兔源产ESBLs大肠杆菌耐药性与优势基因型研究
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摘要
【目的】对四川地区兔源大肠杆菌(Escherichia coli)产ESBLs菌株的耐药特征及ESBLs流行基因型和基因亚型进行研究,为指导临床合理使用抗菌药物、有效控制产ESBLs菌株的感染和流行提供参考。【方法】采用K-B纸片扩散法对97株兔源大肠杆菌进行药物敏感性试验,采用CLSI推荐方法进行ESBLs表型检测,同时采用PCR方法及序列分析,确定其基因型和基因亚型。【结果】兔源大肠杆菌产ESBLs菌的检出率为71.13%(69/97);产ESBLs菌对12种药物的耐药率均高于非产ESBLs菌株,其中对头孢西丁、头孢唑啉、头孢他啶、阿米卡星和诺氟沙星的耐药率在产ESBLs菌和非产ESBLs菌中差异显著(P<0.05);产ESBLs菌多重耐药程度极显著高于非产ESBLs菌(P<0.01),其多重耐药率分别为100%和85.71%。TEM、CTX-M和OXA型基因的检出率分别为62.32%,63.77%和23.19%,未检测到SHV型基因;产酶菌分属于10个基因亚型,其中TEM-1、CTX-M-14和OXA-1亚型分别是各基因型的优势基因亚型。【结论】四川地区兔源产ESBLs菌多重耐药程度显著高于非产ESBLs菌;产ESBLs大肠杆菌中的流行基因型与国内其他地区一致,为TEM型和CTX-M型,但基因亚型存在差异,TEM-1、CTX-M-14和OXA-1亚型分别是各基因型的优势基因亚型。
【Objective】The resistance of ESBLs-producing Escherichia coli strains isolated from rabbits in Sichuan as well as the prevalent genotype and gene subtype of ESBLs were investigated to provide basis for guiding rational use of antimicrobial agents and effective control of infection and spread of ESBLs strains.【Method】The antimicrobial susceptibility of 97 Escherichia coli strains was determined using K-B method,the phenotype of ESBLs was identified and determined by CLSI standard method,and the PCR method and sequencing analysis were used to determine the genotype and subtype of ESBLs gene.【Result】There were 71.13%(69/97)of Escherichia coli strains produced ESBLs.The resistance rate of ESBLsproducing strains against 12 antimicrobial agents was higher than that of non-ESBLs-producing ones,and there was significant difference between them(P<0.05)for FOX,CZO,CAZ,AMI,NOR.The multi-drug resistance rates of ESBLs-producing strains and non-ESBLs-producing strain were 100% and 85.71%,respectively(P<0.01).The detection rates of TEM,CTX-M and OXA genotype were 62.32%,63.77%,and 23.19%,respectively,while SHVgenotype was not detected.The enzyme ESBLs strains belonged to 10 gene subtypes,and the dominant ones were TEM-1,CTX-M-14 and OXA-1.【Conclusion】The multidrug resistance rate of ESBLs-producing strains from Sichuan was significantly higher than that of non-ESBLs-producing strains.The prevalent genotypes in Sichuan(TEM and CTX-M)were same as other areas in China,but the dominant gene subtypes(TEM-1,CTX-M-14 and OXA-1)were different.
【Objective】The resistance of ESBLs-producing Escherichia coli strains isolated from rabbits in Sichuan as well as the prevalent genotype and gene subtype of ESBLs were investigated to provide basis for guiding rational use of antimicrobial agents and effective control of infection and spread of ESBLs strains.【Method】The antimicrobial susceptibility of 97 Escherichia coli strains was determined using K-B method,the phenotype of ESBLs was identified and determined by CLSI standard method,and the PCR method and sequencing analysis were used to determine the genotype and subtype of ESBLs gene.【Result】There were 71.13%(69/97)of Escherichia coli strains produced ESBLs.The resistance rate of ESBLsproducing strains against 12 antimicrobial agents was higher than that of non-ESBLs-producing ones,and there was significant difference between them(P<0.05)for FOX,CZO,CAZ,AMI,NOR.The multi-drug resistance rates of ESBLs-producing strains and non-ESBLs-producing strain were 100% and 85.71%,respectively(P<0.01).The detection rates of TEM,CTX-M and OXA genotype were 62.32%,63.77%,and 23.19%,respectively,while SHVgenotype was not detected.The enzyme ESBLs strains belonged to 10 gene subtypes,and the dominant ones were TEM-1,CTX-M-14 and OXA-1.【Conclusion】The multidrug resistance rate of ESBLs-producing strains from Sichuan was significantly higher than that of non-ESBLs-producing strains.The prevalent genotypes in Sichuan(TEM and CTX-M)were same as other areas in China,but the dominant gene subtypes(TEM-1,CTX-M-14 and OXA-1)were different.
