Abstract:

Objective To discuss the effect of long-term use of chlorhexidine on the resistance of Enterococcusfaecalis （E.faecalis）， and to clarify its mechanism. Methods The standard strain of E.faecalis was repeatedly exposed to chlorhexidine for 10 generations， and the minimum inhibitory concentration （MIC） was recorded at each passage. The bacteria collected from the 10th generation with increased MIC values were designated as the E.faecalis chlorhexidine-resistant strains （E.faecalis-Cs）. The growth curves of two strains were drawn； the morphology of two strains were observed by transmission electron microscope；the number of biofilm formation of two strains was detected by crystal violet staining；the bacterial hydrophobicities of two strains were detected by microbial adhesion to hydrocarbons （MATH） method； the expression levels of S-ribosylhomocysteine lyase （LuxS） mRNA in the bacterial biofilms of two strains were detected by real-time fluorescence quantitative PCR （RT-qPCR） method. Results From the 0th to the 10th generation， the MIC values of E.faecalis were gradually increased. The growth curves of E.faecalis and E.faecalis-Cs showed no significant differences. The transmission electron microscope observation results showed that both E.faecalis and E.faecalis-Cs appeared oval or diplococcal， with intact cell wall structures， smooth edges， and evenly distributed cytoplasm. There were no significant differences in the morphology， size， cell wall thickness， or integrity between two types of bacteria.The crystal violet staining results showed that compared with E.faecalis， the number of biofilm formation of E.faecalis-Cs was significantly increased （P<0.05）. The MATH results showed tha the hydrophobicity of E.faecalis-Cs was significantly higher than that of E.faecalis （P<0.05）. The RT-qPCR results showed that the expression level of LuxS mRNA in the biofilms of E.faecalis-Cs was significantly higher than that of E.faecalis （P<0.05）. Conclusion E.faecalis develops the resistance after repeated exposure to the chlorhexidine，and the pathogenicity of the resistant strain is enhanced. The high expressin of quorum sensing （QS） system LuxS gene and stronger biofilm forming ability of bacteria may be the potential mechanism for E.faecalis to tolerate the chlorhexidine.

Key words: Enterococcus faecalis, Chlorhexidine, Drug resistance, Enterococcus faecalis resistance stain, Quorum sensing system