ppk1基因对尿路致病性大肠埃希菌药物敏感性的影响及其机制

Role of polyphosphate kinase 1 in antibiotics susceptibility of uropathogenic Escherichia coli and its mechanisms

目的探讨多聚磷酸盐激酶1(ppk1)基因缺失对尿路致病性大肠埃希菌药物敏感性的影响及其机制。方法在本团队以往的研究中以尿路致病性大肠埃希菌CFT073为标准野生菌株,并成功构建敲除株△pk1和回补株△pk1-C。分别使用纸片扩散法和微量肉汤法检测CFT073、△pk1和△pk1-C对抗菌药物的敏感性;实时荧光定量PCR法对CFT073和△pk1的耐药性相关基因进行定量分析,对使用β-内酰胺类药物刺激的CFT073和△pk1的耐药性相关基因进行定量分析。结果与CFT073相比,两种药敏实验结果均显示△pk1对头孢噻肟、哌拉西林、他唑巴坦、氯霉素、呋喃妥因、磺胺甲噁唑和多黏菌素B的敏感性增强(P<0.05),而△pk1-C的敏感性无差异;实时荧光定量PCR检测CFT073和△pk1耐药性相关基因结果显示,ompF在△pk1中的表达明显上调,ompC、ompA、mdtA、mdtG、marB、marC和pmrD在△pk1中的表达相对下调,ompW和tolC在两菌株中的表达无差异;分别经哌拉西林和头孢噻肟刺激后,CFT073株ompA、ompW和tolC基因的表达量均升高,△pk1的均无统计学差异。结论ppk1基因的缺失使尿路致病性大肠埃希菌对某些抗菌药物的耐药性降低,并且ppk1基因可以影响其耐药性相关基因的表达。

OBJECTIVE To explore the impact of deletion of polyphosphate kinase 1(ppk1)gene on drug susceptibility of uropathogenic Escherichia coli and analyze its mechanisms.METHODS In the previous studies,the uropathogenic E.coli CFT073 was used as a standard wild strain,and the ppk1 knockout strain(Δpk1)and the complemented strain(Δpk1-C)were successfully constructed.The antibiotic susceptibility of CFT073,△pk1 and△pk1-C strains was determined by using disk diffusion(K-B)method and microbroth dilution method,respectively.Quantitative analysis was performed on the antibiotics resistance-related genes of CFT073 and△pk1 by using real-time fluorescence quantification PCR(qRT-PCR).Quantitative analysis was performed on the antibiotics resistance-related genes of CFT073 and△pk1 that were stimulated withβ-lactam antibiotics.RESULTS As compared with CFT073,the results of antibiotics susceptibility testing showed that the antibiotic susceptibility of the△pk1 to cefotaxime,piperacillin,tazobactam,chloramphenicol,nitrofurantoin,sulfamethoxazole and polymyxin B were increasingly high(P<0.05),there was no difference in the susceptibility of the△pk1-C.The real-time quantitative PCR detection of resistance-related genes in CFT073 and△pk1 showed that the expression of ompF in△pk1 was significantly up-regulated,while the expression of ompC,ompA,mdtA,mdtG,marB,marC and pmrD were down-regulated,there was no difference in the expression of ompW and tolC between the two strains.The levels of expression of ompA,ompW,and tolC in CFT073 were elevated after it was stimulated by piperacillin and cefotaxime,respectively,while there was no significant difference among the△pk1 strains.CONCLUSION The loss of ppk1 gene in uropathogenic E.coli may result in the decrease of resistant to certain antibiotics,and the ppk1 gene can influence the expression of the drug resistance-related genes.