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Bacterial vaginosis (BV) and its recurrence are most commonly associated with the formation of  Gardnerella  species biofilm. Probiotics are typically used to treat BV; however, the optimal period of  Lactobacillus  probiotic application in BV treatment remains uncertain. The present study aimed to explore the effects of  Lactobacillus rhamnosus  and  Lactobacillus casei  on various stages of biofilm formation in  Gardnerella  species. The biofilm-forming ability of seven strains, including one  Gardnerella vaginalis  ATCC 14018 and six clinically isolated  Gardnerella  species, was determined  via  gentian violet staining assay. Moreover, the sensitivity of the planktonic and biofilm forms toward metronidazole and clindamycin was assessed  via  microdilution broth method.  L. rhamnosus  Xbb-LR-1 and  L. casei  Xbb-LC-1 were added during various stages of biofilm formation in  Gardnerella  species and were cocultured for 24 h. The biofilm thickness of each sample was determined  via  confocal laser scanning microscopy (CLSM). The absolute quantities of  Gardnerella  species in each sample was obtained  via  real time polymerase chain reaction method, and the pH value was obtained using a pH indicator paper. Biofilm formation by  Gardnerella  species in a medium with distinct pH values was observed  via  gentian violet staining, CLSM, and scanning electron microscopy (SEM). The biofilm increased the resistance of  Gardnerella  species toward metronidazole and clindamycin.  L. rhamnosus  added at the initial biofilm formation stage in  Gardnerella  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  Gardnerella  species toward antibiotics. Maintaining an acidic vaginal environment with pH <4.5 and a vaginal microbiota dominated by  Lactobacillus  remarkably prevents the formation of  Gardnerella  species biofilm at the initial stage, which further has a significant impact on the treatment and prevention of biofilm-related infections.

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