广西猪源产肠毒素大肠杆菌毒力因子及多重耐药性分析
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摘要
本研究旨在探究广西某猪场可能存在的病原菌及其致病的主要原因。试验采用细菌分离纯化、形态学鉴定、生长曲线测定、生化分析及药敏试验等进行细菌的分离鉴定,应用PCR技术对菌株16S rRNA、毒力基因及耐药基因进行检测。结果表明,该由病料分离的菌株为无芽孢、有菌毛、两端钝圆的粗短杆状的革兰氏阴性菌,其繁殖能力强、生长迅速,疑似为大肠杆菌。16S rRNA扩增结果显示,出现大小为1 474 bp的条带,测序结果表明其与大肠杆菌的同源性可达99%。该菌株对31种常见抗菌药物表现出极强的耐药性,对庆大霉素、替米考星、四环素、恩诺沙星、青霉素、林可霉素、磺胺甲噁唑等均呈现较高的耐药水平,耐药率高达87.10%(27/32),仅对阿米卡星、黏菌素2种药物敏感。毒力因子检测共鉴定致病岛FyuA、肠毒素STb和LT及黏附素F41和K88共5种毒力因子,检出率为35.70%(5/14)。扩增出β-内酰胺酶类bla_(TEM)和bla_(OXA)、四环素类tet(A)、磺胺类Sul2、氨基糖苷类aadA1和aac(3′)-Ⅱa、喹诺酮类GyrA、GyrB和ParC及氯霉素floR共10种耐药基因,检出率为66.70%(10/15),且耐药基因的检出与耐药表型呈正相关。综上,该菌株含多种毒力因子,耐药型复杂,多重耐药性情况严重,需采取相应的预防措施,防止蔓延和传播。
This study was aimed to investigate the possible pathogens and the main causes of disease in a pig farm in Guangxi.The bacteria were isolated and identified by bacterial separation and purification,morphological identification,growth curve measurement,biochemical analysis and drug susceptibility test.The strain 16 S rRNA,virulence genes and drug resistance genes were detected by PCR.The results showed that the strain isolated from the disease material was a Gram-negative bacterium with no spores,pili,and round and short rods at both ends,which had strong reproductive ability and rapid growth,and was suspected to be Escherichia coli(E.coli).The 16 S rRNA amplification results showed that a 1 474 bp band appeared,and the sequencing results showed that the homology of the isolate with E.coli was 99%.The strain showed strong resistance to 31 antibacterial drugs gentamicin,tilmicosin,tetracycline,enrofloxacin,penicillin,lincomycin,sulfamethoxazole,etc.,all showed higher levels of drug resistance,and the drug resistance rate was as high as 87.10%(27/32).It was only sensitive to two drugs,amikacin and colistin.Toxicity factor detection identified a total of five virulence factors,FyuA,enterotoxin STb and LT,adhesin F41 and K88.The detection rate was 35.70%(5/14).It had been amplified β-lactamases bla_(TEM) and bla_(OXA),tetracycline tet(A),sulfonamide Sul2,aminoglycosides aadA1 and aac(3′)-Ⅱa,quinolone GyrA,GyrB and ParC,amide alcohol floR 10 drug resistance genes,the detection rate was 66.70%(10/15),and the detection of drug resistance genes was positively correlated with drug resistance phenotype.In summary,the strain presented a variety of complex virulence factors,complex drug-resistance,and serious multi-drug resistance,and corresponding precautions need to be taken to prevent spread and spread.
This study was aimed to investigate the possible pathogens and the main causes of disease in a pig farm in Guangxi.The bacteria were isolated and identified by bacterial separation and purification,morphological identification,growth curve measurement,biochemical analysis and drug susceptibility test.The strain 16 S rRNA,virulence genes and drug resistance genes were detected by PCR.The results showed that the strain isolated from the disease material was a Gram-negative bacterium with no spores,pili,and round and short rods at both ends,which had strong reproductive ability and rapid growth,and was suspected to be Escherichia coli(E.coli).The 16 S rRNA amplification results showed that a 1 474 bp band appeared,and the sequencing results showed that the homology of the isolate with E.coli was 99%.The strain showed strong resistance to 31 antibacterial drugs gentamicin,tilmicosin,tetracycline,enrofloxacin,penicillin,lincomycin,sulfamethoxazole,etc.,all showed higher levels of drug resistance,and the drug resistance rate was as high as 87.10%(27/32).It was only sensitive to two drugs,amikacin and colistin.Toxicity factor detection identified a total of five virulence factors,FyuA,enterotoxin STb and LT,adhesin F41 and K88.The detection rate was 35.70%(5/14).It had been amplified β-lactamases bla_(TEM) and bla_(OXA),tetracycline tet(A),sulfonamide Sul2,aminoglycosides aadA1 and aac(3′)-Ⅱa,quinolone GyrA,GyrB and ParC,amide alcohol floR 10 drug resistance genes,the detection rate was 66.70%(10/15),and the detection of drug resistance genes was positively correlated with drug resistance phenotype.In summary,the strain presented a variety of complex virulence factors,complex drug-resistance,and serious multi-drug resistance,and corresponding precautions need to be taken to prevent spread and spread.
