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 blaTEM和 blaCTX-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。

abstractsTo 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 of Δpk1-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 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.