某屠宰场大肠杆菌O26的分离与鉴定
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摘要
为了了解屠宰场大肠杆菌O26污染情况,参考美国农业部(united states department of agriculture,USDA)的检测方法,对样品进行选择性增菌,经免疫磁珠富集、选择性显色培养基mRainbow Agar分离纯化后,挑选可疑菌株采用PCR方法鉴定O抗原,进一步采用血清凝集试验进行验证,并对确认的阳性菌株采用多重PCR方法进行毒力基因(stx1、stx2、eae、hly)检测。结果表明,采集的120份样品中共分离到1株大肠杆菌O26,但上述4种毒力基因均为阴性。该屠宰场存在大肠杆菌O26的污染,但分离出的大肠杆菌O26没有携带毒力基因。
Escherichia coli O26 was isolated and identified form a slaughter plant in order to investigate its contamination of E.coli O26.According to USDA detection method,samples were enriched with immunomagnetic separation(IMS)after selective enrichment,and then isolated and purified with selective chromogenic mRainbow agar.Subsequently,the suspected colonies were screened and chose to test the O antigen by PCR and followed by the serum agglutination test.The virulence genes(stx1,stx2,eae and hly)of positive isolations were detected by multiplex PCR.The results showed that only one strain from 120 samples was identified as E.coli O26,however the four virulence genes were found negative.Our results indicated that the E.coli O26 contamination existed in this slaughter house,but these positive bacterium strains did not carry virulence genes.
Escherichia coli O26 was isolated and identified form a slaughter plant in order to investigate its contamination of E.coli O26.According to USDA detection method,samples were enriched with immunomagnetic separation(IMS)after selective enrichment,and then isolated and purified with selective chromogenic mRainbow agar.Subsequently,the suspected colonies were screened and chose to test the O antigen by PCR and followed by the serum agglutination test.The virulence genes(stx1,stx2,eae and hly)of positive isolations were detected by multiplex PCR.The results showed that only one strain from 120 samples was identified as E.coli O26,however the four virulence genes were found negative.Our results indicated that the E.coli O26 contamination existed in this slaughter house,but these positive bacterium strains did not carry virulence genes.
引文
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[17]DEBROY C,ROBERTS E,VALADEZ A M,et al.Detection of shiga toxin-producing Escherichia coli O26,O45,O103,O111,O113,O121,O145,and O157Serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster[J].Foodborne Pathogens and Disease,2011,8(5):651-653.
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[22]MOLINI U,KAMWI S K,KHAISEBB S,et al.Detection of non O157:H7shiga toxin-producing Escherichia coli(STEC)serogroups O26,O45,O103,O111,O121and O145 from beef trim in Namibia[J].Biological and Chemical Research,2016,7:288-294.
[23]MOMTAZ H,SAFARPOOR D F,RAHIMI E,et al.Incidence of shiga toxin-producing Escherichia coli serogroups in ruminant’s meat[J].Meat Science.2013,95(2):381-388.
[24]ARTHUR T M,BARKOCY-GALLAGHER G A,RIV-ERA-BETANCOURT M,et al.Prevalence and characterization of non-O157shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants[J].Applied and Environmental Microbiology,2002,68(10):4 847-4 852.
[25]THOMAS K M,MCCANN M S,COLLERY M M,et al.Tracking verocytotoxigenic Escherichia coli O157,O26,O111,O103and O145in Irish cattle[J].International Journal of Food Microbiology,2012,153(3):288-296.
[26]禹金龙,董晨,王娴静,等.牛源性非O157大肠杆菌的分离与鉴定[J].食品科学,2018,DOI:10.7506/spkx1002-6630-201904000.
[27]SON I,BINET R,LIN A,et al.Identification of five Shiga toxin-producing Escherichia coli genes by Luminex microbead-based suspension array[J].Journal of Microbiological Methods,2015,111:108-110.
[28]BOERLIN P,MCEWEN S A,BOERLIN-PETZOLD F,et al.Associations between virulence factors of shiga toxinproducing Escherichia coli and disease in humans[J].Journal of Clinical Microbiology,1999,37(3):497-503.
[29]MCKEE M L,O’BRIEN A D.Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E.coli[J].Infection and Immunity,1995,63(5):2 070-2 074.
[30]OSWALD E,SCHMIDT H,MORABITO S,et al.Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli:characterization of a new intimin variant[J].Infection and Immunity,2000,68:64-71.
[31]LAW D.Virulence factors of Escherichia coli O157and other shiga toxin-producing E.coli[J].Journal of Applied Microbiology,2000,88(5):729-745.
[2]USDA-FSIS.Compliance guidelines for establishments on the FSIS microbiological testing program and other verification activities for Escherichia coli O157:H7[S].Washington,DC:United States Department of Agriculture Food Safety Inspection Service,2004:1-16.
[3]USDA-FSIS.Shiga toxin-producing Escherichia coli in certain raw beef products[S].Washington,DC:United States Department of Agriculture-Food Safety and Inspection Service,2012:31 975-31 981.
[4]USDA-FSIS.Process for evaluating the equivalence of foreign meat,poultry,and egg products food regulatory systems[S].[S.l.]:United States Department of Agriculture-Food Safety and Inspection Service,2011:1-16.
[5]USDA-FSIS.Ongoing equivalence verifications of foreign food regulatory systems[S].[S.l.]:United States Department of Agriculture-Food Safety and Inspection Service,2015:26 524-26 527.
[6]ETHELBERG S,SMITH B,TORPDAHL M,et al.Outbreak of non-O157shiga toxin-producing Escherichia coli infection from consumption of beef sausage[J].Clinical Infectious Diseases,2009,48(8):78-81.
