1株多重耐药致病性大肠杆菌的分离鉴定与分子分群
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摘要
2017年1月,新疆博乐某牛场犊牛群出现腹泻疫情,本试验旨在调查病因并制定相应的防控措施。采集6头腹泻犊牛的肛拭子和新鲜粪便样本,先用Speed V-Diar4~(TM)试纸进行轮状病毒、冠状病毒、隐孢子虫和大肠杆菌F5(K99)的检测,再对样本进行病原菌的分离鉴定和分子分群,最后对分离菌进行致病力及耐药性的检测。结果显示,所有粪便样本的试纸检测结果均为阴性,从6份样品中共得到6株大肠杆菌和1株爱德华氏菌,分子分群PCR检测结果显示6株大肠杆菌中A群占83.3%(5/6),F群占16.7%(1/6)。半数致死量结果显示,只有1株大肠杆菌(菌株XJ-B1)具有致病性,可致小白鼠腹泻和死亡。药敏试验结果显示,7株分离菌对这18种抗生素产生不同程度的耐药性,其中致病性大肠杆菌XJ-B1耐药程度最严重,对β-内酰胺类、磺胺类和多肽类抗生素耐药,为多重耐药菌。由此可见,这株致病性大肠杆菌可能是引起本次犊牛腹泻的病因之一,也是本试验首次分离到牛源的F群大肠杆菌,该菌的多重耐药性增加了临床治疗的难度,而本试验结果为该牛场治疗此次犊牛腹泻提供了依据。
In January 2017,a diarrhea epidemic occurred in calves of a cattle farm in Bole,Xinjiang.The purpose of this study was to investigate the etiology and formulate corresponding prevention and control measures.Anal swabs and stool samples from 6 diarrhoeal calves were collected,rotavirus,coronavirus,Cryptosporidium and Escherichia coli F5(K99) were detected by Speed V-Diar 4~(TM)test paper.Then the samples were isolated,identified and molecular clustering,the pathogenicity and drug resistance of the isolated bacteria were detected.The test results of all stool samples were negative.6 strains of Escherichia coli and 1 strain of Edwardsiella tarda were obtained from 6 samples.Phylogenetic PCR analysis showed that 6 strains of Escherichia coli A group accounted for 83.3%(5/6),F group accounted for 16.7%(1/6).Half the lethal dose result showed that only one strain of Escherichia coli(strain XJ-B1) was pathogenic,and could cause diarrhea and death in mice.The results of drug sensitivity test showed that 7 isolates had different degrees of resistance to the 18 antibiotics,and Escherichia coli XJ-B1 was the most serious drug-resistant strain.It was multidrug-resistant to beta-lactams,sulfonamides and polypeptide antibiotics.Therefore,this pathogenic Escherichia coli might be one of the causes of this diarrhea in calves,and it was also the first group F Escherichia coli isolated from cattle.The multi-drug resistance of the bacteria increased the difficulty of clinical treatment.This results provided a basis for the cattle farm to treat the calf diarrhea.
In January 2017,a diarrhea epidemic occurred in calves of a cattle farm in Bole,Xinjiang.The purpose of this study was to investigate the etiology and formulate corresponding prevention and control measures.Anal swabs and stool samples from 6 diarrhoeal calves were collected,rotavirus,coronavirus,Cryptosporidium and Escherichia coli F5(K99) were detected by Speed V-Diar 4~(TM)test paper.Then the samples were isolated,identified and molecular clustering,the pathogenicity and drug resistance of the isolated bacteria were detected.The test results of all stool samples were negative.6 strains of Escherichia coli and 1 strain of Edwardsiella tarda were obtained from 6 samples.Phylogenetic PCR analysis showed that 6 strains of Escherichia coli A group accounted for 83.3%(5/6),F group accounted for 16.7%(1/6).Half the lethal dose result showed that only one strain of Escherichia coli(strain XJ-B1) was pathogenic,and could cause diarrhea and death in mice.The results of drug sensitivity test showed that 7 isolates had different degrees of resistance to the 18 antibiotics,and Escherichia coli XJ-B1 was the most serious drug-resistant strain.It was multidrug-resistant to beta-lactams,sulfonamides and polypeptide antibiotics.Therefore,this pathogenic Escherichia coli might be one of the causes of this diarrhea in calves,and it was also the first group F Escherichia coli isolated from cattle.The multi-drug resistance of the bacteria increased the difficulty of clinical treatment.This results provided a basis for the cattle farm to treat the calf diarrhea.
引文
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[15] 陈茂金,黄海玲,付党华,等.圈养虎迟缓爱德华氏菌的分离鉴定及药敏分析[J].野生动物学报,2018,39(1):110-114. CHEN M J,HUANG H L,FU D H,et al.The identification and drug sensitivity analysis of Edwardsiella tarda in captive tiger[J].Chinese Journal of Wildlife,2018,39(1):110-114.(in Chinese)
[16] BLUM S E,LEITNER G.Genotyping and virulence factors assessment of bovine mastitis Escherichia coli[J].Veterinary Microbiol,2013,163(3-4):305-312.
