华东部分地区仔猪腹泻大肠杆菌流行病学调查及氨基糖苷类乙酰转移酶基因的PCR检测
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摘要
肠产毒素性大肠杆菌(ETEC)是引起仔猪黄白痢的重要病原，仔猪黄痢发病率和死亡率均较高；仔猪白痢发病率较高，但死亡率较低，主要影响仔猪生长发育和降低饲料报酬而造成较大经济损失。近年来有关仔猪黄白痢的流行病学的报道较少，本文从华东地区分离鉴定出508株大肠杆菌，应用玻板凝集试验对其O血清型进行鉴定，508株分离株中，除145株未能定型、22株自凝外，341株分离株覆盖了71个血清型，以O101，O9，O20，O149，O107和O91等6个血清型为主，占定型菌株的50.4％，是这次致仔猪黄白痢大肠杆菌调查的主要流行血清型。
从1995年至2006年保存的菌株中选取173株致病性大肠杆菌，进行14种抗菌药物的药敏试验，试验表明敏感率最高的是菌必治，敏感菌株达到161株；其次为多粘菌素B、丁胺卡那霉素敏感菌株分别达到142株和126株；而庆大霉素、环丙沙星、氟哌酸和氯霉素敏感菌株较少；强力霉素、氨苄青、链霉素、四环素、磺胺甲基异噁唑等常用药物均表现强烈的耐药性。受检菌株中只有1998年的一株无耐药性，其余均表现出不同程度的耐药性，且大部分集中于10重-12重耐药。结果显示，随着时间的推延，细菌对大部分抗生素的耐药性会越来越强。为了探讨大肠杆菌氨基糖苷乙酰转移酶(AAC)基因的分布规律及与氨基糖苷类抗生素耐药的相关性，对90株耐庆大霉素的猪大肠杆菌菌株检测，结果显示aac(3)-Ⅱ为主要钝化酶，检出率占71.1％；aac(6′)-Ⅰb其次，检出率占16.7％。这说明对猪源大肠杆菌aac(3)-Ⅱ是氨基糖苷类药物耐药的主要钝化酶之一，是引起相应耐药性的重要因素。
Enterotoxigenic Escherichia coli (ETEC) is main pathogen of the piglet diarrhea, which can cause economic loss due to higher morbidity and mortality in yellow scour of newborn piglets, higher morbidity and lower mortality in while scour of piglets, poor growth rates, and decrease feed efficiency. The first purposes of this study was to determine the present distribution of O-serogroups among pathogenic Escherichia coli isolates collected from piglets with yellow and white diarrhea in partial regions of eastern China. Among 508 E.coli isolates, serogroups of 341 strains were determined while 145 were unable to be classified as certain groups, and 22 were self-agglutinated. These isolates distributed in 71 serogroups and main of which belonging to 6 O-serogroups : O101, 09, 020, O149, O107 and O91.
In order to guide the clinical medication, antibiotic susceptibility of E. coil isolates was determined with t4 kinds of antibiotics. Among 173 E.eoli isolates, which were collected form 1995 to 2006 and stored in the laboratory, 161 strains were sensitive to Ceftriaxone, and 142 and 126 strains were sensitive to Polymyxin B and Kanamycin, respectively. These isolates show little sensitivity to Gentamicin, Ciprofloxacin, Norfloxacin, and Chloramphenicol, whereas resistant to Doxycycline, Ampicillin, Streptomycin, Tetracycline, and Sulfamethoxazole . A novel strain isolated in 1998 shows no drug resistance, meanwhile the others show drug resistance to almost 10～12 antibiotics. These data suggested that there is close correlation between the antibiotic resistance and the frequence of agents prescribed in clinic.
