Toll-like receptor variants and cervical Atopobium vaginae infection in women with pelvic inflammatory disease.

PROBLEM: Toll-like (TLR) receptor genetic variants have been implicated in bacterial vaginosis (BV). We determined whether TLR variants are associated with fastidious BV-associated microbes that are linked with infertility following pelvic inflammatory disease (PID). METHOD OF STUDY: Sneathia spp., Atopobium vaginae, BVAB1, and Ureaplasma urealyticum were measured in 250 women from the PID Evaluation and Clinical Health (PEACH) study. Relative risk (RR) and 95% confidence intervals (CI) were calculated adjusting for chlamydia and gonorrhea. Principal component analysis was used to adjust for population stratification. A false discovery rate q-value of 0.05 was significant. RESULTS: TLR2-1733C>A (P = .003) and TLR2-616A>G (P = .004) were associated with cervical A. vaginae. TLR2-1733C>A and TLR6-438C>T were associated with A. vaginae detection in the endometrium, but this was not significant after adjustment for multiple comparisons (FDR q-value = 0.06). CONCLUSION: Host gene variants in TLR2 signaling pathways were modestly associated with cervical A. vaginae in women with clinical PID.

Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a catastrophic disease of preterm infants, and microbial dysbiosis has been implicated in its pathogenesis. Studies evaluating the microbiome in NEC and preterm infants lack power and have reported inconsistent results. METHODS AND RESULTS: Our objectives were to perform a systematic review and meta-analyses of stool microbiome profiles in preterm infants to discern and describe microbial dysbiosis prior to the onset of NEC and to explore heterogeneity among studies. We searched MEDLINE, PubMed, CINAHL, and conference abstracts from the proceedings of Pediatric Academic Societies and reference lists of relevant identified articles in April 2016. Studies comparing the intestinal microbiome in preterm infants who developed NEC to those of controls, using culture-independent molecular techniques and reported α and ß-diversity metrics, and microbial profiles were included. In addition, 16S ribosomal ribonucleic acid (rRNA) sequence data with clinical meta-data were requested from the authors of included studies or searched in public data repositories. We reprocessed the 16S rRNA sequence data through a uniform analysis pipeline, which were then synthesized by meta-analysis. We included 14 studies in this review, and data from eight studies were available for quantitative synthesis (106 NEC cases, 278 controls, 2944 samples). The age of NEC onset was at a mean ± SD of 30.1 ± 2.4 weeks post-conception (n = 61). Fecal microbiome from preterm infants with NEC had increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes prior to NEC onset. Alpha- or beta-diversity indices in preterm infants with NEC were not consistently different from controls, but we found differences in taxonomic profiles related to antibiotic exposure, formula feeding, and mode of delivery. Exploring heterogeneity revealed differences in microbial profiles by study and the target region of the 16S rRNA gene (V1-V3 or V3-V5). CONCLUSIONS: Microbial dysbiosis preceding NEC in preterm infants is characterized by increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes. Microbiome optimization may provide a novel strategy for preventing NEC.

Bacterial communities in penile skin, male urethra, and vaginas of heterosexual couples with and without bacterial vaginosis.

BACKGROUND: The epidemiology of bacterial vaginosis (BV) suggests it is sexually transmissible, yet no transmissible agent has been identified. It is probable that BV-associated bacterial communities are transferred from male to female partners during intercourse; however, the microbiota of sexual partners has not been well-studied. RESULTS: Pyrosequencing analysis of PCR-amplified 16S rDNA was used to examine BV-associated bacteria in monogamous couples with and without BV using vaginal, male urethral, and penile skin specimens. The penile skin and urethral microbiota of male partners of women with BV was significantly more similar to the vaginal microbiota of their female partner compared to the vaginal microbiota of non-partner women with BV. This was not the case for male partners of women with normal vaginal microbiota. Specific BV-associated species were concordant in women with BV and their male partners. CONCLUSIONS: In monogamous heterosexual couples in which the woman has BV, the significantly higher similarity between the vaginal microbiota and the penile skin and urethral microbiota of the male partner, supports the hypothesis that sexual exchange of BV-associated bacterial taxa is common.

Identification of novel microbes associated with pelvic inflammatory disease and infertility.