[17] 宋康,王静梅,蒋建军,等.新疆部分地区致犊牛腹泻大肠杆菌的分离鉴定及药物敏感性测定[J].畜牧与兽医,2016,48(7):89-92. SONG K,WANG J M,JIANG J J,et al.Isolation,identification and drug sensitivity of Escherichia coli causing calf diarrhea in parts of Xinjiang[J].Animal Husbandry & Veterinary Medicine,2016,48(7):89-92.(in Chinese)
[18] 王姣姣.东北地区致犊牛腹泻大肠杆菌的分离鉴定与HPI毒力岛相关序列分析[D].长春:吉林农业大学,2016. WANG J J.Isolation,identification,and analysis of related genes of HPI pathogenicity island of Escherichia coli in calves diarrhea disease in Northeast of China[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[19] 刘少昆.京津部分地区犊牛腹泻大肠杆菌分离鉴定及耐药性分析[D].长春:吉林农业大学,2016. LIU S K.Identification of calf diarrhea in some areas of Beijing and Tianjin and drug resistance of E.coli isolated[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[20] ZHUGE X K,JIANG J W,PAN Z H,et al.Comparative genomic analysis shows that avian pathogenic Escherichia coli isolate IMT5155 (O2∶K1∶H5;ST Complex 95,ST140) shares close relationship with ST95 APEC O1∶K1 and HumanEx PEC O18∶K1 strains[J].PLoS One,2014,9(11):e112048.
[21] 李金朋,温文彦,靳曼玉,等.河南地区猪源大肠杆菌分子分群、耐药性及生物被膜表型分析[J].中国预防兽医学报,2018,3:190-194. LI J P,WEN W Y,JIN M Y,et al.Phylogenetic group,drug resistance and biofilm formation of Escherichia coli isolated from pigs in Henan regions[J].Chinese Journal of Preventive Veterinary Medicine,2018,3:190-194.(in Chinese)
[22] 张星星,乔军,孟庆玲,等.新疆犊牛源大肠杆菌系统进化及其耐药特性[J].家畜生态学报,2016,37(6):60-64. ZHANG X X,QIAO J,MENG Q L,et al.Phylogenetic analysis and resistance characteristics of Escherichia coli from calves in Xinjiang[J].Acta Ecologiae Animalis Domastici,2016,37(6):60-64.(in Chinese)
[23] MUSTANK H K,GUNAYDIN E,INCI BASAK K,et al.Phylotyping of clinical isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR[J].Veterinary Quarterly,2015,35(4):194-199.
[2] CHO Y,YOON K.An overview of calf diarrhea infectious etiology,diagnostics,and intervention[J].Journal of Veterinary Science,2014,15(1):1-17.
[3] MAINIL J.Escherichia coli virulence factors[J].Veterinary Immunology & Immunopathology,2013,152(1-2):2-12.
[4] 崔冰冰.34株致犊牛腹泻的大肠杆菌耐药性分析及合理用药建议[D].长春:吉林农业大学,2015. CUI B B.Resistance analysis and rational drug recommendations of 34 calves with diarrhea caused by E.coli[D].Changchun:Jilin Agricultural University,2015.(in Chinese)
[5] 李复煌.北京地区犊牛腹泻主要病原调查及综合防控[D].北京:中国农业大学,2016. LI F H.The investigation of major pathogens causin calf diarrhea and heir control Beijing[D].Beijing:China Agricultural University,2016.(in Chinese)
[6] 张坤,剡根强,王静梅,等.新疆北疆部分地区致犊牛腹泻冠状病毒的分子流行病学调查[J].中国兽医科学,2015,45(12):1270-1276. ZHANG K,YAN G Q,WANG J M,et al.Molecular epidemiological investigation of calf diarrhea coronavirusin parts of Northern Xinjiang[J].Chinese Veterinary Science,2015,45(12):1270-1276.(in Chinese)
[7] 郝彦龙,剡根强,王静梅,等.新疆北疆地区致犊牛腹泻大肠杆菌的分离鉴定及部分生物学特性[J].中国兽医学报,2012,32(6):848-851. HAO Y L,YAN G Q,WANG J M,et al.Isolation and identification of E.coli inducing diarrhea of calf and its biological characteristics in Northern Xinjiang[J].China Journal of Veterinary Science,2012,32(6):848-851.(in Chinese)
[8] CLERMONT O,CHRISTENSON J K,DENAMUR E,et al.The Clermont Escherichia coli phylo-typing method revisited:Improvement of specificity and detection of new phylo-groups[J].Environmental Microbiology Reports,2013,5(1):58-65.
[9] CLINICAL AND LABORATORY STANDARDS INSTITUTE.Performance standards for antimicrobial susceptibility testing,18th informational supplement M100-S18[S].Wayne,PA.2008.
