Lactobacillus rhamnosus and Lactobacillus casei Affect Various Stages of Gardnerella Species Biofilm Formation

Abstract

Bacterial vaginosis (BV) and its recurrence are most commonly associated with the formation of <i>Gardnerella</i> species biofilm. Probiotics are typically used to treat BV; however, the optimal period of <i>Lactobacillus</i> probiotic application in BV treatment remains uncertain. The present study aimed to explore the effects of <i>Lactobacillus rhamnosus</i> and <i>Lactobacillus casei</i> on various stages of biofilm formation in <i>Gardnerella</i> species. The biofilm-forming ability of seven strains, including one <i>Gardnerella vaginalis</i> ATCC 14018 and six clinically isolated <i>Gardnerella</i> species, was determined <i>via</i> gentian violet staining assay. Moreover, the sensitivity of the planktonic and biofilm forms toward metronidazole and clindamycin was assessed <i>via</i> microdilution broth method. <i>L. rhamnosus</i> Xbb-LR-1 and <i>L. casei</i> Xbb-LC-1 were added during various stages of biofilm formation in <i>Gardnerella</i> species and were cocultured for 24 h. The biofilm thickness of each sample was determined <i>via</i> confocal laser scanning microscopy (CLSM). The absolute quantities of <i>Gardnerella</i> species in each sample was obtained <i>via</i> real time polymerase chain reaction method, and the pH value was obtained using a pH indicator paper. Biofilm formation by <i>Gardnerella</i> species in a medium with distinct pH values was observed <i>via</i> gentian violet staining, CLSM, and scanning electron microscopy (SEM). The biofilm increased the resistance of <i>Gardnerella</i> species toward metronidazole and clindamycin. <i>L. rhamnosus</i> added at the initial biofilm formation stage in <i>Gardnerella</i> species exhibited highest inhibitory effect, with a percentage inhibition of 38.17% ± 1.35%. When the pH value of the culture medium was <4.5 or >6.5, ATCC 14018 could hardly form a biofilm; however, at pH ≥4.5 and ≤6.5, it was able to form a stronger biofilm. The amount of biofilm attained maximum value at optical density of 3.29 ± 0.28 (595 nm), pH 5.5, and at 36 h. Biofilm formation increases the resistance of <i>Gardnerella</i> species toward antibiotics. Maintaining an acidic vaginal environment with pH <4.5 and a vaginal microbiota dominated by <i>Lactobacillus</i> remarkably prevents the formation of <i>Gardnerella</i> species biofilm at the initial stage, which further has a significant impact on the treatment and prevention of biofilm-related infections.