OBJECTIVES: As pelvic inflammatory disease (PID) aetiology is not completely understood, we examined the relationship between select novel bacteria, PID and long-term sequelae. METHODS: Fastidious bacterial vaginosis (BV)-associated bacteria (Sneathia (Leptotrichia) sanguinegens, Sneathia amnionii, Atopobium vaginae and BV-associated bacteria 1 (BVAB1)), as well as Ureaplasma urealyticum and Ureaplasma parvum were identified in cervical and endometrial specimens using organism-specific PCR assays among 545 women enrolled in the PID Evaluation and Clinical Health study. Risk ratios and 95% CIs were constructed to determine associations between bacteria, histologically confirmed endometritis, recurrent PID and infertility, adjusting for age, race, gonorrhoea and chlamydia. Infertility models were additionally adjusted for baseline infertility. RESULTS: Persistent detection of BV-associated bacteria was common (range 58% for A. vaginae to 82% for BVAB1) and elevated the risk for persistent endometritis (RRadj 8.5, 95% CI 1.6 to 44.6) 30 days post-cefoxitin/doxycycline treatment, independent of gonorrhoea and chlamydia. In models adjusted for gonorrhoea and chlamydia, endometrial BV-associated bacteria were associated with recurrent PID (RRadj 4.7, 95% CI 1.7 to 12.8), and women who tested positive in the cervix and/or endometrium were more likely to develop infertility (RRadj 3.4, 95% CI 1.1 to 10.4). Associations between ureaplasmas and PID sequelae were modest. CONCLUSIONS: To our knowledge, this is the first prospective study to demonstrate that S. sanguinegens, S. amnionii, BVAB1 and A. vaginae are associated with PID, failure of the Centers for Disease Control and Prevention-recommended treatment to eliminate short-term endometritis, recurrent PID and infertility. Optimal antibiotic regimens for PID may require coverage of novel BV-associated microbes.

Bacterial diversity and Clostridia abundance decrease with increasing severity of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating neonatal gastrointestinal disease that primarily affects premature infants. It is characterized by bowel inflammation and necrosis. In spite of extensive research, there has been little progress in decreasing the incidence or mortality of NEC over the past three decades. The exact etiology of NEC has not been identified. However, it is believed to result from an inappropriate immune response to gut microbiota. Using 454-pyrosequencing analyses of 16S rRNA genes that were PCR-amplified from stool DNA specimens, we compared the gut microbiota of infants with NEC to matched controls without NEC. The infants with NEC were then categorized into three subgroups based on severity: mild, severe, and lethal. We compared the microbiota among these subgroups and between each severity group and appropriate controls. RESULTS: Bacterial diversity and the relative abundance of Actinobacteria and Clostridia were significantly lower in NEC specimens compared to controls. The absence of Clostridia was significantly associated with NEC. Microbial diversity and Clostridia abundance and prevalence decreased with increasing severity of NEC. CONCLUSIONS: Low bacterial diversity in stool specimens may be indicative of NEC and the severity of NEC. The low bacterial diversity, and the lack of Clostridia in lethal specimens, could indicate that the presence of a diverse bacterial population in the gut as well as the presence of taxa such as Clostridia may play a role in attenuating inflammation leading to NEC.

Unique vaginal microbiota that includes an unknown Mycoplasma-like organism is associated with Trichomonas vaginalis infection.

BACKGROUND: The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis-infected women differs from that in T. vaginalis-uninfected women. METHODS: Vaginal samples from 30 T. vaginalis-infected women were matched by Nugent score to those from 30 T. vaginalis-uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction-amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. RESULTS: Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis-infected and T. vaginalis-uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis-infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. CONCLUSIONS: T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response.

Environmental distribution and seasonal prevalence of Mycobacterium ulcerans in Southern Louisiana.

Mycobacterium ulcerans is an emerging environmental pathogen that causes debilitating, ulcerative disease in humans and other vertebrates. The majority of human cases occur in tropical and temperate regions of Africa and Australia, and outbreaks of piscine mycobacteriosis caused by M. ulcerans have been reported in disparate geographic locations spanning the globe. While exposure to a natural body of water is the most common risk factor for human infection, the environmental distribution of M. ulcerans in aquatic habitats has not been extensively studied. Although no human cases have been reported in the United States, a strain of M. ulcerans has been identified as the cause of a piscine mycobacteriosis in Striped bass (Morone saxatilis) within the Chesapeake Bay. Infected fish exhibit bright red ventral and lateral dermal lesions. We observed a possible outbreak causing similar lesions on red drum (Sciaenops ocellatus) in wetlands of southern Louisiana and detected M. ulcerans-specific genetic markers in lesion samples from these fish. Based on these findings, we studied the geographic and seasonal prevalence of these markers across southern Louisiana. M. ulcerans was detected in each of the nine areas sampled across the state. M. ulcerans prevalence was significantly lower in the fall samples, and the low prevalence coincided with decreased nutrient levels and an increase in water temperature. To our knowledge, this is the first study of M. ulcerans biomarkers in the southern United States.

