基于MMR缺陷的高频突变食源性沙门氏菌环丙沙星耐药机制

doi:10.7506/spkx1002-6630-20200710-145

食品科学 ›› 2021, Vol. 42 ›› Issue (22): 162-170.doi: 10.7506/spkx1002-6630-20200710-145

• 生物工程 • 上一篇

基于MMR缺陷的高频突变食源性沙门氏菌环丙沙星耐药机制

王银，杨保伟，盛焕精，李怡澜，施春雷，史贤明，肖英平，杨华

（1.西北大学食品科学与工程学院，陕西西安 710069；2.上海交通大学农业与生物学院，中美食品安全联合研究中心，上海 200240；3.西北农林科技大学食品科学与工程学院，陕西杨凌 712100；4.浙江省农业科学院农产品质量标准研究所，浙江省植物有害生物防治省部共建国家重点实验室培育基地，浙江杭州 310021）

发布日期:2021-11-23
基金资助:
“十三五”国家重点研发计划重点专项（2017YFC1600100）；国家自然科学基金面上项目（31671956）；省部共建农产品质量安全危害因子与风险防控国家重点实验室培育项目（2010DS700124-ZZ1905）；陕西省教育厅专项（18JK0776）

Effect of Methyl-directed Mismatch Repair Gene Deficiency on Ciprofloxacin Resistance in Salmonella Hypermutators

WANG Yin, YANG Baowei, SHENG Huanjing, LI Yilan, SHI Chunlei, SHI Xianming, XIAO Yingping, YANG Hua

(1. College of Food Science and Engineering, Northwest University, Xi’an 710069, China;2. MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; 3. College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China;4. Institute of Quality and Standard for Agro-products, State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China)

Published:2021-11-23

摘要/Abstract

摘要： 目的：通过研究甲基导向错配修复系统（methyl-directed mismatch repair，MMR）主要调控基因缺陷对食源性沙门氏菌高频突变子突变频率和环丙沙星药敏性的影响，揭示其调控机制。方法：采用萘啶酮酸和利福平平板测定90 株食源性沙门氏菌高频突变子的突变频率；聚合酶链式反应结合DNA测序法检测耐药基因突变；琼脂稀释法测定环丙沙星对沙门氏菌高频突变子的最小抑菌浓度（minimal inhibitory concentration，MIC），确定MMR缺陷与沙门氏菌高频突变子突变频率、MIC及耐药基因间的关系；通过电转化法将野生mutS、mutH、mutL和uvrD基因分别转入高频突变子103D2，采用实时聚合酶链式反应法测定野生型基因转入前后103D2耐药相关基因表达差异，分析MMR基因缺陷对沙门氏菌高频突变子环丙沙星耐药性的影响及调控机制。结果：90 株沙门氏菌高频突变子中共有89 株对环丙沙星耐药，49 株MutS编码基因发生突变（MutS－），且相较于MutS非突变株，在MutS突变株中GyrA（67.3%）及ParC（87.8%）蛋白突变检出率更高。103D2转入野生型mutS、mutH、mutL 及uvrD基因质粒后，103D2:P-mutS突变频率显著下降，103D2:P-mutL和103D2:P-uvrD突变频率极显著下降。4 株互补菌株中marA基因显著下调，marR、tolC（除103D2:P-uvrD外）基因上调，降低103D2对环丙沙星耐药性。结论：MMR缺陷可通过影响外排泵和膜编码基因marA、marR和tolC的表达改变对沙门氏菌高频突变子的耐药性及突变频率产生影响。

关键词: 沙门氏菌；高频突变子；耐药基因；实时聚合酶链式反应；甲基导向错配修复系统

Abstract: Objective: In this study, the relationship among methyl-directed mismatch repair (MMR) deficiency, mutation frequency, and the minimal inhibitory concentration (MIC) of ciprofloxacin was investigated to reveal the antimicrobial resistance mechanism of Salmonella hypermutators (SHs). Methods: Ninety SHs were investigated for their susceptibility to ciprofloxacin via agar dilution method. Mutation frequencies were determined by using agar plates containing nalidixic acid and rifampicin. Mutations related to antibiotic resistance were tested by polymerase chain reaction (PCR). The wild-type mutant genes mutS, mutH, mutL, and uvrD were separately transferred into the representative hypermutator 103D2 by electrotransformation. The expression of antibiotic resistance genes in 103D2 and the resulting transformants were tested by real-time PCR so as to determine the effect of MMR gene deficiency on ciprofloxacin resistance. Results: Forty-nine of the 90 SHs (89 of which were ciprofloxacin resistant) were deficient in the MutS (MutS-), and GyrA (67.3%) and ParC (87.8%) mutations were more likely (P = 0.007) to occur in SHs with MutS- compared with the non-MutS wild-type strains. The mutation frequency of the transformants on Luria-Bertani-agar plates with nalidixic acid (20 μg/mL) was significantly (103D2:P-mutS) or extremely significantly (103D2:P-mutL and 103D2:P-uvrD) decreased compared with the original strains. In all transformants, the expression of marA was significantly down-regulated whereas the expression of marR and tolC were significantly upregulated except for 103D2:P-uvrD, leading to deceased ciprofloxacin resistance in 103D2. Conclusion: MMR gene deficiency may be a factor contributing to antibiotic resistance in SHs by affecting the efflux pumps and the expression of marA, tolC, and marR, associated with antibiotic susceptibility.

Key words: Salmonella; hypermutator; antimicrobial resistance; real-time polymerase chain reaction; methyl-directed mismatch repair genes

中图分类号:

TS201.6

王银，杨保伟，盛焕精，李怡澜，施春雷，史贤明，肖英平，杨华. 基于MMR缺陷的高频突变食源性沙门氏菌环丙沙星耐药机制[J]. 食品科学, 2021, 42(22): 162-170.

WANG Yin, YANG Baowei, SHENG Huanjing, LI Yilan, SHI Chunlei, SHI Xianming, XIAO Yingping, YANG Hua. Effect of Methyl-directed Mismatch Repair Gene Deficiency on Ciprofloxacin Resistance in Salmonella Hypermutators[J]. FOOD SCIENCE, 2021, 42(22): 162-170.

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基于MMR缺陷的高频突变食源性沙门氏菌环丙沙星耐药机制

Effect of Methyl-directed Mismatch Repair Gene Deficiency on Ciprofloxacin Resistance in Salmonella Hypermutators

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