牛源性非O157大肠杆菌的分离与鉴定
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摘要
目的:对牛粪及牛肉中的非O157大肠杆菌进行分离、鉴定和毒力基因携带情况进行检测,以了解非O157大肠杆菌的污染状况。方法:参考USDA检测方法,对样品进行选择性增菌,用多重聚合酶链式反应(polymerase chain reaction,PCR)方法进行初步筛选,检测O抗原(O157、O121、O111、O103、O26),阳性样本用选择性显色培养基rainbow agar分离纯化,可疑菌株用多重PCR方法鉴定O抗原,PCR-限制性片段长度多态性鉴定H抗原,并采用血清凝集实验进行验证,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:在153份牛粪和49份牛肉样本中,共40份样品检出1个或多个O血清型阳性,牛粪检出率高于牛肉,非O157阳性率为19.3%,O157的阳性率为0.50%;经分离纯化后,共鉴定出阳性菌株30株,其中O26最多占73.3%,O121、O103、O157分别占16.7%、6.7%、3.3%。毒力基因检测结果显示,分离自牛肉的2株O26:H11,一株stx1、hly阳性,另一株hly阳性,分离自牛粪的1株O26:H11携带hly基因,因此30株菌株中带毒菌株阳性率为10.0%,非O157产志贺毒素大肠杆菌阳性率为3.4%。结论:牛粪和牛肉中非O157大肠杆菌的污染率明显高于O157,尤其是O26:H11血清型最高,且检出含志贺毒素基因的大肠杆菌,这提示零售牛肉市场存在非O157大肠杆菌污染的安全风险,我国应该加强对非O157大肠杆菌的检测和监控。
Objectives: The aims of this study were to isolate and identify non-O157 Escherichia coli strains from bovine fecal and beef samples and to detect the virulence genes of the isolates, in order to understand the contamination status of non-O157 E. coli. Methods: After selective enrichment of beef and fecal samples by the USDA method with slight modifications, multiplex PCR was used as a prescreening method to determine the O-groups(O157, O121, O111, O103 and O26). The enriched positive sample was streaked for isolation and purification on chromogenic rainbow agar, and tested for the O-groups. The flagellar antigens were identified by PCR-restriction fragment length polymorphism(PCR-RFLP) and the positive isolates were further verified by means of serum agglutination test. The virulence genes(stx1, stx2, eae, and hly) of the positive isolates were detected by PCR. Results: A total of 153 fecal samples and 49 beef samples were collected.Overall, 40 samples were tested positive for one O-group, and the detection rate in feces was higher than that in beef, 19.3%of which were tested positive for non-O157, and 0.50% positive for O157. After purification, 30 positive isolates were identified. O26 was detected most frequently, accounting for 73.3% of the total isolates, followed by O26, O121, O103,and O157, accounted for 73.3%, 16.7%, 6.7% and 3.3%, respectively. The results of virulence gene analysis showed that two strains of O26:H11 were isolated from beef, one being positive for stx1 and hly and the other being positive for hly.One strain of O26:H11 harboring the hly gene was obtained from feces. Therefore, 10.0% of the 30 strains were positive for virulence genes, and 3.4% were non-O157 STEC. Conclusion: The contamination rate of non-O157 E. coli, especially O26:H11, in feces and beef was significantly higher than that of O157. Shiga toxin-producing E. coli was even detected in the beef samples, which suggested that there is a potential risk of non-O157 E. coli contamination in the retail beef market.The detection and monitoring of non-O157 E. coli should be strengthened in China.