Histamine-2 receptor blockers alter the fecal microbiota in premature infants.

OBJECTIVES: Bacterial colonization is considered a major risk factor for necrotizing enterocolitis (NEC). The objective of the present study was to test the hypothesis that histamine-2 receptor (H2-) blockers alter colonic bacterial colonization by analyzing and comparing the fecal microbiota in premature infants with and without H2-blocker therapy using sensitive molecular biological techniques. METHODS: Seventy-six premature infants ≤1500 g or <34 weeks gestation were enrolled in this case-controlled, cross-sectional study. Stool samples were collected from 25 infants receiving H2-blockers and 51 babies who had never received them. Following DNA extraction and PCR amplification of 16S rRNA, 454 pyrosequencing was undertaken and the resulting sequences were subjected to comparison with published sequence libraries. RESULTS: Proteobacteria and Firmicutes were the major phyla contributing to fecal microbial communities. Microbial diversity was lower, relative abundance of Proteobacteria (primarily of the family Enterobacteriaceae) was increased, whereas that of Firmicutes was decreased in the stools of infants receiving H2-blockers compared with those who had never received them. CONCLUSIONS: Although not designed to look specifically at the effect of H2-blockers on the incidence of NEC, our study suggests that their use lowers fecal microbial diversity and shifts the microfloral pattern toward Proteobacteria. These alterations in fecal microbiota may predispose the vulnerable immature gut to necrotizing enterocolitis and suggest prudence in the use of H2-blockers in the premature infant.

Safety and tolerability of Lactobacillus reuteri DSM 17938 and effects on biomarkers in healthy adults: results from a randomized masked trial.

BACKGROUND: There are few carefully-designed studies investigating the safety of individual probiotics approved under Investigational New Drug policies. OBJECTIVES: The primary aim of this prospective, double-blind placebo-controlled trial was to investigate if daily treatment of adults with Lactobacillus reuteri DSM 17938 (LR) for 2 months is safe and well-tolerated. Our secondary aim was to determine if LR treatment has immune effects as determined by regulatory T cell percentages, expression of toll-like receptors (TLR)-2 and -4 on circulating peripheral blood mononuclear cells (PMBCs), cytokine expression by stimulated PBMC, and intestinal inflammation as measured by fecal calprotectin. METHODS: Forty healthy adults were randomized to a daily dose of 5 × 10(8) CFUs of LR (n = 30) or placebo (n = 10) for 2 months. Participants completed a daily diary card and had 7 clinic visits during treatment and observation. RESULTS: There were no severe adverse events (SAEs) and no significant differences in adverse events (AEs). There were no differences in PBMC subclasses, TLRs, or cytokine expression after treatment. The probiotic-treated group had a significantly higher fecal calprotectin level than the placebo group after 2 months of treatment: 50 µg/g (IQR 24-127 µg/g) vs. 17 µg/g (IQR 11-26 µg/g), p = 0.03, although values remained in the normal clinical range (0-162.9 µg/g). LR vials retained >10(8) CFUs viable organisms/ml. CONCLUSIONS: LR is safe and well tolerated in adults, without significant changes in immunologic markers. There was a small but significant increase in fecal calprotectin, perhaps indicating some element of immune recognition at the intestinal level. TRIAL REGISTRATION: Clinical Trials.gov NCT00922727.

The microbiota of the human genitourinary tract: trying to see the forest through the trees.

