中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2014年
2期
83-90
,共8页
王银环%葛玉梅%孙爱华%严杰%程东庆
王銀環%葛玉梅%孫愛華%嚴傑%程東慶
왕은배%갈옥매%손애화%엄걸%정동경
大肠埃希菌%超广谱β-内酰胺酶%β-内酰胺类抗生素%蛋白磷酸化%组氨酸激酶
大腸埃希菌%超廣譜β-內酰胺酶%β-內酰胺類抗生素%蛋白燐痠化%組氨痠激酶
대장애희균%초엄보β-내선알매%β-내선알류항생소%단백린산화%조안산격매
Escherichia coli%Extended spectrum β-lactamases%β-lactam antibiotics%Protein phosphorylation%Histidine kinase
目的 了解大肠埃希菌临床菌株超广谱β-内酰胺酶(ESBLs)基因及其携带模式、β-内酰胺类抗生素诱导以及组氨酸激酶抑制剂抑制细菌蛋白磷酸化和ESBLs基因表达的作用机制.方法 采用PCR和测序法确定大肠埃希菌主要ESBLs基因(KPC、TEM、SHV、CTX-M)及其携带模式.采用E-test和微量试管稀释法分别测定最低抑菌浓度(MIC)和最低杀菌浓度(MBC).采用固定化金属螯合层析法、细菌蛋白磷酸化检测试剂盒及实时荧光定量RT-PCR分别检测1/4 MIC青霉素和头孢噻肟诱导或组氨酸激酶抑制剂(氯氰碘柳胺、自行设计的溴化或碘化甲基咪唑)抑制大肠埃希菌耐药菌株蛋白磷酸化和ESBLs基因mRNA水平升高的作用.结果 183株β-内酰胺类抗生素耐药大肠埃希菌临床菌株中,TEM和CTX-M基因检出率(83.1％和77.1％)高于其他基因且以两者同时携带模式为主(65.0％)(P＜0.05).1/4 MIC青霉素和头孢噻肟钠能诱导大肠埃希菌耐药菌株蛋白磷酸化及TEM、SHV、CTX-M基因mRNA水平显著升高(P＜0.05).200 μmol氯氰碘柳胺、2或10μmol溴化甲基咪唑、20或50 μmol碘化甲基咪唑无抑制或杀灭大肠埃希菌的作用,但能抑制上述抗生素诱导的细菌蛋白磷酸化和ESBLs基因mRNA水平升高(P＜0.05),也能提高大肠埃希菌耐药菌株对青霉素和头孢噻肟的敏感性(P＜0.05).结论 TEM和CTX-M是本地区β-内酰胺类抗生素耐药大肠埃希菌临床菌株优势ESBLs基因,TEM+CTX-M是ESBLs基因优势携带模式.亚致死量β-内酰胺类抗生素可经TCSS上调大肠埃希菌ESBLs基因表达水平,但可被组氨酸激酶抑制剂所抑制.
目的 瞭解大腸埃希菌臨床菌株超廣譜β-內酰胺酶(ESBLs)基因及其攜帶模式、β-內酰胺類抗生素誘導以及組氨痠激酶抑製劑抑製細菌蛋白燐痠化和ESBLs基因錶達的作用機製.方法 採用PCR和測序法確定大腸埃希菌主要ESBLs基因(KPC、TEM、SHV、CTX-M)及其攜帶模式.採用E-test和微量試管稀釋法分彆測定最低抑菌濃度(MIC)和最低殺菌濃度(MBC).採用固定化金屬螯閤層析法、細菌蛋白燐痠化檢測試劑盒及實時熒光定量RT-PCR分彆檢測1/4 MIC青黴素和頭孢噻肟誘導或組氨痠激酶抑製劑(氯氰碘柳胺、自行設計的溴化或碘化甲基咪唑)抑製大腸埃希菌耐藥菌株蛋白燐痠化和ESBLs基因mRNA水平升高的作用.結果 183株β-內酰胺類抗生素耐藥大腸埃希菌臨床菌株中,TEM和CTX-M基因檢齣率(83.1％和77.1％)高于其他基因且以兩者同時攜帶模式為主(65.0％)(P＜0.05).1/4 MIC青黴素和頭孢噻肟鈉能誘導大腸埃希菌耐藥菌株蛋白燐痠化及TEM、SHV、CTX-M基因mRNA水平顯著升高(P＜0.05).200 μmol氯氰碘柳胺、2或10μmol溴化甲基咪唑、20或50 μmol碘化甲基咪唑無抑製或殺滅大腸埃希菌的作用,但能抑製上述抗生素誘導的細菌蛋白燐痠化和ESBLs基因mRNA水平升高(P＜0.05),也能提高大腸埃希菌耐藥菌株對青黴素和頭孢噻肟的敏感性(P＜0.05).結論 TEM和CTX-M是本地區β-內酰胺類抗生素耐藥大腸埃希菌臨床菌株優勢ESBLs基因,TEM+CTX-M是ESBLs基因優勢攜帶模式.亞緻死量β-內酰胺類抗生素可經TCSS上調大腸埃希菌ESBLs基因錶達水平,但可被組氨痠激酶抑製劑所抑製.