Objectives: The aims of this study were to isolate and identify non-O157 Escherichia coli strains from bovine fecal and beef samples and to detect the virulence genes of the isolates, in order to understand the contamination status of non-O157 E. coli. Methods: After selective enrichment of beef and fecal samples by the USDA method with slight modifications, multiplex PCR was used as a prescreening method to determine the O-groups(O157, O121, O111, O103 and O26). The enriched positive sample was streaked for isolation and purification on chromogenic rainbow agar, and tested for the O-groups. The flagellar antigens were identified by PCR-restriction fragment length polymorphism(PCR-RFLP) and the positive isolates were further verified by means of serum agglutination test. The virulence genes(stx1, stx2, eae, and hly) of the positive isolates were detected by PCR. Results: A total of 153 fecal samples and 49 beef samples were collected.Overall, 40 samples were tested positive for one O-group, and the detection rate in feces was higher than that in beef, 19.3%of which were tested positive for non-O157, and 0.50% positive for O157. After purification, 30 positive isolates were identified. O26 was detected most frequently, accounting for 73.3% of the total isolates, followed by O26, O121, O103,and O157, accounted for 73.3%, 16.7%, 6.7% and 3.3%, respectively. The results of virulence gene analysis showed that two strains of O26:H11 were isolated from beef, one being positive for stx1 and hly and the other being positive for hly.One strain of O26:H11 harboring the hly gene was obtained from feces. Therefore, 10.0% of the 30 strains were positive for virulence genes, and 3.4% were non-O157 STEC. Conclusion: The contamination rate of non-O157 E. coli, especially O26:H11, in feces and beef was significantly higher than that of O157. Shiga toxin-producing E. coli was even detected in the beef samples, which suggested that there is a potential risk of non-O157 E. coli contamination in the retail beef market.The detection and monitoring of non-O157 E. coli should be strengthened in China.
引文
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[12]USDA/FSIS.Shiga toxin-producing Escherichia coli in certain raw beef products[J].Federal Register,2011,76(182):58157-58165.DOI:10.1017/CBO9781107415324.004.
[13]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.DOI:10.7506/spkx1002-6630-201417057.
[14]白莉,王伟,胡豫杰,等.免疫磁珠富集联合荧光定量PCR快速检测牛肉馅中产志贺毒素大肠埃希菌O26:H11[J].中国食品卫生杂志,2014,26(1):30-35.DOI:10.13590/j.cjfh.2014.01.001.
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[17]USDA-FSIS.Detection and isolation of non-O157 Shiga toxinproducing Escherichia coli(STEC)from meat products and carcass and environmental sponges:MLG 5B.05[S].Washington DC:United States Department of Agriculture Food Safety Inspection Service,2011:1-15.
[18]SVOBODA A L,DUDLEY E G,DEBROY C,et al.Presence of Shiga toxin-producing Escherichia coli O-groups in small and verysmall beef-processing plants and resulting ground beef detected by a multiplex polymerase chain reaction assay[J].Foodborne Pathogens&Disease,2013,10(9):789-795.DOI:10.1089/fpd.2012.1445.
[19]DEBROY C,ROBERTS E,VALADEZ A M,et al.Detection of Shiga toxin-producing Escherichia coli O26,O45,O103,O111,O113,O121,O145,and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster[J].Foodborne Pathogens&Disease,2011,8(5):651-652.DOI:10.1089/fpd.2010.0769.
[20]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.DOI:10.1016/S0923-2508(00)00223-0.
[21]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].BMC Microbiology,2013,13(1):1-11.DOI:10.1186/1471-2180-13-19.
[22]郭喜玲,史智扬.应用多重引物PCR技术检测O157:H7毒力基因[J].中华流行病学杂志,2000,21(6):410-412.DOI:10.3760/j.issn:0254-6450.2000.06.003.
[23]KARMALI M A,GANNON V,SARGEANT J M.Verocytotoxinproducing Escherichia coli(VTEC)[J].Veterinary Microbiology,2010,140(3/4):360-370.DOI:10.1016/j.vetmic.2009.04.011.
[24]PEARCE M C,JENKINS C,VALI L,et al.Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26,O103,O111,O145,and O157 in a cohort of beef calves and their dams[J].Applied&Environmental Microbiology,2004,70(3):1708-1716.DOI:10.1128/AEM.70.3.1708-1716.2004.
[25]MENRATH A,WIELER L H,HEIDEMANNS K,et al.Shiga toxin producing Escherichia coli:identification of non-O157:H7-supershedding cows and related risk factors[J].Gut Pathogens,2010,2(1):2-7.DOI:10.1186/1757-4749-2-7.
