乳源耐甲氧西林金黄色葡萄球菌icaA/D基因缺失株的构建及其生物被膜形成能力与耐药性分析

Construction of icaA/D gene knock-out in methicillin resistant Staphylococcus aureus isolated from milk for analyzing its biofilm-forming ability and drug resistance

【背景】耐甲氧西林金黄色葡萄球菌(Methicillin Resistant Staphylococcus aureus,MRSA)是一种具有多重耐药性的人畜共患病原菌,常引起奶牛乳房炎等疾病。形成生物被膜是MRSA重要的耐药机制之一。研究发现ica操纵子调控的胞间多糖黏附素(Polysaccharide Intercellular Adhesin,PIA)通过促进MRSA的黏附与聚集介导生物被膜形成,icaA和icaD基因共表达可显著提高MRSA的N-乙酰葡聚糖转移酶活性,但icaA/D蛋白对MRSA生物被膜形成和耐药性的影响仍不清楚。【目的】探讨icaA/D基因、MRSA生物被膜形成及耐药性三者之间的相关性,为寻找药物作用新靶点提供科学依据。【方法】以具有多重耐药性且生物被膜形成能力强的乳源MRSA M5分离株为研究对象,利用同源重组技术构建其icaA/D基因缺失株;利用FITC-ConA染色结合激光共聚焦显微镜观察野生株与icaA/D基因缺失株的生物被膜形成过程与能力;采用微量肉汤稀释法测定14种抗菌药物对野生株与icaA/D基因缺失株的最小抑菌浓度(MinimumInhibitoryConcentration,MIC)。【结果】构建了MRSA M5的icaA/D基因缺失株。激光共聚焦显微镜下观察到野生株在培养16 h后形成了一层厚的成熟生物被膜,随后开始解离,直至120 h完全解离;而icaA/D基因缺失株在培养后16 h仅形成一薄层生物被膜,48h完全解离。10种受试抗菌药物对缺失株的MIC较野生株减小,而且缺失株对8种药物的敏感性由原来的耐药或中介转变为中介或敏感。【结论】icaA/D基因缺失可明显降低MRSA的生物被膜形成能力与耐药性。

[Background]Methicillin resistant Staphylococcus aureus(MRSA)is a multidrug-resistant zoonotic pathogen that often causes many diseases such as cow mastitis.Bacterial biofilm-formation is one of the important drug-resistant mechanisms of MRSA.It has been found that the polysaccharide intercellular adhesion(PIA)regulated by ica operon,which mediates the biofilm-formation by promoting the adhesion and aggregation of MRSA,and the co-expression of the icaA and icaD genes can significantly increase the activity of N-acetylglucan transferase in S.aureus.However,it is unclear that the effect of the icaA/D protein on the biofilm-formation and drug-resistance of MRSA.[Objective]To study the relationship among icaA/D gene,biofilm-formation and drug-resistance of MRSA and provide a scientific basis for finding new drug targets.[Methods]MRSA M5 isolated from mastitis milk,which exhibited a multidrug-resistant and strong biofilm-forming ability,was used to construct the icaA/D genes deletion strain by homologous recombination technology.The biofilm-forming ability and process of the MRSA wild strain and icaA/D genes deletion strain were assessed by FITC-ConA staining combined with laser confocal microscopy.Finally,the minimum inhibitory concentrations(MICs)of 14 antimicrobial agents to wild strain and icaA/D genes deletion strain were detected by broth microdilution method.[Results]The icaA/D genes deletion strain was successfully constructed.Through observation under a laser confocal microscopy,it was found that the wild strain formed a thick layer of mature biofilm at 16 h.Subsequently,the formed biofilm began to dissociate until 120 h;In contrast,a thin biofilm was formed by icaA/D genes deletion strain at 16 h after culture,and completely dissociated at 48 h.Compared with the wild strain,the MICs of 10 tested antimicrobial agents to icaA/D genes deletion strain were decreased,and the drug sensitivity of icaA/D genes deletion strain to 8 tested antimicrobial agents changed from resistance or intermediation to intermediation or sensitivity.[Conclusion]icaA/D gene deletion can significantly reduce the biofilm-formation ability and drug-resistance of MRSA.