重庆市主城区动物携带大肠杆菌O157的流行病学研究
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摘要
目的：掌握本地区动物携带大肠杆菌O157的基本情况，菌株的基本特点以及相关流行病学资料，为防制大肠杆菌O157感染提供科学依据。
方法：2001年6月至9月间，对主城区中5个区进行了动物粪便及其环境标本的采集和流行病学资料的收集。对所采集的570份标本进行了细菌分离鉴定、药物敏感试验、质粒图谱及限制性内切酶图谱分析以及小鼠毒性试验。
结果：
* 从奶牛、鸡、羊粪便及饲养场周围环境中分离到大肠杆菌O157共计17株，其中O157：H7 14株、O157：NM 3株，平均分离率为2.98%。动物粪便分离率分别为1.65%、1.97%、12.5%。动物带菌似有明显的季节性，6至8月能分离出大肠杆菌O157，9月则未能分离。
分离菌基本符合大肠杆菌生化特点，但部分能发酵山梨醇，有些不能利用赖氨酸和鸟氨酸，有一株吲哚试验阴性。所有菌株对14种抗生素中的AM/SU、CEC、CRO、AZT呈现100%敏感，而对SMX、TC耐药，耐药率分别为88.24%和70.59%。对两重及以上耐药者占
* 69%，其中有3株对8种抗生素耐药。此外有4株菌产生(-内酰胺酶，经耐药质粒分析发现一3Kb质粒可能编码对氨苄西林的抗性。
* 分离株均带有质粒，可分为8种模式，其中共有的质粒为95Kb和24Kb。经质粒图谱和限制性内切酶图谱分析发现，来自于一奶牛场的菌株与来自于该奶牛场旁污水的菌株属同一克隆株。
* 各分离株对小鼠均表现不同程度的毒性。有7株菌的培养上清液能致小鼠死亡。另外10株细菌的培养物也能使小鼠出现感染中毒症状最终死亡。Vero细胞毒性试验表明17株分离株中有4株能产生Vero毒素。病理切片证实各脏器发生不同程度的病理改变。
结论：重庆市主城区的养殖场动物较为广泛地存在携带大肠杆菌O157的情况。病原体已经从宿主排出，对环境造成了污染，大肠杆菌O157感染流行的环节已基本具备。需要提高对大肠杆菌O157感染的认识和防制水平。
Object:To understand the distribution of E.coli O157 in domestic animals and fowl in chongqing urban district, the traits of isolates and associated epidemiological informations. To provide scientific basis for the prevention and control of E.coli O157 infections.
Methods:Specimens of animal feces and enviromental samples were collected from stock farms in five districts from June to September in 2001.E.coli O157 isolated from total 570 specimens by IMS were tested for antibiotic susceptibility,plasmid profiles,plasmid restriction endonuclease profiles and pathogenic factors.
Results:17 strains of E.coli O157 were isolated,the mean detection rate was 2.98%.14 strains belong to O157:H7,3 strains O157:NM.The bacteria were isolated from cow,chickens,goats and enviroment ,but none from pigs.The corresponding detection rates were 1.65%,1.97%,12.5% respectively.Among them,3 strains were detected from
enviroment.E.coli O157 was not detected from Shapingba and Jiulongpo districts and only isolated from June to Augest.
All isolates were of basic biochemistry traits of E.coli,but partial strains couldn't ferment sorbitol,some shew negtive results of Lysine and IND tests.All strains were susceptible to AM/SU,CEC,CRO,AZT,and the resistance to SMX and TX was severe as drug-resistance rate was respectively 88.24%,70.59%.There were about 69% of isolates resisted to no less than two kinds of antibiotics and three of them shew resistance to eight.Four strains could synthesize β-Lactamase which was most likely encoded on a plamid of 3 Kb.All strains harboured plasmids. The plasmid profiles detected in this study were divided into eight types, A to H, which carried 95 and 24Kb plasmids in common. The plasmid profiles and plasmid restriction endonuclease profiles of two strains, one isolated from cow feces and the other sewage near the same farm, were identical to each other, so they might belong to the same clone and have epidemiological relation.
Culture suspension of 7 strains could cause rats die,four of which could produce Vero-toxin.Culture broth of the other ten strains also could cause pathological changes in rats and resulted in death.So the isolated
strains might have some new pathogenic factor.
