建立ERIC-PCR技术快速分析不同耐药性鲍曼不动杆菌的同源性
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摘要
目的建立肠杆菌科基因间重复序列引物PCR技术(ERIC-PCR技术)用于分析亚胺培南耐药和亚胺培南敏感鲍曼不动杆菌同源性,为判断医院内感染发生提供一种简便的新方法。方法收集2018年3-6月江苏大学附属医院重症监护室患者痰液中分离的非重复鲍曼不动杆菌80株,其中包含50株亚胺培南耐药菌株,30株亚胺培南敏感菌株。使用细菌基因组DNA提取试剂盒提取细菌基因组DNA,PCR扩增菌株ERIC序列,Quantity One软件分析不同耐药性菌株电泳条带以判断菌株同源性。结果 50株亚胺培南耐药的鲍曼不动杆菌可分为6种基因型,其中最多的一种基因型有24株;30株亚胺培南敏感的鲍曼不动杆菌可分为20种基因型,其中最多的一种基因型仅有3株。结论该院ICU来源的亚胺培南耐药的鲍曼不动杆菌的基因型较少,提示该菌株可能发生了医院内感染。ERIC-PCR技术简单、方便,可以快速分析鲍曼不动杆菌同源性,适合于在各级医院中推广。
Objective To establish the ERIC-PCR technique for analyzing the homology of Acinetobacter baumannii with imipenem-resistant and imipenem-sensitive activity so as to provide a simple and convenient method for judging the occurrence of nosocomial infection.Methods Eighty strains of non-repeated Acinetobacter baumannii isolated from the sputum in ICU of this hospital during March to June 2018 were collected,including 50 strains were imipenem resistant and 30 strains were imipenem sensitive.The bacterial genomic DNA extraction kit was used to extract the bacterial genomic DNA.The bacterial strain ERIC gene sequence was amplified by PCR.The Quantity One software was used to analyze the electrophoresis bands in different drug-resistant bacterial strains for judging its homology.Results Fifty strains of imipenem-resistant Acinetobacter baumannii could be classified into 6 genotypes,in which the maximum genotype had 24 strains;thirty strains of imipenem-sensitive Acinetobacter baumannii could be classified into 20 genotypes,in which the maximum genotypes only had 3 strains.Conclusion The imipenem-resistant Acinetobacter baumannii derived from ICU has fewer genotypes,suggesting that the strain may develop the nosocomial infection.The ERICPCR method is simple and convenient,can rapidly analyze the homology of Acinetobacter baumannii and is suitable for promotion in various levels of hospital.
Objective To establish the ERIC-PCR technique for analyzing the homology of Acinetobacter baumannii with imipenem-resistant and imipenem-sensitive activity so as to provide a simple and convenient method for judging the occurrence of nosocomial infection.Methods Eighty strains of non-repeated Acinetobacter baumannii isolated from the sputum in ICU of this hospital during March to June 2018 were collected,including 50 strains were imipenem resistant and 30 strains were imipenem sensitive.The bacterial genomic DNA extraction kit was used to extract the bacterial genomic DNA.The bacterial strain ERIC gene sequence was amplified by PCR.The Quantity One software was used to analyze the electrophoresis bands in different drug-resistant bacterial strains for judging its homology.Results Fifty strains of imipenem-resistant Acinetobacter baumannii could be classified into 6 genotypes,in which the maximum genotype had 24 strains;thirty strains of imipenem-sensitive Acinetobacter baumannii could be classified into 20 genotypes,in which the maximum genotypes only had 3 strains.Conclusion The imipenem-resistant Acinetobacter baumannii derived from ICU has fewer genotypes,suggesting that the strain may develop the nosocomial infection.The ERICPCR method is simple and convenient,can rapidly analyze the homology of Acinetobacter baumannii and is suitable for promotion in various levels of hospital.
引文
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[15]钟海波,伍哓锋,曾瑜,等.多重耐药鲍曼不动杆菌ERIC-PCR的分子流行病学研究[J].现代医院,2013,13(12):19-22.
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[17]LOWINGS M,EHLERS M M,DREYER A W,et al.High prevalence of oxacillinases in clinical multidrug-resistant Acinetobacter baumannii isolates from the Tshwane region,South Africa:an update[J].BMC Infect Dis,2015,15:521-524.