[26]CONRAD C C,STANFORD K,MCALLISTER T A,et al.Further development of sample preparation and detection methods for O157and the top 6 non-O157 STEC serogroups in cattle feces[J].Journal of Microbiological Methods,2014,105:22-30.DOI:10.1016/j.mimet.2014.06.020.
[27]ZELYAS N,POON A,PATTERSON-FORTIN L,et al.Assessment of commercial chromogenic solid media for the detection of non-O157 Shiga toxin-producing Escherichia coli(STEC)[J].Diagnostic Microbiology&Infectious Disease,2016,85(3):302-308.DOI:10.1016/j.diagmicrobio.2016.03.013.
[28]ELDER R O.Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces,hides,and carcasses of beef cattle during processing[J].Proceedings of the National Academy of Sciences 2000,97(7):2999-3003.DOI:10.1073/pnas.060024897.
[29]DEWSBURY D M,RENTER D G,SHRIDHAR P B,et al.Summer and winter prevalence of Shiga toxin-producing Escherichia coli(STEC)O26,O45,O103,O111,O121,O145,and O157 in feces of feedlot cattle[J].Foodborne Pathogens&Disease,2015,12(8):726-732.DOI:10.1089/fpd.2015.1987.
[30]BALTASAR P,MILTON S,SWECKER W,et al.Shiga toxin-producing Escherichia coli distribution and characterization in a pasture-based cow-calf production system[J].Journal of Food Protection,2014,77(5):722-731.DOI:10.4315/0362-028X.JFP-13-420.
[31]EKIRI A B,LANDBLOM D,DOETKOTT D,et al.Isolation and characterization of Shiga toxin-producing Escherichia coli serogroups O26,O45,O103,O111,O113,O121,O145,and O157 shed from range and feedlot cattle from postweaning to slaughter[J].Journal of Food Protection,2014,77(7):1052-1061.DOI:10.4315/0362-028X.JFP-13-373.
[32]KOOHMARAIE M.Prevalence and control of non-O157 STEC in beef and lamb processing plants[C]//The public health significance of Non-O157 Shiga Toxin-producing Escherichia coli(STEC)Public Meeting.Arlington:2007.
[33]BARLOW R S,GOBIUS K S,DESMARCHELIER P M,et al.Shiga toxin-producing Escherichia coli in ground beef and lamb cuts:results of a one-year study[J].International Journal of Food Microbiology,2006,111(1):1-5.DOI:10.1016/j.ijfoodmicro.2006.04.039.
[34]AUVRAY F,LECUREUIL C,TACHéJ,et al.Detection,isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques,immunoassays and colony hybridization[J].Letters in Applied Microbiology,2007,45(6):646-651.DOI:10.1111/j.1472-765X.2007.02239.x.
[35]JIANG 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.DOI:10.1016/j.ijfoodmicro.2015.03.025.
[36]European Food Safety Authority(EFSA).The community summary report on trends and sources of zoonoses,zoonotic agents,antimicrobial resistance and food borne outbreaks in the European Union in 2005[J].EFSA Journal,2006,4(12):175-185.DOI:10.2903/j.efsa.2006.94r.
[37]HUSSEIN H S.Prevalence and pathogenicity of Shiga toxin-producing Escherichia coli in beef cattle and their products[J].Journal of Animal Science,2007,85(13):63-72.DOI:10.2527/jas.2006-421.
[2]BELL C,KYRIAKIDES A.17-Pathogenic Escherichia coli[J].Foodborne Pathogens,2009,295(1):581-626.DOI:10.1533/9781845696337.2.581.
[3]JR P W,MILLER M,BINDER H J,et al.Enteric infections,diarrhea,and their impact on function and development[J].Journal of Clinical Investigation,2008,118(4):1277-1290.DOI:10.1172/JCI34005.
[4]DURSO L M,KEEN J E.Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E.coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces[J].Journal of Applied Microbiology,2007,103(6):2457-2464.DOI:10.1111/j.1365-2672.2007.03473.x.