Conclusion:There were two types of E.coli O157 from domestic animals and fowl in chongqing urban district. The toxic factors of the isolated strains were different from the reference strains in China and foreign country. The bacteria excreted by host had already polluted surroundings, especially water-medium, and caused threats to public health. Suggestions for prevention and control were discussed too.
方法：2001年6月至9月间，对主城区中5个区进行了动物粪便及其环境标本的采集和流行病学资料的收集。对所采集的570份标本进行了细菌分离鉴定、药物敏感试验、质粒图谱及限制性内切酶图谱分析以及小鼠毒性试验。
结果：
* 从奶牛、鸡、羊粪便及饲养场周围环境中分离到大肠杆菌O157共计17株，其中O157：H7 14株、O157：NM 3株，平均分离率为2.98%。动物粪便分离率分别为1.65%、1.97%、12.5%。动物带菌似有明显的季节性，6至8月能分离出大肠杆菌O157，9月则未能分离。
分离菌基本符合大肠杆菌生化特点，但部分能发酵山梨醇，有些不能利用赖氨酸和鸟氨酸，有一株吲哚试验阴性。所有菌株对14种抗生素中的AM/SU、CEC、CRO、AZT呈现100%敏感，而对SMX、TC耐药，耐药率分别为88.24%和70.59%。对两重及以上耐药者占
* 69%，其中有3株对8种抗生素耐药。此外有4株菌产生(-内酰胺酶，经耐药质粒分析发现一3Kb质粒可能编码对氨苄西林的抗性。
* 分离株均带有质粒，可分为8种模式，其中共有的质粒为95Kb和24Kb。经质粒图谱和限制性内切酶图谱分析发现，来自于一奶牛场的菌株与来自于该奶牛场旁污水的菌株属同一克隆株。
* 各分离株对小鼠均表现不同程度的毒性。有7株菌的培养上清液能致小鼠死亡。另外10株细菌的培养物也能使小鼠出现感染中毒症状最终死亡。Vero细胞毒性试验表明17株分离株中有4株能产生Vero毒素。病理切片证实各脏器发生不同程度的病理改变。
结论：重庆市主城区的养殖场动物较为广泛地存在携带大肠杆菌O157的情况。病原体已经从宿主排出，对环境造成了污染，大肠杆菌O157感染流行的环节已基本具备。需要提高对大肠杆菌O157感染的认识和防制水平。
Object:To understand the distribution of E.coli O157 in domestic animals and fowl in chongqing urban district, the traits of isolates and associated epidemiological informations. To provide scientific basis for the prevention and control of E.coli O157 infections.
Methods:Specimens of animal feces and enviromental samples were collected from stock farms in five districts from June to September in 2001.E.coli O157 isolated from total 570 specimens by IMS were tested for antibiotic susceptibility,plasmid profiles,plasmid restriction endonuclease profiles and pathogenic factors.
Results:17 strains of E.coli O157 were isolated,the mean detection rate was 2.98%.14 strains belong to O157:H7,3 strains O157:NM.The bacteria were isolated from cow,chickens,goats and enviroment ,but none from pigs.The corresponding detection rates were 1.65%,1.97%,12.5% respectively.Among them,3 strains were detected from
enviroment.E.coli O157 was not detected from Shapingba and Jiulongpo districts and only isolated from June to Augest.
All isolates were of basic biochemistry traits of E.coli,but partial strains couldn't ferment sorbitol,some shew negtive results of Lysine and IND tests.All strains were susceptible to AM/SU,CEC,CRO,AZT,and the resistance to SMX and TX was severe as drug-resistance rate was respectively 88.24%,70.59%.There were about 69% of isolates resisted to no less than two kinds of antibiotics and three of them shew resistance to eight.Four strains could synthesize β-Lactamase which was most likely encoded on a plamid of 3 Kb.All strains harboured plasmids. The plasmid profiles detected in this study were divided into eight types, A to H, which carried 95 and 24Kb plasmids in common. The plasmid profiles and plasmid restriction endonuclease profiles of two strains, one isolated from cow feces and the other sewage near the same farm, were identical to each other, so they might belong to the same clone and have epidemiological relation.
Culture suspension of 7 strains could cause rats die,four of which could produce Vero-toxin.Culture broth of the other ten strains also could cause pathological changes in rats and resulted in death.So the isolated
strains might have some new pathogenic factor.