引文
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[22] 岳山.黑龙江部分地区肉牛大肠杆菌耐药基因与毒力基因检测及耐药性分析[D].大庆:黑龙江八一农垦大学,2017. YUE S.Detection and drug resistance analysis of drug resistance genes and virulence genes in beef cattle in parts of Heilongjiang province[D].Daqing:Heilongjiang Bayi Agricultural University,2017.(in Chinese)
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[2] 周虹,朱军,朱国强.动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展[J].微生物报,2012,52(6):679-686. ZHOU H,ZHU J,ZHU G Q.Fimbriae of animal-originated enterotoxigenic Escherichia coli—A re-view[J].Acta Microbiologica Sinica,2012,52(6):679-686.(in Chinese)
[3] 李海花,朱琪,王世琼,等.产肠毒素大肠杆菌K88诱导仔猪炎症反应的分子机制[J].中国畜牧兽医,2017,44(1):262-267. LI H H,ZHU Q,WANG S Q,et al.Molecular mechanism of the inflammatory response induced by enterotrxigenic E.coli K88 in piglets[J].China Animal Husbandry & Veterinary Medicine,2017,44(1):262-267.(in Chinese)
[4] 张雪寒,何孔旺,张书霞.产肠毒素性大肠杆菌肠毒素的研究概况[J].动物医学进展,2003,24(3):38-40. ZHANG X H,HE K W,ZHANG S X.Research survey of enterotoxin in enterotoxigenic Escherichia coli[J].Progress in Veterinary Medicine,2003,24(3):38-40.(in Chinese)
[5] 吕阳,张林,李雪妮,等.表达耐热肠毒素的重组大肠杆菌对7日龄仔猪肠道形态结构及抗氧化功能的影响[J].中国畜牧兽医,2017,44(9):2816-2821. LYU Y,ZHANG L,LI X N,et al.Effects of recombinant E.coli expressing heat-stable enterotoxin on intestinal morphology and antioxidant capacity in seven days old piglets[J].China Animal Husbandry & Veterinary Medicine,2017,44(9):2816-2821.(in Chinese)
[6] 贺秀媛,李玉峰,王建华.大肠杆菌耐热性肠毒素的研究进展[J].动物医学进展,2000,21(13):175-179. HE X Y,LI Y F,WANG J H.Research progress in heat-resistant enterotoxin of Escherichia coli[J].Progress in Veterinary Medicine,2000,21(13):175-179.(in Chinese)
[7] 尹业师,陈华海,曹林艳,等.细菌耐药性应对策略研究进展[J].生物工程学报,2018,34(8):1346-1360. YIN Y S,CHEN H H,CAO L Y,et al.Progress in strategies to combat antimicrobial resistance[J].Chinese Journal of Biotechnology,2018,34(8):1346-1360.(in Chinese)
[8] 高海涛,韩俊丽,关道明.大肠杆菌耐药现状的严峻性[J].生命科学,2017,29(5):514-520. GAO H T,HAN J L,GUAN D M.The grim situation of drug resistance in Escherichia coli[J].Chinese Bulletin of Life Sciences,2017,29(5):514-520.(in Chinese)
[9] 朱阵,曹明泽,张吉丽,等.细菌耐药性研究进展[J].中国畜牧兽医,2015,42(12):3371-3376. ZHU Z,CAO M Z,ZHANG J L,et al.Research progress on bacterial resistance[J].China Animal Husbandry & Veterinary Medicine,2015,42(12):3371-3376.(in Chinese)
[10] 马超.奶牛乳腺炎大肠杆菌优势血清型相关毒力基因与耐药基因的检测与序列分析[D].银川:宁夏大学,2014. MA C.Detection and sequence analysis of correlative virulence genes and drug resistance genes of superiority Escherichia coli serotypes isolated from cow mastitis[D].Yinchuan:Ningxia University,2014.
