: Franco Vicariotto, MD, Gynaecology Unit, “San Pio X” Nursing Home, Milan, Italy Via Francesco Nava, Milan 31-20159, Italy (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
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Results: L. fermentum LF15 showed the strongest in vitro inhibitory activity towards G. vaginalis American Type Culture Collection (ATCC) 10231 after both 24 and 48 hours. In the human trial, the 2 lactobacilli selected, namely L. fermentum LF15 and L. plantarum LP01, significantly reduced the Nugent score below the threshold of 7 after 28 days in 22 patients of 24 in the active group (91.7%, P < 0.001). Eight women (33.3%) recorded a Nugent score between 4 and 6, evidence of an intermediate situation, whereas the remaining 14 (58.3%) showed a score 7, definable as BV (16.7%, P = 0.065 compared with d28). In the placebo group, no significant differences were recorded at any time. Conclusions: BV, also known as vaginal bacteriosis is the most common cause of vaginal infection in women of childbearing age. Furthermore, BV is often asymptomatic as about 50% of women with this condition have no symptoms at all and the prevalence rate in apparently healthy women is around 10%. This study suggests the ability of the 2 strains L. fermentum LF15 and L. plantarum LP01 to counteract acute Gardnerella infections effectively and significantly improve the related uncomfortable symptoms in a very high percentage of women. This could be partially attributed to the presence of tara gum, which is able to create a mechanical barrier against Gardnerella on the surface of vaginal mucosa as a primary mechanism. Furthermore, long-term physiological protection seems to be established, thanks to the integration of the 2 lactobacilli into the vaginal microbiota and to their adhesion to the epithelial cells of the mucosa. In the light of the additional in vitro inhibitory activity against E. coli, their prospective use in AV could also prove interesting. Key Words: bacterial vaginosis, aerobic vaginitis, vaginal microbiota, lactobacilli, barrier effect
(J Clin Gastroenterol 2014;48:S106–S112)
T
he bacterial biota of the human vagina can have a profound impact on the health of women and their neonates.1 Lactobacilli are believed to promote a healthy ecosystem by producing lactic acid, hydrogen peroxide, and bacteriocins with antimicrobial features, thereby excluding pathogens from this niche.2 Vaginitis is an inflammation of the vagina, and affects women of all ages. Of the millions of cases of vaginitis each year, most are caused by bacterial vaginosis (BV) (about 40% to 50%), followed by yeast infections (20% to 25%) and then trichomoniasis (15% to 20%). BV is a polymicrobial syndrome involving replacement of normal vaginal hydrogen peroxide-producing lactobacilli by a variety of gram-negative rods and mycoplasmas.3
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Microbiologically, BV is associated with noticeable changes in microbial flora, characterized by a loss or reduction of lactobacilli, and the concurrent high concentration of numerous other bacterial species, mainly G. vaginalis, but also anaerobes such as Prevotella spp. and Mobiluncus spp.4 It is not clear whether the primary event triggering BV is the loss of key lactobacilli or the acquisition of a complex bacterial community typically found in this syndrome. However, these may be simultaneous processes leading to a significant imbalance. BV is highly prevalent, affecting on average from 10% to 30% of women.5 Although BV is an important medical condition itself, it is associated with several more serious adverse outcomes including preterm birth,6 pelvic inflammatory disease,7 and infection with genital herpes, gonorrhea, or chlamydia.8 Women with BV may have a malodorous vaginal discharge or local irritation, but about half of the women with diagnosable BV have no clear symptoms.9 The diagnosis of BV is usually performed using a series of clinical criteria evaluated by a practitioner performing a pelvic examination, or by interpretation of vaginal fluid Gram stains. Amsel clinical criteria are usually employed for the diagnosis of BV in the clinical setting.9 At least 3 of 4 Amsel criteria must be present to establish a diagnosis of BV, in detail: (1) vaginal fluid pH > 4.5; (2) a positive “whiff test” which consists in detecting a fishy odor upon addition of 10% potassium hydroxide to a slide containing vaginal fluid; (3) the presence of clue cells (>20%) in vaginal fluid which are shed vaginal epithelial cells coated with bacteria creating shadowy borders; (4) a homogeneous, milky vaginal discharge. An alternative method for diagnosis of BV relies on analysis of Gram stains performed on vaginal fluid smears. The method of Nugent et al10 assesses the presence and relative amounts of 3 bacterial morphotypes, including gram-positive rods (lactobacilli), gram-negative and gramvariable rods (G. vaginalis and Bacteroides species), and curved rods (Mobiluncus species). Current treatment strategies for BV include the administration of antibiotics either orally or topically. The use of oral metronidazole for 7 days or vaginal metronidazole for 5 days mediated an improvement of symptoms in 83% to 87% of women within 2 to 3 weeks.11,12 Similar response rates are observed with the use of vaginal clindamycin. Vaginal recolonization rates with lactobacilli are similar with both antibiotics, as defined by detection of lactobacilli on Gram stain 21 to 30 days after initiation of antibiotic treatment.13,14 Even though antibiotic medication is effective in up to 90% of cases, about 25% of women will develop BV again within 4 weeks.15,16 Moreover, long-term recurrence rates have been shown to be >70%.17–19 With this regard, it is interesting to point out that there is emerging new evidence that biofilms are associated with BV and it has been suggested that they may be critical in pathogenesis and recurrences. Swidsinski et al20 demonstrated the presence of adherent bacterial biofilms in 90% of subjects with BV, whereas they were detectable in only 10% of subjects without BV. Adherent biofilms were defined as lawns of bacteria that were tightly attached to the vaginal epithelial surface and contained specific bacterial groups. Bacteria in biofilms respond differently to antibiotic treatment when compared with their planktonic counterparts.21 r
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To avoid issues of antibiotic resistance in bacterial biofilms, 1 study has tested a probiotic approach to attempt clearance of the G. vaginalis biofilm.22 G. vaginalis biofilms grown in vitro were displaced with Lactobacillus reuteri RC-14 and to a limited extent with Lactobacillus iners, commonly found in the vaginal niche. In BV relapses, but also as prophylaxis and prevention, the use of specific lactic acid bacteria, especially lactobacilli, is consolidating, with the aim of restoring the physiological balance of the vaginal ecosystem, mainly attributed to the presence of the so-called Do¨derlein’s complex.23 The use of live microorganisms with a probiotic value was, in fact, indicated some years ago by many gynecologists as an alternative or complementary therapy to the use of traditional topical medicines with antibacterial or antimycotic activity.24 The oral and vaginal administration of lactobacilli is currently considered an effective approach, including during pregnancy.25 Probiotics are well known for their ability to lower intravaginal pH, thus establishing a barrier effect against many pathogens.26 Some strains are also able to create additional and more focused antagonistic activities mediated by specific molecules such as hydrogen peroxide, extracellular proteins, and bacteriocins.27 In any case, despite some undeniable positive evidence, other intervention studies have at least partially failed to highlight a statistically significant alleviation of BV symptoms,28 and this is most likely attributable to the lack of a specific inhibitory activity of the strains used towards the bacteria commonly causing BV, such as G. vaginalis. In the light of the evidence above, this study was undertaken firstly to select at least 1 Lactobacillus that is able to directly in vitro antagonize G. vaginalis so that it can be associated with another microorganism exerting an inhibitory activity on E. coli. The inhibition of 5 E. coli strains, including the diarrheagenic biotype O157:H7, was determined in a previous study by Mogna et al,29 leading to the selection of a very effective Lactobacillus, namely L. plantarum LP01. As a second step, a human pilot trial in women diagnosed with BV was performed using the most promising bacteria to assess their ability to create and maintain a vaginal microenvironment unfavorable to the establishment, propagation, or persistence of BV.
