CarO蛋白与鲍曼不动杆菌耐药性的相关性研究
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摘要
目的:研究CarO蛋白的表达与临床鲍曼不动杆菌耐环丙沙星及亚胺培南的相关性,了解CarO蛋白的结构与鲍曼不动杆菌耐药的相关性。
方法:①收集长沙地区四家医院的56株鲍曼不动杆菌进行环丙沙星及亚胺培南药敏实验;②对临床敏感株进行环丙沙星体外诱导耐药实验;③PCR扩增CarO基因并测序;④实时荧光定量PCR测定各组CarO蛋白mRNA表达情况;⑤提取外膜蛋白并用SDS-PAGE分析CarO蛋白的表达水平;⑥利用生物信息学分析技术比对敏感菌株和耐药菌株CarO蛋白的结构与功能。
结果:①所收集的临床鲍曼不动杆菌对环丙沙星的耐率为85.7%,对亚胺培南的耐药率为12.5%;②无论是临床耐药菌株与临床敏感菌株比较,还是临床敏感菌株被诱导成耐环丙沙星菌株以后,CarO mRNA的表达和蛋白的表达都有下降;③信息学分析发现耐药菌株与敏感菌株相比,氨基酸突变多发生在胞外区域;④信息学分析比对发现,耐药菌株CarO蛋白氨基酸序列在177位比敏感菌株多一个蛋白激酶C磷酸化位点,该处位点与细菌耐药的关系有待证明;⑤耐药菌株CarO蛋白的内在氢键网络可能与敏感株有差别。
结论:①收集的临床鲍曼不动杆菌对环丙沙星的耐药情况较亚胺培南严重;②CarO蛋白的表达下调可能与临床鲍曼不动杆菌的耐药性相关;③CarO蛋白与鲍曼不动杆菌耐环丙沙星有一定相关性,佐证了CarO蛋白可能与细菌多重耐药有关;④CarO蛋白可能与细胞信号传导有关;⑤敏感菌株与耐药菌株的CarO蛋白功能位点和内部结构有差异,可能与细菌耐药有关。
Objective:To investigate the correlation between the expression of CarO protein and the antibiotic resistance of clinical Acinetobacter baumannii isolates to imipenem and ciprofloxacin;To explore the relationship between the structure of CarO and the antibiotic resistance of Acinetobacter baumannii.
Methods:①56 of Clinical Acinetobacter baumannii isolates were collected from four hospitals in Changsha.The antimicrobial susceptibility test was carried out by means of K-B method and microdilution method;②Some clinical ciprofloxacin-sensitive strains were induced into ciprofloxacin-resistant ones by successive selection in media contained increasing concentrations of ciprofloxacin;③The CarO gene was amplified by polymerase chain reaction(PCR) and the PCR products were sequenced;④The gene expression of CarO mRNA was analyzed by real-time PCR;⑤Out membrane protein extracted from Acinetobacter baumannii isolates was analyzed by SDS-PAGE;⑥To contrast the CarO protein structure and function of the sensitive strains with ones of the resistant strains by methods of Bioinformatics.
Results:①Of all the collected clinical Acinetobacter baumannii isolates in Changsha,85.7%are ciprofloxacin-resistant strains and 12.5% are imipenem-resistant strains;②Compared with clinical sensitive strains,the CarO gene coding sequences of the clinical resistant strains were very different,and the expression of CarO mRNA and CarO protein is also significantly dissimilar,the same situation happend after inducing some clinical sensitive strains into ciprofloxacin-resistant strains;③Comparing the resistant strains with the sensitive strains by methods of Bioinformatics,amino acid mutations occurred in the extracellular region even more;④There is one more protein kinase C phosphorylation site in the number of 177 in CarO protein amino acid sequences of the resistant strains than in ones of the sensitive strains,it should be proved that this situation relates with bacterial resistance;⑤There may be some difference between the inside hydrogen bond network of the resistant strains and one of the sensitive strains.
Conclusions:①The collected clinical Acinetobacter baumannii isolates were more seriously resistable to Ciprofloxacin than to Imipenem;②The antibiotic resistance of clinical Acinetobacter baumannii isolates may be associated with the decline of CarO protein expression;③There is some relationship between CarO protein and ciprofloxacin-resistant Acinetobacter baumannii strains,it proves that CarO protein supports bacteria on multi-drug resistance;④CarO protein may be related to cell signalling;⑤The difference in CarO protein functional sites and structure between the sensitive strains and the resistant strains may be related to bacterial resistance.