[5]SMITH J L,FRATAMICO P M,GUNTHER N W.Shiga toxin-producing Escherichia coli[J].Advances in Applied Microbiology,2014,86(5):145-197.DOI:10.1016/B978-0-12-800262-9.00003-2.
[6]KAPER J B.Pathogenic Escherichia coli[J].Nature Reviews:Microbiology,2005,295(6):355-356.DOI:10.1038/nrmicro818.
[7]BACH S J,MCALLISTER T A,VEIRA D M,et al.Transmission and control of Escherichia coli O157:H7:a review[J].Canadian Journal of Animal Science,2002,82(4):475-490.DOI:10.4141/A02-021.
[8]BITZAN M.Treatment options for HUS secondary to Escherichia coli O157:H7[J].Kidney International,2009,75(112):62-66.DOI:10.1038/ki.2008.624.
[9]Enters of Disease Control and Prevention(CDC).Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food;10 states,2007[J].Morbidity and Mortality Weekly Report,2008,57(14):366-370.DOI:10.15585/mmwr.mm6711a3.
[10]BOSILEVAC J M,KOOHMARAIE M.Predicting the presence of non-O157 Shiga toxin-producing Escherichia coli in ground beef by using molecular tests for Shiga toxins,intimin,and O serogroups[J].Applied&Environmental Microbiology,2012,78(19):7152-7155.DOI:10.1128/AEM.01508-12.
[11]BROOKS J T,SOWERS E G,WELLS J G,et al.Non-O157 Shiga toxin-producing Escherichia coli infections in the United States,1983-2002[J].Journal of Infectious Diseases,2005,192(8):1422-1429.DOI:10.1086/466536.
[12]USDA/FSIS.Shiga toxin-producing Escherichia coli in certain raw beef products[J].Federal Register,2011,76(182):58157-58165.DOI:10.1017/CBO9781107415324.004.
[13]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.DOI:10.7506/spkx1002-6630-201417057.
[14]白莉,王伟,胡豫杰,等.免疫磁珠富集联合荧光定量PCR快速检测牛肉馅中产志贺毒素大肠埃希菌O26:H11[J].中国食品卫生杂志,2014,26(1):30-35.DOI:10.13590/j.cjfh.2014.01.001.
[15]巴鹏斌,孟琼,白向宁,等.非O157产志贺毒素大肠埃希菌研究进展[J].中国人兽共患病学报,2017,33(2):156-162.DOI:10.3969/j.issn.1002-2694.2017.02.012.
[16]申进玲.非O157产志贺毒素大肠杆菌和单增李斯特菌的分子分型及毒力分析[D].杨凌:西北农林科技大学,2013.
[17]USDA-FSIS.Detection and isolation of non-O157 Shiga toxinproducing Escherichia coli(STEC)from meat products and carcass and environmental sponges:MLG 5B.05[S].Washington DC:United States Department of Agriculture Food Safety Inspection Service,2011:1-15.
[18]SVOBODA A L,DUDLEY E G,DEBROY C,et al.Presence of Shiga toxin-producing Escherichia coli O-groups in small and verysmall beef-processing plants and resulting ground beef detected by a multiplex polymerase chain reaction assay[J].Foodborne Pathogens&Disease,2013,10(9):789-795.DOI:10.1089/fpd.2012.1445.
[19]DEBROY C,ROBERTS E,VALADEZ A M,et al.Detection of Shiga toxin-producing Escherichia coli O26,O45,O103,O111,O113,O121,O145,and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster[J].Foodborne Pathogens&Disease,2011,8(5):651-652.DOI:10.1089/fpd.2010.0769.
[20]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.DOI:10.1016/S0923-2508(00)00223-0.
[21]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].BMC Microbiology,2013,13(1):1-11.DOI:10.1186/1471-2180-13-19.
[22]郭喜玲,史智扬.应用多重引物PCR技术检测O157:H7毒力基因[J].中华流行病学杂志,2000,21(6):410-412.DOI:10.3760/j.issn:0254-6450.2000.06.003.