MATERIALS AND METHODS In Vitro Study Bacterial Strains and Growth Conditions The lactobacilli strains used in this study, all isolated either from the feces of healthy humans or from vaginal swabs of healthy female subjects, were classified based on their phenotypic and genotypic characteristics. The G. vaginalis strain employed was purchased from the ATCC. The in vitro inhibitory activity of different lactobacilli against G. vaginalis was quantified at Biolab Research laboratories (Novara, Italy). Before being used in the experiment, the G. vaginalis ATCC 10231 strain was grown in brain-heart infusion broth (BHI) (Oxoid, Milan, Italy) containing 2% (wt/wt) gelatin, 0.5% yeast extract, 0.1% starch, and 0.1% glucose,30 whereas individual lactobacilli were cultured in liquid de Man, Rogosa and Sharpe (MRS) medium (BD Difco, Milan, Italy). www.jcge.com |
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All the plates were incubated at 371C for 48 hours under anaerobic conditions (GasPak system) with Anaerocult A kits (Merck Millipore, Milan, Italy).
Assessment of the Antagonistic Activity of Selected Lactobacilli Toward G. vaginalis Culture supernatants of the different lactobacilli tested (2 L. crispatus, 2 L. paracasei, and 4 L. fermentum) were prepared as follows: an overnight culture of each isolate was centrifuged at 5000g. The resulting supernatant was neutralized at pH 6.5 with NaOH 1 N, sterilized by filtration through syringe filters (Ministart pore size: 0.22 mm), and assayed for the presence of any inhibitory molecule in the broth. Neutralized supernatants of individual Lactobacillus were then added in different percentages to fresh BHI broth, prepared as described above, inoculated with G. vaginalis. The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants ranging from 0.5% to 4%, after 24 and 48 hours of incubation at 371C in microaerophilic conditions, was quantified by means of optical density at 600 nm (OD600). The growth of positive control (Gardnerella alone) was assessed in BHI broth added to a quantity of fresh MRS broth ranging from 0.5% to 4%. Thus, the positive controls were as similar as possible to test tubes inoculated with individual lactobacilli. This test was repeated 3 times for each strain to ensure reliability and reproducibility.
Human Trial Study Design Thirty-five female patients (aged between 18 and 50, mean 34.7 ± 8.9, no menopausal women) were initially enrolled in this pilot, randomized, placebo-controlled study between September and October 2012 at the Gynecological Associated Medical Practice “Vicariotto-De Maria” (Milan, Italy). Informed written consent was obtained from all participants involved in the study. This study was carried out in accordance with the Helsinki Declaration (2000) of the World Medical Association. Eligible subjects were at least 18 years old and had active, symptomatic acute BV according to the Amsel criteria and diagnosed according to the Nugent score,10 involving the microscopic quantitation of bacterial morphotypes yielding a score between 0 and 10. Patients were excluded from the study if the Nugent score was 7 and definable as BV (16.7%, P = 0.065 compared with d28). In the placebo group, no significant differences were recorded at any time. The statistical comparison between the 2 groups (unpaired t test) at the same times highlighted statistically significant differences (P < 0.001 at both d28 and d56), whereas at baseline, the subjects were comparable (P = 0.137). Figure 1 shows the average Nugent score recorded in the placebo group and in the group being given the 2 lactobacilli at the different times. At baseline, all the subjects www.jcge.com |
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TABLE 2. Incidence of Bacterial Vaginosis (Nugent Score Z7) at the Beginning of the Study (Time Zero), After 4 Weeks of Treatment (Day 28), and at the End of the Second Month of the Study Protocol (Day 56)
Group A P (d28 and d56 Versus d0) Time zero No. subjects with bacterial vaginosis (Z7) No. subjects with an intermediate situation (4-6) No. healthy subjects (r3) Day 28 No. subjects with bacterial vaginosis (Z7) No. subjects with an intermediate situation (4-6) No. healthy subjects (r3) Percentage of healing (Nugent score 7, whereas in the active group, mean values of 3.50 and 4.25 were recorded, respectively.
DISCUSSION It is a recognized fact that microbial communities have a robust influence on human health and quality of life. It is not astounding that the bacterial community within the human vagina has a profound impact on a woman’s health, as the microbiota present in this organ plays a crucial role in determining the biochemical profile of the vagina and its inflammatory profile.31
FIGURE 1. Nugent score in group A and in the placebo group at baseline (day 0), at the end of the acute treatment (day 28), and at the end of the study (day 56). The mean values ± SD are reported. BV indicates bacterial vaginosis.