[10] 王丹阳,王旭荣,张康,等.牛病毒性腹泻病毒、大肠杆菌和奇异变形杆菌混合感染致犊牛腹泻的研究[J].中国畜牧兽医,2018,45(1):189-195. WANG D Y,WANG X R,ZHANG K,et al.Study on calves diarrhea caused by mixed infection of bovine viral diarrhea virus,Escherichia coli and Proteus mirabilis[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):189-195.(in Chinese)
[11] 李艳婷,孙光野,张露,等.犊牛轮状病毒和大肠杆菌混合感染检测[J].中国畜牧兽医,2017,44(1):248-253. LI Y T,SUN G Y,ZHANG L,et al.The test of polyinfection of bovine rotavirus and Escherichia coli[J].China Animal Husbandry & Veterinary Medicine,2017,44(1):248-253.(in Chinese)
[12] 王海瑞.甘肃等13省区犊牛腹泻主要病原调查及大肠杆菌生物学特性分析[D].北京:中国农业科学院,2017. WANG H R.The investigation of major pathogens of the calf diarrhea and the analysis of biological characteristics of E.coli in 13 provinces,China[D].Beijing:Chinese Academy of Agricultural Sciences,2017.(in Chinese)
[13] JANDE J M,ABBOTT S I,KROSKE B S,et al.Pathogenmic properties of Edwardsiella species[J].Journal of Clinical Microbiology,1991,29:1997-2001.
[14] 刘春,李凯彬,王庆,等.斑马鱼迟缓爱德华氏菌的鉴定、致病性及药物敏感性[J].华中农业大学学报,2013,32(3):105-111. LIU C,LI K B,WANG Q,et al.Identification,pathogenicity and drug sensitivity of Edwardsiella tarda from Danio rerio[J].Journal of Huazhong Agricultural University,2013,32(3):105-111.(in Chinese)
[15] 陈茂金,黄海玲,付党华,等.圈养虎迟缓爱德华氏菌的分离鉴定及药敏分析[J].野生动物学报,2018,39(1):110-114. CHEN M J,HUANG H L,FU D H,et al.The identification and drug sensitivity analysis of Edwardsiella tarda in captive tiger[J].Chinese Journal of Wildlife,2018,39(1):110-114.(in Chinese)
[16] BLUM S E,LEITNER G.Genotyping and virulence factors assessment of bovine mastitis Escherichia coli[J].Veterinary Microbiol,2013,163(3-4):305-312.
[17] 宋康,王静梅,蒋建军,等.新疆部分地区致犊牛腹泻大肠杆菌的分离鉴定及药物敏感性测定[J].畜牧与兽医,2016,48(7):89-92. SONG K,WANG J M,JIANG J J,et al.Isolation,identification and drug sensitivity of Escherichia coli causing calf diarrhea in parts of Xinjiang[J].Animal Husbandry & Veterinary Medicine,2016,48(7):89-92.(in Chinese)
[18] 王姣姣.东北地区致犊牛腹泻大肠杆菌的分离鉴定与HPI毒力岛相关序列分析[D].长春:吉林农业大学,2016. WANG J J.Isolation,identification,and analysis of related genes of HPI pathogenicity island of Escherichia coli in calves diarrhea disease in Northeast of China[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[19] 刘少昆.京津部分地区犊牛腹泻大肠杆菌分离鉴定及耐药性分析[D].长春:吉林农业大学,2016. LIU S K.Identification of calf diarrhea in some areas of Beijing and Tianjin and drug resistance of E.coli isolated[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[20] ZHUGE X K,JIANG J W,PAN Z H,et al.Comparative genomic analysis shows that avian pathogenic Escherichia coli isolate IMT5155 (O2∶K1∶H5;ST Complex 95,ST140) shares close relationship with ST95 APEC O1∶K1 and HumanEx PEC O18∶K1 strains[J].PLoS One,2014,9(11):e112048.
[21] 李金朋,温文彦,靳曼玉,等.河南地区猪源大肠杆菌分子分群、耐药性及生物被膜表型分析[J].中国预防兽医学报,2018,3:190-194. LI J P,WEN W Y,JIN M Y,et al.Phylogenetic group,drug resistance and biofilm formation of Escherichia coli isolated from pigs in Henan regions[J].Chinese Journal of Preventive Veterinary Medicine,2018,3:190-194.(in Chinese)
[22] 张星星,乔军,孟庆玲,等.新疆犊牛源大肠杆菌系统进化及其耐药特性[J].家畜生态学报,2016,37(6):60-64. ZHANG X X,QIAO J,MENG Q L,et al.Phylogenetic analysis and resistance characteristics of Escherichia coli from calves in Xinjiang[J].Acta Ecologiae Animalis Domastici,2016,37(6):60-64.(in Chinese)
[23] MUSTANK H K,GUNAYDIN E,INCI BASAK K,et al.Phylotyping of clinical isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR[J].Veterinary Quarterly,2015,35(4):194-199.