Cultivation-Independent Analysis of Changes in Bacterial Vaginosis Flora Following Metronidazole Treatment

Abstract

PCR was used to survey bacterial vaginosis flora before and after metronidazole treatment. The species composition for pretreatment patients was variable. Lactobacillus iners was prominent in all patients posttreatment. Atopobium vaginae concentrations were highest for patients who failed or responded incompletely to treatment and lowest for patients who were cured.Bacterial vaginosis (BV) is the most common cause of vaginal irritation and is associated with adverse pregnancy outcomes (4, 13) and an increased risk of human immunodeficiency virus infection (14,15). BV results when the normal, predominantly Lactobacillus vaginal flora shifts to one dominated by Gardnerella vaginalis, Mycoplasma hominis, and a variety of anaerobic organisms. However, no specific pathogen has been identified, and the cause of BV is unknown (6). Metronidazole is the most commonly prescribed antibiotic for treatment of BV, but failure and recurrence rates are high (7). Recent cultivation-independent analyses of PCR-amplified 16S rRNA gene sequences reveal that there are bacterial genera associated with BV that were not previously recognized, including a metronidozole-resistant anaerobe, Atopobium vaginae (8,9,17,19). We examined the species composition of the vaginal flora of BV patients before and 1 month after metronidazole treatment by using PCR assays directed toward a broad range of bacterial genera and a quantitative PCR assay targeting A. vaginae.Clinical assessments of BV were made just prior to treatment and at 4 weeks posttreatment. All six BV patients in the study met all Amsel criteria (2) and had Nugent scores of Ն4 (12). Treatment was a 0.75% topical metronidazole gel applied once daily for 5 days. The study was approved by the Louisiana State University Health Sciences Center institutional review board, and informed consent was obtained from each participant. Vaginal swabs were collected prior to treatment and 30 days posttreatment by using a standard protocol and were stored frozen. To isolate DNA, swabs were agitated in 0.5 ml of molecular-biology-grade water, suspensions were centrifuged, and nucleic acids were isolated from pellets by using an AquaPure genomic DNA kit (Bio-Rad, Hercules, CA). Primers for quantitative PCR assays of A. vaginae 16S rRNA genes were designed using Primrose (3); primers were 5Ј-GTTAGG TCAGGAGTTAAATCTG-3Ј and 5Ј-TCATGGCCCAGAC C-3Ј. Real-time amplifications were performed on an iCycler (Bio-Rad) using iQ-SYBR Green Supermix (Bio-Rad). Thermal cycling consisted of 95°C for 2.5 min, followed by 40 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 30 s; 10 ng of template DNA was used in each amplification. PCR amplification of vaginal DNA using primers targeting conserved regions of the16S rRNA gene were used to generate clone libraries with a TOPO

Vaginal microbiome of reproductive-age women

“…The remaining communities found in 27% of the women formed a large heterogeneous group (IV) and were typified by higher proportions of strictly anaerobic bacteria, including Prevotella, Dialister, Atopobium, Gardnerella, Megasphaera, Peptoniphilus, Sneathia, Eggerthella, Aerococcus, Finegoldia, and Mobiluncus. This finding is consistent with those of previous studies wherein the species composition of vaginal communities was investigated by cloning and sequencing of 16S rRNA genes (17)(18)(19)(20)(21)(22)(23)(24). It should be pointed out that although communities of group IV seem to be diverse relative to the other groups ( Fig.…”

“…These species are thought to play key protective roles by lowering the environmental pH through lactic acid production (12,13), by producing various bacteriostatic and bacteriocidal compounds, or through competitive exclusion (13)(14)(15)(16). The advent of culture-independent molecular approaches based on the cloning and sequencing of 16S rRNA genes has furthered our understanding of the vaginal microbiota by identifying taxa that had not been cultured (17)(18)(19)(20)(21)(22)(23)(24). However, this technique is limited by high cost and low throughput, hence only small numbers of samples have usually been analyzed, and the depth of sample analysis was not great.…”

Vaginal microbiome and sexually transmitted infections: an epidemiologic perspective

“…were not the dominant species in 27% of the women who were surveyed (30). This was an intriguing result because the participants did not report any vaginal symptoms (including malodor and discharge) on direct questioning despite having a microbial community that was similar in composition to that of women diagnosed with clinically defined BV (28,81). Interestingly, Klebanoff et al demonstrated in a larger study of 2,888 women that as many as 50% of women with high Nugent scores (indicative of BV) are asymptomatic (24).…”

Qualitative and semiquantitative analysis of Lactobacillus species in the vaginas of healthy fertile and postmenopausal Chinese women

“…Today, most studies apply culture-independent techniques to evaluate microbial communities such as the vaginal microbiota (Antonio et al, 1999;Heilig et al, 2002). These techniques are based on the analysis of DNA without relying on cultivation of the bacteria and can yield valuable information about the complexity of the microbial community (Byun et al, 2004;Bradshaw et al, 2006b;Ferris et al, 2007).…”