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The healthy vagina is populated predominantly by lactobacilli. The species differ with regard to their ability to maintain a stable population through environmental modifications such as pH changes due to sexual intercourse or menstruation and with respect to their ability to counteract the growth of other bacteria. All of the species produce lactic acid and selected biotypes also secrete hydrogen peroxide and bacteriocins, molecules that are known for their ability to hinder the growth and persistence of specific target bacterial species. BV is the most common vaginal syndrome afflicting fertile, premenopausal, and pregnant women.31 Furthermore, BV is often asymptomatic, as about 50% of women with this condition have no symptoms at all and the prevalence in apparently healthy women is around 10%. In cases of BV, beneficial lactic acid-producing bacteria, with particular reference to lactobacilli, are replaced by amineproducing anaerobic bacteria. Microbiological analysis of BV has shown the gram-variable bacterium G. vaginalis to be the most frequent causative organism. This bacterium has been detected in >98% of BV cases. However, it is not clear if some events induce a drop in the population of lactobacilli, which in turn makes local conditions tolerant to the growth of other bacteria, if BVassociated species such as G. vaginalis displace the lactobacilli according to certain kinetics, or whether these 2 factors are not directly related.32 Treatment of BV involves oral or local administration of metronidazole or clindamycin, and varies in efficacy (48% to 85% for absence of infection, Z4 wk after treatment).33 The overall long-term cure rate is low, as BV recurs in up to 40% of women within 3 months after initiation of antibiotic therapy and in up to 50% of women r
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after 6 months.19 There are several unpleasant side effects and disadvantages associated with these therapies, including superinfections by pathogenic microorganisms18 and susceptibility of lactobacilli to clindamycin,34 a fact that could expose the vaginal milieu to possible reinfections with harmful microbes. Moreover, vaginal pathogens, particularly G. vaginalis and anaerobic bacteria, are showing increased drug resistance.35,36 Many studies have provided evidence of the valuable functions of a healthy microbiota and suggested the selection of bacterial strains, recognized as probiotics, with beneficial effects for the prevention or treatment of conditions involving a disruption of indigenous microbiota. Lactobacilli use in BV is supported by positive results obtained in some clinical trials. The majority of the studies yielding positive results have been performed using probiotic preparations containing high doses of lactobacilli, thus suggesting that the amount of exogenously applied lactobacilli could have a role in the effectiveness of the product.31 However, it is well known that only selected strains could exert significant effectiveness, not limited to the nonspecific mechanism of action attributable to the secretion of organic acids such as lactic and acetic acids. This study suggests that L. fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) are able to effectively counteract acute Gardnerella infections and significantly improve the uncomfortable symptoms reported by women with BV. This evidence may be at least partially attributed to the presence of tara gum, an ingredient that is able to create a mechanical barrier against the adhesion of Gardnerella and of other possible harmful microbes to the surface of vaginal mucosa. The overall compliance and tolerability were very good, with only 1 dropout in the placebo group recorded, due to protocol deviation and not to any adverse events. The very limited percentage of recurrences recorded in the active group during the second month of the protocol suggests a long-term physiological protection thanks to the integration of the 2 lactobacilli into the vaginal microbiota and their adhesion to the epithelial cells of the mucosa. In this regard, the use of arabinogalactan and FOS, 2 fibers that are able to enhance vaginal colonization by the 2 lactobacilli, could have a role in the long-term barrier against reinfections recorded in the second month of the study protocol. Even though the placebo group included a smaller number of subjects compared with the active group, the placebo effect was very low, as no significant statistical differences were recorded either after 28 days (P = 0.619) or at the end of the protocol (P = 0.823), thus suggesting that the improvement recorded in the subjects being given the 2 lactobacilli is specific and attributable to the overall effect of the product. It is also interesting to point out the fact that an in vitro functionality exerted by a selected Lactobacillus, such as the inhibition of Gardnerella growth by means of specific mechanisms of action, was successfully confirmed during this pilot trial in female subjects diagnosed with BV. This could suggest that the in vitro antagonism toward G. vaginalis could be valuable as a criterion to effectively select lactobacilli with a protective action in cases of BV, both in terms of an acute treatment and for long-term prevention of recurrences. In the in vitro part of the study, the selection of a proper growth medium suitable for the inhibition test for r
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(Selected) Lactobacilli in Bacterial Vaginosis
Gardnerella was another key point, as presumably some components present in MRS, the medium commonly used to grow lactobacilli before the inhibition assessment, could potentially promote the growth of Gardnerella. In fact, the growth of Gardnerella in BHI alone was relatively poor, especially after 48 hours. In the light of the in vitro inhibitory activity against E. coli exerted mainly by L. plantarum LP01,29 the prospective use of its association with L. fermentum LF15 in aerobic vaginitis (AV) could prove interesting. In fact, AV is predominantly associated with E. coli, but S. agalactiae and S. aureus are also involved. Standard treatment of AV consists of oral or intravaginal antibiotics, although these cannot automatically restore normal flora characterized by a high concentration of lactobacilli, thus facilitating relapses and reinfections. In conclusion, this study investigates a possible innovative approach aimed at counteracting acute vaginal microbiota imbalance and preventing possible recurrences either caused by G. vaginalis (BV) or by E. coli (aerobic vaginitis), although future additional investigations will be needed to quantify the effectiveness of L. fermentum LF15 and L. plantarum LP01 in that condition. REFERENCES 1. Srinivasan S, Fredricks DN. The human vaginal bacterial biota and bacterial vaginosis. Interdiscip Perspect Infect Dis. 2008;2008:750479. 2. Aroutcheva AA, Simoes JA, Faro S. Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardnerella vaginalis. Infect Dis Obstet Gynecol. 2001;9:33–39. 3. Tamonud M, Preeti A, Charan A, et al. Diagnosis of bacterial vaginosis in cases of abnormal vaginal discharge: comparison of clinical and microbiological criteria. J Infect Develop Countries. 2011;5:353–360. 4. Coste I, Judlin P, Lepargneur JP, et al. Safety and efficacy of an intravaginal prebiotic gel in the prevention of recurrent bacterial vaginosis: a randomized double-blind study. Obstet Gynecol Int. 2012;2012:147867. 5. Koumans EH, Sternberg M, Bruce C, et al. The prevalence of bacterial vaginosis in the United States, 2001-2004; associations with symptoms, sexual behaviours, and reproductive health. Sex Transm Dis. 2007;34:864–869. 6. Hillier SL, Nugent RP, Eschenbach DA, et al. Association between bacterial vaginosis and preterm delivery of a lowbirth-weight infant. The Vaginal Infections and Prematurity Study Group. N Engl J Med. 1995;333:1737–1742. 7. Haggerty CL, Hillier SL, Bass DC, et al. Bacterial vaginosis and anaerobic bacteria are associated with endometritis. Clin Infect Dis. 2004;39:990–995. 8. Marrazzo JM, Martin DH. Management of women with cervicitis. Clin Infect Dis. 2007;44(suppl 3):S102–S110. 9. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. Am J Med. 1983;74:14–22. 10. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol. 1991;29:297–301. 11. Hanson JM, McGregor JA, Hillier SL, et al. Metronidazole for bacterial vaginosis: a comparison of vaginal gel vs. oral therapy. J Reprod Med. 2000;45:889–896. 12. Livengood CH III, Soper DE, Sheehan KL, et al. Comparison of once-daily and twice-daily dosing of 0.75% metronidazole gel in the treatment of bacterial vaginosis. Sex Transm Dis. 1999;26:137–142. 13. Agnew KJ, Hillier SL. The effect of treatment regimens for vaginitis and cervicitis on vaginal colonization by lactobacilli. Sex Transm Dis. 1995;22:269–273.
