金黄色葡萄球菌细胞壁变化与β-内酰胺类抗生素耐药的关系

doi:10.11843/j.issn.0366-6964.2020.03.020

摘要/Abstract

摘要： 旨在揭示金黄色葡萄球菌针对β-内酰胺类抗生素可能存在细胞壁增厚的耐药机制。2016-2018年间，采集宁夏地区部分奶牛养殖场临床及亚临床型乳腺炎的乳样，通过显色培养基鉴别、镜检及PCR方法，分离鉴定牛乳源金黄色葡萄球菌；利用微量肉汤稀释法测定细菌对14种抗菌药物的耐药性，了解本地区金黄色葡萄球菌的耐药率及多重耐药情况；通过qRT-PCR方法检测细胞壁增厚相关的pbpB、murG、glmU、atlR基因转录丰度，并结合透射电镜进行形态观察，以确定增厚及发生原因。结果显示，分离鉴定出261株牛乳源金黄色葡萄球菌，其中包括9株耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）。药敏试验结果显示，金黄色葡萄球菌对β-内酰胺类抗生素具有较高的耐药率，其中氨苄西林为79.69%，青霉素为78.54%。多重耐药情况是以3、7和8重耐药的菌株居多；其中1株耐药种数达14种之多。qRT-PCR结果表明，4种相关基因的转录丰度均极显著上调（P<0.001或P<0.01）。透射电镜观察发现，甲氧西林敏感的金黄色葡萄球菌（methicillin sensitive Staphylococcus aureus，MSSA）JY21菌株的细胞壁在64和128 μg·mL^-1的青霉素浓度下，较对照组均极显著增厚（P<0.001），并可见细胞壁表面粗糙，有结节状凸起；但药物浓度从64 μg·mL^-1升高至128 μg·mL^-1细胞壁不再显著增厚（P>0.05）。MRSA WLD10菌株细胞壁未出现明显增厚（P>0.05）。综上所述，本地区牛乳源金黄色葡萄球菌针对β-内酰胺类抗生素，存在细胞壁增厚的耐药机制；增厚的原因主要是肽聚糖的过度合成及细胞自溶的减少。与MSSA JY21菌株相比，细胞壁增厚并非MRSA WLD10重要的耐药机制。

关键词: 金黄色葡萄球菌, 耐药性, 细胞壁, β-内酰胺类抗生素

Abstract: This study was conducted to reveal the resistance mechanism of Staphylococcus aureus to β-lactams, which may associate with thickening of the cell wall. From 2016 to 2018, we collected milk samples from clinical and subclinical mastitis in some dairy farms in Ningxia region. Separation and identification of S. aureus were conducted by using chromogenic medium, microscopic examination and PCR. The resistance in S. aureus isolates to 14 antibacterials was detected by micro-dilution method, to understand the resistance rate and multi-drug resistance of S. aureus isolates in the region. Transcription levels of pbpB, murG, glmU and atlR genes that are related to cell wall thickening were detected by qRT-PCR. Furthermore, phenotypes were examined by the transmission electron microscopy. Our goal was to discover the mechanism of cell wall thickening. The results indicated that we isolated and identified 261 strains of S. aureus, including 9 strains of Methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial susceptibility determination showed that the isolates have high resistance rate to β-lactams, the resistance rate to ampicillin was 79.69% and that to penicillin was 78.54%. Multi-drug resistance was distributed by isolates with 3, 7 and 8 resistance, in particular, one of the isolates could tolerate 14 antibacterials. The results of qRT-PCR showed that 4 related genes were significantly up-regulated (P<0.01 or P<0.001). The cell wall of JY21 (Methicillin sensitive Staphylococcus aureus, MSSA) was significantly thicker than the control group at the concentration of 64 and 128 μg·mL^-1 penicillin (P<0.001), and the cell wall was rough with nodular processes. Whereas, with the penicillin concentration increased from 64 μg·mL^-1 to 128 μg·mL^-1, and the cell wall was no longer significantly thickened (P>0.05). MRSA WLD10 showed no significant cell wall thickening (P>0.05). In conclusion, these data illustrated that cell wall thickening is a resistance mechanism for S.aureus to β-lactams in Ningxia. Simultaneously, the reason for cell wall thickening is mainly the excessive synthesis of peptidoglycan and the decrease of cell autolysis. Cell wall thickening is a critical resistance mechanism for the MSSA JY21. However, for MRSA WLD10, it's not the case.

Key words: Staphylococcus aureus, resistance, cell wall, β-lactams

中图分类号:

S852.611

马强, 王艺晖, 常佳伟, 万佳宏, 魏彦琴, 王桂琴. 金黄色葡萄球菌细胞壁变化与β-内酰胺类抗生素耐药的关系[J]. 畜牧兽医学报, 2020, 51(3): 602-611.

MA Qiang, WANG Yihui, CHANG Jiawei, WAN Jiahong, WEI Yanqin, WANG Guiqin. The Relationship between Cell Wall Thickening of Staphylococcus aureus and Resistance to β-lactams[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 602-611.

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