方法:①收集长沙地区四家医院的56株鲍曼不动杆菌进行环丙沙星及亚胺培南药敏实验;②对临床敏感株进行环丙沙星体外诱导耐药实验;③PCR扩增CarO基因并测序;④实时荧光定量PCR测定各组CarO蛋白mRNA表达情况;⑤提取外膜蛋白并用SDS-PAGE分析CarO蛋白的表达水平;⑥利用生物信息学分析技术比对敏感菌株和耐药菌株CarO蛋白的结构与功能。
结果:①所收集的临床鲍曼不动杆菌对环丙沙星的耐率为85.7%,对亚胺培南的耐药率为12.5%;②无论是临床耐药菌株与临床敏感菌株比较,还是临床敏感菌株被诱导成耐环丙沙星菌株以后,CarO mRNA的表达和蛋白的表达都有下降;③信息学分析发现耐药菌株与敏感菌株相比,氨基酸突变多发生在胞外区域;④信息学分析比对发现,耐药菌株CarO蛋白氨基酸序列在177位比敏感菌株多一个蛋白激酶C磷酸化位点,该处位点与细菌耐药的关系有待证明;⑤耐药菌株CarO蛋白的内在氢键网络可能与敏感株有差别。
结论:①收集的临床鲍曼不动杆菌对环丙沙星的耐药情况较亚胺培南严重;②CarO蛋白的表达下调可能与临床鲍曼不动杆菌的耐药性相关;③CarO蛋白与鲍曼不动杆菌耐环丙沙星有一定相关性,佐证了CarO蛋白可能与细菌多重耐药有关;④CarO蛋白可能与细胞信号传导有关;⑤敏感菌株与耐药菌株的CarO蛋白功能位点和内部结构有差异,可能与细菌耐药有关。
Objective:To investigate the correlation between the expression of CarO protein and the antibiotic resistance of clinical Acinetobacter baumannii isolates to imipenem and ciprofloxacin;To explore the relationship between the structure of CarO and the antibiotic resistance of Acinetobacter baumannii.
Methods:①56 of Clinical Acinetobacter baumannii isolates were collected from four hospitals in Changsha.The antimicrobial susceptibility test was carried out by means of K-B method and microdilution method;②Some clinical ciprofloxacin-sensitive strains were induced into ciprofloxacin-resistant ones by successive selection in media contained increasing concentrations of ciprofloxacin;③The CarO gene was amplified by polymerase chain reaction(PCR) and the PCR products were sequenced;④The gene expression of CarO mRNA was analyzed by real-time PCR;⑤Out membrane protein extracted from Acinetobacter baumannii isolates was analyzed by SDS-PAGE;⑥To contrast the CarO protein structure and function of the sensitive strains with ones of the resistant strains by methods of Bioinformatics.
Results:①Of all the collected clinical Acinetobacter baumannii isolates in Changsha,85.7%are ciprofloxacin-resistant strains and 12.5% are imipenem-resistant strains;②Compared with clinical sensitive strains,the CarO gene coding sequences of the clinical resistant strains were very different,and the expression of CarO mRNA and CarO protein is also significantly dissimilar,the same situation happend after inducing some clinical sensitive strains into ciprofloxacin-resistant strains;③Comparing the resistant strains with the sensitive strains by methods of Bioinformatics,amino acid mutations occurred in the extracellular region even more;④There is one more protein kinase C phosphorylation site in the number of 177 in CarO protein amino acid sequences of the resistant strains than in ones of the sensitive strains,it should be proved that this situation relates with bacterial resistance;⑤There may be some difference between the inside hydrogen bond network of the resistant strains and one of the sensitive strains.
Conclusions:①The collected clinical Acinetobacter baumannii isolates were more seriously resistable to Ciprofloxacin than to Imipenem;②The antibiotic resistance of clinical Acinetobacter baumannii isolates may be associated with the decline of CarO protein expression;③There is some relationship between CarO protein and ciprofloxacin-resistant Acinetobacter baumannii strains,it proves that CarO protein supports bacteria on multi-drug resistance;④CarO protein may be related to cell signalling;⑤The difference in CarO protein functional sites and structure between the sensitive strains and the resistant strains may be related to bacterial resistance.
引文
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[4]El Shafie,M.Alishaq,M.Leni Garcia.Investigation of an outbreak of multidrug-resistant Acinetobacter baumannii in trauma intensive care unit[J].Hosp.Infect,2004,56:101-105.
[5]崔生辉,李景云,马越.2007.细菌对氟喹诺酮类药物的耐药机制[J].中国药房.18(2):148-150.
[6]黄艳飞,陈群.2004.鲍曼不动杆菌对喹诺酮类耐药机制[J].中国公共卫生.20(8):959-960.
[7]刘丁,陈萍,俞志海,陈伟.2003.鲍氏不动杆菌耐喹诺酮类药物的机理研究[J].中国抗生素杂志.28(3):168-171.
[8]Bou,G.,A.Oliver,and J.Martinez-Beltran.2000.OXA-24,a novel class D-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain[J].Antimicrob.Agents Chemother.44:1556-1561.
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