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14. Nyirjesy P, McIntosh MJ, Gattermeir DJ, et al. The effects of intravaginal clindamycin and metronidazole therapy on vaginal lactobacilli in patients with bacterial vaginosis. Am J Obstet Gynecol. 2006;194:1277–1282. 15. Hillier SL, Lipinsky C, Briselden AM, et al. Efficacy of intravaginal 0.75% metronidazole gel for the treatment of bacterial vaginosis. Obstet Gynecol. 1993;81:963–967. 16. Livengood CH III, McGregor JA, Soper DE, et al. Bacterial vaginosis: efficacy and safety of intravaginal metronidazole treatment. Am J Obstet Gynecol. 1994;170:759–764. 17. Sa´nchez J, Campos PE, Courtois B, et al. Prevention of sexually transmitted diseases (STDs) in female sex workers: prospective evaluation of condom promotion and strengthened STD services. Sex Transm Dis. 2003;30:273–279. 18. Sobel JD, Ferris D, Schwebke J, et al. Suppressive antibacterial therapy with 0.75% metronidazole vaginal gel to prevent recurrent bacterial vaginosis. Am J Obstet Gynecol. 2006;194: 1283–1289. 19. Bradshaw CS, Morton AN, Hocking J, et al. High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. J Infect Dis. 2006;193:1478–1486. 20. Swidsinski A, Mendling W, Loening-Baucke V, et al. Adherent biofilms in bacterial vaginosis. Obstet Gynecol. 2005;106(5 pt 1):1013–1023. 21. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284: 1318–1322. 22. Saunders S, Bocking A, Challis J, et al. Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms. Colloids Surf B Biointerfaces. 2007;55:138–142. 23. Do¨derlein A. Das Scheidensekret und seine Bedeutung fu¨r das Puerperalfieber. Leipzig: Garrison-Morton (5th ed.) 6279; 1892. 24. MacPhee RA, Hummelen R, Bisanz JE, et al. Probiotic strategies for the treatment and prevention of bacterial vaginosis. Expert Opin Pharmacother. 2010;11:2985–2995. 25. Nishijima K, Shukunami K, Kotsuji F. Probiotics affects vaginal flora in pregnant women, suggesting the possibility of preventing preterm labor. J Clin Gastroenterol. 2005;39:447–448.
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26. Ro¨nnqvist PD, Forsgren-Brusk UB, Grahn-Ha˚kansson EE. Lactobacilli in the female genital tract in relation to other genital microbes and vaginal pH. Acta Obstet Gynecol Scand. 2006;85:726–735. 27. Falagas M, Betsi GI, Athanasiou S. Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infect. 2007;13:657–664. 28. Senok AC, Verstraelen H, Temmerman M, et al. Probiotics for the treatment of bacterial vaginosis. Cochrane Database Syst Rev. 2009;4:CD006289. 29. Mogna L, Del Piano M, Deidda F, et al. Assessment of the in vitro inhibitory activity of specific probiotic bacteria against different Escherichia coli strains. J Clin Gastroenterol. 2012;46(suppl):S29–S32. 30. Castro J, Henriques A, Machado A, et al. Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. Int J Med Sci. 2013;10:1193–1198. 31. Mastromarino P, Vitali B, Mosca L. Bacterial vaginosis: a review on clinical trials with probiotics. New Microbiol. 2013;36:229–238. 32. Leppa¨luoto PA. Bacterial vaginosis: what is physiological in vaginal bacteriology? An update and opinion. Acta Obstet Gynecol Scand. 2011;90:1302–1306. 33. Koumans EH, Markowitz LE, Hogan V, CDC BV Working Group. Indications for therapy and treatment recommendations for bacterial vaginosis in nonpregnant and pregnant women: a synthesis of data. Clin Infect Dis. 2002;35(suppl 2):S152–S172. 34. Bayer AS, Chow AW, Concepcion N, et al. Susceptibility of 40 lactobacilli to six antimicrobial agents with broad grampositive anaerobic spectra. Antimicrob Agents Chemother. 1978;14:720–722. 35. McLean NW, McGroarty JA. Growth inhibition of metronidazole-susceptible and metronidazole-resistant strains of Gardnerella vaginalis by lactobacilli in vitro. Appl Environ Microbiol. 1996;62:1089–1092. 36. Beigi RH, Austin MN, Meyn LA, et al. Antimicrobial resistance associated with the treatment of bacterial vaginosis. Am J Obstet Gynecol. 2004;191:1124–1129.
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Effectiveness of the Two Microorganisms Lactobacillus fermentum LF15 and Lactobacillus plantarum LP01, Formulated in Slow-release Vaginal Tablets, in Women Affected by Bacterial Vaginosis A Pilot Study Franco Vicariotto, MD,* Luca Mogna, PhD,w and Mario Del Piano, MDz
Background: Bacterial vaginosis (BV) is the most common reason for abnormal vaginal discharge in reproductive-age women and one of its most important causative agents is the gram-variable bacterium Gardnerella vaginalis. BV is not accompanied by significant local inflammation, whereas the “fishy odor” test is always positive. In contrast, aerobic vaginitis (AV) is predominantly associated with Escherichia coli, but Streptococcus agalactiae and Staphylococcus aureus are also involved. Standard treatment of BV consists of oral or intravaginal antibiotics, although these are unable to spontaneously restore normal flora characterized by a high concentration of lactobacilli. The main limitation is the inability to offer a long-term defensive barrier, thus facilitating relapses and recurrences. This study was undertaken firstly to assess the ability of selected lactobacilli to in vitro antagonize G. vaginalis to determine an association with a strain able to inhibit E. coli, thus identifying a possible use in AV. The second step of the study was to conduct a human pilot trial in women affected by BV using an association of the most promising and active bacteria. Materials and Methods: For this purpose, neutralized supernatants of individual lactobacilli were tested at percentages ranging from 0.5% to 4% to determine their ability to hinder the growth of G. vaginalis American Type Culture Collection 10231. The bacterium that was able to exert the strongest inhibition was subsequently tested with Lactobacillus plantarum LP01 in a human intervention, placebo-controlled, pilot trial involving 34 female subjects (aged between 18 and 50, mean 34.7 ± 8.9, no menopausal women) diagnosed with BV. The 2 microorganisms Lactobacillus fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) were delivered to the vagina by means of slow-release vaginal tablets, also containing 50 mg of tara gum. The amount of each strain was 400 million live cells per dose. The women were instructed to apply a vaginal tablet once a day for 7 consecutive nights, followed by 1 tablet every 3 nights for a further 3-week application (acute phase) and, finally, 1 tablet per week to maintain a long-term vaginal colonization against possible recurrences. A clinical examination was performed and the Nugent score was quantified for each patient at enrollment (d0), after 28 days (d28), and at the end of the second month of relapse prevention (d56). A statistical comparison was made between d28, or d56, and d0, and between d56 and d28 to quantify the efficacy against possible recurrences.