Conclusion:There were two types of E.coli O157 from domestic animals and fowl in chongqing urban district. The toxic factors of the isolated strains were different from the reference strains in China and foreign country. The bacteria excreted by host had already polluted surroundings, especially water-medium, and caused threats to public health. Suggestions for prevention and control were discussed too.
引文
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2. Xu JG,Quan TS,Xiao DL et al. Isolation and charicterisation of Escherichia coli O157:H7 strain in china.Curr Microbiol.1990,20:299-303
3. 郭维植，林成水，程发稷等．福建省家禽家畜O157大肠杆菌初查．中国人兽共患病杂志．1998,14(2):3-64.
4. 姜桂香,张守平,安静等.沈阳市动物携带O157:H7大肠杆菌检测的初步结果.疾病监测.1999,14(5):178-179
5. 顾宝柯,王春宴,李生等.家禽家畜肠出血性大肠杆菌O157:H7的监测.疾病监测.2000,15(6):219-220
6. 杨晋川,景怀奇,李洪卫等.用免疫胶体金快速诊断卡初筛粪便中的O157:H7大肠杆菌.疾病监测.2001,16(9):327-329
7. Karch,-H; Janetzki-Mittmann,-C; Aleksic,-S et al .Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture.J-Clin-Microbiol. 1996 ,34(3): 516-9
8. Onoue,-Y; Konuma,-H; Nakagawa,-H et al.Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef..Int-J-Food-Microbiol. 1999,46(1): 27-36
9. Bettelheim,-K-A.Reliability of CHROMagar O157 for the detection of enterohaemorrhagic Escherichia coli (EHEC) O157 but not EHEC belonging to other serogroups.J-Appl-Microbiol. 1998,85(3): 425-8
10. WHO Guidelines for antimicrobiol suspectibility testing Lab .1979,3
11. 李影林主编.中华医学检验全书.北京,人民卫生出版社.1996:1250-1251
12. 金冬雁,黎孟枫译.分子克隆实验指南.第二版,北京,科学出版社,1993
13. DominicoP. Rapid plasmid DNA isolation from mucoid gram-negative bacteria.J Clin Microbiol.1992,30(11):2859-2862
14. Portnoy, -D-A; Moseley, -S-L; Falkow, -S.Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis.Infect-Immun. 1981,31(2): 775-82
15. 颜子颖,王海林译.精编分子生物学实验指南.第三版,北京,科学出版社,1998
16. 卢耀增主编.实验动物学.北京:北京医科大学中国协和医科大学联合出版社.1995
17. 隋燕萍,彭奕冰,支立民等.Vero细胞毒性实验法检测大肠杆菌O157:H7毒素.微生物学杂志.1999,19(3):17-19
18. FlorbesBA,SchaberyOR.Transfer of resistance plasmid from staphylococas epiderimids to staphylococas aureas evidence for conjugative exchange of resistance. J Bacterial, 1983,153(1): 627
19. RobertsJA, UptonPA, AzeneG et al. Escherichia coli O157:H7; an economic assessment of an outbreak. J Public Health Med.2000, 22(1): 99-107
20. TarrPI.Escherchia coli O157:H7 clinical, diagnosis, and epidemiological aspects of human, Infection.1995, 20(1): 9-10
21. CDC.University outbreak of calicivirus infection mistakenly attributed to Shiga toxin-producing Escherichia coli O157: H7--Virginia, 2000.MMWR-Morb-Mortal-Wkly-Rep. 2001, 50(23): 489-91
22. 徐建国,景怀奇.中国1997-1998年O157:H7大肠杆菌检测情况分析.疾病监测.1999,14(5):176-178
23. Michel,-P; Wilson,-J-B; Martin,-S-W.et al.Temporal and geographical distributions of reported cases of Escherichia coli O157:H7 infection in Ontario.Epidemiol-Infect. 1999 ,122(2): 193-200
24. PritchardGC,WillshawGA,BaileyJR et al. Verocytotoxin-producing Escherichia coli 0157 on a farm open to the public: outbreak investigation and longitudinal bacteriological study. Vet Rec 2000,147(10):259-264