[18]ELLIS D,COHEN B,LIU J,et al.Risk factors for hospital-acquired antimicrobial-resistant infection caused by Acinetobacter baumannii[J].Antimicrob Resist Infect Control,2015,4:40-43.
[19]SOUZA A V,MOREIRA C R,PASTERNAK J,et al.Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit[J].JMed Microbiol,2004,53(Pt 10):999-1005.
[20]PETTIGREW M M,FOXMAN B,ECEVIT Z,et al.Use of pulsed-field gel electrophoresis,enterobacterial repetitive intergenic consensus typing,and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae[J].J Clin Microbiol,2002,40(2):660-662.
[21]M′ZALI F H,HERITAGE J,GASCOYNE-BINZI D M,et al.PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7extendedspectrum beta-lactamase and to identify different SHVgenes within the same strain[J].J Antimicrob Chemother,1998,41(1):123-125.
[22]SILBERT S,PFALLER M A,HOLLIS R J,et al.Evaluation of three molecular typing techniques for nonfermentative Gram-negative bacilli[J].Infect Control Hosp Epidemiol,2004,25(10):847-851.
[23]YING C,LI Y,WANG Y,et al.Investigation of the molecular epidemiology of Acinetobacter baumannii isolated from patients and environmental contamination[J].J Antibiot(Tokyo),2015,68(9):562-567.
[24]王晓辉,宗志勇,吕晓菊.携带bla_(OXA-58)的鲍曼不动杆菌的克隆相关性分析[J].四川大学学报(医学版),2013,44(3):405-409.
[25]TSAI H C,CHOU M Y,WU C C,et al.Seasonal Distribution and genotyping of antibiotic resistant strains of listeriaInnocua isolated from a river basin categorized by ERIC-PCR[J].Int J Environ Res Public Health,2018,15(7):E1559.
[26]DORNELES E M,SANTANA J A,RIBEIRO D,et al.E-valuation of ERIC-PCR as genotyping method for Corynebacterium pseudotuberculosis isolates[J].PLoS One,2014,9(6):e98758.
[27]MO Q H,WANG H B,TAN H,et al.Optimization and head-to-head comparison of MISSR-PCR,ERIC-PCR,RAPD and 16SrRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China[J].Indian J Med Microbiol,2015,33(4):516-523.
[28]GILLINGS M,HOLLEY M.Repetitive element PCR fingerprinting(rep-PCR)using enterobacterial repetitive intergenic consensus(ERIC)primers is not necessarily directed at ERIC elements[J].Lett Appl Microbiol,1997,25(1):17-21.
[29]GILLINGS M,HOLLEY M.Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation[J].Electrophoresis,1997,18(9):1512-1518.
[30]ZOWAWI H M,SYRMIS M W,KIDD T J,et al.Identification of carbapenem-resistant Pseudomonas aeruginosa in selected hospitals of the Gulf Cooperation Council States:dominance of high-risk clones in the region[J/OL].J Med Microbiol,2018,67(6):846-853.
[31]SILVA JUNIOR V V D,FERREIRA L D,ALVES L R,et al.Detection of multidrug-resistant Pseudomonas aeruginosa harboring bla GES-1and bla GES-11in Recife,Brazil[J].Rev Soc Bras Med Trop,2017,50(6):764-768.
[32]DAVANDEH I,ERAC B,AYDEMIR S S.Investigation of class-d beta-lactamases causing carbapenem resistance in clinical Acinetobacter baumannii isolates[J].Turk JMed Sci,2017,47(5):1661-1666.
[33]CARTELLE GESTAL M,ZURITA J,GUALPA G,et al.Early detection and control of an Acinetobacter baumannii multi-resistant outbreak in a hospital in Quito,Ecuador[J].J Infect Dev Ctries,2016,10(12):1294-1298.
[34]MAMISHI S,MAHMOUDI S,SADEGHI R H,et al.Genotyping of Staphylococcus aureus strains among healthcare workers and patients in the tertiary referral children′s medical hospital in Tehran,Iran[J].Br J Biomed Sci,2012,69(4):173-177.
