致病性大肠杆菌和蜡样芽胞杆菌的分离鉴定
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摘要
【背景】猪只消化道疾病是养猪业上一大重要疾病,给养猪业带来一定的经济损失。大肠杆菌是引起猪腹泻的一种常见病原菌,可以引起不同日龄的猪腹泻,但主要以幼龄猪为主。【目的】旨在分离鉴定引起四川省眉山市一规模化养猪场病猪大规模腹泻的病原菌。【方法】采用常规细菌分离方法结合16S rRNA基因序列的分析方法从发病猪肝脏、胃以及污染的饲料分离鉴定细菌,并对分离株进行小鼠致病性试验、16S rRNA基因遗传进化树分析、毒力基因的检测、药物敏感试验。【结果】从腹泻猪肝脏中分离到一株致病性大肠杆菌,胃中分离到一株蜡样芽胞杆菌,并且追溯到传染源是该猪场饲料。通过检测这两株菌相应的毒力基因发现大肠杆菌不属于肠外致病性型,蜡样芽胞杆菌检测到了nheA、nheB、nheC、bceT、entFM 5种毒力基因;药敏试验表明常规的氨基糖苷类和头孢类抗生素对大肠杆菌抑菌效果较好,红霉素、氟苯尼考、头孢氨苄、头孢哌酮对蜡样芽胞杆菌抑菌效果较好,而蜡样芽胞杆菌对青霉素、阿莫西林等常规药物不敏感。【结论】饲料存在大肠杆菌和蜡样芽胞杆菌的混合污染。
[Background] Pig digestive tract disease is one of the most important diseases in the pig industry and brings certain economic losses to the swine industry. Escherichia coli is a common pathogen causing diarrhea of pigs at different ages, but mainly in young pigs. [Objective] The purpose of this study was to isolate and identify pathogens causing large-scale diarrhea in pigs on a large-scale pig farm in Meishan City, Sichuan Province. [Methods] Bacteria were isolated and identified from the infected pig liver, stomach and contaminated feed using conventional bacterial isolation methods combined with 16S rRNA gene sequence analysis. The isolates were tested for pathogenicity in mice, 16S rRNA gene phylogenetic analysis, virulence genes, and drug sensitivity testing. [Results] One pathogenic Escherichia coli was isolated from the liver of diarrhea pigs, one strain of Bacillus cereus was isolated from the stomach, and the source of infection was traced back to the feed on the farm. By detecting the corresponding virulence genes of these two strains, it was found that Escherichia coli is not extra-intestinal pathogenic type. Bacillus cereus was detected to have five virulence genes: nheA, nheB, nheC, bceT,entFM; Aminoglycosides and cephalosporins have good antibacterial effects on Escherichia coli.Erythromycin, florfenicol, cephalexin, cefoperazone have good antibacterial effect on Bacillus cereus, but the bacteria were not sensitive to conventional antibiotics such as penicillin and amoxicillin. [Conclusion] Feed is contaminated by Escherichia coli and Bacillus cereus.
[Background] Pig digestive tract disease is one of the most important diseases in the pig industry and brings certain economic losses to the swine industry. Escherichia coli is a common pathogen causing diarrhea of pigs at different ages, but mainly in young pigs. [Objective] The purpose of this study was to isolate and identify pathogens causing large-scale diarrhea in pigs on a large-scale pig farm in Meishan City, Sichuan Province. [Methods] Bacteria were isolated and identified from the infected pig liver, stomach and contaminated feed using conventional bacterial isolation methods combined with 16S rRNA gene sequence analysis. The isolates were tested for pathogenicity in mice, 16S rRNA gene phylogenetic analysis, virulence genes, and drug sensitivity testing. [Results] One pathogenic Escherichia coli was isolated from the liver of diarrhea pigs, one strain of Bacillus cereus was isolated from the stomach, and the source of infection was traced back to the feed on the farm. By detecting the corresponding virulence genes of these two strains, it was found that Escherichia coli is not extra-intestinal pathogenic type. Bacillus cereus was detected to have five virulence genes: nheA, nheB, nheC, bceT,entFM; Aminoglycosides and cephalosporins have good antibacterial effects on Escherichia coli.Erythromycin, florfenicol, cephalexin, cefoperazone have good antibacterial effect on Bacillus cereus, but the bacteria were not sensitive to conventional antibiotics such as penicillin and amoxicillin. [Conclusion] Feed is contaminated by Escherichia coli and Bacillus cereus.