From the *Gynaecology Unit, “San Pio X” Nursing Home, Milan; wBiolab Research Ltd.; and zGastroenterology Unit, Maggiore della Carita` Hospital, Novara, Italy. L.M. is an employee of Biolab Research Ltd. The remaining authors declare that they have nothing to disclose. Reprints: Franco Vicariotto, MD, Gynaecology Unit, “San Pio X” Nursing Home, Milan, Italy Via Francesco Nava, Milan 31-20159, Italy (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
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Results: L. fermentum LF15 showed the strongest in vitro inhibitory activity towards G. vaginalis American Type Culture Collection (ATCC) 10231 after both 24 and 48 hours. In the human trial, the 2 lactobacilli selected, namely L. fermentum LF15 and L. plantarum LP01, significantly reduced the Nugent score below the threshold of 7 after 28 days in 22 patients of 24 in the active group (91.7%, P < 0.001). Eight women (33.3%) recorded a Nugent score between 4 and 6, evidence of an intermediate situation, whereas the remaining 14 (58.3%) showed a score 7, definable as BV (16.7%, P = 0.065 compared with d28). In the placebo group, no significant differences were recorded at any time. Conclusions: BV, also known as vaginal bacteriosis is the most common cause of vaginal infection in women of childbearing age. Furthermore, BV is often asymptomatic as about 50% of women with this condition have no symptoms at all and the prevalence rate in apparently healthy women is around 10%. This study suggests the ability of the 2 strains L. fermentum LF15 and L. plantarum LP01 to counteract acute Gardnerella infections effectively and significantly improve the related uncomfortable symptoms in a very high percentage of women. This could be partially attributed to the presence of tara gum, which is able to create a mechanical barrier against Gardnerella on the surface of vaginal mucosa as a primary mechanism. Furthermore, long-term physiological protection seems to be established, thanks to the integration of the 2 lactobacilli into the vaginal microbiota and to their adhesion to the epithelial cells of the mucosa. In the light of the additional in vitro inhibitory activity against E. coli, their prospective use in AV could also prove interesting. Key Words: bacterial vaginosis, aerobic vaginitis, vaginal microbiota, lactobacilli, barrier effect
(J Clin Gastroenterol 2014;48:S106–S112)
T
he bacterial biota of the human vagina can have a profound impact on the health of women and their neonates.1 Lactobacilli are believed to promote a healthy ecosystem by producing lactic acid, hydrogen peroxide, and bacteriocins with antimicrobial features, thereby excluding pathogens from this niche.2 Vaginitis is an inflammation of the vagina, and affects women of all ages. Of the millions of cases of vaginitis each year, most are caused by bacterial vaginosis (BV) (about 40% to 50%), followed by yeast infections (20% to 25%) and then trichomoniasis (15% to 20%). BV is a polymicrobial syndrome involving replacement of normal vaginal hydrogen peroxide-producing lactobacilli by a variety of gram-negative rods and mycoplasmas.3
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Microbiologically, BV is associated with noticeable changes in microbial flora, characterized by a loss or reduction of lactobacilli, and the concurrent high concentration of numerous other bacterial species, mainly G. vaginalis, but also anaerobes such as Prevotella spp. and Mobiluncus spp.4 It is not clear whether the primary event triggering BV is the loss of key lactobacilli or the acquisition of a complex bacterial community typically found in this syndrome. However, these may be simultaneous processes leading to a significant imbalance. BV is highly prevalent, affecting on average from 10% to 30% of women.5 Although BV is an important medical condition itself, it is associated with several more serious adverse outcomes including preterm birth,6 pelvic inflammatory disease,7 and infection with genital herpes, gonorrhea, or chlamydia.8 Women with BV may have a malodorous vaginal discharge or local irritation, but about half of the women with diagnosable BV have no clear symptoms.9 The diagnosis of BV is usually performed using a series of clinical criteria evaluated by a practitioner performing a pelvic examination, or by interpretation of vaginal fluid Gram stains. Amsel clinical criteria are usually employed for the diagnosis of BV in the clinical setting.9 At least 3 of 4 Amsel criteria must be present to establish a diagnosis of BV, in detail: (1) vaginal fluid pH > 4.5; (2) a positive “whiff test” which consists in detecting a fishy odor upon addition of 10% potassium hydroxide to a slide containing vaginal fluid; (3) the presence of clue cells (>20%) in vaginal fluid which are shed vaginal epithelial cells coated with bacteria creating shadowy borders; (4) a homogeneous, milky vaginal discharge. An alternative method for diagnosis of BV relies on analysis of Gram stains performed on vaginal fluid smears. The method of Nugent et al10 assesses the presence and relative amounts of 3 bacterial morphotypes, including gram-positive rods (lactobacilli), gram-negative and gramvariable rods (G. vaginalis and Bacteroides species), and curved rods (Mobiluncus species). Current treatment strategies for BV include the administration of antibiotics either orally or topically. The use of oral metronidazole for 7 days or vaginal metronidazole for 5 days mediated an improvement of symptoms in 83% to 87% of women within 2 to 3 weeks.11,12 Similar response rates are observed with the use of vaginal clindamycin. Vaginal recolonization rates with lactobacilli are similar with both antibiotics, as defined by detection of lactobacilli on Gram stain 21 to 30 days after initiation of antibiotic treatment.13,14 Even though antibiotic medication is effective in up to 90% of cases, about 25% of women will develop BV again within 4 weeks.15,16 Moreover, long-term recurrence rates have been shown to be >70%.17–19 With this regard, it is interesting to point out that there is emerging new evidence that biofilms are associated with BV and it has been suggested that they may be critical in pathogenesis and recurrences. Swidsinski et al20 demonstrated the presence of adherent bacterial biofilms in 90% of subjects with BV, whereas they were detectable in only 10% of subjects without BV. Adherent biofilms were defined as lawns of bacteria that were tightly attached to the vaginal epithelial surface and contained specific bacterial groups. Bacteria in biofilms respond differently to antibiotic treatment when compared with their planktonic counterparts.21 r
