The role of lactobacillus casei rhamnosus lcr35 in restoring the normal vaginal flora after antibiotic treatment of bacterial vaginosis

The role of Lactobacillus casei rhamnosus Lcr35in restoring the normal vaginal flora afterantibiotic treatment of bacterial vaginosis Department of Obstetrics and Fetomaternal Medicine, Medical University of Vienna, Vienna, AustriaCorrespondence: Dr A Witt, Department of Obstetrics and Fetomaternal Medicine, Medical University of Vienna, Waehringer Guertel 18-20,1090 Vienna, Austria. Email [email protected] Objective To evaluate the efficacy of additional topical Main outcome measures The primary efficacy variable was Lactobacillus casei rhamnosus (Lcr35) subsequent to antibiotic a change in the Nugent score between the baseline and the end of treatment of bacterial vaginosis (BV) to restore the normal the study of at least 5 grades in each individual woman.
Results Sixty-nine of the 83 women (83%) in the intervention Study design Single-centre, randomised, observerblinded study.
group and 31 of the 88 women (35%) in the control group showeda reduction of the Nugent score by at least 5 grades. The difference Setting Population-based study in Vienna over 1 year.
in the number of women with improvement was highly significant Sample 190 women were enrolled in the study.
(P < 0.001). The median difference in Nugent scores betweeninitial and final swabs was 6.61 in the intervention group and Methods Women with Nugent scores between 7 and 10 on initial 4.13 in the control group (P < 0.001).
vaginal swab were randomised to the one of two groups. Allwomen were treated with standard antibiotic therapy for 7 days.
Conclusion Our data show that the restoration of the vaginal flora Only women in the intervention group received vaginal capsules after antibiotic treatment of BV can be significantly enhanced by containing 109 colony-forming units of live Lcr35 for 7 days after antibiotic treatment. Final vaginal swabs for Nugent scoring were Keywords Bacterial vaginosis, lactobacilli, vaginal flora.
taken 4 weeks after the last administration of the study medication.
Please cite this paper as: Petricevic L, Witt A. The role of Lactobacillus casei rhamnosus Lcr35 in restoring the normal vaginal flora after antibiotic treatmentof bacterial vaginosis. BJOG 2008;115:1369–1374.
spp.3 Metronidazole and clindamycin have been shown tobe effective treatments for BV and to promote similar levels Common reproductive tract infections and the associated in- of restoration of vaginal lactobacilli.4,5 Although anti-infective flammatory responses are the most frequent gynaecological treatment is available and usually highly efficient in eradicat- complaints, representing a central problem in modern clinical ing pathogenic microorganisms, its long-term efficiency is care. Vaginitis is usually characterised by vaginal discharge, often limited by relapses, which are most likely, due to an vulval itching, irritation, or malodour. Bacterial vaginosis (BV), inability to re-establish the normal lactobacillus-dominated vulvovaginal candidiasis, and trichomoniasis are regarded as the most common vaginal infections worldwide.1,2 To understand the treatment failures frequently occurring BV is a condition of the female genital tract characterised in vaginal infections, it is important to consider the vaginal by a malodorous vaginal discharge, a vaginal pH of >4.5, and ecosystem in health and disease. The normal microflora is depletion of Lactobacillus spp. BV does not follow Koch’s dominated by lactobacilli capable of inhibiting the adhesion postulate, whereby a single pathogen is responsible for a spe- and growth of pathogens, depleting nutrients otherwise avail- cific disease but is characterised by an overgrowth of diverse able to pathogens, and modulating the host immune response aerobic, anaerobic, and micro-aerophilic species, such as Gar- and microenvironment.8,9 There are three proposed mecha- dnerella vaginalis, Prevotella spp., Peptostreptococcus spp., Myco- nisms that elucidate the protective role of lactobacilli. First, plasma hominis, Ureaplasma urealyticum, and Mobiluncus they help to produce lactic acid as a by-product of glycogen ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology metabolism in the cells of the vaginal vault, thus acidifying the and neoplasia from among outpatients of the Department healthy vagina to a pH of 4.0–4.5, a level at which many of Obstetrics and Gynaecology. To determine the effect of pathologic microbes cannot flourish. Second, many species lactobacilli in the absence of estrogen, women receiving oral of Lactobacillus produce hydrogen peroxide (H2O2), which contraceptives or other estrogen-containing treatments were further inhibits microbial growth.10–12 Third, lactobacilli com- excluded from the study, as were women having received pete with pathogenic microorganisms for adherence on epithe- antibiotic treatment in the past 4 weeks prior to study entry.
