利用MLST技术探讨不同地区致病性耐药鸡源大肠杆菌的遗传进化关系
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摘要
选取来自山东等10省698株鸡源大肠杆菌为研究对象,分别利用PCR、微量肉汤稀释法、MLST技术方法进行系统进化群分群、耐药表型检测、分子分型,并采用Bio Numerics v4.0软件和SPSS20.0软件进行数据处理分析。结果显示,低致病群B_1群为48.85%(341/698)和非致病群A群24.64%(172/698)分布较多,高致病群D群20.20%(141/698)和B_2群为6.31%(44/698)。鸡源大肠杆菌耐药严重,12种药物的耐药率约在50%以上。B_2和D群菌株对8种抗菌药的耐药率高于A和B_1群菌株,其中对庆大霉素、大观霉素、头孢噻呋、氨苄西林和奥格门丁5种药物极显著性差异(P<0.01),对氟苯尼考和复方新诺明显著性差异(P<0.05)。135株高致病菌株经MLST分型得到48个ST型,ST117(12株)和ST354(12株)最多,其次是ST115(8株),ST2309(8株),ST10型(7株),ST69(6株),ST1011(5株),ST1158(5株)。有29个ST型各1株,18株菌株未分出序列型。不同地区大肠杆菌ST型分布存在差异,ST69和ST354型分布最为广泛(4个省),其次是ST115、ST117和ST2309型(3个省),ST10、ST48、ST57、ST362、ST371、ST1011和ST1158(2个省),其余36个ST型菌株仅在1个省市有检出。135株菌株分为35个聚类簇,其中23个聚类簇的遗传相似性在40%以下,但有6个聚类簇菌株间的遗传相似性均在80%以上。48个ST型菌株共获得了76种耐药谱。我国各地健康鸡群隐性携带相对较高比例的高致病菌株,且耐药程度相对严重,耐药谱较广。鸡源大肠杆菌菌株分布具有多态性和地域性,ST型与耐药谱之间无相关性。
To investigate the phylogenetic group distribution,and reveal the relationship between drug-resistance and the genetic evolutionary of Escherichia coli(E.coli)strains originated from chicken in different regions with a view of providing evidence for molecular tracing and control of this bacteria.A total of 698 E.coli strains were isolated from Shandong and other 9provinces in China.PCR technology was adopted to detect the phylogenetic clustering,and micro broth dilution method was used to detect drug-resistance phenotype,while MLST technique was applied to carry out molecular type,and Bio Numerics v4.0 and SPSS20.0 software were employed to analyze the experimental data.Low pathogenic group B_1 and the nonpathogenic group A distributed widely,accounting for 48.85%(341/698)and 24.64%(172/698),respectively.Next to them were highly pathogenic group D making up 20.20%(172/698)and group B_2 making up 6.31%(44/698).The resistance of chicken originated E.coli was serious and the resistant rate of 12 kinds of drugs was all more than 50.0%.The resistant rate of group B_2 and D strains for 8 kinds of drugs was higher than that of A and B_1 strains.For the two categories of strains,there was extremely significant difference for 5 kinds of drugs such as gentamicin,spectinomycin,ceftiofur,ampicillin,augmentin(P<0.01);and significant differences for florfenicol,sulfamethoxazole(P<0.05).48 STs were obtained from 135 highly pathogenic strains by MLST.ST117 and ST354were most popular,both from 12 strains,then followed by ST115(8),ST2309(8),ST10(7),ST69(6),ST1011(5),ST1158(5)29ST types were from 1strain,respectively.And ST types of 18 strains were uncertain.There was difference of E.coli ST type distribution in different areas.ST69 and ST354distributed most widely(4 provinces),and then ST115,ST117 and ST2309(3 provinces),next were ST10,ST48,ST57,ST362,ST371,ST1011 and ST1158(2 provinces),the remaining 36 ST types strains were detected only in one province.135 strains were divided into 35 clustering groups.Among them,the genetic similarity of 23 clustering groups was under 40%,but the genetic similarity of 6 clustering groups strains were above 80%.76 kinds of drug-resistant spectrum were obtained from these 48 ST types of strains.Healthy chickens in our country silently carry a relatively high proportion of highly pathogenic strains,and their drug resistance problem is serious,with a wide drug-resistant spectrum.The distribution of E.coli strains originated from chicken is quiet diversified and has distinct regional features.There is no correlation between ST types and drug-resistant spectrums.
