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

Michael J Ferris 1 2 3 Alicia Masztal 1 Kenneth E Aldridge 0 J Dennis Fortenberry 4 Paul L Fidel Jr 3 David H Martin 5 0 Dept. of Infectious Disease, Louisiana State University Health Sciences Center , 1542 Tulane Ave, New Orleans, LA 70112 , USA 1 Research and Education Bldg., The Research Institute for Children , 200 Henry Clay Ave, New Orleans, LA 70118 , USA 2 Dept. of Pediatrics, Louisiana State University Health Sciences Center , New Orleans, LA, 70112 , USA 3 Dept. of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center , 1901 Perdido St., New Orleans, LA 70112 , USA 4 Department of Pediatrics, Indiana University School of Medicine, Section of Adolescent Medicine , 575 N West St., Room 070, Indianapolis, IN 46202 , USA 5 Dept. of Internal Medicine, Louisiana State University Health Sciences Center , New Orleans, LA, 70112 , USA 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 3) and abnormal flora (Nugent score 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 (~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. - Background Bacterial vaginosis (BV) is a syndrome that appears to represent a disturbance of the vaginal ecosystem associated with a shift in the microflora [1,2]. Several adverse medical outcomes have been associated with BV [3-11]. While the cause of BV is not understood, some strong associations have been made between the syndrome and the presence of particular bacterial species, such as Gardnerella vaginalis and Prevotella sp. [2,12]. Most of these, however, represent bacteria that have been detected by cultivation and identified by traditional morphological and biochemical methods. Cultivation-independent (molecular) analyses, most notably sequencing of 16S rRNA genes PCR-amplified from microbial community DNA, offer a more systematic approach to detecting microbes in natural habitats [13] and a more concrete method of identification and classification [14,15]. Molecular surveys of microbes in environmental samples have repeatedly shown that cultivated species do not represent the full complement of microbes in most habitats [16] and molecular analyses are now commonly used to survey bacterial flora in mammalian systems [17,18]. One technique, denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene segments, offers the possibility of revealing a significant portion of the bacterial flora in a sample as a pattern of bands on an acrylamide gel [19,20]. The bands are separated based on their denaturing characteristics, i.e. sequences and the sequences of individual bands can be obtained allowing the identification of bacteria in the sample. DGGE patterns have been used to reveal changes in microflora and to identify microbes in a variety of habitats [2026] including human vaginal flora [12,27,28] and other mammalian systems [29-32]. In the course of DGGE analyses of vaginal bacterial flora of normal and BV-positive patients, we detected a band whose sequence matched that of Atopobium vaginae (GenBank reference no. AF325325 and American Type Culture Collection [ATCC] #BAA 55) in 55% of BV positive patients. A. vaginae is a recently recognized species [33] whose clinical significance is unknown, though it has been identified recently as the cause of a tuboovarian abscess [34]. The isolate described in this case report was found to be highly resistant to metronidazole. We isolated two A. vaginae strains from BV patients. Antibiotic susceptibility studies were performed using these two strains and the ATCC #BAA 44 strain. Details of these studies are presented below. Methods Subjects Subjects participated in two different protocols in two locations. In Indianapolis, adolescent females attending one of three adolescent health clinics were enrolled in a prospective cohort study of risk factors for the acquisition of STD's between May, 1999, and September, 2001. They were predominantly African-American (85%) ranging in age from 14 to 17. Having a sexually transmitted disease was not a prerequisite for enrolment but 84% of the cohort were sexually active at the time of testing. Informed consent was obtained from all participants as well as permission from the accompanying parent/guardian for entry into the study. Specimens were collected at the clinics in Indianapolis and were subsequently shipped overnight using cold packs to LSUHSC where they were processed and analyzed. In New Orleans healthy women between the ages of 18 55 were enrolled into a study of the immune response to Candida albicans vaginitis through the Obstetrics and Gynecology Clinic at the Louisiana State University Health Sciences Center (LSUHSC), New Orleans, Louisiana. HIV infected and pregnant women were not eligible for participation in the study. The specimens used in this study were obtained prior to inoculation of C. albicans. All clinical research protocols we (...truncated)