Defining the relationships between Gardnerella biofilm composition, antimicrobial resistance and recurrent vaginal dysbiosis

Abstract

Bacterial vaginosis (BV) is the most common vaginal condition to prompt healthcare, impacting 80% of women at some point in their lifetime. Although treatable with antimicrobials, BV is hallmarked by high rates of recurrence due the involvement of a tissue-adherent biofilm dominated by Gardnerella species. Limited species-level information on BV biofilms is available, as the taxonomic grouping formerly referred to as a single species, Gardnerella vaginalis, was recently emended to define a genus (Gardnerella) with three newly named species and up to 7 yet-unnamed genomospecies. Cataloguing the differences between newly discovered genomospecies has become a research priority, but there is still a lack of optimized protocols for growing and phenotyping these organisms in liquid culture conditions. Thus, the first major objective of this thesis was to identify a culture medium capable of supporting robust growth and condition-matched phenotyping of Gardnerella genomospecies. My results showed that the growth medium PYG-mod, when supplemented with heat inactivated human serum, optimally supported the growth of diverse Gardnerella genomospecies and promoted biofilm development. I further established a unique OD600 based methodology that quantifies the fraction of culture biomass that is adherent (biofilm), non-adherent (planktonic), and loosely adherent in a high throughput 96-well plate format. Using the OD600 based methodology in conjunction with traditional safranin staining, I showed that all Gardnerella genomospecies tested were strong biofilm producers and differences in the genomospecies’ ability to form biofilms are minimal when accounting for the high degree of phenotypic variance across biological replicates that is probably caused by this clade’s strong propensity for phase variation. The next objective of this thesis was to quantify each genomospecies’ intrinsic resistance to metronidazole and observe if antibiotics can affect biofilm development. Using liquid culture MIC50 experiments and a new OD600 based method, I showed that Genomospecies 07 may have higher intrinsic resistance to metronidazole and that metronidazole exposure does not affect biofilm development of any genomospecies. Together, these experiments have advanced our understanding of Gardnerella genomospecies and will allow for improved experimental design and more appropriate interpretation of future in vitro studies that may wish to assess their phenotypic differences to determine whether certain genomospecies may be linked to BV treatment success, failure, or recurrence.