[11] 姜中其,王娜,方维焕,等.集约化猪场PWD病原性大肠杆菌毒力因子分析[J].浙江大学学报(农业与生命科学版),2006,5:520-526. JIANG Z Q,WANG N,FANG W H,et al.Serological and virulence factors analysis of Escherichia coli isolates causing post-weaning diarrhea in intensive pig farms[J].Journal of Zhejiang University (Agriculture and Life Sciences),2006,5:520-526.(in Chinese)
[12] WOODWARD M J,CARROLL P J,WRAY C.Detection of entero- and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction[J].Veterinary Microbiology,1992:31(2-3):251-261.
[13] FAGAN P K,HORNITZKY M A,BETTELHEIM K A,et al.Detection of shiga-like toxin (stx1 and stx2),intimin (eaeA),and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR[J].Applied & Environmental Microbiology,1999,65(2):868-872.
[14] NGELEKA M,PRITCHARD J,APPLEYARD Q,et al.Isolation and association of Escherichia coli AIDA-I/STb,rather than EAST1 pathotype,with diarrhea in piglets and antibiotic sensitivity of isolates[J].Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc,2003,15(3):242-252.
[15] LORTIE L A,DUBREUIL J D,HAREL J.Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea[J].Journal of Clinical Microbiology,1991,29(3):656-659.
[16] 姚媛媛.致犊牛腹泻病肠道大肠杆菌的分离鉴定及蒙药复方对其抗菌效果研究[D].呼和浩特:内蒙古农业大学,2014. YAO Y Y.A study on the isolation and identification of E.coli from calf diarrhea and antimicrobial effect of Mongolian medicine on they[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[17] 张金宝.宁夏地区牛源大肠杆菌优势血清型和毒力基因检测及耐药性分析[D].银川:宁夏大学,2015. ZHANG J B.Detection of superiority serotypes and virulence genes and antimicrobials resistance analysis of Escherichia coli isolated from cow in Ningxia[D].Yinchuan:Ningxia University,2015.(in Chinese)
[18] PATON A W,PATON J C.Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1,stx2,eaeA,enterohemorrhagic E.coli h1yA,rfb0111,and rfb0157[J].Journal of Clinical Microbiology,1998,36(2):598-602.
[19] 武瑞兵,高玉敏,王鹏翔,等.牛肉源大肠杆菌的耐药性检测及相关耐药基因分布[J].中国畜牧兽医,2015,42(2):452-458. WU R B,GAO Y M,WANG P X,et al.Detection of antibiotic resistance and distribution of resistance determinants in Escherichia coli isolated from beef[J].China Animal Husbandry & Veterinary Medicine,2015,42(2):452-458.(in Chinese)
[20] 王利勤.鸡源致病性大肠埃希菌耐药基因及毒力基因检测研究[D].杨凌:西北农林科技大学,2012. WANG L Q.Study on resistance genes and virulence genes of pathogenic Escherichia coli isolates from chickens[D].Yangling:Northwest A & F University,2012.(in Chinese)
[21] 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.Resistant analysis and rational drug recommendations of 34 calves with diarrhea caused by E.coli[D].Changchun:Jilin Agricultural University,2015.(in Chinese)
[22] 岳山.黑龙江部分地区肉牛大肠杆菌耐药基因与毒力基因检测及耐药性分析[D].大庆:黑龙江八一农垦大学,2017. YUE S.Detection and drug resistance analysis of drug resistance genes and virulence genes in beef cattle in parts of Heilongjiang province[D].Daqing:Heilongjiang Bayi Agricultural University,2017.(in Chinese)
[23] 张震,宋美英,只勇,等.黑龙江省部分牛场致病性大肠杆菌毒力因子的调查与分析[J].黑龙江畜牧兽医,2017,3:160-162. ZHANG Z,SONG M Y,ZHI Y,et al.Investigation and analysis of virulence factors of pathogenic Escherichia coli in some cattle farms in Heilongjiang province[J].Heilongjiang Animal Science and Veterinary Medicine,2017,3:160-162.(in Chinese)
[24] 沈莉萍,徐锋,王建.上海地区猪源大肠杆菌血清型及其毒力因子分布调查[J].畜牧与兽医,2014,46(8):94-97. SHEN L P,XU F,WANG J.Investigation on the distribution of serotypes and virulence factors of Escherichia coli in pigs in Shanghai[J].Animal Husbandry and Veterinary Medicine,2014,46(8):94-97.(in Chinese)
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