引文
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[10]Lucia P,Monica M,Antonia M,et al.Detection of CTX-M typeβ-lactamase genes in fecal Escherichia coli isolates from healthy children in bolivia and peru[J].Antimicrobial Ggents and Chemotherapy,2004,48(12):4556-4561.
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[12]Lahlaouia M H,Ben Haj Khalifab A,Ben Moussa M.Epidemiology of enterobacteriaceae producing CTX-M type extendedspectrumβ-lactamase(ESBL)[J].Medecine et Maladies Infectieuses,2004,44(9):400-404.
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[14]刘保光,世洪勋,格桑顿旦,等.西藏地区藏鸡大肠杆菌超广谱β-内酰胺酶检测及药物敏感性分析[J].家畜生态学报,2013,34(3):5-8.Liu B G,Shi H X,Ge S D D,et al.Deternimation of ESBLs and antibiotics susceptibility test analysis for Escherichia coli from the chickens in Xizang[J].Acta Ecologiae Animalis Domastici,2013,34(3):5-8.
[15]汤电,张浩吉,纪雪筱,等.广东地区患病食品动物源大肠杆菌ESBLs和AmpC酶流行分布调查[J].中国兽医学报,2012,32(2):242-248.Tang D,Zhang H J,Ji X X,et al.Prevalence of plasmid mediated ESBLs and AmpC-typeβ-lactamases among E.coliisolated from sick food-producing animals in Guangdong[J].Chinese Journal of Veterinary Science,2012,32(2):242-248.
[16]刘保光,刘建华,吴华,等.舒巴坦钠和EDTA使产ESBLs鸡大肠杆菌恢复对抗菌药物敏感性[J].湖南农业大学学报(自然科学版),2010,32(2):242-248.Liu B G,Liu J H,Wu H,et al.Recovery of antibacterial sensitivity of ESBLs-producing fowl E.coli by sulbactam and EDTA[J].Journal of Hunan Agricultural University(Nat Sci),2010,32(2):242-248.
[17]Yuan L,Liu J H,Hu G Z,et al.Molecular characterization of extended-spectrumβ-lactamase-producing Escherichia coli isolates from chickens in Henan Province,China[J].Journal of Medical Microbiology,2009,58(11):1449-1453.
[18]Paterson D L,Bonomo R A.Extended-spectrum beta-lactamases:a clinical update[J].Clinical Microbiology Reviews,2005,18(4):657-686.
[19]Kiratisin P,Apisarnthanarak A,Laesripa C,et al.Molecular characterization and epidemiology of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in Thailand,where the CTX-M family is endemic[J].Antimicrobial Agents and Chemotherapy,2008,52(8):2818-2824.
[20]Ahmed A M,Shimabukuro H,Shimamoto T.Isolation and molecular characterization of multidrug-resistant strains of Escherichia coli and Salmonellafrom retail chicken meat in Japan[J].Journal of Food Science,2009,74(7):405-410.
[21]苑丽,刘建华,胡功政,等.鸡大肠杆菌TEM和CTX-M型超广谱β-内酰胺酶基因分型研究[J].中国农业科学,2010,43(20):4310-4316.Yuan L,Liu J H,Hu G Z,et al.Genotypes of TEM-type and CTX-M-type extended-spectrumβ-lactamases produced by Escherichia coli isolated from the fowl[J].Scientia Agricultura Sinica,2010,43(20):4310-4316.