To investigate the relationship between the presence of N-acetyltransferases (AAC) genes and the resistance of aminoglycoside of E.coli isolates, two pairs of primers of AAC genes were designed and PCR was performed. Among ninety strains of E.coli isolates which resistant to Gentamicin, 71.1% of isolates were aac(3)-Ⅱpositive, while 16.7% of isolates were aac(6')-Ⅰb positive. The results suggested that aae(3)-Ⅱmaybe the main modifying enzyme of aminoglycoside in E.coli.
从1995年至2006年保存的菌株中选取173株致病性大肠杆菌，进行14种抗菌药物的药敏试验，试验表明敏感率最高的是菌必治，敏感菌株达到161株；其次为多粘菌素B、丁胺卡那霉素敏感菌株分别达到142株和126株；而庆大霉素、环丙沙星、氟哌酸和氯霉素敏感菌株较少；强力霉素、氨苄青、链霉素、四环素、磺胺甲基异噁唑等常用药物均表现强烈的耐药性。受检菌株中只有1998年的一株无耐药性，其余均表现出不同程度的耐药性，且大部分集中于10重-12重耐药。结果显示，随着时间的推延，细菌对大部分抗生素的耐药性会越来越强。为了探讨大肠杆菌氨基糖苷乙酰转移酶(AAC)基因的分布规律及与氨基糖苷类抗生素耐药的相关性，对90株耐庆大霉素的猪大肠杆菌菌株检测，结果显示aac(3)-Ⅱ为主要钝化酶，检出率占71.1％；aac(6′)-Ⅰb其次，检出率占16.7％。这说明对猪源大肠杆菌aac(3)-Ⅱ是氨基糖苷类药物耐药的主要钝化酶之一，是引起相应耐药性的重要因素。
Enterotoxigenic Escherichia coli (ETEC) is main pathogen of the piglet diarrhea, which can cause economic loss due to higher morbidity and mortality in yellow scour of newborn piglets, higher morbidity and lower mortality in while scour of piglets, poor growth rates, and decrease feed efficiency. The first purposes of this study was to determine the present distribution of O-serogroups among pathogenic Escherichia coli isolates collected from piglets with yellow and white diarrhea in partial regions of eastern China. Among 508 E.coli isolates, serogroups of 341 strains were determined while 145 were unable to be classified as certain groups, and 22 were self-agglutinated. These isolates distributed in 71 serogroups and main of which belonging to 6 O-serogroups : O101, 09, 020, O149, O107 and O91.
In order to guide the clinical medication, antibiotic susceptibility of E. coil isolates was determined with t4 kinds of antibiotics. Among 173 E.eoli isolates, which were collected form 1995 to 2006 and stored in the laboratory, 161 strains were sensitive to Ceftriaxone, and 142 and 126 strains were sensitive to Polymyxin B and Kanamycin, respectively. These isolates show little sensitivity to Gentamicin, Ciprofloxacin, Norfloxacin, and Chloramphenicol, whereas resistant to Doxycycline, Ampicillin, Streptomycin, Tetracycline, and Sulfamethoxazole . A novel strain isolated in 1998 shows no drug resistance, meanwhile the others show drug resistance to almost 10～12 antibiotics. These data suggested that there is close correlation between the antibiotic resistance and the frequence of agents prescribed in clinic.
To investigate the relationship between the presence of N-acetyltransferases (AAC) genes and the resistance of aminoglycoside of E.coli isolates, two pairs of primers of AAC genes were designed and PCR was performed. Among ninety strains of E.coli isolates which resistant to Gentamicin, 71.1% of isolates were aac(3)-Ⅱpositive, while 16.7% of isolates were aac(6')-Ⅰb positive. The results suggested that aae(3)-Ⅱmaybe the main modifying enzyme of aminoglycoside in E.coli.
引文
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2．杨正时．动物病原性大肠杆菌与大肠杆菌病．中国兽医科技，1987，6：25-293
3．陈怀青，陆承平．中国兽医科技EJ]，1989，(5)：38-40．
4. Parry S H, Porter P. Immunology[J], 1987, 34: 41-49.
5. Turnes C G, Aleixo J A, Monteiro A V, etal. DNA inoculation with a plasmid vector carrying the faeG adhesin gene of Eseherichia coli K88ab induced immune responses in miee andpigs[J]. Vaccine. 1999, 17(15-16): 2089-2095.