Based on traditional microbiological methods, namely cultivation and microscopic analyses, the vaginal microbiota (VMB) has been defined as healthy when it is predominated by hydrogen peroxide-producing Lactobacillus spp., most prominently Lactobacillis crispatus. Similarly, the VMB has been defined as bacterial vaginosis (BV) when it is predominated by Gardnerella vaginalis as well as a number of other anaerobic bacterial species. BV is associated with a distinct vaginal discharge syndrome, poor pregnancy outcomes, pelvic inflammatory disease, post-operative wound infections, and endometritis after elective abortions. Additionally, BV predisposes women to infection by HIV as well as other sexually transmitted diseases (STDs). The application of molecular techniques over the last decade to studies of the VMB has significantly advanced our understanding of its structure and variation. It is now clear that the diversity of the VMB is far more complex than previously recognized; it is comprised of many heretofore unknown bacteria in addition to those previously identified by culture. Here we describe the application of 454 pyrosequencing technology to a study of vaginal specimens from 92 women attending the New Orleans STD clinic in an effort to obtain a more precise view of how different types of "trees" (bacteria) assemble to form a recognizable "forest" (VMB). This knowledge will be useful in the design of future clinical studies that investigate the mechanisms by which the vaginal microbiome influences human health and disease.

Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation.

BACKGROUND: Bacterial vaginosis (BV) is an enigmatic disease of unknown origin that affects a large percentage of women. The vaginal microbiota of women with BV is associated with serious sequelae, including abnormal pregnancies. The etiology of BV is not fully understood, however, it has been suggested that it is transmissible, and that G. vaginalis may be an etiological agent. Studies using enzymatic assays to define G. vaginalis biotypes, as well as more recent genomic comparisons of G. vaginalis isolates from symptomatic and asymptomatic women, suggest that particular G. vaginalis strains may play a key role in the pathogenesis of BV. METHODOLOGY/PRINCIPAL FINDINGS: To explore G. vaginalis diversity, distribution and sexual transmission, we developed a Shannon entropy-based method to analyze low-level sequence variation in 65,710 G. vaginalis 16S rRNA gene segments that were PCR-amplified from vaginal samples of 53 monogamous women and from urethral and penile skin samples of their male partners. We observed a high degree of low-level diversity among G. vaginalis sequences with a total of 46 unique sequence variants (oligotypes), and also found strong correlations of these oligotypes between sexual partners. Even though Gram stain-defined normal and some Gram stain-defined intermediate oligotype profiles clustered together in UniFrac analysis, no single G. vaginalis oligotype was found to be specific to BV or normal vaginal samples. CONCLUSIONS: This study describes a novel method for investigating G. vaginalis diversity at a low level of taxonomic discrimination. The findings support cultivation-based studies that indicate sexual partners harbor the same strains of G. vaginalis. This study also highlights the fact that a few, reproducible nucleotide variations within the 16S rRNA gene can reveal clinical or epidemiological associations that would be missed by genus-level or species-level categorization of 16S rRNA data.

A framework for analysis of metagenomic sequencing data.

The human body is home to a diverse assemblage of microbial species. In fact, the number of microbial cells in each person is an order of magnitude greater than the number of cells that make up the body itself. Changes in the composition and relative abundance of these microbial species are highly associated with intestinal and respiratory disorders and diseases of the skin and mucus membranes. While cultivation-independent methods employing PCR-amplification, cloning and sequence analysis of 16S rRNA or other phylogenetically informative genes have made it possible to assess the composition of microbial species in natural environments, until recently this approach has been too time consuming and expensive for routine use. Advances in high throughput pyrosequencing have largely eliminated these obstacles, reducing cost and increasing sequencing capacity by orders of magnitude. In fact, although numerous arithmetic and statistical measurements are available to assess the composition and diversity of microbial communities, the limiting factor has become applying these analyses to millions of sequences and visualizing the results. We introduce a new, easy-to-use, extensible visualization and analysis software framework that facilitates the manipulation and interpretation of large amounts of metagenomic sequence data. The framework automatically performs an array of standard metagenomic analyses using FASTA files that contain 16S rRNA sequences as input. The framework has been used to reveal differences between the composition of the microbiota in healthy individuals and individuals with diseases such as bacterial vaginosis and necrotizing enterocolitis.

Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis.