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To avoid issues of antibiotic resistance in bacterial biofilms, 1 study has tested a probiotic approach to attempt clearance of the G. vaginalis biofilm.22 G. vaginalis biofilms grown in vitro were displaced with Lactobacillus reuteri RC-14 and to a limited extent with Lactobacillus iners, commonly found in the vaginal niche. In BV relapses, but also as prophylaxis and prevention, the use of specific lactic acid bacteria, especially lactobacilli, is consolidating, with the aim of restoring the physiological balance of the vaginal ecosystem, mainly attributed to the presence of the so-called Do¨derlein’s complex.23 The use of live microorganisms with a probiotic value was, in fact, indicated some years ago by many gynecologists as an alternative or complementary therapy to the use of traditional topical medicines with antibacterial or antimycotic activity.24 The oral and vaginal administration of lactobacilli is currently considered an effective approach, including during pregnancy.25 Probiotics are well known for their ability to lower intravaginal pH, thus establishing a barrier effect against many pathogens.26 Some strains are also able to create additional and more focused antagonistic activities mediated by specific molecules such as hydrogen peroxide, extracellular proteins, and bacteriocins.27 In any case, despite some undeniable positive evidence, other intervention studies have at least partially failed to highlight a statistically significant alleviation of BV symptoms,28 and this is most likely attributable to the lack of a specific inhibitory activity of the strains used towards the bacteria commonly causing BV, such as G. vaginalis. In the light of the evidence above, this study was undertaken firstly to select at least 1 Lactobacillus that is able to directly in vitro antagonize G. vaginalis so that it can be associated with another microorganism exerting an inhibitory activity on E. coli. The inhibition of 5 E. coli strains, including the diarrheagenic biotype O157:H7, was determined in a previous study by Mogna et al,29 leading to the selection of a very effective Lactobacillus, namely L. plantarum LP01. As a second step, a human pilot trial in women diagnosed with BV was performed using the most promising bacteria to assess their ability to create and maintain a vaginal microenvironment unfavorable to the establishment, propagation, or persistence of BV.
MATERIALS AND METHODS In Vitro Study Bacterial Strains and Growth Conditions The lactobacilli strains used in this study, all isolated either from the feces of healthy humans or from vaginal swabs of healthy female subjects, were classified based on their phenotypic and genotypic characteristics. The G. vaginalis strain employed was purchased from the ATCC. The in vitro inhibitory activity of different lactobacilli against G. vaginalis was quantified at Biolab Research laboratories (Novara, Italy). Before being used in the experiment, the G. vaginalis ATCC 10231 strain was grown in brain-heart infusion broth (BHI) (Oxoid, Milan, Italy) containing 2% (wt/wt) gelatin, 0.5% yeast extract, 0.1% starch, and 0.1% glucose,30 whereas individual lactobacilli were cultured in liquid de Man, Rogosa and Sharpe (MRS) medium (BD Difco, Milan, Italy). www.jcge.com |
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All the plates were incubated at 371C for 48 hours under anaerobic conditions (GasPak system) with Anaerocult A kits (Merck Millipore, Milan, Italy).
Assessment of the Antagonistic Activity of Selected Lactobacilli Toward G. vaginalis Culture supernatants of the different lactobacilli tested (2 L. crispatus, 2 L. paracasei, and 4 L. fermentum) were prepared as follows: an overnight culture of each isolate was centrifuged at 5000g. The resulting supernatant was neutralized at pH 6.5 with NaOH 1 N, sterilized by filtration through syringe filters (Ministart pore size: 0.22 mm), and assayed for the presence of any inhibitory molecule in the broth. Neutralized supernatants of individual Lactobacillus were then added in different percentages to fresh BHI broth, prepared as described above, inoculated with G. vaginalis. The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants ranging from 0.5% to 4%, after 24 and 48 hours of incubation at 371C in microaerophilic conditions, was quantified by means of optical density at 600 nm (OD600). The growth of positive control (Gardnerella alone) was assessed in BHI broth added to a quantity of fresh MRS broth ranging from 0.5% to 4%. Thus, the positive controls were as similar as possible to test tubes inoculated with individual lactobacilli. This test was repeated 3 times for each strain to ensure reliability and reproducibility.
Human Trial Study Design Thirty-five female patients (aged between 18 and 50, mean 34.7 ± 8.9, no menopausal women) were initially enrolled in this pilot, randomized, placebo-controlled study between September and October 2012 at the Gynecological Associated Medical Practice “Vicariotto-De Maria” (Milan, Italy). Informed written consent was obtained from all participants involved in the study. This study was carried out in accordance with the Helsinki Declaration (2000) of the World Medical Association. Eligible subjects were at least 18 years old and had active, symptomatic acute BV according to the Amsel criteria and diagnosed according to the Nugent score,10 involving the microscopic quantitation of bacterial morphotypes yielding a score between 0 and 10. Patients were excluded from the study if the Nugent score was 7 and definable as BV (16.7%, P = 0.065 compared with d28). In the placebo group, no significant differences were recorded at any time. The statistical comparison between the 2 groups (unpaired t test) at the same times highlighted statistically significant differences (P < 0.001 at both d28 and d56), whereas at baseline, the subjects were comparable (P = 0.137). Figure 1 shows the average Nugent score recorded in the placebo group and in the group being given the 2 lactobacilli at the different times. At baseline, all the subjects www.jcge.com |
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TABLE 2. Incidence of Bacterial Vaginosis (Nugent Score Z7) at the Beginning of the Study (Time Zero), After 4 Weeks of Treatment (Day 28), and at the End of the Second Month of the Study Protocol (Day 56)
Group A P (d28 and d56 Versus d0) Time zero No. subjects with bacterial vaginosis (Z7) No. subjects with an intermediate situation (4-6) No. healthy subjects (r3) Day 28 No. subjects with bacterial vaginosis (Z7) No. subjects with an intermediate situation (4-6) No. healthy subjects (r3) Percentage of healing (Nugent score 7, whereas in the active group, mean values of 3.50 and 4.25 were recorded, respectively.