To investigate the phylogenetic group distribution,and reveal the relationship between drug-resistance and the genetic evolutionary of Escherichia coli(E.coli)strains originated from chicken in different regions with a view of providing evidence for molecular tracing and control of this bacteria.A total of 698 E.coli strains were isolated from Shandong and other 9provinces in China.PCR technology was adopted to detect the phylogenetic clustering,and micro broth dilution method was used to detect drug-resistance phenotype,while MLST technique was applied to carry out molecular type,and Bio Numerics v4.0 and SPSS20.0 software were employed to analyze the experimental data.Low pathogenic group B_1 and the nonpathogenic group A distributed widely,accounting for 48.85%(341/698)and 24.64%(172/698),respectively.Next to them were highly pathogenic group D making up 20.20%(172/698)and group B_2 making up 6.31%(44/698).The resistance of chicken originated E.coli was serious and the resistant rate of 12 kinds of drugs was all more than 50.0%.The resistant rate of group B_2 and D strains for 8 kinds of drugs was higher than that of A and B_1 strains.For the two categories of strains,there was extremely significant difference for 5 kinds of drugs such as gentamicin,spectinomycin,ceftiofur,ampicillin,augmentin(P<0.01);and significant differences for florfenicol,sulfamethoxazole(P<0.05).48 STs were obtained from 135 highly pathogenic strains by MLST.ST117 and ST354were most popular,both from 12 strains,then followed by ST115(8),ST2309(8),ST10(7),ST69(6),ST1011(5),ST1158(5)29ST types were from 1strain,respectively.And ST types of 18 strains were uncertain.There was difference of E.coli ST type distribution in different areas.ST69 and ST354distributed most widely(4 provinces),and then ST115,ST117 and ST2309(3 provinces),next were ST10,ST48,ST57,ST362,ST371,ST1011 and ST1158(2 provinces),the remaining 36 ST types strains were detected only in one province.135 strains were divided into 35 clustering groups.Among them,the genetic similarity of 23 clustering groups was under 40%,but the genetic similarity of 6 clustering groups strains were above 80%.76 kinds of drug-resistant spectrum were obtained from these 48 ST types of strains.Healthy chickens in our country silently carry a relatively high proportion of highly pathogenic strains,and their drug resistance problem is serious,with a wide drug-resistant spectrum.The distribution of E.coli strains originated from chicken is quiet diversified and has distinct regional features.There is no correlation between ST types and drug-resistant spectrums.
引文
[1]王少辉,戴建君,陆承平.禽致病性大肠杆菌IMT5155疑似毒力基因在人源及动物源大肠杆菌中的分布[J].微生物学报,2011,51(7):972-978.
[2]KOCZURA R,MOKRACKA J,BARCZAK A,et al.Association between the presence of class lintegrons,virulence genes,and phylogenetic groups of Escherichia coli isolates from river water[J].Microbial Ecol,2013,65(1):84-90.
[3]CLERMONT O,BONACORSI S,BINGEN E.Rapid and simple determination of the Escherichia coli phylogenetic group[J].Appl Environm Microbiol,2000,66(10):4555-4558.
[4]JAUREGUY F,LANDRAUD L,PASSET V,et al.Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains[J].BMC Genom,2008,26(9):560-573.
[5]胡林,刘晓燕,王颢锦,等.华东部分地区禽致病性大肠杆菌系统进化分群及毒力相关基因的检测[J].畜牧与兽医,2014,46(1):9-13.
[6]RODRIGUESIEK K E,GIDDINGS C W,DOETKOTT C,et al.Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis[J].Microbiology,2005,151(6):2097-2110.
[7]CARLOS C,PIRES M M,STOPPE N C,et al.Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination[J].BMC Microbiol,2010,10(1):1-10.
[8]栾鹏,冷丽,徐国锋,等.鸡源大肠杆菌ESBLs基因型检测及耐药性分析研究[J].中国家禽,2014,36(15):19-22.
[9]杨泽晓,庞歌,王印,等.四川省部分地区大肠杆菌的耐药性监测[J].畜牧与兽医,2011,43(2):84-87.
[10]DAVID S,ARNAUD L M,DAVID Z,et al.Integronassociated antibiotic resistance and phylogenetic grouping of Escherichia coli isolates from healthy subjects free of recent antibiotic exposure[J].Antimicrob Agents Chemoth,2005,49(7):3062-3065.