Participants and Gram-stain readers were blinded to the There is a considerable geographical variation in the com- treatment assignment. The diagnosis of BV was established position of the normal vaginal Lactobacillus flora. Several spe- by the investigator at the initial examination based on each cies of lactobacilli have been described to populate the vagina woman’s history and clinical findings, such as a whitish-grey, to varying degrees. Vasquez et al.15 found that the vaginal homogeneous discharge, or fishy smell. The final decision flora of most participants was dominated by a single Lacto- about participation in the study was based on microscopic bacillus species among others, while another study showed that the most frequent Lactobacillus species were Lactobacillus From each potential participant, an initial vaginal smear crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lac- was taken and smeared on microscopy slide. Smears were Gram-stained and evaluated at a central laboratory with staff Lactobacillus casei rhamnosus has been successfully ex- experienced in the use of the Spiegel criteria.24 To detect even ploited commercially as a pharmaceutical product for more minor treatment effects, scoring was performed using the than 20 years. Its beneficial effects include the treatment and entire 10-grade Nugent scale.25 Only women with evident prevention of diarrhoea. Thus, Forestier et al.17 showed that BV as evidenced by a Nugent score between 7 and 10 were in- this strain has probiotic activities, such as the ability to adhere vited to participate in the study after giving written informed to intestinal cells and also exert antibacterial activity against consent and were randomised to one of two study groups several pathogens. Furthermore, Lcr35 has been shown to be using a computer-generated randomisation list. All women effective in treating children with chronic constipation.18 were treated orally for BV with a standard antibiotic regime, Also, a pilot study by Forestier et al. showed that oral admin- that is 2 · 300 mg clindamycin for 7 days.
istration of a probiotic delayed respiratory tract colonisation Participants assigned to the intervention group also received by and infection with by Pseudomonas aeruginosa.19 In addi- a vaginal Lactobacillus capsule (Gynophilus; Laboratoires Lyo- tion to its positive probiotic activity, L. rhamnosus is known centre, Aurillac Cedex, France) for 7 days after antibiotic treat- to produce the bacteriocin lactocin 160, which has been ment. Each capsule contained at least 109 colony-forming units shown to inhibit growth of G. vaginalis.20 Lactocin 160 is of live L. casei rhamnosus (Lcr35), 5.59 mg lactose, and 3.41 mg highly specific in its antimicrobial action. It disturbs the cel- magnesium stearate. Women in the control group did not lular membrane and induces ATP efflux, most likely because of pore formation, in this way killing microorganisms asso- Final vaginal swabs for Nugent scoring were taken 4 weeks ciated with BV but leaving the healthy vaginal microflora after the last administration of the medication in both groups.
intact.21 Lactocin 160 causes minimal irritation and has a good Compliance was assessed by interview and by having women potential for intravaginal application.22 While depletion of lactobacilli and an overgrowth of patho- gens are distinct features of BV, antibiotic therapy further low- ers the numbers of lactobacilli.23 The topical administration oflactobacilli preparations in addition to antibiotic therapy may The primary efficacy variable was a change in the Nugent help restore the normal vaginal flora and avoid the relapses of score between the baseline and the end of the study of at least infection. Therefore, the aim of this study was to evaluate the 5 grades in each individual woman. In addition, we assessed efficacy of topical L. casei rhamnosus Lcr35 in restoring the whether treatment improved the Nugent score grade, regard- normal vaginal flora after antibiotic treatment of BV.
