猪、鸡与貂源肠外致病性大肠杆菌群型、耐药性与致病性分析
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摘要
为检测不同动物源肠外致病性大肠杆菌(ExPEC)的耐药及致病性差异,本研究从132份鸡、猪和水貂等动物肠道外组织中分离鉴定大肠杆菌,并对分离菌株进行了16S rRNA基因测序、毒力基因检测、药敏试验、O抗原鉴定、系统进化群分析及动物致病性试验。实验结果显示:分离出53株ExPEC,且至少携带特异性毒力基因pap、sfa/foc、afa/dra、iutA、kpsMTII中的两种基因,并携带fimH、ibeA、hlyA、malX、iss、iroN等其它相关毒力基因。53株ExPEC分离株对氯霉素、卡那霉素、头孢噻吩、四环素、多粘菌素B、氨苄青霉素等呈现不同程度的多重耐药性;O抗原主要分布于O1、O2、O8、O11、O38、O78、O120血清型;系统进化群分析结果显示:28株属于A群,19株属于B1群,1株为B2群,5株属于D群。选取20株ExPEC分离菌株按照6×10~6cfu/只对小鼠进行腹腔注射,验证其致病性;并测定ExPEC6、ExPEC42、ExPEC52、ExPEC53菌株对BALB/c小鼠的LD_(50),其结果分别为3.49×10~6cfu/mL、9.49×10~5cfu/mL、9.48×10~6cfu/mL、2.38×10~7cfu/mL。本研究为动物源性ExPEC的预防诊断提供依据,并为下一步研究其致病机理等奠定基础。
To examine the antimicrobial resistance and pathogenicity differences of extraintestinal pathogenic Escherichia coli(ExPEC) from different animals, the ExPECs were isolated and identified from 132 extra-intestinal tissues of infected animals, such as swine, chicken and mink. Then these ExPECs were subjected to examination of the 16 S rRNA gene, virulence genes,antimicrobial resistance, O-antigen, phylogenetic classification, and pathogenicity to mice. The results showed that 53 of ExPECs isolates possessed at least two specific virulence genes of pap, sfa/foc, afa/dra, iutA, kpsMTII, and carried other virulence factor genes such as fimH, ibeA, hlyA, malX, iss, iroN and so on. The 53 ExPECs displayed different multiple antimicrobial resistances to Chloramphenicol, Kanamycin, Cefotaxime, Tetracycline, Ampicillin, Polymyxin B and so on. The serogroups of O-antigen were mainly distributed in O1, O2, O8, O11, O38, O78 and O120. Phylogenetic analysis showed that the classifications included group A of 28 isolates, group B1 of 19 isolates, group B2 of 1 isolates and group D of 5 isolates. The pathogenicity of 20 ExPEC were determined by intraperitoneally injecting mice at dose of 6 ×10~6 cfu/mice. Then the LD_(50) of ExPEC6, ExPEC42, ExPEC52,ExPEC53 isolates in mice were 3.49 ×10~6 cfu/mL, 9.49 ×10~5 cfu/mL, 9.48 ×10~6 cfu/mL and 2.38 ×10~7 cfu/mL, respectively. This study provided a basis for the prevention and diagnosis of animal ExPEC, and the foundation to further study bacteria pathogenesis.
To examine the antimicrobial resistance and pathogenicity differences of extraintestinal pathogenic Escherichia coli(ExPEC) from different animals, the ExPECs were isolated and identified from 132 extra-intestinal tissues of infected animals, such as swine, chicken and mink. Then these ExPECs were subjected to examination of the 16 S rRNA gene, virulence genes,antimicrobial resistance, O-antigen, phylogenetic classification, and pathogenicity to mice. The results showed that 53 of ExPECs isolates possessed at least two specific virulence genes of pap, sfa/foc, afa/dra, iutA, kpsMTII, and carried other virulence factor genes such as fimH, ibeA, hlyA, malX, iss, iroN and so on. The 53 ExPECs displayed different multiple antimicrobial resistances to Chloramphenicol, Kanamycin, Cefotaxime, Tetracycline, Ampicillin, Polymyxin B and so on. The serogroups of O-antigen were mainly distributed in O1, O2, O8, O11, O38, O78 and O120. Phylogenetic analysis showed that the classifications included group A of 28 isolates, group B1 of 19 isolates, group B2 of 1 isolates and group D of 5 isolates. The pathogenicity of 20 ExPEC were determined by intraperitoneally injecting mice at dose of 6 ×10~6 cfu/mice. Then the LD_(50) of ExPEC6, ExPEC42, ExPEC52,ExPEC53 isolates in mice were 3.49 ×10~6 cfu/mL, 9.49 ×10~5 cfu/mL, 9.48 ×10~6 cfu/mL and 2.38 ×10~7 cfu/mL, respectively. This study provided a basis for the prevention and diagnosis of animal ExPEC, and the foundation to further study bacteria pathogenesis.
