Morphotypic variation of vaginal clue cells broadens understanding of vaginal biofilms Laura K. Sycuro*,1,2, Shaelen Konschuh1, George Kuo2, and David N. Fredricks2,3 1International Microbiome Centre, University of Calgary; 2Fred Hutchinson Cancer Research Center; 3University of Washington *Corresponding author
Bacterial vaginosis (BV) is a dysbiotic state of the vaginal microbiota that affects 10-60% of premenopausal women globally. BV increases a woman's risk of acquiring sexually transmitted infections (STI) and experiencing pregnancy complications such as preterm birth. One of the pivotal problems in the clinical management of BV is its high rate of recurrence. A primary hypothesis for why BV recurs is the presence of an adherent vaginal biofilm that enables microbes to survive exposure to antimicrobials. Here we report a longitudinal sub-study aimed at describing the morphotypic appearance of vaginal epithelial cells coated in adherent biofilm (clue cells) from diagnosis and treatment through 3-6 months of follow-up. Of 85 mostly African American (58%) and White (34%) women enrolled through the Public Health Seattle and King County Sexually Transmitted Diseases Clinic, 38 (45%) completed all 6 months of follow-up and 60% experienced BV recurrence. Most participants (73/74) who were BV-positive at the baseline visit had vaginal clue cells observed in their vaginal fluid. Freshly collected cervicovaginal lavage fluid from the study's 374 patient visits was microscopically observed, Gram stained, and fixed for downstream analysis. Using microscopy and molecular probing we observed that in addition to the established clue cell morphotype consisting predominantly of Gardnerella vaginalis and Atopobium vaginae, there was a second, although rare morphotype dominated by curved rod bacteria. We confirmed the identity of the abundantly adherent bacteria in some samples was Mobiluncus mulieris, and subsequent in vitro studies suggested this species' ability to form adherent biofilms is strain-variable. To our knowledge, this study is the first to report bacteria other than G. vaginalis and A. vaginae can dominate the vaginal biofilm and contribute a distinct morphotypic variation of vaginal clue cells. Efforts to identify the other curved rod bacteria contributing to the 'fuzzy' appearance of vaginal clue cells are on-going, as is a systematic assessment of how variable clue cell morphotypes are linked to patient outcomes.
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