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

Abstract

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.

Figure 1

Normal vs. BV positive DGGE banding patterns Examples of DGGE banding patterns obtained from 16S rRNA gene segments PCR-amplified from nucleic acids isolated from vaginal lavage samples of patients clinically described as having BV-positive (A) or normal (B) vaginal flora. The boxed area encloses examples of A. vaginae bands. These were found in 55% of BV-positive samples.

Figure 2

Identification of two A. vaginae strains by DGGE using PCR amplified segments of the 16S rRNA gene Panel A, lane 1 is from vaginal lavage sample #7189A obtained from a patient with BV. Note the closely opposed A. vaginae bands in the boxed area. Lanes 2 and 3 show the individual A. vaginae bands from the same sample after extraction and re-amplification of the two bands in lane 1. Panel B, lane 1 is from a vaginal lavage sample #7200A obtained from another BV positive patient #7200. It also shows the double A. vaginae band pattern (boxed area). Lane 2 shows the band obtained from an A. vaginae strain isolated from patient #7200. This band co-migrates with the lower of the two bands obtained from this patient's vaginal lavage sample. Lane 3 shows the band obtained from the ATCC A. vaginae culture collection strain. It co-migrates with the upper of the two A. vaginae bands shown in lane 1. The slight difference in the electrophoretic migration of the A. vaginae bands correlates with a single nucleotide difference in their amplicons.