6. Mol 0, Oudhuis W C, Oud R P,et al. Biosynthesis of K88 fimbriae in Escherichia coli: interaction oftip-subunit FaeC with the periplasmic chaperone Face and the outer membrane usher FaeD[J].J Mol Microbiol Biotechnol, 2001, 3(1): 135-142.
7. Jin L Z. Zhaao X. Intestinal receptors for adhesive fimbriae of Enterotoxigenic Escherichia coli(ETEC)K88 in swine-areview[J]. Appl MicrobiolBiotechnol. 2000, 54(3): 311-318.
8. Kyogashima M, Ginsburg V, Krivan HC, Escheriehia coli K99 binds to N-glyeolylisaloparagloboside and N-glycolyl-GM3 found in piglet small intestine, Arch Biochem Biophos 1989, 270(1):391-397
9. Roosendaal E, Damoiscaux J, Jordi W, et al, Transcriptional organization of the DNA region controlling expression of the K99 gene cluster, Mol Gen Genet, 1989, 215:250-256.
10. Lo-Tseng T, Lee J, Isaacson R E. Regulators of Escherichia coli K99 region 1 genes[J]. Adv Exp Med Biol, 1997, 412:303-310.
11. Robet A Edwards, Linda H Keller, Dieter M Schifferli. Gene[J], 1998, 207: 49-157.
12．于涟，王淦，蔡渭明，等．浙江农业大学学报[J]，1992，18(4)：54-58．
13．董国雄，张英，江美娟，等．对Fc株猪源性肠毒性大肠杆菌致病因子的研究[J]．江苏农学院学报，1984，7(3)；1．6．
14．陆承平 主编，兽医微生物学(第三版)[M]，北京：中国农业出版社，2001，219-221
15．刘瑞田．大肠杆菌耐热性肠毒素的特性及检测．中国畜禽传染病，1992，64(3)：64-65
16. H Sunji, Y Fujii, H Yamanaka et al. Some properties of purified coli heat-laible enterotoxin Ⅱ.Infect Immun.1992,60:4468—4474
17. J D Dubreuil, J M Fairbrother, R Lallier et al. Production and purification of heat-stable enterotoxin from a porcine Escheriehia coli.strain. Infect Immun. 1991,59:198-203
18．潘玲，吴信法．细菌的粘附与粘附素．畜牧与兽医，1997，29(2)：86-88
19．高明远，周应志等．甘肃省仔猪腹泻的流行情况调查[J]．中国兽医科技，1998，28(3)：13-15
20．杨恒威，金吕市郊仔猪黄白痢发病情况调杳[J]．中国兽医科技，1998，28(2)：38
21．王孝友，曾秀，孔路军等，仔猪黄白痢大肠杆菌的分离鉴定[J]．湖北畜牧兽医，2004，23(6)：27-29
22．孙广力，孙刚，李书华等．黑龙江省规模化养猪场大肠杆菌病血清型流行病学调查[J]，黑龙江动物科学与动物医学，2004，32(7)：90-92
23．李文刚，甘孟侯．猪病诊断与防治[M]，北京：中国农业大学出版社，2002，198-200
24．蔡蒸蒸，靳国琴等．仔猪人肠杆菌性腹泻病的调杏及病原性的研究[[J]．动物医学进展，2002 23(5)：101-103
25．张永红，崔德风等．北京地区猪致病性大肠埃希氏菌的分离鉴定[J]．中国兽医科技，1999，29(4)：29-30
26．刘梦元，吴斌等．规模化猪场大肠杆菌的耐药性监测及血清流行病学调查[J]．中国兽医学报，2004，24(1)：16-18
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