[35]HETSA B A,KUMAR A,ATEBA C N.Characterization of multiple antibiotic resistant clinical strains of Staphylococcus isolated from pregnant women vagina[J].Folia Microbiol(Praha),2018,63(5):607-617.
[36]ABDOLLAHI S,RAMAZANZADEH R,KHIABANI ZD,et al.Epidemiological and Inducible Resistance in Coagulase Negative Staphylococci[J].Glob J Health Sci,2015,8(4):109-119.
[2]ANTUNES L C,VISCA P,TOWNER K J.Acinetobacter baumannii:evolution of a global pathogen[J].Pathog Dis,2014,71(3):292-301.
[3]ZHANG H Z,ZHANG J S,QIAO L.The Acinetobacter baumannii group:a systemic review[J].World J Emerg Med,2013,4(3):169-174.
[4]胡付品,郭燕,朱德妹,等.2016年中国CHINET细菌耐药性监测[J].中国感染与化疗杂志,2017,17(5):481-491.
[5]LI H,LIU F,ZHANG Y,et al.Evolution of carbapenemresistant Acinetobacter baumannii revealed through whole-genome sequencing and comparative genomic analysis[J].Antimicrob Agents Chemother,2015,59(2):1168-1176.
[6]RAHMAN M,PRASAD K N,GUPTA S,et al.Prevalence and molecular characterization of new delhi metallobeta-lactamases in multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii from India[J].Microb Drug Resist,2018,24(6):792-798.
[7]SINGKHAM-IN U,CHATSUWAN T.In vitro activities of carbapenems in combination with amikacin,colistin,or fosfomycin against carbapenem-resistant Acinetobacter baumannii clinical isolates[J].Diagn Microbiol Infect Dis,2018,91(2):169-174.
[8]QURESHI Z A,HITTLE L E,O′HARA J A,et al.Colistin-resistant Acinetobacter baumannii:beyond carbapenem resistance[J].Clin Infect Dis,2015,60(9):1295-1303.
[9]GOSIEWSKI T,BRZYCHCZY-WLOCH M.The use of PF-GE method in genotyping of selected bacteria species of the Lactobacillus genus[J].Methods Mol Biol,2015,1301:225-240.
[10]FENDRI I,BEN HASSENA A,GROSSET N,et al.Genetic diversity of food-isolated Salmonella strains through pulsed field gel electrophoresis(PFGE)and Enterobacterial Repetitive Intergenic Consensus(ERIC-PCR)[J].PLoS One,2013,8(12):e81315.
[11]SHARPLES G J,LLOYD R G.A novel repeated DNAsequence located in the intergenic regions of bacterial chromosomes[J].Nucleic Acids Res,1990,18(22):6503-6508.
[12]HULTON C S,HIGGINS C F,SHARP P M.ERIC sequences:a novel family of repetitive elements in the genomes of Escherichia coli,Salmonella typhimurium and other enterobacteria[J].Mol Microbiol,1991,5(4):825-834.
[13]VERSALOVIC J,KOEUTH T,LUPSKI J R.Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes[J].Nucleic Acids Res,1991,19(24):6823-6831.
[14]陈迎春,曹又方,赵立平.大肠杆菌MG1655菌株ERIC-PCR图谱主带序列组成分析[J].微生物学通报,2002,29(6):28-32.
[15]钟海波,伍哓锋,曾瑜,等.多重耐药鲍曼不动杆菌ERIC-PCR的分子流行病学研究[J].现代医院,2013,13(12):19-22.
[16]MOLTER G,SEIFERT H,MANDRAKA F,et al.Outbreak of carbapenem-resistant Acinetobacter baumannii in the intensive care unit:a multi-level strategic management approach[J].J Hosp Infect,2016,92(2):194-198.
[17]LOWINGS M,EHLERS M M,DREYER A W,et al.High prevalence of oxacillinases in clinical multidrug-resistant Acinetobacter baumannii isolates from the Tshwane region,South Africa:an update[J].BMC Infect Dis,2015,15:521-524.
[18]ELLIS D,COHEN B,LIU J,et al.Risk factors for hospital-acquired antimicrobial-resistant infection caused by Acinetobacter baumannii[J].Antimicrob Resist Infect Control,2015,4:40-43.
[19]SOUZA A V,MOREIRA C R,PASTERNAK J,et al.Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit[J].JMed Microbiol,2004,53(Pt 10):999-1005.