DISCUSSION It is a recognized fact that microbial communities have a robust influence on human health and quality of life. It is not astounding that the bacterial community within the human vagina has a profound impact on a woman’s health, as the microbiota present in this organ plays a crucial role in determining the biochemical profile of the vagina and its inflammatory profile.31
FIGURE 1. Nugent score in group A and in the placebo group at baseline (day 0), at the end of the acute treatment (day 28), and at the end of the study (day 56). The mean values ± SD are reported. BV indicates bacterial vaginosis.
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The healthy vagina is populated predominantly by lactobacilli. The species differ with regard to their ability to maintain a stable population through environmental modifications such as pH changes due to sexual intercourse or menstruation and with respect to their ability to counteract the growth of other bacteria. All of the species produce lactic acid and selected biotypes also secrete hydrogen peroxide and bacteriocins, molecules that are known for their ability to hinder the growth and persistence of specific target bacterial species. BV is the most common vaginal syndrome afflicting fertile, premenopausal, and pregnant women.31 Furthermore, BV is often asymptomatic, as about 50% of women with this condition have no symptoms at all and the prevalence in apparently healthy women is around 10%. In cases of BV, beneficial lactic acid-producing bacteria, with particular reference to lactobacilli, are replaced by amineproducing anaerobic bacteria. Microbiological analysis of BV has shown the gram-variable bacterium G. vaginalis to be the most frequent causative organism. This bacterium has been detected in >98% of BV cases. However, it is not clear if some events induce a drop in the population of lactobacilli, which in turn makes local conditions tolerant to the growth of other bacteria, if BVassociated species such as G. vaginalis displace the lactobacilli according to certain kinetics, or whether these 2 factors are not directly related.32 Treatment of BV involves oral or local administration of metronidazole or clindamycin, and varies in efficacy (48% to 85% for absence of infection, Z4 wk after treatment).33 The overall long-term cure rate is low, as BV recurs in up to 40% of women within 3 months after initiation of antibiotic therapy and in up to 50% of women r
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after 6 months.19 There are several unpleasant side effects and disadvantages associated with these therapies, including superinfections by pathogenic microorganisms18 and susceptibility of lactobacilli to clindamycin,34 a fact that could expose the vaginal milieu to possible reinfections with harmful microbes. Moreover, vaginal pathogens, particularly G. vaginalis and anaerobic bacteria, are showing increased drug resistance.35,36 Many studies have provided evidence of the valuable functions of a healthy microbiota and suggested the selection of bacterial strains, recognized as probiotics, with beneficial effects for the prevention or treatment of conditions involving a disruption of indigenous microbiota. Lactobacilli use in BV is supported by positive results obtained in some clinical trials. The majority of the studies yielding positive results have been performed using probiotic preparations containing high doses of lactobacilli, thus suggesting that the amount of exogenously applied lactobacilli could have a role in the effectiveness of the product.31 However, it is well known that only selected strains could exert significant effectiveness, not limited to the nonspecific mechanism of action attributable to the secretion of organic acids such as lactic and acetic acids. This study suggests that L. fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) are able to effectively counteract acute Gardnerella infections and significantly improve the uncomfortable symptoms reported by women with BV. This evidence may be at least partially attributed to the presence of tara gum, an ingredient that is able to create a mechanical barrier against the adhesion of Gardnerella and of other possible harmful microbes to the surface of vaginal mucosa. The overall compliance and tolerability were very good, with only 1 dropout in the placebo group recorded, due to protocol deviation and not to any adverse events. The very limited percentage of recurrences recorded in the active group during the second month of the protocol suggests a long-term physiological protection thanks to the integration of the 2 lactobacilli into the vaginal microbiota and their adhesion to the epithelial cells of the mucosa. In this regard, the use of arabinogalactan and FOS, 2 fibers that are able to enhance vaginal colonization by the 2 lactobacilli, could have a role in the long-term barrier against reinfections recorded in the second month of the study protocol. Even though the placebo group included a smaller number of subjects compared with the active group, the placebo effect was very low, as no significant statistical differences were recorded either after 28 days (P = 0.619) or at the end of the protocol (P = 0.823), thus suggesting that the improvement recorded in the subjects being given the 2 lactobacilli is specific and attributable to the overall effect of the product. It is also interesting to point out the fact that an in vitro functionality exerted by a selected Lactobacillus, such as the inhibition of Gardnerella growth by means of specific mechanisms of action, was successfully confirmed during this pilot trial in female subjects diagnosed with BV. This could suggest that the in vitro antagonism toward G. vaginalis could be valuable as a criterion to effectively select lactobacilli with a protective action in cases of BV, both in terms of an acute treatment and for long-term prevention of recurrences. In the in vitro part of the study, the selection of a proper growth medium suitable for the inhibition test for r
