Egyptian Journal of Medical Microbiology, October 2007 Vol. 16, No 4

Topical Clindamycin or Metronidazole for Treatment of : Microbiologic-Clinical Study

Amal A. Wafy, M.D.; Ahmed T. Soliman, M.D. * and Safaa K. Marei, M.D. *

Departments of Microbiology& Immunology and Obstetrics & Gynecology *, Faculty of Medicine, Tanta University

BACKGROUND: Aim: To compare the frequencies, concentrations, and antimicrobial susceptibilities of vaginal microbes isolated from women with bacterial vaginosis (BV) before and after therapy with intra-vaginal clindamycin or metronidazole. DESIGN: Prospective randomized controlled study. PATIENTS & METHODS: 119 non-pregnant women aged 20 - 45 with clinical and Gram stain evidence of BV were randomized to receive intra-vaginal clindamycin or metronidazole. Vaginal swabs were collected at baseline and 7 to 12 days, 35 to 45 days, and 70 to 90 days following therapy for quantitative vaginal culture. For the 99 women completing all four visits, statistical analyses were performed comparing differences in vaginal microflora between the two treatment arms and between visits in the same treatment group. Antimicrobial susceptibility testing using the agar dilution method was performed for anaerobic gram-negative rods. RESULTS: Although both therapies resulted in decreased colonization by Gardnerella vaginalis and , only metronidazole treatment resulted in a significant decrease in the frequency and concentration of bivia and black-pigmented Prevotella species. Of the 865 anaerobic gram-negative rods evaluated for susceptibility, only 3 (0.3%) were resistant to metronidazole, whereas clindamycin resistance increased significantly for P. bivia and black-pigmented anaerobic gram-negative rods persisting following clindamycin therapy. Clindamycin-resistant subpopulations of P. bivia and black-pigmented Prevotella species emerged 7 to 12 days after therapy even among women colonized initially by clindamycin- susceptible strains. These resistant subpopulations persisted at high frequencies (42 to 50%) 70 to 90 days following therapy. CONCLUSION: The two topical agents for treatment of BV have differing microbiologic effects on the vaginal microflora. The emergence of clindamycin-resistant anaerobic gram-negative rods following therapy is of concern.

INTRODUCTION for the diagnosis of bacterial vaginosis have included culture for G. vaginalis 7, 8, direct Gram Bacterial vaginosis (BV) is a common stain of vaginal secretions 9, biochemical tests for lower genital tract syndrome affecting women of metabolic by-products of vaginal (gas reproductive age. Microbes associated with BV are chromatography) 10, and more recently, the proline part of the endogenous flora of the vagina, and the aminopeptidase test 11. acquisition of BV results when there are changes The treatments recommended by the of the normal flora of the vagina, causing an Centers for Disease Control (CDC) for BV are increased prevalence of Gardnerella vaginalis, either metronidazole or clindamycin administered Mycoplasma hominis, and anaerobic organisms orally or intravaginally 12. Metronidazole is a and a decreased prevalence of the dominant nitroimidazole with activity against anaerobic Lactobacillus species. Acquisition of BV is organisms, while clindamycin, a macrolide, has a associated with adverse outcomes among non- broad spectrum of activity against a variety of pregnant and pregnant women. Previous studies microbes including aerobic and anaerobic have shown that the alteration of the normal flora organisms. could increase the risk of acquiring BV, human Despite treatment with either immunodeficiency type 1, or sexually metronidazole or clindamycin, similar percentages transmitted diseases 1, 2 . Furthermore, BV has of women (approximately 10 to 15%) fail therapy been found to be associated with preterm labor, after 1 month 13. The proportion of women who preterm delivery, low birth weight, post-cesarean relapse also increases over time. The recurrence , and post-abortion pelvic rate of BV is approximately 30% at 3 months and inflammatory disease 3-5 . approximately 50 to 80% at 1 year following Because bacterial vaginosis is a clinical therapy with either drug 14-16. Current therapy for syndrome which has been associated with a group managing recurrent BV is repeated treatment with of genital microorganisms rather than a single antibiotics. An obvious problem and important etiologic agent, it has been defined primarily by health issue associated with repeated exposure to the following clinical signs: vaginal pH >4.5, the the same antibiotic is resistance of those microbes presence of adherent white discharge, detection of targeted by the drug, which can result in an "clue cells," and the presence of an amine odor alteration of flora and possible persistence of BV- after the addition of KOH 6. Laboratory methods associated pathogens. Resistance to metronidazole,

