ppk1基因缺失对产ESBLs尿路感染大肠埃希菌药物敏感性的影响  

Effects of ppk1 deletion on the drug susceptibility of uropathogenic Escherichia coli producing ESBLs

作　　者：区静怡[1] 陈万山[1] 陈美君[1] 赵令斋[1] 李凌华[2] 彭亮[3] 梁烂 石亚玲 Ou Jingyi;Chen Wanshan;Chen Meijun;Zhao Lingzhai;Li Linghua;Peng Liang;Liang Lan;Shi Yaling(Department of Clinical Laboratory,Guangzhou Eighth People's Hospital,Guangzhou Medical University,Guangzhou 510440 China;Infectious Department,Guangzhou Eighth People's Hospital,Guangzhou Medical University,Guangzhou 510440,China;Department of Clinical Laboratory,The Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou 510700,China;The KingMed College of Laboratory Medicine,Guangzhou Medical University,Guangzhou 511436,China)

机构地区：[1]广州医科大学附属市八医院检验科,广州510440 [2]广州医科大学附属市八医院感染科,广州510440 [3]广州医科大学附属第五医院检验科,广州510700 [4]广州医科大学金域学院,广州511436

出　　处：《中华预防医学杂志》2023年第8期1238-1245,共8页Chinese Journal of Preventive Medicine

基　　金：2020年广州市卫生健康科技一般引导项目(20201A010024);广州市基础研究计划民生科技专题(202002020005);国家自然科学基金课题(82072265)。

摘　　要：探讨ppk1基因缺失对产超广谱β-内酰胺酶尿路感染大肠埃希菌(ESBLs-UPEC)药物敏感性的影响及其作用机制。本课题为实验性研究,2021年3至4月期间从广州医科大学附属市八医院检验科临床尿路感染患者尿液标本中分离1株产超广谱β-内酰胺酶(耐药基因型为TEM合并CTX-M-14型)的大肠埃希菌,命名为UE210113,并采用自杀质粒同源重组技术构建ppk1基因敲除株Δpk1并同时构建回补株Δpk1-C,用于开展研究ppk1基因对ESBLs-UPEC药物敏感性的影响及作用机制。使用Vitek2 Compact和微量肉汤稀释法分别检测UE210113、Δpk1及Δpk1-C对抗菌药物的敏感性;同时采用实时荧光定量PCR法对UE210113、Δpk1及Δpk1-C的ESBLs、外膜蛋白以及主动外排系统基因进行定量分析。通过两独立样本秩和检验,药物敏感性实验结果显示,与UE210113相比,Δpk1对头孢他啶、头孢吡肟、妥布霉素、米诺环素和复方新诺明的敏感性增强(Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05),Δpk1-C的药敏性恢复与UE210113一致(Z=0,P>0.05);实时荧光定量PCR法检测耐药性相关基因的结果显示,Δpk1中编码ESBLs基因bla_(TEM)和bla_(CTX-M-14)表达量较UE210113明显下调,但在Δpk1-C未恢复表达;外膜蛋白基因omp F在Δpk1中的表达明显上调而omp A、omp C则表达下调,主动外排系统基因tol C、mdt A和mdt G在Δpk1中的表达相对下调,外膜蛋白和主动外排系统基因在Δpk1-C均能恢复表达。综上,ppk1基因的缺失影响了ESBLs-UPEC的外膜蛋白基因和主动外排系统蛋白基因的表达,使ESBLs-UPEC对多种药物的敏感性提高。To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases(ESBLs-UPEC).The study was an experimental study.From March to April 2021,a strain of ESBLs-UPEC(genotype was TEM combined with CTX-M-14)named as UE210113,was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital,meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique,which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism.The drug susceptibility of UE210113,Δpk1,and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method.The quantitative expression of ESBLs,outer membrane protein and multidrug efflux systems encoding genes of UE210113,Δpk1 and Δpk1-C were performed by using qRT-PCR analysis.By using two independent sample Mann-Whitney U test,the drug susceptibility results showed that,compared with UE210113 strain,the sensitivities of Δpk1 to ceftazidime,cefepime,tobramycin,minocycline and cotrimoxazole were enhanced(Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05),and the drug susceptibility ofΔpk1-C restored to the same as which of UE210113(Z=0,P>0.05).The expression levels of ESBLs-enconding genes blaTEM and blaCTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113,but the expression was not restored in Δpk1-C.The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated,while the expression of omp A and omp C were down-regulated.The results showed that the expression of multidrug efflux systems encoding genes tol C,mdt A and mdtG were down-regulated in Δpk1 compared with UE210113.The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C.In conclusion,the lost of ppk1 gen

关 键 词：ppk1基因 尿路感染 大肠埃希菌 超广谱Β-内酰胺酶 药物敏感性 

分 类 号：R691.3[医药卫生—泌尿科学] R446.5[医药卫生—外科学]