引文
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[15]Yang X,Lu ZH,Chen LY,et al.Isolation and identification of enteropathogenic E.coli in newborn piglets[J].Animal Husbandry and Veterinary,2005,37(9):34-35(in Chinese)杨霞,卢中华,陈丽颖,等.仔猪致病性大肠杆菌的分离鉴定[J].畜牧与兽医,2005,37(9):34-35
[16]Li L,Chen Y,Zhang C,et al.A review:Identification of aeromonas in aquaculture[J].Fisheries Science,2015,34(2):128-134(in Chinese)李莉,陈颖,张超,等.水产动物气单胞菌鉴定方法研究进展[J].水产科学,2015,34(2):128-134
[17]Kamat AS,Nair CKK.Evidence for plasmid-mediated toxin production in Bacillus cereus BIS-59[J].World Journal of Microbiology&Biotechnology,1992,8(2):210-211
[18]Ehling-Schulz M,Fricker M,Scherer S.Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay[J].FEMS Microbiology Letters,2004,232(2):189-195
[19]Johnson JR,Russo TA.Molecular epidemiology of extraintestinal pathogenic(uropathogenic)Escherichia coli[J].International Journal of Medical Microbiology,2005,295(6/7):383-404
[2]Seong SJ,Lim JS,Lee KG,et al.Toxin gene profiling of Bacillus cereus food isolates by PCR[J].Journal of the Korean Society for Applied Biological Chemistry,2008,51(4):263-268
[3]Ehling-Schulz M,Vukov N,Schulz A,et al.Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus[J].Applied&Environmental Microbiology,2005,71(1):105-113
[4]Bennett RW,Harmon SM.Bacillus cereus food poisoning[A]//Balows A,Hausler WJ,Ohashi M,et al.Laboratory Diagnosis of Infectious Diseases[M].New York:Springer,1988:83-93
[5]Yang SS,Wang XR,Han J.Part of changes in the US CLSIantimicrobial susceptibility test operating standard(2010edition)[J].Chinese Journal of Infection Control,2010,9(4):303-304(in Chinese)杨沙沙,王喜仁,韩杰.美国CLSI抗菌药物敏感试验操作标准(2010年版)部分变更内容[J].中国感染控制杂志,2010,9(4):303-304
[6]Berthold-Pluta A,Pluta A,Garbowska M.The effect of selected factors on the survival of Bacillus cereus in the human gastrointestinal tract[J].Microbial Pathogenesis,2015,82:7-14
[7]Wang J.Study on contamination distribution and genetic diversity of Bacillus cereus isolated from food in China[D].Guangzhou:Master’s Thesis of Guangdong University of Technology,2013(in Chinese)王君.全国食品中蜡样芽孢杆菌的污染分布规律及遗传多样性研究[D].广州:广东工业大学硕士学位论文,2013
[8]Dierick K,Van Coillie E,Swiecicka I,et al.Fatal family outbreak of Bacillus cereus-associated food poisoning[J].Journal of Clinical Microbiology,2005,43(8):4277-4279
[9]Chon JW,Kim JH,Lee SJ,et al.Toxin profile,antibiotic resistance,and phenotypic and molecular characterization of Bacillus cereus in Sunsik[J].Food Microbiology,2012,32(1):217-222
[10]Cao FY.Research on the virulence genes and antibiotic resistance phenotypes of Bacillus cereus detected from fermented bean curd in Beijing[D].Harbin:Master’s Thesis of Northeast Agricultural University,2017(in Chinese)曹飞扬.北京市腐乳中蜡样芽胞杆菌检测及毒力基因和耐药性研究[D].哈尔滨:东北农业大学硕士学位论文,2017
[11]Zhuang ZH.Study on biochemical type,virulence gene and drug sensitivity of food-derived Bacillus cereus in China[D].Beijing:Master’s Thesis of Chinese Center for Disease Control and Prevention,2013(in Chinese)庄子慧.我国食源性蜡样芽胞杆菌生化型、毒力基因及药物敏感性研究[D].北京:中国疾病预防控制中心硕士学位论文,2013
[12]Kim JY,Kim SH,Kwon NH,et al.Isolation and identification of Escherichia coli O157:H7 using different detection methods and molecular determination by multiplex PCR and RAPD[J].Journal of Veterinary Science,2005,6(1):7-19
[13]Liu CY,Zheng HJ,Yang MJ,et al.Genome analysis and in vivo virulence of porcine extraintestinal pathogenic Escherichia coli strain PCN033[J].BMC Genomics,2015,16(1):717
[14]Zhou HL,Xiong JL,Wang CY,et al.Identification and drug sensitivity test of swine pathogenic Escherichia coli[J].China Animal Husbandry and Veterinary Medicine,2009,36(1):109-111(in Chinese)周华林,熊江林,王长义,等.猪致病性大肠杆菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2009,36(1):109-111
[15]Yang X,Lu ZH,Chen LY,et al.Isolation and identification of enteropathogenic E.coli in newborn piglets[J].Animal Husbandry and Veterinary,2005,37(9):34-35(in Chinese)杨霞,卢中华,陈丽颖,等.仔猪致病性大肠杆菌的分离鉴定[J].畜牧与兽医,2005,37(9):34-35
[16]Li L,Chen Y,Zhang C,et al.A review:Identification of aeromonas in aquaculture[J].Fisheries Science,2015,34(2):128-134(in Chinese)李莉,陈颖,张超,等.水产动物气单胞菌鉴定方法研究进展[J].水产科学,2015,34(2):128-134
[17]Kamat AS,Nair CKK.Evidence for plasmid-mediated toxin production in Bacillus cereus BIS-59[J].World Journal of Microbiology&Biotechnology,1992,8(2):210-211
[18]Ehling-Schulz M,Fricker M,Scherer S.Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay[J].FEMS Microbiology Letters,2004,232(2):189-195
[19]Johnson JR,Russo TA.Molecular epidemiology of extraintestinal pathogenic(uropathogenic)Escherichia coli[J].International Journal of Medical Microbiology,2005,295(6/7):383-404