Knowledge of the abundance of bacterial species in vaginal communities will help us to better understand their role in health and disease. However, progress in this field has been limited because quantifying bacteria in natural specimens is an arduous process. We developed quantitative real-time PCR (qPCR) assays to facilitate assessments of bacterial abundance in vaginal specimens and evaluated the utility of these assays by measuring species abundance in patients whose vaginal floras were clinically described as normal, intermediate, or bacterial vaginosis (BV) as defined by Nugent's criteria. The qPCR measurements showed that Lactobacillus species were predominant in normal vaginal specimens and that high Lactobacillus crispatus and Lactobacillus jensenii abundance was specific to normal specimens, while Lactobacillus iners abundance was high in all categories including BV. The abundances of all non-Lactobacillus species were higher in BV specimens than in normal specimens. Prevotella species were prevalent in all specimens and represented a high percentage of total species in BV specimens. qPCR assays can be a useful tool for describing the structure of vaginal communities and elucidating their role in health and disease.

Altered fecal microflora and increased fecal calprotectin in infants with colic.

OBJECTIVE: We explored whether gut inflammation, colonic fermentation, and/or an altered colonic flora could provide a pathophysiological mechanism for colic. STUDY DESIGN: The study population consisted of 36 term infants ranging in age from 14 to 81 days. We measured fecal calprotectin (a marker of neutrophil infiltration) by ELISA; stool microorganisms by denaturing gradient gel electrophoresis, cloning, and sequencing; and breath hydrogen levels using gas chromatography. RESULTS: During 24 hours, infants with colic (n = 19) cried and fussed for a mean of 314 +/- 36 (SEM) minutes, compared with control infants (n = 17, 103 +/- 17 minutes). Fecal calprotectin levels were 2-fold higher in infants with colic than in control infants (413 +/- 71 vs 197 +/- 46 microg/g, P = .042). Stools of infants with colic had fewer identifiable bands on denaturing gradient gel electrophoresis. Klebsiella species were detected in more colic patients than in control patients (8 vs 1, P = .02), whereas Enterobacter/Pantoea species were detected only in the control patients. These differences could not be attributed to differences in formula versus breast milk feeding, consumption of elemental formula, or exposure to antibiotics. CONCLUSIONS: Infants with colic, a condition previously believed to be nonorganic in nature, have evidence of intestinal neutrophilic infiltration and a less diverse fecal microflora.

Prevalence and abundance of uncultivated Megasphaera-like bacteria in the human vaginal environment.

Cultivation-independent analysis of 16S rRNA gene sequences in vaginal samples revealed two previously unrecognized, uncultivated Megasphaera-like phylotypes. Phylogenetic analysis and environmental distribution suggest that these Megasphaera types may be unique to the vaginal environment. Quantitative PCR suggests that both phylotypes are present in higher concentrations in women with bacterial vaginosis.

Cultivation-independent analysis of changes in bacterial vaginosis flora following metronidazole treatment.

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.

Cyanobacterial ecotypes in the microbial mat community of Mushroom Spring (Yellowstone National Park, Wyoming) as species-like units linking microbial community composition, structure and function.

We have investigated microbial mats of alkaline siliceous hot springs in Yellowstone National Park as natural model communities to learn how microbial populations group into species-like fundamental units. Here, we bring together empirical patterns of the distribution of molecular variation in predominant mat cyanobacterial populations, theory-based modelling of how to demarcate phylogenetic clusters that correspond to ecological species and the dynamic patterns of the physical and chemical microenvironments these populations inhabit and towards which they have evolved adaptations. We show that putative ecotypes predicted by the theory-based model correspond well with distribution patterns, suggesting populations with distinct ecologies, as expected of ecological species. Further, we show that increased molecular resolution enhances our ability to detect ecotypes in this way, though yet higher molecular resolution is probably needed to detect all ecotypes in this microbial community.

Legionella species diversity in an acidic biofilm community in Yellowstone National Park.

Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30 degrees C) and higher (35 to 38 degrees C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

Use of species-directed 16S rRNA gene PCR primers for detection of Atopobium vaginae in patients with bacterial vaginosis.

Recent studies suggest that the association between a metronidazole-resistant anaerobe, Atopobium vaginae, and bacterial vaginosis (BV) warrants further investigation. In the present study, specific primers enhanced detection of A. vaginae and provided additional evidence that this bacterium is prevalent among patients with BV but absent among patients with normal vaginal flora.

Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess. METHODS: Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score < or = 3) and abnormal flora (Nugent score > or = 4). These women ranged in age from 14 to 48 and 82% were African American. RESULTS: The DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (approximately 300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant. CONCLUSION: The results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant.