氯己定对粪肠球菌耐药性和致病性的影响及其机制

Effect of chlorhexidine on drug resistance and pathogenicity of Enterococcus faecalis and its mechanism

摘要

目的 探讨长期使用氯己定对粪肠球菌（E.faecalis）耐药性的影响，并阐明其作用机制。 方法 将E.faecalis标准菌株反复暴露于氯己定中传代10代，记录每一次传代时的最低抑菌浓度（MIC）值。收集第10代且MIC值增加的细菌，即为E.faecalis氯己定耐药菌株（E.faecalis-Cs）。绘制2种菌株生长曲线，透射电子显微镜观察2种菌株形态表现，结晶紫染色法检测2种菌株细菌生物膜形成量，微生物黏着碳氢化合物（MATH）法检测2种菌株细菌疏水率，实时荧光定量PCR（RT-qPCR）法检测2种菌株细菌生物膜中S-核糖基高半胱氨酸裂解酶（LuxS） mRNA表达水平。 结果 随着传代次数（第0~10代）的增加，E.faecalis的MIC值逐渐升高。E.faecalis和E.faecalis-Cs的生长曲线无明显差异。透射电镜，E.faecalis和E.faecalis-Cs均呈椭圆形或双球型，细胞壁结构完整，边缘光滑，细胞质分布均匀，2种细菌形态大小和细胞壁厚度及完整性方面无明显差异。结晶紫染色法，与E.faecalis比较，E.faecalis-Cs生物膜形成量明显增加（P<0.05）。MATH法，与E.faecalis比较，E.faecalis-Cs的细菌疏水率明显升高（P<0.05）。RT-qPCR法，与E.faecalis比较，E.faecalis-Cs细菌生物膜中LuxS mRNA表达水平明显升高（P<0.05）。 结论 E.faecalis在反复暴露于氯己定后会产生耐药性，耐药菌株致病能力增强。群体感应（QS）系统 LuxS 基因高表达和更强的细菌生物膜形成能力可能是E.faecalis对氯己定产生耐药性的潜在机制。

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.

关键词

粪肠球菌 / 氯己定 / 耐药性 / 粪肠球菌氯己定耐药菌株 / 群体感应系统

Key words

Enterococcus faecalis / Chlorhexidine / Drug resistance / Enterococcus faecalis resistance stain / Quorum sensing system

Author summay

徐智博（1998-），男，安徽省滁州市人，在读硕士研究生，主要从事牙体牙髓病基础和临床方面的研究。