[23]KARMALI M A,GANNON V,SARGEANT J M.Verocytotoxinproducing Escherichia coli(VTEC)[J].Veterinary Microbiology,2010,140(3/4):360-370.DOI:10.1016/j.vetmic.2009.04.011.
[24]PEARCE M C,JENKINS C,VALI L,et al.Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26,O103,O111,O145,and O157 in a cohort of beef calves and their dams[J].Applied&Environmental Microbiology,2004,70(3):1708-1716.DOI:10.1128/AEM.70.3.1708-1716.2004.
[25]MENRATH A,WIELER L H,HEIDEMANNS K,et al.Shiga toxin producing Escherichia coli:identification of non-O157:H7-supershedding cows and related risk factors[J].Gut Pathogens,2010,2(1):2-7.DOI:10.1186/1757-4749-2-7.
[26]CONRAD C C,STANFORD K,MCALLISTER T A,et al.Further development of sample preparation and detection methods for O157and the top 6 non-O157 STEC serogroups in cattle feces[J].Journal of Microbiological Methods,2014,105:22-30.DOI:10.1016/j.mimet.2014.06.020.
[27]ZELYAS N,POON A,PATTERSON-FORTIN L,et al.Assessment of commercial chromogenic solid media for the detection of non-O157 Shiga toxin-producing Escherichia coli(STEC)[J].Diagnostic Microbiology&Infectious Disease,2016,85(3):302-308.DOI:10.1016/j.diagmicrobio.2016.03.013.
[28]ELDER R O.Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces,hides,and carcasses of beef cattle during processing[J].Proceedings of the National Academy of Sciences 2000,97(7):2999-3003.DOI:10.1073/pnas.060024897.
[29]DEWSBURY D M,RENTER D G,SHRIDHAR P B,et al.Summer and winter prevalence of Shiga toxin-producing Escherichia coli(STEC)O26,O45,O103,O111,O121,O145,and O157 in feces of feedlot cattle[J].Foodborne Pathogens&Disease,2015,12(8):726-732.DOI:10.1089/fpd.2015.1987.
[30]BALTASAR P,MILTON S,SWECKER W,et al.Shiga toxin-producing Escherichia coli distribution and characterization in a pasture-based cow-calf production system[J].Journal of Food Protection,2014,77(5):722-731.DOI:10.4315/0362-028X.JFP-13-420.
[31]EKIRI A B,LANDBLOM D,DOETKOTT D,et al.Isolation and characterization of Shiga toxin-producing Escherichia coli serogroups O26,O45,O103,O111,O113,O121,O145,and O157 shed from range and feedlot cattle from postweaning to slaughter[J].Journal of Food Protection,2014,77(7):1052-1061.DOI:10.4315/0362-028X.JFP-13-373.
[32]KOOHMARAIE M.Prevalence and control of non-O157 STEC in beef and lamb processing plants[C]//The public health significance of Non-O157 Shiga Toxin-producing Escherichia coli(STEC)Public Meeting.Arlington:2007.
[33]BARLOW R S,GOBIUS K S,DESMARCHELIER P M,et al.Shiga toxin-producing Escherichia coli in ground beef and lamb cuts:results of a one-year study[J].International Journal of Food Microbiology,2006,111(1):1-5.DOI:10.1016/j.ijfoodmicro.2006.04.039.
[34]AUVRAY F,LECUREUIL C,TACHéJ,et al.Detection,isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques,immunoassays and colony hybridization[J].Letters in Applied Microbiology,2007,45(6):646-651.DOI:10.1111/j.1472-765X.2007.02239.x.
[35]JIANG 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.DOI:10.1016/j.ijfoodmicro.2015.03.025.
[36]European Food Safety Authority(EFSA).The community summary report on trends and sources of zoonoses,zoonotic agents,antimicrobial resistance and food borne outbreaks in the European Union in 2005[J].EFSA Journal,2006,4(12):175-185.DOI:10.2903/j.efsa.2006.94r.
[37]HUSSEIN H S.Prevalence and pathogenicity of Shiga toxin-producing Escherichia coli in beef cattle and their products[J].Journal of Animal Science,2007,85(13):63-72.DOI:10.2527/jas.2006-421.
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