Assuming a response rate of 50% in the control group, including drop-outs, 95 women had to be randomised to eachgroup to detect a difference in response rates of 20% with This single-centre, randomised, observer-blinded study was a statistical power of 80% at a two-sided significance level performed with the approval of the ethics committee of the Medical University of Vienna and in accordance with the The statistical analysis software used is a validated, propri- Declaration of Helsinki and the guidelines of Good Clinical etary development written in IBM APL 2, version 2, Service Practice. Between May 2005 and April 2007, we enrolled non- level 6. Rates between groups were compared in 2 · 2 con- pregnant women aged 18–45 years without vaginal bleeding tingency tables using the chi-square test, and average score ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology Efficacy of Lactobacillus casei rhamnosus ratings between groups were compared using the t test for and 35% in the control group) using the chi-square test independent samples. The Bowker test for symmetry in score changes was used for the intraindividual pre- versus post- In women with an intermediate vaginal flora (11% in the intervention group and 55% in the control group), the sameprocedure showed a P value of <0.001 (x2 for the proportions of women with BV, no statisticallysignificant difference between study groups was found (P < Of the 1550 women screened for enrolment in the study, 1360 did not meet the eligibility criteria. Therefore, 95 The median difference in Nugent scores between initial and women were randomised to the intervention group and 95 final swabs was 6.61 (SD 2.44) in the intervention group and women were randomised to the control group (Figure 1). All 4.13 (SD 2.37) in the control group, a highly significant dif- women were of Caucasian origin. In the intervention and ference between study groups (P < 0.001). Table 2 shows the control groups, 12 and 7 women were lost to follow up, so shifts in Nugent scores between initial and final swabs in indi- that outcome data were available from 83 women in the vidual women. Sixty-nine of the 83 women (83%) in the inter- intervention group and 88 women in the control group.
vention group and 31 of the 88 women (35%) in the control The mean age of the study participants was 32.6 (SD 8.8) group showed a reduction in the Nugent score by at least 5 years, mean weight was 59.6 (SD 8.2) kg, and mean height grades. In both study groups, the intraindividual improvements was 164.6 (SD 7.5) cm, with no significant differences evaluated using Bowker’s symmetry test were found to be between the study groups. Likewise, there were no between- highly significant (P < 0.001). By the end of the study, Nugent group differences in terms of smoking habits, alcohol con- scores had decreased by even 8 grades in 60% of women in the sumption, or the numbers of previous pregnancies or deliv- intervention group and in 14% of women in the control group, eries. Only 5% of women in both groups had reported a this difference again being significant (P < 0.001).
The distributions of initial and final Nugent scores in the intervention and control groups are shown in Table 1. Thepost-treatment comparison of the proportions of women The results of this study suggest that the restoration of the with a normal vaginal flora (83% in the intervention group vaginal flora after antibiotic treatment of BV can be ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology Table 1. Distribution of the Spiegel grades and Nugent scores for initial and final swabs in the intervention and control groups (chi-square test) significantly enhanced by exogenously applied live lactoba- flora’ not adequately responding to standard anti-infective cilli. The decision to use Lcr35 as a test preparation was based therapy.6,7,23,26 In our study, a normal vaginal flora could not on the multiple beneficial effects of L. casei rhamnosus.
be restored in 65% of women in the control group.
Besides its proven positive role as a probiotic,17 it is one of Although metronidazole is recommended as first-line four lactobacilli frequently isolated from the vagina.16 Also, it treatment of BV, clinical practice at our department has produces proteinaceous substances exhibiting antibacterial shown that clindamycin is better tolerated by our women.
activity, such as lactocin 160, a bacteriocin that is highly spe- Metronidazole and clindamycin have been shown to be cific in its antimicrobial action, eliminating microorganisms effective treatments for BV and to promote similar levels of associated with BV but leaving the healthy vaginal microflora restoration of vaginal lactobacilli.4,5 Nyirjesy et al. found that intact.21 Furthermore, lactocin 160 causes minimal irritation clindamycin eradicated an abnormal vaginal flora in 90% of and has a good potential for intravaginal application.22 pregnant women. Also, clindamycin has been shown to be Although modern anti-infective treatment is usually highly more effective than metronidazole at reducing vaginal efficient in eradicating pathogenic microorganisms, numerous Mobiluncus morphotypes in women with BV, correlating with authors have reported treatment failures in BV, particularly in terms of long-term success,6,7 with BV and an ‘intermediate On the downside, treatment of BV with clindamycin is associated with marked evidence of antimicrobial resistanceamong vaginal anaerobic bacteria.7 Table 2. Difference in Nugent scores between initial and final One limitation of this study is that we did not obtain vag- swabs in the intervention and control groups (chi-square test) inal swabs for assessment of the vaginal flora after treatmentwith clindamycin and before administration of lactobacilli.