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Egyptian Journal of Medical Microbiology, October 2007 Vol. 16, No 4 despite its use for over 3 decades, is rare 17, 18. days after finishing the last dose of medication and Recent studies have shown an emergence of 35 to 45 days and 70 to 90 days after treatment. If clindamycin-resistant genital organisms among women were diagnosed with BV at a follow-up clinically relevant bacteria, including group B visit, they were retreated with the same therapy streptococci 19, 20. they initially received. Although no comparative studies have Clinical evaluation evaluated the microbiologic responses, separate A clinical cure was defined as the absence of clue placebo-controlled trials suggest that there may be cells on wet mount in addition to the absence of different microbiological responses following two or more clinical signs (no homogenous therapy with metronidazole and clindamycin 13. discharge, pH less than 4.7, no amine odor upon The objectives of the present study were addition of potassium hydroxide). Only the subset to compare the frequencies and median of 99 women completing all four visits and having concentrations of major constituents of the vaginal valuable culture results from those visits was microflora before and after treatment to assess for included in the present analysis. metronidazole and clindamycin resistance among Specimen collection anaerobic components of the microflora before and Vaginal swabs were obtained at baseline after therapy and to evaluate whether (pretreatment) and at the three consecutive follow- antimicrobial susceptibility accounts for the up visits following treatment. Vaginal swabs were persistence of anaerobic gram-negative rods obtained by placing a non-lubricated speculum following therapy. into the vagina and swabbing the lateral wall discharge with a Dacron polyester fiber-tipped MATERIALS AND METHODS swab (Fisher Scientific, Pittsburgh, PA). The swabs were transported to the laboratory, where Patient population. A total of 119 non-pregnant they were processed within 24 hours of collection. women aged 20 to 45 years with a clinical and

Gram stain diagnosis of BV were recruited from Microbiological evaluation the outpatient clinic of Tanta University Hospital. Isolation and identification. Quantitative vaginal Written informed consent of the protocol was culturing was performed. The Lactobacillus spp. obtained from all patients prior to enrollment. isolated were identified by Gram stain showing Exclusion criteria gram-positive rods, characteristic ground glass <20 years of age • colony morphology, and negative catalase • pregnancy reaction. Hydrogen peroxide (H2O2) detection of • known allergy to metronidazole or clindamycin the Lactobacillus spp. isolated was performed by a • concurrent antibiotic use, qualitative method in which isolates are inoculated • menstruation, onto a brucella agar base supplemented with • presence of an intrauterine device, 3,3',5,5'-tetramethylbenzidine and horseradish • known active infection due to , peroxidase. Anaerobic incubation at 37°C for 48 , or Trichomonas, or clinically to 72 h followed by exposure to air for 30 min

apparent infection. results in a blue pigment if there is H2O2

Clinical diagnosis of BV was determined using production and no pigment if H2O2 production is Amsel's criteria 6. Three or more of these criteria absent 22. The Gardnerella vaginalis isolates were must be present to ensure the clinical diagnosis: identified by their characteristic colony 1. Homogenous , morphology, beta hemolysis on human bilayer 2. >20% clue cells on wet mount preparation, agar with Tween (Becton Dickinson, Rockville, 3. Elevated pH of vaginal discharge ( 4.7), MD), Gram stain showing gram-variable 4. Release of a fishy amine odor upon addition of pleomorphic rods, and negative catalase reaction. 10% potassium hydroxide solution to vaginal The Mycoplasma hominis and Ureaplasma fluid. urealyticum isolates were identified by their A Gram stain score of 4 based on the criteria for characteristic colony morphology on A-8 agar. BV assessment developed by Nugent et al. was The Escherichia coli isolates were identified by required for Gram stain diagnosis 21 . their colony morphology, Gram stain showing Treatment: Upon enrollment, women were gram-negative rods, and positive indole (Sigma, randomized using a computer-generated list to St. Louis, MO) test. The anaerobic gram-negative receive one of two treatments, clindamycin vaginal bacteria isolated were identified by Gram stain, a cream (Dalacin vaginal cream 2%®, Pfizer, Egypt) lack of growth on aerobically incubated media, once daily for 3 days or metronidazole vaginal susceptibility to a 20% bile disk (bile and disk, suppository (Metronidazole vaginal suppository Becton Dickinson), susceptibility to colistin

® (Becton Dickinson), and 4-methylumbelliferone 500 mg , Amrya Pharmaceuticals, Egypt) once 23 daily for 5 days. Following enrollment and spot testing . treatment, women were to follow up at 7 to 12

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A study comparing oligonucleotide frequencies of microbes and clinical efficacy probes and spot testing for the other non- between the clindamycin and metronidazole pigmented Prevotella spp. has not yet been treatment groups. Fisher's exact test was also used undertaken; therefore, reliable phenotypic tests for to compare frequencies of loss and acquisition of identification do not exist, and this results in the microbes between treatment groups among those grouping of these organisms 24. The anaerobic whose colonization status changed between the gram-negative rods selected for antimicrobial enrollment and first follow-up visit. A chi-square susceptibility testing were those colony types test for linear trend was used to evaluate the isolated in the highest concentrations. These change in the percentage of resistant isolates from isolates were stock-frozen in litmus milk (Becton enrollment through the third follow-up visit within Dickinson) at –70°C until susceptibility testing the clindamycin group. A chi-square test was used was performed. to evaluate differences in colonization frequencies of microbes at the first follow-up visit between Antimicrobial susceptibility testing. those who were not colonized, those who were Antimicrobial susceptibility testing using the agar colonized with a clindamycin-susceptible isolate, dilution method approved by the NCCLS and as and those who were colonized with a clindamycin- previously described was performed to determine resistant isolate at enrollment. The Mann-Whitney the MICs of 865 isolates of anaerobic gram- U test was used to evaluate differences in the negative rods belonging to the following groups of median concentration of microbes between bacteria: Prevotella bivia, non-pigmented treatment groups among those who were colonized Prevotella species, black-pigmented Prevotella at each visit. Cochran's Q and Friedman's tests species, Porphyromonas species, and Bacteroides were used to evaluate differences in colonization species 25, 26. The technologists selecting the frequencies and median concentrations of isolates for susceptibility testing and performing microbes between visits in each subject within the susceptibility testing were blind as to the treatment groups. All statistical tests were antibiotic treatment group and clinical response to evaluated at the 0.05 significance level. therapy. MICs were determined using the NCCLS RESULTS guidelines. The breakpoints of resistance are 32 The clinical responses to BV treatment µg/ml for metronidazole and 8 µg/ml for did not differ for the two treatment groups clindamycin. following therapy. At 7 to 12 days, cure rates were

79% and 88% for metronidazole and clindamycin Statistical analyses. (P = 0.3). At 35 to 45 days, the cure rates were All statistical analyses were performed 62% and 55%, respectively (P = 0.5), and rates using SPSS statistical software release 12.0.1 were 58% and 55% (P = 0.8) 70 to 90 days after (SPSS Inc., Chicago, IL). Fisher's exact test was therapy (Table 1). used to evaluate differences in colonization

TABLE 1. clinical responses to BV treatment with either clindamycin or metronidazole

Metronidazole group Clindamycin group P- value 7 - 12 days 79% 88% NS 35 - 45 days 62% 55% NS 70 - 90 days 58% 55% NS

A graphical analysis comparing the following therapy with either clindamycin or changes observed among the vaginal metronidazole. Women treated with metronidazole microorganisms associated with BV isolated at had a significant decrease in colonization by U. baseline and 7 to 12 days, 35 to 45 days, and 70 to urealyticum (94% to 79%, P < 0.001) compared 90 days following therapy with metronidazole or with clindamycin-treated women (73% to 65%, P clindamycin is presented in Fig. 1. = 0.3). Following therapy with metronidazole, The presence of H2O2-producing lactobacilli is there was a significant decrease in colonization considered an indicator of optimal vaginal among two groups of anaerobic gram-negative ecology, and a significant increase in colonization rods associated with BV, P. bivia (P = 0.03) and by H2O2-producing Lactobacillus species was black-pigmented Prevotella spp. (P = 0.02), but a observed among women following therapy with significant decrease in these organisms was not either metronidazole or clindamycin over the 90 observed among women who were treated with days of follow-up (P < 0.001). A significant clindamycin (Fig. 1). Both Porphyromonas spp. decrease in colonization by G. vaginalis and M. and nonpigmented Prevotella spp. decreased hominis (P < 0.001) was observed among women