[11]JOHNSON J R,KUSKOWSKI M A,KRISTA O,et al.Phylogenetic origin and virulence genotype in relation to resistance to fluoroquinolones and/or extended-spectrum cephalosporins and cephamycins among Escherichia coli isolates from animals and humans[J].J Infect Dis,2003,188(5):759-768.
[12]PICARD B,GOULLET P.Correlation between electrophoretic types B1and B2of carboxylesterase B and sex of patients in Escherichia coli urinary tract infections[J].Epidemiol Infect,1989,103(1):97-103.
[13]郭潇木,杨铜,贺丹丹,等.广东地区猪源大肠杆菌耐药性及其系统发育群的研究[J].中国畜牧兽医,2014,41(1):182-186.
[14]潘伟光,李一凡,钟海萍,等.产超广谱β内酰胺酶大肠埃希菌多位点序列分型研究[J].中国感染与化疗杂志,2013,13(4):302-306.
[15]ADIRI R S,GOPHNA U,RON E Z.Multilocus sequence typing(MLST)of Escherichia coli O78strains[J].Fems Microbiol Lett,2003,222(2):199-203.
[16]ZURFLUH K,WANG J,KLUMPP J,et al.Vertical transmission of highly similar bla CTX-M-1-harboring IncI1plasmids in Escherichia coli with different MLST types in the poultry production pyramid[J].Front Microbiol,2014(5):519.
[17]GUILLOUZOUIC A,CAROFF N,DAUVERGNE S,et al.MLST typing of Escherichia coli isolates overproducing AmpCβ-lactamase[J].J Antimicrob Chemoth,2009,63(6):1290-1292.
[18]ZHANG S H,YANG G Z,LAI Z B,et al.Distribution and molecular typing(MLST)of Non-O157diarrheagenic Escherichia coli isolated from retail foods in South China[J].Modern Food Sci Technol,2017,33(3):266-273.
[19]叶菊莲,占利,梅玲玲,等.利用MLST技术对浙江省大肠杆菌O157的分子流行病学研究[J].中国人兽共患病学报,2011,27(10):901-904.
[20]MAIDEN M C.Multilocus sequence typing of bacteria[J].Ann Rev Microbiol,2006,60(60):561-588.
[21]孙晖,白向宁,赵爱兰,等.牦牛携带的产志贺毒素大肠杆菌分离株的多位点序列分型研究[J].中国人兽共患病学报,2013,12(5):1137-1142.
[22]逄波,景怀琦,郑翰,等.我国部分地区产生志贺毒素肠出血性大肠埃希菌O157:H7脉冲电场凝胶电泳分型[J].中华流行病学杂志,2002,23(2):123-126.
[23]孙晖,赵爱兰,白向宁,等.不同来源肠致病性大肠杆菌(EPEC)分离株eae基因分析[J].实用预防医学,2014,21(7):773-776.
[24]孙晖,刘学通,赵爱兰,等.肠集聚性大肠埃希菌分离株脉冲场凝胶电泳分析[J].疾病监测,2013,28(10):807-810.
[25]盖文燕,王娟,曲志娜,等.山东地区大肠杆菌的耐药性及分子分型研究[J].中国食品卫生杂志,2015,27(2):109-114.
[26]王娟,黄秀梅,翟海华,等.鸡大肠杆菌毒力因子流行病学调查及耐药性分析[J].中国兽医杂志,2013,49(10):38-41.
[27]盖文燕,李文卉,王鸿盛,等.旋毛虫丝氨酸蛋白酶抑制因子基因的克隆与原核表达[J].中国兽医科学,2010(5):470-474.
[2]KOCZURA R,MOKRACKA J,BARCZAK A,et al.Association between the presence of class lintegrons,virulence genes,and phylogenetic groups of Escherichia coli isolates from river water[J].Microbial Ecol,2013,65(1):84-90.
[3]CLERMONT O,BONACORSI S,BINGEN E.Rapid and simple determination of the Escherichia coli phylogenetic group[J].Appl Environm Microbiol,2000,66(10):4555-4558.
[4]JAUREGUY F,LANDRAUD L,PASSET V,et al.Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains[J].BMC Genom,2008,26(9):560-573.
[5]胡林,刘晓燕,王颢锦,等.华东部分地区禽致病性大肠杆菌系统进化分群及毒力相关基因的检测[J].畜牧与兽医,2014,46(1):9-13.
[6]RODRIGUESIEK K E,GIDDINGS C W,DOETKOTT C,et al.Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis[J].Microbiology,2005,151(6):2097-2110.