Based on results reported by Aroutcheva et al.,28 clindamycin most likely had the same effect in both study groups, not onlyeliminating pathogenic microorganisms but also lowering the number of lactobacilli. Therefore, the vaginal flora in the intervention group after antibiotic treatment and before administration of lactobacilli is likely to have been similar At the end of the study, there was a significant difference between study groups with respect to both grades 1 and 2 (P < 0.001; Table 1). Thus, 11% of women in the intervention group had an intermediate flora compared with 55% in the con-trol group. This finding is important in that approximately ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology Efficacy of Lactobacillus casei rhamnosus half of the women with an intermediate flora have been found to develop BV, suggesting that in the control group, the likeli-hood for women to progress to BV is higher than in the The authors thank DI Kurt Neumann, Executive Information Service Ltd, Vienna, Austria, for statistical analyses, Birgit Although lactobacilli have been shown to dominate the Fu¨ssl, Germania Pharmazeutika, Vienna, Austria, for provid- healthy vaginal flora,29 data on the role of lactobacilli in the ing the test preparation, and Gabriele Berghammer, the text treatment or prevention of urogenital infection are scarce. In clinic, for medical writing services. j 2005, Ozkinay et al.30 reported that restoration of the vaginalflora can be significantly enhanced by the administration oflive lactobacilli in combination with low-dose estriol. Uehara et al.31 found that local application of lactobacilli may help 1 Eschenbach DA. History and review of bacterial vaginosis. Am J Obstet prevent recurrent urinary tract infections.
Because estrogen encourages the vaginal colonisation with 2 Sobel JD. Vaginitis. N Engl J Med 1997;337:1896–903.
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and maintain a low vaginal pH that inhibits the growth of Bacterial vaginosis in pregnancy: distribution of bacterial species indifferent gram-stain categories of the vaginal flora. J Med Microbiol many pathogens, we excluded women receiving oral contra- ceptives or other estrogen-containing medications to deter- 4 Simoes JA, Aroutcheva AA, Shott S, Faro S. Effect of metronidazole on mine the effect of lactobacilli in the absence of estrogen. In the growth of vaginal lactobacilli in vitro. Infect Dis Obstet Gynecol our study, local application of Lcr35 after antibiotic treatment of BV significantly improved the vaginal flora as demon- 5 Nyirjesy P, McIntosh MJ, Gattermeir DJ, Schumacher RJ, Steinmetz JI, Joffrion JL. The effects of intravaginal clindamycin and metronidazole strated on the basis of Nugent scores, a scoring system gen- therapy on vaginal lactobacilli in patients with bacterial vaginosis. Am J erally considered an adequate and objective method for the 6 Papanikolau EG, Tsanadis G, Dalkalitis N, Lolis D. Recurrent bacterial Local Lcr35 restored a normal flora in 83% of women in the vaginosis in virgin adolescent: a new method of treatment. Infection intervention group. In the control group, 35% of women had 7 Beigi RH, Austin MN, Meyn LA, Krohn MA, Hillier SL. Antimicrobial a normal vaginal flora at the end of the study. This difference resistance associated with the treatment of bacterial vaginosis. Am J between groups was highly significant (Table 2). Moreover, every single woman in the intervention group had a significant 8 Erickson KL, Hubbard NE. Probiotic immunomodulation in health and shift in the Nugent score of at least 5 grades between the base- disease. J Nutr 2000;130 (2S Suppl):S403–9.
line and the end of the study. Therefore, the results of our 9 Reid G, Cook RL, Bruce AW. Examination of strains of lactobacilli for properties that may influence bacterial interference in the urinary tract.
study suggest that topical L. casei rhamnosus LCR 35 may help restore the normal vaginal flora after antibiotic therapy of BV.
10 Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, Critchlow CM, et al. Prevalence of hydrogen peroxide-producingLactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 1989;27:251–6.
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University (EK Nr. 099/2005) and supported by the head of 12 Hillier SL, Krohn MA, Rabe LK, Klebanoff SJ, Eschenbach DA.
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ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology

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