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Egyptian Journal of Medical Microbiology, October 2007 Vol. 16, No 4 significantly following treatment with either clindamycin, the percentages of clindamycin- regimen (Table 2). resistant isolates were 51%, 43%, 68%, 14%, and Antimicrobial susceptibility testing of 0%, respectively. Isolates recovered 35 to 45 days anaerobic gram-negative rods recovered from the and 70 to 90 days following therapy also exhibited women before and after treatment (Table 3) increased resistance. While clindamycin-resistant revealed a marked and sustained increase in the anaerobic gram-negative rods were isolated from proportion of clindamycin-resistant anaerobic the women who were treated with topical gram-negative rods from women following metronidazole before therapy, the proportion of therapy with clindamycin but not metronidazole. isolates expressing clindamycin resistance did not The proportions of P. bivia, non-pigmented increase following metronidazole therapy. These Prevotella, black-pigmented Prevotella, data suggest that the increase in clindamycin- Porphyromonas, and Bacteroides isolates resistant resistant isolates following clindamycin therapy is to clindamycin before treatment with clindamycin attributable to clindamycin exposure rather than were 8%, 14%, 11%, 3%, and 0%, respectively. treatment of BV per se However, at 7 to 12 days following therapy with

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Table 2. Anaerobic gram-negative rods resistant to clindamycin and metronidazole before and after therapy No. (%) of drug-resistant isolates from patients Group Visit No. of treated witha: isolates Clindamycin Metronidazole tested Clindamycin Metronidazole Clindamycin Metronidazole resistant resistant resistant resistant P. bivia Pretreatment 60 5 (8) 0 10 (17) 0 7-12 days 39 20 (51) 0 2 (5) 0 35-45 days 61 23 (38) 0 7 (11) 0 70-90 days 53 22 (42) 0 5 (9) 0 Non-pigmented Pretreatment 108 15 (14) 0 7 (6) 1 (1) Prevotella spp. 7-12 days 40 17 (43) 0 3 (8) 1 (3) 35-45 days 85 24 (28) 0 7 (8) 0 70-90 days 68 17 (25) 0 9 (13) 0 Black- Pretreatment 56 6 (11) 0 7 (13) 0 pigmented Prevotella spp. 7-12 days 22 15 (68) 0 2 (9) 0 35-45 days 44 16 (36) 0 3 (7) 1 (2) 70-90 days 36 17 (47) 0 4 (11) 0 Porphyromonas Pretreatment 89 3 (3) 0 2 (2) 0 spp. 7-12 days 14 2 (14) 0 2 (14) 0 35-45 days 41 15 (37) 0 2 (5) 0 70-90 days 31 13 (50) 0 3 (10) 0 Bacteroides Pretreatment 5 0 0 0 0 spp. 7-12 days 3 0 0 0 0 35-45 days 5 1 (20) 0 2 (40) 0 70-90 days 5 1 (20) 0 1 (20) 0 a P values are from chi-square tests for linear trend. P values for clindamycin results are as follows: for P. bivia, <0.001; for nonpigmented Prevotella spp., 0.08; for black-pigmented Prevotella spp., <0001; for Porphyromonas spp., <0.001; and for Bacteroides spp., 0.2. P values for metronidazole results are as follows: for P. bivia, 0.3; for nonpigmented Prevotella spp., 0.2; for black-pigmented Prevotella spp., 0.6; for Porphyromonas spp., 0.1; and for Bacteroides spp., 0.2.

Table 3. Frequency of Prevotella bivia and black-pigmented Prevotella species before & after topical clindamycin therapy, stratified by clindamycin susceptibility Pretreatment culture or statistic No. (%) of isolates No. (%) of isolates resistant positiveb for given to clindamycin /total no. of organism/total no. of isolatesb isolates P. bivia present Clindamycin susceptible (n = 19) 10/19 (52%) 9/10 (90%) Clindamycin resistant (n = 5) 3/5 (60%) 3/3 (100%) P. bivia absent (n = 22) 6/22 (27%) 6/6 (100%) P valuea P = 0.2 P = 0.6 Black-pigmented Prevotella present Clindamycin susceptible (n = 15) 1/15 (7%) 1/1 (100%) Clindamycin resistant (n = 6) 1/6 (17%) 1/1 (100%) Black-pigmented Prevotella absent (n = 20) P valuea 11/20 (55%) 10/11 (91%) P = 0.007 P = 0.9 a P values are from chi-square tests. b The numbers of positive and clindamycin-resistant cultures were determined 7-12 days post-treatment.