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Gardnerella was another key point, as presumably some components present in MRS, the medium commonly used to grow lactobacilli before the inhibition assessment, could potentially promote the growth of Gardnerella. In fact, the growth of Gardnerella in BHI alone was relatively poor, especially after 48 hours. In the light of the in vitro inhibitory activity against E. coli exerted mainly by L. plantarum LP01,29 the prospective use of its association with L. fermentum LF15 in aerobic vaginitis (AV) could prove interesting. In fact, AV is predominantly associated with E. coli, but S. agalactiae and S. aureus are also involved. Standard treatment of AV consists of oral or intravaginal antibiotics, although these cannot automatically restore normal flora characterized by a high concentration of lactobacilli, thus facilitating relapses and reinfections. In conclusion, this study investigates a possible innovative approach aimed at counteracting acute vaginal microbiota imbalance and preventing possible recurrences either caused by G. vaginalis (BV) or by E. coli (aerobic vaginitis), although future additional investigations will be needed to quantify the effectiveness of L. fermentum LF15 and L. plantarum LP01 in that condition. REFERENCES 1. Srinivasan S, Fredricks DN. The human vaginal bacterial biota and bacterial vaginosis. Interdiscip Perspect Infect Dis. 2008;2008:750479. 2. Aroutcheva AA, Simoes JA, Faro S. Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardnerella vaginalis. Infect Dis Obstet Gynecol. 2001;9:33–39. 3. Tamonud M, Preeti A, Charan A, et al. Diagnosis of bacterial vaginosis in cases of abnormal vaginal discharge: comparison of clinical and microbiological criteria. J Infect Develop Countries. 2011;5:353–360. 4. Coste I, Judlin P, Lepargneur JP, et al. Safety and efficacy of an intravaginal prebiotic gel in the prevention of recurrent bacterial vaginosis: a randomized double-blind study. Obstet Gynecol Int. 2012;2012:147867. 5. Koumans EH, Sternberg M, Bruce C, et al. The prevalence of bacterial vaginosis in the United States, 2001-2004; associations with symptoms, sexual behaviours, and reproductive health. Sex Transm Dis. 2007;34:864–869. 6. Hillier SL, Nugent RP, Eschenbach DA, et al. Association between bacterial vaginosis and preterm delivery of a lowbirth-weight infant. The Vaginal Infections and Prematurity Study Group. N Engl J Med. 1995;333:1737–1742. 7. Haggerty CL, Hillier SL, Bass DC, et al. Bacterial vaginosis and anaerobic bacteria are associated with endometritis. Clin Infect Dis. 2004;39:990–995. 8. Marrazzo JM, Martin DH. Management of women with cervicitis. Clin Infect Dis. 2007;44(suppl 3):S102–S110. 9. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. Am J Med. 1983;74:14–22. 10. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol. 1991;29:297–301. 11. Hanson JM, McGregor JA, Hillier SL, et al. Metronidazole for bacterial vaginosis: a comparison of vaginal gel vs. oral therapy. J Reprod Med. 2000;45:889–896. 12. Livengood CH III, Soper DE, Sheehan KL, et al. Comparison of once-daily and twice-daily dosing of 0.75% metronidazole gel in the treatment of bacterial vaginosis. Sex Transm Dis. 1999;26:137–142. 13. Agnew KJ, Hillier SL. The effect of treatment regimens for vaginitis and cervicitis on vaginal colonization by lactobacilli. Sex Transm Dis. 1995;22:269–273.
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14. Nyirjesy P, McIntosh MJ, Gattermeir DJ, et al. The effects of intravaginal clindamycin and metronidazole therapy on vaginal lactobacilli in patients with bacterial vaginosis. Am J Obstet Gynecol. 2006;194:1277–1282. 15. Hillier SL, Lipinsky C, Briselden AM, et al. Efficacy of intravaginal 0.75% metronidazole gel for the treatment of bacterial vaginosis. Obstet Gynecol. 1993;81:963–967. 16. Livengood CH III, McGregor JA, Soper DE, et al. Bacterial vaginosis: efficacy and safety of intravaginal metronidazole treatment. Am J Obstet Gynecol. 1994;170:759–764. 17. Sa´nchez J, Campos PE, Courtois B, et al. Prevention of sexually transmitted diseases (STDs) in female sex workers: prospective evaluation of condom promotion and strengthened STD services. Sex Transm Dis. 2003;30:273–279. 18. Sobel JD, Ferris D, Schwebke J, et al. Suppressive antibacterial therapy with 0.75% metronidazole vaginal gel to prevent recurrent bacterial vaginosis. Am J Obstet Gynecol. 2006;194: 1283–1289. 19. Bradshaw CS, Morton AN, Hocking J, et al. High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. J Infect Dis. 2006;193:1478–1486. 20. Swidsinski A, Mendling W, Loening-Baucke V, et al. Adherent biofilms in bacterial vaginosis. Obstet Gynecol. 2005;106(5 pt 1):1013–1023. 21. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284: 1318–1322. 22. Saunders S, Bocking A, Challis J, et al. Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms. Colloids Surf B Biointerfaces. 2007;55:138–142. 23. Do¨derlein A. Das Scheidensekret und seine Bedeutung fu¨r das Puerperalfieber. Leipzig: Garrison-Morton (5th ed.) 6279; 1892. 24. MacPhee RA, Hummelen R, Bisanz JE, et al. Probiotic strategies for the treatment and prevention of bacterial vaginosis. Expert Opin Pharmacother. 2010;11:2985–2995. 25. Nishijima K, Shukunami K, Kotsuji F. Probiotics affects vaginal flora in pregnant women, suggesting the possibility of preventing preterm labor. J Clin Gastroenterol. 2005;39:447–448.
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26. Ro¨nnqvist PD, Forsgren-Brusk UB, Grahn-Ha˚kansson EE. Lactobacilli in the female genital tract in relation to other genital microbes and vaginal pH. Acta Obstet Gynecol Scand. 2006;85:726–735. 27. Falagas M, Betsi GI, Athanasiou S. Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infect. 2007;13:657–664. 28. Senok AC, Verstraelen H, Temmerman M, et al. Probiotics for the treatment of bacterial vaginosis. Cochrane Database Syst Rev. 2009;4:CD006289. 29. Mogna L, Del Piano M, Deidda F, et al. Assessment of the in vitro inhibitory activity of specific probiotic bacteria against different Escherichia coli strains. J Clin Gastroenterol. 2012;46(suppl):S29–S32. 30. Castro J, Henriques A, Machado A, et al. Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. Int J Med Sci. 2013;10:1193–1198. 31. Mastromarino P, Vitali B, Mosca L. Bacterial vaginosis: a review on clinical trials with probiotics. New Microbiol. 2013;36:229–238. 32. Leppa¨luoto PA. Bacterial vaginosis: what is physiological in vaginal bacteriology? An update and opinion. Acta Obstet Gynecol Scand. 2011;90:1302–1306. 33. Koumans EH, Markowitz LE, Hogan V, CDC BV Working Group. Indications for therapy and treatment recommendations for bacterial vaginosis in nonpregnant and pregnant women: a synthesis of data. Clin Infect Dis. 2002;35(suppl 2):S152–S172. 34. Bayer AS, Chow AW, Concepcion N, et al. Susceptibility of 40 lactobacilli to six antimicrobial agents with broad grampositive anaerobic spectra. Antimicrob Agents Chemother. 1978;14:720–722. 35. McLean NW, McGroarty JA. Growth inhibition of metronidazole-susceptible and metronidazole-resistant strains of Gardnerella vaginalis by lactobacilli in vitro. Appl Environ Microbiol. 1996;62:1089–1092. 36. Beigi RH, Austin MN, Meyn LA, et al. Antimicrobial resistance associated with the treatment of bacterial vaginosis. Am J Obstet Gynecol. 2004;191:1124–1129.
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