[7]CHASE-TOPPING M E,ROSSER T,ALLISON L J,et al.Pathogenic potential to humans of bovine Escherichia coli O26,Scotland[J].Emerging Infectious Diseases,2012,18:439-448.
[8]JIANGIANG Y,YIN S,DUDLEY E G,et al.Diversity of CRISPR loci and virulence genes in pathogenic Escherichia coli isolates from various sources[J].International Journal of Food Microbiology,2015,204(1):41-46.
[9]KARMALI M A,GANNON V,SARGEANT J M,et al.Verocytotoxin-producing Escherichia coli(VTEC)[J].Veterinary Microbiology,2010,140(3/4):360-370.
[10]BIELASZEWSKA M,PRAGER R,KCK R,et al.Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26infection in humans[J].Applied&Environmental Microbiology,2007,73(10):3 144-3 150.
[11]BIELASZEWSKA M,MIDDENDORF B,KOCK R,et al.Shiga toxin-negative attaching and effacing Escherichia coli:distinct clinical associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution[J].Clinical Infectious Diseases,2008,47(2):208-217.
[12]SUBELZU N,BARTESAGHI S,BEM A D,et al.Evolution of enterohemorrhagic Escherichia coli O26based on single-nucleotide polymorphisms[J].Genome Biology and Evolution,2013,5(10):1 807-1 816.
[13]ERICKSON M C,DOYLE M P.Food as a vehicle for transmission of Shiga toxin-producing Escherichia coli[J].Journal of Food Protection,2007,70:2 426-2 449.
[14]夏诗琪,赖卫华,刘道峰,等.O26等产志贺毒素的6种血清型大肠杆菌的暴发流行情况及检测研究现状[J].食品科学,2014,35(17):301-305.
[15]LQAS/FSIS.Detection and isolation of non-O157shiga toxin-producing Escherichia coli(STEC)from meat products and carcass and environmental sponges:MLG 5B.04[S].Washington,DC:United States Department of Agriculture Food Safety Inspection Service,2013:1-16.
[16]SVOBODA A L,DUDLEY E G,DEBROY C,et al.Presence of shiga toxin-producing Escherichia coli O-groups in small and very-small beef-processing plants and resulting ground beef detected by a multiplex polymerase chain reaction assay[J].Foodborne Pathogens and Disease,2013,10(9):789-795.
[17]DEBROY C,ROBERTS E,VALADEZ A M,et al.Detection of shiga toxin-producing Escherichia coli O26,O45,O103,O111,O113,O121,O145,and O157Serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster[J].Foodborne Pathogens and Disease,2011,8(5):651-653.
[18]MACHADO J,GRIMONT F,GRIMONT P A.Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene[J].Research in Microbiology,2000,151(7):535-546.
[19]WANG Y,AMETAJ B N,AMBROSE D J,et al.Characterisation of the bacterial microbiota of the vagina of dairy cows and isolation of pediocin-producing Pediococcus acidilactici[J].Biomed Central Microbiology,2013,13(1):1-11.
[20]郭喜玲,史智扬,顾玲,等.应用多重引物PCR技术检测O157H7毒力基因[J].中华流行病学杂志,2000,21(6):410-412.
[21]BOSILEVAC J M,WANG R,LUEDTKE B E,et al.Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses[J].Journal of Food Protection,2017,80(1):136-145.
[22]MOLINI U,KAMWI S K,KHAISEBB S,et al.Detection of non O157:H7shiga toxin-producing Escherichia coli(STEC)serogroups O26,O45,O103,O111,O121and O145 from beef trim in Namibia[J].Biological and Chemical Research,2016,7:288-294.
[23]MOMTAZ H,SAFARPOOR D F,RAHIMI E,et al.Incidence of shiga toxin-producing Escherichia coli serogroups in ruminant’s meat[J].Meat Science.2013,95(2):381-388.
[24]ARTHUR T M,BARKOCY-GALLAGHER G A,RIV-ERA-BETANCOURT M,et al.Prevalence and characterization of non-O157shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants[J].Applied and Environmental Microbiology,2002,68(10):4 847-4 852.
[25]THOMAS K M,MCCANN M S,COLLERY M M,et al.Tracking verocytotoxigenic Escherichia coli O157,O26,O111,O103and O145in Irish cattle[J].International Journal of Food Microbiology,2012,153(3):288-296.
[26]禹金龙,董晨,王娴静,等.牛源性非O157大肠杆菌的分离与鉴定[J].食品科学,2018,DOI:10.7506/spkx1002-6630-201904000.
[27]SON I,BINET R,LIN A,et al.Identification of five Shiga toxin-producing Escherichia coli genes by Luminex microbead-based suspension array[J].Journal of Microbiological Methods,2015,111:108-110.
[28]BOERLIN P,MCEWEN S A,BOERLIN-PETZOLD F,et al.Associations between virulence factors of shiga toxinproducing Escherichia coli and disease in humans[J].Journal of Clinical Microbiology,1999,37(3):497-503.
[29]MCKEE M L,O’BRIEN A D.Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E.coli[J].Infection and Immunity,1995,63(5):2 070-2 074.
[30]OSWALD E,SCHMIDT H,MORABITO S,et al.Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli:characterization of a new intimin variant[J].Infection and Immunity,2000,68:64-71.
[31]LAW D.Virulence factors of Escherichia coli O157and other shiga toxin-producing E.coli[J].Journal of Applied Microbiology,2000,88(5):729-745.