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DISCUSSION bacteria colonizing the vagina, and treatment with clindamycin results in selection for clindamycin- Despite similarities in clinical responses resistant anaerobic gram-negative rods following observed among women treated with therapy. This was not observed following therapy metronidazole and clindamycin, the present study with metronidazole. shows that treatment with clindamycin yields a Surprisingly, there was no difference different microbial flora pattern following therapy observed in the proportion of clindamycin- for BV and that the anaerobic gram-negative rods resistant Bacteroides species isolated before or persisting after therapy are usually resistant to after therapy with clindamycin or metronidazole. clindamycin but not metronidazole. Clindamycin resistance is highly associated with The increase in colonization by the Bacteroides fragilis group, and previous Lactobacillus spp. during the week following studies have shown increased resistance among therapy with metronidazole but not clindamycin is this group occurring over the past decade 33, 34. A expected, since clindamycin has a broad spectrum possible reason increased resistance was not of activity against anaerobic as well as aerobic and observed was because the B. fragilis group isolates facultative organisms, like Lactobacillus spp. 27. were generally recovered at densities of 103 CFU Metronidazole is a nitroimidazole with activity per gram of vaginal fluid and were not the against obligatory anaerobic organisms. Aerobic predominant anaerobic species chosen for and facultative organisms are generally not susceptibility testing. inhibited by nitroimidazoles because their The emergence of clindamycin resistance mechanism of action, the reduction of the nitro following treatment is not a novel finding. In 1986, group, is dependent upon the absence of oxygen Ohm-Smith et al. reported on the emergence of 28. clindamycin-resistant anaerobic bacteria from the Surprisingly, there was a similar endometria of women with pelvic soft tissue reduction observed in vaginal colonization by G. infections 35. As in the present study, they found vaginalis and M. hominis after therapy with either no correlation between antibiotic resistance and metronidazole or clindamycin and a significant clinical response to therapy. Further, they reported decrease in vaginal colonization by U. urealyticum that four of seven (57%) of the anaerobic gram- among women treated with metronidazole but not negative rods recovered following treatment were clindamycin. Clindamycin has activity against resistant to clindamycin. The present study differs these organisms, while metronidazole generally from that 1986 study in that the frequency of does not. A possible explanation for metronidazole clindamycin resistance at baseline has increased having activity against these organisms is that over the past 2 decades, a larger number of isolates previous studies have reported the was evaluated, and women were monitored for hydroxymetabolite of metronidazole, a compound persistence of resistant organisms for 70 to 90 formed when metronidazole is metabolized by the days. The emergence and persistence of liver or from the breakdown of metronidazole by clindamycin-resistant populations of anaerobes in anaerobic bacteria during its action, has the vagina following topical therapy may have antimicrobial activity and may have activity implications for the future use of clindamycin for against G. vaginalis 13, 17, 29, 30, 31. Anaerobic gram- treatment of pelvic infections, because most negative rods are reported BV pathogens and are organisms causing pelvic infections are derived isolated from virtually all women with BV. A from the lower reproductive tract. previous study has found that P. bivia and the

Prevotella corporis/Bacteroides levii group, a Conclusion black-pigmented group of organisms, are In summary, treatment with clindamycin and significantly associated with BV 32. There was a treatment with metronidazole for BV result in significant decrease in colonization among these different microbiological patterns following two groups of anaerobic gram-negative rods in therapy. While both topical metronidazole and women after therapy with metronidazole clindamycin yielded similar clinical responses to compared with women who were treated with treatment, metronidazole may be superior to clindamycin. Antimicrobial susceptibility testing clindamycin based on the low level of resistance of P. bivia and black-pigmented Prevotella spp. and its capacity to eradicate anaerobic gram- revealed that 51% and 68% of the isolates, negative rods from the vagina. respectively, were resistant to clindamycin following therapy with clindamycin but not REFERENCES metronidazole. Increased metronidazole resistance was not observed among these groups of bacteria 1. Hawes, S. E., S. L. Hillier, J. Benedetti, et al. 1996. regardless of whether women were treated with Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J. Infect. Dis. 174:1058-1063. clindamycin or metronidazole. This suggests that 2. Martin, H. L., B. A. Richardson, P. M. Nyange, et al. clindamycin-resistant anaerobic gram-negative 1999. Vaginal lactobacilli, microbial flora and risk of rods are present among the mixed population of human immunodeficiency virus type 1 and sexually

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