黏菌素体外诱导耐药对肠炎沙门菌的主要耐药机制  

Main resistance mechanism of polymyxin induced drug resistance to Salmonella enteritidis in vitro

作　　者：王舒博 周杰 牟航 李艳[1] 王靖淳 邱丽雯 安凯 魏述永 WANG Shubo;ZHOU Jie;MU Hang;LI Yan;WANG Jingchun;QIU Liwen;AN Kai;WEI Shuyong(College of Veterinary Medicine,Southwest University,Rongchang,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460

出　　处：《中国兽医学报》2024年第9期1940-1947,共8页Chinese Journal of Veterinary Science

基　　金：中央高校基本科研业务费资助项目(XDJK2020C021)。

摘　　要：为了分析黏菌素体外诱导肠炎沙门菌耐药的主要耐药机制。本研究针对肠炎沙门菌CMCC(B)50335(ZK),通过体外诱导其对黏菌素耐药,并采用比浊法、半固体琼脂法、透射电镜、纸片扩散法测定诱导前后生长特性、运动能力、超微结构及对16种抗菌药物敏感性变化,Illumina NovaSeq PE150法检测其全基因组及单核苷酸多态性(SNP),RT-qPCR检测6种耐药相关基因表达量差异,Red大肠杆菌同源重组法构建诱导耐药株E1-128-1的phoP基因重组株△phoPE1-128-1,并检测对黏菌素的敏感性变化。结果表明,筛选出3株诱导耐药菌E1-128-1、E1-128-3、E2-128-3,耐药稳定性检测后最小抑菌浓度(minimal inhibit concentration,MIC)分别升高128、64、64倍;诱导耐药对受试菌生长能力及16种抗菌药物敏感性无显著性影响,E1-128-1运动能力显著升高,细胞壁及质膜明显增厚;诱导前后E1-128-1基因组组分无明显差异,但检测出phoP/phoQ、cpxP、lptD、csrA、acrB等6个耐药相关基因的8个错义突变,包括phoP错义突变位点4个,分别为Leu185Trp、His189Ser、Thr190Tyr和Ile191His,对相应基因进行PCR测序,结果与SNP结果相符;RT-qPCR结果表明3株诱导菌突变基因表达量均显著性上升;△phoPE1-128-1的黏菌素MIC值较E1-128-1下降至1 mg/L。提示phoP基因突变及表达量升高是体外诱导沙门菌CMCC(B)50335多黏菌素耐药的重要因素。This study aims to analyze the main drug resistance mechanism of Salmonella enteritidis induced by polymyxin in vitro.In this study,the resistance of Salmonella enteritidis CMCC(B)50335(ZK)to polymyxin was induced in vitro,and the growth characteristics,exercise ability,ultrastructure and sensitivity to 16 antimicrobial agents before and after induction were determined by turbidimetry,semi-solid agar method,transmission electron microscope and disk diffusion method,and the whole genome and single nucleotide polymorphism(SNP)were detected by Illumina NovaSeq PE150 method.RT-qPCR was used to detect the differences in the expression levels of six drug-resistant related genes.The recombinant strain△phoP E1-128-1,which induced drugresistant strain E1-128-1,was constructed by homologous recombination of Red E-scherichia coli,and its sensitivity to polymyxin was detected.The results showed that three strains of induced drug-resistant bacteria E1-128-1,E1-128-3 and E2-128-3 were screened out,and the MIC increased by 128,64 and 64 times respectively after drug resistance stability test.Induced drug resistance had no significant effect on the growth ability of the tested bacteria and the sensitivity of 16 antibacterial drugs.The exercise ability of E1-128-1 was significantly increased,and the cell wall and plasma membrane obviously thicken.There was no significant difference in the genome components of E1-128-1 before and after induction,but eight missense mutations of six drug-resistance related genes,including phoP/phoQ,cpxP lptD,csrA and acrB,were detected,including four missense mutation sites of phoP,namely Leu185Trp,His189Ser,Thr190Tyr and Ile191His.The corresponding genes were sequenced by PCR,and the results were consistent with those of SNP.RT-qPCR results showed that the expression levels of mutant genes of the three induced strains increased significantly.Compared with E1-128-1,the MIC of△phoPE1-128-1 decreased to 1 mg/L.It is suggested that the mutation and increased expression of phoP gene are important fa

关 键 词：沙门菌 黏菌素 诱导耐药 PHOP Red大肠杆菌同源重组 

分 类 号：S852.61[农业科学—基础兽医学]