[22]Zhao W H,Hu Z Q.Epidemiology and genetics of CTX-M extended-spectrumβ-lactamases in gram-negative bacteria[J].Critical Reviews in Microbiology,2013,39(1):79-101.
[23]Lahlaour H,Ben Haj Khalifa A,Ben Moussa M.Epidemiology of enterobacteriaceae producing CTX-M type extended spectrumβ-lactamase(ESBLs)[J].Medecine et Maladies Infectieuses,2014,44(9):400-404.
[24]苑丽,莫娟,胡功政,等.49株鸡大肠杆菌OXA型β-内酰胺酶的检测[J].江西农业学报,2009,21(8):1-3.Yuan L,Mo J,Hu G Z,et al.Detection of OXA-typeβ-lactamases among 49 strains of Escherichia coli isolated from chickens[J].Acta Agriculturae Jiangxi,2009,21(8):1-3.
[2]Nikaido H.Multidrug resistance in bacteria[J].Annual Review of Biochemistry,2009,78:119-146.
[3]曲志娜,刘红玉,王娟,等.青岛地区产ESBLs鸡源大肠杆菌耐药性调查与优势基因型分析[J].中国农业科学,2015,48(10):2058-2066.Qu Z N,Liu H Y,Wang J,et al.Antimicrobial resistance investigation and dominant denotype analysis of ESBLs-producing Escherichia coil strains from chicken in Qingdao[J].Scientia Agricultura Sinica,2015,48(10):2058-2066.
[4]曲志娜,张颖,李玉清,等.鸡、猪大肠杆菌ESBLs基因型检测及耐药性分析[J].中国农学通报,2013,29(8):50-54.Qu Z N,Zhang Y,Li Y Q,et al.Genotype detection of ESBLs and antibiotic resistance analysis in E.coli strains from chickens and pigs[J].Chinese Agricultural Science Bulletin,2013,29(8):50-54.
[5]舒刚,侯蓉,林居纯,等.畜禽源大肠杆菌质粒介导β-内酰胺酶检测及其耐药基因特性研究[J].中国农业大学学报,2016,21(8):92-97.Shu G,Hou R,Lin J C,et al.Detection of Escherichia coli plasmid-mediatedβ-lactamases genotype from livestock and poultry and characterization of antimicrobial resistance[J].Journal of China Agricultural University,2016,21(8):92-97.
[6]Randall L P,Clouting C,Horton R A,et al.Prevalence of Escherichia coli carrying extended-spectrum beta-lactamases(CTXM and TEM-52)from broiler chickens and turkeys in Great Britain between 2006and 2009[J].The Journal of Antimicrobial Chemotherapy,2011,66(1):86-95.
[7]M100-S22Performance standarda for antimicrobial susceptility testing:twentieth information supplements[S].Pennsylvania19087USA:Clinical and Laboratory Standards Institute,2012.
[8]高彤彤.大熊猫粪便及环境中大肠杆菌耐药性和质粒介导ESBLs酶耐药基因检测[D].四川雅安:四川农业大学,2014.Gao T T.The detection of resistance and plasmid-mediated ESBLs in Escherichia coil strains from feces of pandas and living environment[D].Ya’an,Sichuan:Sichuan Agricultural University,2014.
[9]佟盼盼.鸡粪便菌耐药基因及其产ESBLs大肠杆菌耐药性的研究[D].吉林长春:吉林大学,2015.Tong P P.Study on fecal microbiota resistant gene and antibiotics resistance of ESBLs-producing Escherichia coli chicken origin[D].Changchun,Jilin:Jilin University,2015.
[10]Lucia P,Monica M,Antonia M,et al.Detection of CTX-M typeβ-lactamase genes in fecal Escherichia coli isolates from healthy children in bolivia and peru[J].Antimicrobial Ggents and Chemotherapy,2004,48(12):4556-4561.
[11]Jacob S,Shiri N V,Inna C,et al.Extended-spectrum beta-lacta-mases among enterobacter isolates obtained in Tel Aviv,Israel[J].Antimicrobial Agents and Chemotherapy,2005,49(3):1150-1156.