[7]CARLOS C,PIRES M M,STOPPE N C,et al.Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination[J].BMC Microbiol,2010,10(1):1-10.
[8]栾鹏,冷丽,徐国锋,等.鸡源大肠杆菌ESBLs基因型检测及耐药性分析研究[J].中国家禽,2014,36(15):19-22.
[9]杨泽晓,庞歌,王印,等.四川省部分地区大肠杆菌的耐药性监测[J].畜牧与兽医,2011,43(2):84-87.
[10]DAVID S,ARNAUD L M,DAVID Z,et al.Integronassociated antibiotic resistance and phylogenetic grouping of Escherichia coli isolates from healthy subjects free of recent antibiotic exposure[J].Antimicrob Agents Chemoth,2005,49(7):3062-3065.
[11]JOHNSON J R,KUSKOWSKI M A,KRISTA O,et al.Phylogenetic origin and virulence genotype in relation to resistance to fluoroquinolones and/or extended-spectrum cephalosporins and cephamycins among Escherichia coli isolates from animals and humans[J].J Infect Dis,2003,188(5):759-768.
[12]PICARD B,GOULLET P.Correlation between electrophoretic types B1and B2of carboxylesterase B and sex of patients in Escherichia coli urinary tract infections[J].Epidemiol Infect,1989,103(1):97-103.
[13]郭潇木,杨铜,贺丹丹,等.广东地区猪源大肠杆菌耐药性及其系统发育群的研究[J].中国畜牧兽医,2014,41(1):182-186.
[14]潘伟光,李一凡,钟海萍,等.产超广谱β内酰胺酶大肠埃希菌多位点序列分型研究[J].中国感染与化疗杂志,2013,13(4):302-306.
[15]ADIRI R S,GOPHNA U,RON E Z.Multilocus sequence typing(MLST)of Escherichia coli O78strains[J].Fems Microbiol Lett,2003,222(2):199-203.
[16]ZURFLUH K,WANG J,KLUMPP J,et al.Vertical transmission of highly similar bla CTX-M-1-harboring IncI1plasmids in Escherichia coli with different MLST types in the poultry production pyramid[J].Front Microbiol,2014(5):519.
[17]GUILLOUZOUIC A,CAROFF N,DAUVERGNE S,et al.MLST typing of Escherichia coli isolates overproducing AmpCβ-lactamase[J].J Antimicrob Chemoth,2009,63(6):1290-1292.
[18]ZHANG S H,YANG G Z,LAI Z B,et al.Distribution and molecular typing(MLST)of Non-O157diarrheagenic Escherichia coli isolated from retail foods in South China[J].Modern Food Sci Technol,2017,33(3):266-273.
[19]叶菊莲,占利,梅玲玲,等.利用MLST技术对浙江省大肠杆菌O157的分子流行病学研究[J].中国人兽共患病学报,2011,27(10):901-904.
[20]MAIDEN M C.Multilocus sequence typing of bacteria[J].Ann Rev Microbiol,2006,60(60):561-588.
[21]孙晖,白向宁,赵爱兰,等.牦牛携带的产志贺毒素大肠杆菌分离株的多位点序列分型研究[J].中国人兽共患病学报,2013,12(5):1137-1142.
[22]逄波,景怀琦,郑翰,等.我国部分地区产生志贺毒素肠出血性大肠埃希菌O157:H7脉冲电场凝胶电泳分型[J].中华流行病学杂志,2002,23(2):123-126.
[23]孙晖,赵爱兰,白向宁,等.不同来源肠致病性大肠杆菌(EPEC)分离株eae基因分析[J].实用预防医学,2014,21(7):773-776.
[24]孙晖,刘学通,赵爱兰,等.肠集聚性大肠埃希菌分离株脉冲场凝胶电泳分析[J].疾病监测,2013,28(10):807-810.
[25]盖文燕,王娟,曲志娜,等.山东地区大肠杆菌的耐药性及分子分型研究[J].中国食品卫生杂志,2015,27(2):109-114.
[26]王娟,黄秀梅,翟海华,等.鸡大肠杆菌毒力因子流行病学调查及耐药性分析[J].中国兽医杂志,2013,49(10):38-41.
[27]盖文燕,李文卉,王鸿盛,等.旋毛虫丝氨酸蛋白酶抑制因子基因的克隆与原核表达[J].中国兽医科学,2010(5):470-474.