Investigating antimicrobial resistance patterns in Gardnerella vaginalis isolates.

Abstract

Background: Bacterial vaginosis (BV) is gaining interest due to its problematic outcomes in women of reproductive age. Common symptoms are abnormal grey discharge that has a fishy odour accompanied by itchiness and pain. BV is characterized by the overall decrease in Lactobacilli and an increase in anaerobic facultative bacteria such as Gardnerella vaginalis. G. vaginalis is said to be the main species of bacteria involved in BV and is responsible for its pathogenicity. G. vaginalis has many virulence factors (formation of biofilms, mucus degrading sialidase and the production of the protein toxin, vaginolysin) that influence its pathogenicity, aiding in establishing infection. Treatment of BV includes the use of broad spectrum antibiotics such as metronidazole and clindamycin, which can be administered orally or in the form of an ointment. However, in clinical settings, antimicrobial resistance has increased due to resistant bacteria left behind after initial treatment, resulting in recurrent BV. The aim of the current study was to determine the susceptibility patterns of isolates of G. vaginalis to various antibiotics. This study also investigated the genetic diversity of isolates and linked genetic data with antimicrobial resistance patterns. attending care at the antenatal clinic at the King Edward VIII hospital in Durban, KwaZulu-Natal, South Africa. A total of 150 pregnant women, 18 years and older, who were willing to provide written informed consent and willing to provide two self-collected vaginal swabs were enrolled in this study. Following sample collection, the first swab was placed in Amies transport media (Copan, Brescia, Italy) for the cultivation of G. vaginalis. The second swab was rolled onto a glass slide to diagnose BV by Nugent scoring on gram-stained vaginal smears. The genetic diversity assessments of the isolates were based on genetic differences in the tuf gene using clade specific primers on a quantitative polymerase chain reaction (PCR) platform. The antimicrobial susceptibility profiles were generated using the Sensititre™ Anaerobe MIC Plate (Thermo Fischer Scientific, United States). Results: Sixteen isolates of G. vaginalis were cultured from pregnant women. Of the 16 isolates, five isolates were obtained from BV positive women, nine from BV intermediate women, and two were from BV negative women. The 16S rRNA gene specific to G. vaginalis was amplified in all isolates, confirming the identity of the isolates. The genotyping/clade PCR revealed the presence of three clades. The frequencies for each clade were as follows; 100% for clade 1, 37.5% for clade 2 and 43.75% for clade 4. Multiple clades were found in 56.25% of the isolates. For antimicrobial susceptibility testing, only 14 isolates were viable for analysis. Of the 14 isolates that were successfully cultured and tested, 8/14 (57.1%) were susceptible to metronidazole (MIC of ≤8μg/ml) and 6/14 (42.9%) were resistant to metronidazole (MIC of ≥32μg/ml). Of the G. vaginalis isolates (n=5) cultured from BV positive women, 60% (3/5) of the isolates were susceptible to metronidazole whilst 40% (2/5) were resistant. There was no link between resistant patterns and infection symptoms, since one isolate was from an asymptomatic woman and the other isolate from a symptomatic woman. Of the G. vaginalis isolates (n=7) cultured from intermediate BV women, 43% (3/7) of the isolates were resistant to metronidazole while 57% (4/7) were susceptible. For intermediate women, there was a link between resistant patterns and infection symptoms, since all resistant isolates were cultured from symptomatic women. The G. vaginalis isolate cultured from the BV-negative women was susceptible to metronidazole and the woman was asymptomatic. Of the six resistant isolates, five isolates (83%) were assigned to clade 1 alone. The remaining resistant isolate was a mixed clade, it was assigned clade 1 and 4. The eight susceptible isolates belonged to mixed clades such as 1 and 2, 1, 2 and 4 and 1 and 4. The resistant isolates were more homogeneous in terms of the clades to which they belonged. However, it cannot be suggested that a particular clade is driving resistance since the clades were distributed among susceptible and resistant isolates. Conclusion: The current study provided information on the resistance patterns of clinical isolates of G. vaginalis. In this study a high level of resistance to metronidazole was observed. Approaches to approve the administration of clindamycin and vancomycin should be taken into consideration for the future treatment of BV in our current setting.