[12]Lahlaouia M H,Ben Haj Khalifab A,Ben Moussa M.Epidemiology of enterobacteriaceae producing CTX-M type extendedspectrumβ-lactamase(ESBL)[J].Medecine et Maladies Infectieuses,2004,44(9):400-404.
[13]Trott D.β-lactam resistance in gramnegatuve pathogens isolated from animals[J].Current Pharmaceutical Design,2013,9(2):4239-4249.
[14]刘保光,世洪勋,格桑顿旦,等.西藏地区藏鸡大肠杆菌超广谱β-内酰胺酶检测及药物敏感性分析[J].家畜生态学报,2013,34(3):5-8.Liu B G,Shi H X,Ge S D D,et al.Deternimation of ESBLs and antibiotics susceptibility test analysis for Escherichia coli from the chickens in Xizang[J].Acta Ecologiae Animalis Domastici,2013,34(3):5-8.
[15]汤电,张浩吉,纪雪筱,等.广东地区患病食品动物源大肠杆菌ESBLs和AmpC酶流行分布调查[J].中国兽医学报,2012,32(2):242-248.Tang D,Zhang H J,Ji X X,et al.Prevalence of plasmid mediated ESBLs and AmpC-typeβ-lactamases among E.coliisolated from sick food-producing animals in Guangdong[J].Chinese Journal of Veterinary Science,2012,32(2):242-248.
[16]刘保光,刘建华,吴华,等.舒巴坦钠和EDTA使产ESBLs鸡大肠杆菌恢复对抗菌药物敏感性[J].湖南农业大学学报(自然科学版),2010,32(2):242-248.Liu B G,Liu J H,Wu H,et al.Recovery of antibacterial sensitivity of ESBLs-producing fowl E.coli by sulbactam and EDTA[J].Journal of Hunan Agricultural University(Nat Sci),2010,32(2):242-248.
[17]Yuan L,Liu J H,Hu G Z,et al.Molecular characterization of extended-spectrumβ-lactamase-producing Escherichia coli isolates from chickens in Henan Province,China[J].Journal of Medical Microbiology,2009,58(11):1449-1453.
[18]Paterson D L,Bonomo R A.Extended-spectrum beta-lactamases:a clinical update[J].Clinical Microbiology Reviews,2005,18(4):657-686.
[19]Kiratisin P,Apisarnthanarak A,Laesripa C,et al.Molecular characterization and epidemiology of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in Thailand,where the CTX-M family is endemic[J].Antimicrobial Agents and Chemotherapy,2008,52(8):2818-2824.
[20]Ahmed A M,Shimabukuro H,Shimamoto T.Isolation and molecular characterization of multidrug-resistant strains of Escherichia coli and Salmonellafrom retail chicken meat in Japan[J].Journal of Food Science,2009,74(7):405-410.
[21]苑丽,刘建华,胡功政,等.鸡大肠杆菌TEM和CTX-M型超广谱β-内酰胺酶基因分型研究[J].中国农业科学,2010,43(20):4310-4316.Yuan L,Liu J H,Hu G Z,et al.Genotypes of TEM-type and CTX-M-type extended-spectrumβ-lactamases produced by Escherichia coli isolated from the fowl[J].Scientia Agricultura Sinica,2010,43(20):4310-4316.
[22]Zhao W H,Hu Z Q.Epidemiology and genetics of CTX-M extended-spectrumβ-lactamases in gram-negative bacteria[J].Critical Reviews in Microbiology,2013,39(1):79-101.
[23]Lahlaour H,Ben Haj Khalifa A,Ben Moussa M.Epidemiology of enterobacteriaceae producing CTX-M type extended spectrumβ-lactamase(ESBLs)[J].Medecine et Maladies Infectieuses,2014,44(9):400-404.
[24]苑丽,莫娟,胡功政,等.49株鸡大肠杆菌OXA型β-内酰胺酶的检测[J].江西农业学报,2009,21(8):1-3.Yuan L,Mo J,Hu G Z,et al.Detection of OXA-typeβ-lactamases among 49 strains of Escherichia coli isolated from chickens[J].Acta Agriculturae Jiangxi,2009,21(8):1-3.