Examining the impact of strain-level differences of Gardnerella vaginalis function relevant to vaginal health

Abstract

Bacterial vaginosis (BV) affects up to 30% of women in the United States, increasing risk for preterm birth, infertility, and sexually transmitted infections. A healthy vaginal microbiome is typically considered one dominated by a Lactobacillus spp. These microbes produce lactic acid and hydrogen peroxide which maintain an acidic pH and prevent the growth of pathogenic bacteria. In contrast, the overgrowth of pathogenic bacteria, mainly facultative anaerobes, leads to increased levels of sialidases and ammonia which degrade the protective mucin layer and raise the pH of the vaginal microbiome, leading to BV. The gold standard for BV treatment is antibiotic treatment with either metronidazole (met) or clindamycin. However, Gardnerella vaginalis, one of the most crucial players in BV, exhibits resistance genes to antibiotics in some strains. This resistance contributes to high rates of BV recurrence, often reported as high as ~60% within a year of antibiotic treatment. Antibiotic resistance can be developed through a variety of mechanisms; a more recent non-canonical form is through extracellular vesicle (EV) uptake. Bacterial EVs (bEVs) are lipid-bound nanoparticles that are naturally released by bacteria during external stress or death, participating in microbe-microbe and microbe-host communication. This work demonstrates that clinical and type strain G. vaginalis strains significantly respond to metronidazole treatment and form biofilms differently. In addition, bEVs from a type strain (TS) G. vaginalis strain (175.8 ± 8.1 nm) are larger than bEVs from clinical metronidazole-resistant (MR) G. vaginalis bEVs (157.7 ± 5.8 nm, p = 0.0383) and contain more protein per particle particles (4.07 ± 1.07 µg/108 particles) than metronidazole-susceptible (MS) (0.839 ± 0.160 µg/108 particles, p = 0.0065) and MR (0.961 ± 0.150 µg/108 particles, p = 0.0086) bEVs. While there are no significant metronidazole-level differences in morphology, concentration, or surface charge, MR G. vaginalis bEVs elicited a high inflammatory response (56.40 ± 7.62 pg/mL) in vaginal epithelial cells compared to a negative vehicle control (19.93 ± 1.66 pg/mL, p < 0.0001) and trended higher than its metronidazole-treated (Met-EV) counterpart (36.28 ± 4.05 pg/mL, p = 0.0600), suggesting a functional difference in the bEVs cargoes in microbe-host communication. Investigating how bEVs are affected by metronidazole treatment or strain-level differences advances how microbe-microbe and microbe-host communication play a role in the treatment of BV.