引文
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[15]Kim K S.Pathogenesis of bacterial meningitis:from bacteraemia to neuronal injury[J].Nat Rev Neurosci,2003,4(5):376-385.
[16]Pitout J D.Extraintestinalpathogenic Escherichia coli:acombination of virulence with antibiotic resistance[J].Front Microbiol,2012,3:9.
[17]Clermont O,Bonacorsi S,Bingen E.The Yersinia highpathogenicity island is highly predominant in virulence as sociated phylogenetic groups of Escherichia coli[J].FEMS MicrobiolLett,2001,196(2):153-157.
[18]Johnson T J,Siek K E,Johnson S J,et al.K.DNA sequence of a Col V plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains[J].J Bacteriol,2006,188(2):745-758.
[2]Russo T A,Johnson J R.Medical and economic impact of extraintestinal infections due to Escherichia coli:focus on an increasingly important endemic problem[J].Microbes Infect,2003,5(5):449-456.
[3]陈祥.禽病原性大肠杆菌的生物学特性及其可能毒力相关基因的鉴定[D].江苏:扬州大学,2006.
[4]Tan Chen,Tang Xi-biao,Zhang Xuan,et al.Serotypes and virulence genes of extraintestinal pathogenic Escherichia coli isolates from diseased pigs in China[J].Vet J,2012,192(3):483-488.
[5]杨青.山东省貂源大肠杆菌分离鉴定及相关生物学特性研究[D].泰安:山东农业大学,2016.
[6]张翠翠.禽致病性大肠杆菌的分离鉴定与O1、O2、O78菌株lpx L、lpx M基因缺失灭活疫苗的初步研究[D].江苏:扬州大学,2016.
[7]刘璨颖.猪源肠外致病性大肠杆菌比较基因组学和亚单位疫苗的研究[D].武汉:华中农业大学,2014.
[8]Johnson J R,Murray A C,Gajewski A,et al.Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chicken products[J].Antimicrob Agents Chemother,2003,47(7):2161-2168.
[9]Koga V L,Tomazetto G,CyoiaP S,et al.Molecular screening of virulence genes in extraintestinal pathogenic Escherichia coli isolated from human blood culture in Brazil[J].Biomed Res Int,2014,10:465-474.
[10]Clermont O,Bonacorsi S,Bingen E.Rapid and simple determination of the Escherichia coli phylogenetic group[J].Appl Environ Microbiol,2000,66(10):4555-4558.
[11]Moreno E,Prats G,Planells I,et al.Characterization of Escherichia coli isolates derived from phylogenetic groups A and B1 causing extraintestinal infection[J].Enferm Infect Microbiol Clin,2006,24(8):483-489.
[12]Rodriguez-Siek K E,Giddings C W,Doetkott C,et al.Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis[J].Microbiol,2005,151(6):2097-2110.
[13]Ewers C,Li Gan-wu,Wilking H,et al.Avian pathogenic,uropathogenic,and newborn meningitis-causing Escherichia coli:How closely related are they?[J].Int J Med Microbiol,2007,297(3):163-176.
[14]Tivendale K A,Logue C M,Kariyawasam S,et al.Avianpathogenic Escherichia coli strains are similar to neonatal meningitis E.coli strains and are able to cause meningitis in the rat model of human disease[J].Infect Immun,2010,78(8):3412-3419.
[15]Kim K S.Pathogenesis of bacterial meningitis:from bacteraemia to neuronal injury[J].Nat Rev Neurosci,2003,4(5):376-385.
[16]Pitout J D.Extraintestinalpathogenic Escherichia coli:acombination of virulence with antibiotic resistance[J].Front Microbiol,2012,3:9.
[17]Clermont O,Bonacorsi S,Bingen E.The Yersinia highpathogenicity island is highly predominant in virulence as sociated phylogenetic groups of Escherichia coli[J].FEMS MicrobiolLett,2001,196(2):153-157.
[18]Johnson T J,Siek K E,Johnson S J,et al.K.DNA sequence of a Col V plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains[J].J Bacteriol,2006,188(2):745-758.