목적 료해대장애희균림상균주초엄보β-내선알매(ESBLs)기인급기휴대모식、β-내선알류항생소유도이급조안산격매억제제억제세균단백린산화화ESBLs기인표체적작용궤제.방법 채용PCR화측서법학정대장애희균주요ESBLs기인(KPC、TEM、SHV、CTX-M)급기휴대모식.채용E-test화미량시관희석법분별측정최저억균농도(MIC)화최저살균농도(MBC).채용고정화금속오합층석법、세균단백린산화검측시제합급실시형광정량RT-PCR분별검측1/4 MIC청매소화두포새우유도혹조안산격매억제제(록청전류알、자행설계적추화혹전화갑기미서)억제대장애희균내약균주단백린산화화ESBLs기인mRNA수평승고적작용.결과 183주β-내선알류항생소내약대장애희균림상균주중,TEM화CTX-M기인검출솔(83.1％화77.1％)고우기타기인차이량자동시휴대모식위주(65.0％)(P＜0.05).1/4 MIC청매소화두포새우납능유도대장애희균내약균주단백린산화급TEM、SHV、CTX-M기인mRNA수평현저승고(P＜0.05).200 μmol록청전류알、2혹10μmol추화갑기미서、20혹50 μmol전화갑기미서무억제혹살멸대장애희균적작용,단능억제상술항생소유도적세균단백린산화화ESBLs기인mRNA수평승고(P＜0.05),야능제고대장애희균내약균주대청매소화두포새우적민감성(P＜0.05).결론 TEM화CTX-M시본지구β-내선알류항생소내약대장애희균림상균주우세ESBLs기인,TEM+CTX-M시ESBLs기인우세휴대모식.아치사량β-내선알류항생소가경TCSS상조대장애희균ESBLs기인표체수평,단가피조안산격매억제제소억제.
Objective To investigate the genotypes of extended spectrum β-lactamases (ESBLs) and their carrying modes in Escherichia coli (E.coli) isolates,and to analyze the mechanism of protein phosphorylation and ESBLs gene expression induced by β-lactam antibiotics or inhibited by histidine kinase inhibitors.Methods The predominant genotypes of ESBLs (KPC,TEM,SHV and CTX-M) and their carrying modes were identified by PCR and sequencing analysis.E-test and micro-tube dilution method were applied to measure minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs).Immobilized metal ion affinity chromatography,bacterial protein phosphorylation detection kit and real-time fluorescent quantitation RT-PCR were performed to analyze the enhancing effects of 1/4 MIC penicillin or cefotaxime or the inhibitory effects of histidine kinase inhibitors (closantel,bromized or iodized methylimidazol) on protein phosphorylation and the expression of ESBLs at mRNA level in E.coli isolates.Results In 183 β-lactam antibiotics-resistant E.coli isolates,TEM and CTX-M genes (83.1％ and 77.1％) were highly expressed than other two ESBLs genes with a prevalent carrying mode of coexisting (65.0％) (P＜0.05).Penicillin or cefotaxime at 1/4 MIC induced the protein phosphorylation and promoted the expression of TEM,SHV and CTX-M at mRNA level (P＜0.05).Closantel (200 μmol),bromized methylimidazol (2 or 10 μmol) or iodized methylimidazol (20 or 50 μmol) could neither kill E.coli isolates nor inhibit their growth,but could inhibit the protein phosphorylation induced by above mentioned antibiotics and enhance the expression of ESBLs at mRNA level (P＜0.05).Moreover,the susceptibility of antibioticresistant E.coli strains to penicillin and cefotaxime were increased (P＜0.05).Conclusion TEM and CTX-M were the predominant genotypes of ESBLs carried by β-lactam antibiotics-resistant E.coli strains isolated from Zhejiang province,which were mostly found in a TEM plus CTX-M carrying mode.Sublethal dose of β-lactam antibiotics could up-regulate the expression of ESBLs genes in E.coli isolates via TCSS,but it could be inhibited by histidine kinase inhibitors.
