Zoliflodacin inhibits Streptococcus mutans biofilms

Zoliflodacin (ZFD) is a new type of antibiotic whose anticaries potential through controlling biofilms remains unknown. This study aimed to explore the effects of ZFD on the conditional formation of cariogenic Streptococcus mutans (S. mutans) biofilms.

Crystal violet analysis, colony forming unit (CFU) counting, and MTT assays were used to determine biofilm biomass, viable bacterial cell counts, and bacterial metabolic viability, respectively, while acid generation by biofilms was monitored by measuring pH and lactic acid production. The anthrone-sulfuric acid method and polysaccharide/bacterial staining were used to investigate extracellular polysaccharide (EPS) production. The microstructure of S. mutans biofilms was observed using scanning electron microscopy (SEM). Growth curves were used to visualize bacterial growth, while live/dead bacterial staining was used to determine microbial activity. Furthermore, the cytotoxicity of ZFD was evaluated by live/dead cellular staining together with the CCK-8 assay, while drug resistance was induced using bacterial reduplicative exposure to ZFD. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to calculate gene expression levels. In addition, checkerboard microdilution assays were performed to evaluate potential synergistic or antagonistic interactions of ZFD in combination with NaF or chlorhexidine (CHX) against bacterial growth and biofilm formation.

We found ZFD significantly inhibited S. mutans biofilm formation, decreasing overall biofilm biomass, viable bacterial cell counts within biofilms, and biofilm metabolic viability. ZFD not only markedly suppressed S. mutans cariogenic virulence involving acid and EPS synthesis, but also affected bacterial morphology within biofilms. Furthermore, ZFD inhibited bacterial growth without significantly suppressing the survival rate of bacterial cells within biofilms. Moreover, ZFD demonstrated no significant cytotoxicity and did not trigger drug resistance in S. mutans after 20 passages. qRT-PCR analysis revealed significant differences in the expression of virulence-, quorum sensing-, and oxidative stress protection-associated genes. Indifferent effects were found when ZFD was combined with NaF or CHX on S. mutans growth and biofilm formation.

In summary, ZFD is a potential antibacterial agent for caries control because of its antibiofilm effects, inconspicuous cytotoxicity, low risk of resistance induction and antibacterial activity against S. mutans.