[20]PETTIGREW M M,FOXMAN B,ECEVIT Z,et al.Use of pulsed-field gel electrophoresis,enterobacterial repetitive intergenic consensus typing,and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae[J].J Clin Microbiol,2002,40(2):660-662.
[21]M′ZALI F H,HERITAGE J,GASCOYNE-BINZI D M,et al.PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7extendedspectrum beta-lactamase and to identify different SHVgenes within the same strain[J].J Antimicrob Chemother,1998,41(1):123-125.
[22]SILBERT S,PFALLER M A,HOLLIS R J,et al.Evaluation of three molecular typing techniques for nonfermentative Gram-negative bacilli[J].Infect Control Hosp Epidemiol,2004,25(10):847-851.
[23]YING C,LI Y,WANG Y,et al.Investigation of the molecular epidemiology of Acinetobacter baumannii isolated from patients and environmental contamination[J].J Antibiot(Tokyo),2015,68(9):562-567.
[24]王晓辉,宗志勇,吕晓菊.携带bla_(OXA-58)的鲍曼不动杆菌的克隆相关性分析[J].四川大学学报(医学版),2013,44(3):405-409.
[25]TSAI H C,CHOU M Y,WU C C,et al.Seasonal Distribution and genotyping of antibiotic resistant strains of listeriaInnocua isolated from a river basin categorized by ERIC-PCR[J].Int J Environ Res Public Health,2018,15(7):E1559.
[26]DORNELES E M,SANTANA J A,RIBEIRO D,et al.E-valuation of ERIC-PCR as genotyping method for Corynebacterium pseudotuberculosis isolates[J].PLoS One,2014,9(6):e98758.
[27]MO Q H,WANG H B,TAN H,et al.Optimization and head-to-head comparison of MISSR-PCR,ERIC-PCR,RAPD and 16SrRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China[J].Indian J Med Microbiol,2015,33(4):516-523.
[28]GILLINGS M,HOLLEY M.Repetitive element PCR fingerprinting(rep-PCR)using enterobacterial repetitive intergenic consensus(ERIC)primers is not necessarily directed at ERIC elements[J].Lett Appl Microbiol,1997,25(1):17-21.
[29]GILLINGS M,HOLLEY M.Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation[J].Electrophoresis,1997,18(9):1512-1518.
[30]ZOWAWI H M,SYRMIS M W,KIDD T J,et al.Identification of carbapenem-resistant Pseudomonas aeruginosa in selected hospitals of the Gulf Cooperation Council States:dominance of high-risk clones in the region[J/OL].J Med Microbiol,2018,67(6):846-853.
[31]SILVA JUNIOR V V D,FERREIRA L D,ALVES L R,et al.Detection of multidrug-resistant Pseudomonas aeruginosa harboring bla GES-1and bla GES-11in Recife,Brazil[J].Rev Soc Bras Med Trop,2017,50(6):764-768.
[32]DAVANDEH I,ERAC B,AYDEMIR S S.Investigation of class-d beta-lactamases causing carbapenem resistance in clinical Acinetobacter baumannii isolates[J].Turk JMed Sci,2017,47(5):1661-1666.
[33]CARTELLE GESTAL M,ZURITA J,GUALPA G,et al.Early detection and control of an Acinetobacter baumannii multi-resistant outbreak in a hospital in Quito,Ecuador[J].J Infect Dev Ctries,2016,10(12):1294-1298.
[34]MAMISHI S,MAHMOUDI S,SADEGHI R H,et al.Genotyping of Staphylococcus aureus strains among healthcare workers and patients in the tertiary referral children′s medical hospital in Tehran,Iran[J].Br J Biomed Sci,2012,69(4):173-177.
[35]HETSA B A,KUMAR A,ATEBA C N.Characterization of multiple antibiotic resistant clinical strains of Staphylococcus isolated from pregnant women vagina[J].Folia Microbiol(Praha),2018,63(5):607-617.
[36]ABDOLLAHI S,RAMAZANZADEH R,KHIABANI ZD,et al.Epidemiological and Inducible Resistance in Coagulase Negative Staphylococci[J].Glob J Health Sci,2015,8(4):109-119.