BielaszewskaM,SchmidtH,LiesegangA et al . Cattle can be a reservoir of sorbitol-fermenting shiga toxin-producing Escherichia coli O157:H(-) strains and
25. a source of human diseases.J Clin Microbiol.2000,38(9):3470-3473
26. CerqueiraAM,GuthBE,JoaquimRM. High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil. Vet Mirobiol.1999,70(1-2):111-121
27. CobboldR, DesmarchelierP. A longitudinal study of Shiga-toxigenic Escherichia coli (STEC) prevalence in three Australian diary herds. Vet Microbiol .2000,71(1-2): 125-137
28. HancockD,BesserT,LejeaneJ et al . The control of VTEC in the animal reservoir.Int J Food Microbiol.2001,66(1-2):71-78
29. VersalovicJ .DNA-based identification and epidemiologic typing of bacteriol pathogens.Arch Pathol Lab Med.1993,117(11):1088
30. FrataminoPM,BhaduriS,BuchananRL et al. Studies on Escherichia coli serotype O157:H7 strains containing a 60MDa plasmid and on 60MDa plasmid-cured derivatives.J Med Microbiol.1993,39(5):371-381
31. MakinoSI,AsakuraH,ShirahataT et al.Entero-hemorrhagic Escherichia coli O157 outbreak in Obihiro city-Study on antibiotic suspectivity and plasmid profile.J Japn Assoc Infect Dis,1998,72(2):89-96
32. 田口真澄, 户和子,小林一宽.STEC O157家族内感染の疫学マ—カ—解析:感染症学杂志.1999,74(2):104-111
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2. Xu JG,Quan TS,Xiao DL et al. Isolation and charicterisation of Escherichia coli O157:H7 strain in china.Curr Microbiol.1990,20:299-303
3. 郭维植，林成水，程发稷等．福建省家禽家畜O157大肠杆菌初查．中国人兽共患病杂志．1998,14(2):3-64.
4. 姜桂香,张守平,安静等.沈阳市动物携带O157:H7大肠杆菌检测的初步结果.疾病监测.1999,14(5):178-179
5. 顾宝柯,王春宴,李生等.家禽家畜肠出血性大肠杆菌O157:H7的监测.疾病监测.2000,15(6):219-220
6. 杨晋川,景怀奇,李洪卫等.用免疫胶体金快速诊断卡初筛粪便中的O157:H7大肠杆菌.疾病监测.2001,16(9):327-329
7. Karch,-H; Janetzki-Mittmann,-C; Aleksic,-S et al .Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture.J-Clin-Microbiol. 1996 ,34(3): 516-9
8. Onoue,-Y; Konuma,-H; Nakagawa,-H et al.Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef..Int-J-Food-Microbiol. 1999,46(1): 27-36
9. Bettelheim,-K-A.Reliability of CHROMagar O157 for the detection of enterohaemorrhagic Escherichia coli (EHEC) O157 but not EHEC belonging to other serogroups.J-Appl-Microbiol. 1998,85(3): 425-8
10. WHO Guidelines for antimicrobiol suspectibility testing Lab .1979,3
11. 李影林主编.中华医学检验全书.北京,人民卫生出版社.1996:1250-1251
12. 金冬雁,黎孟枫译.分子克隆实验指南.第二版,北京,科学出版社,1993
13. DominicoP. Rapid plasmid DNA isolation from mucoid gram-negative bacteria.J Clin Microbiol.1992,30(11):2859-2862
14. Portnoy, -D-A; Moseley, -S-L; Falkow, -S.Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis.Infect-Immun. 1981,31(2): 775-82
15. 颜子颖,王海林译.精编分子生物学实验指南.第三版,北京,科学出版社,1998
16. 卢耀增主编.实验动物学.北京:北京医科大学中国协和医科大学联合出版社.1995
17. 隋燕萍,彭奕冰,支立民等.Vero细胞毒性实验法检测大肠杆菌O157:H7毒素.微生物学杂志.1999,19(3):17-19
18. FlorbesBA,SchaberyOR.Transfer of resistance plasmid from staphylococas epiderimids to staphylococas aureas evidence for conjugative exchange of resistance. J Bacterial, 1983,153(1): 627
19. RobertsJA, UptonPA, AzeneG et al. Escherichia coli O157:H7; an economic assessment of an outbreak. J Public Health Med.2000, 22(1): 99-107
20. TarrPI.Escherchia coli O157:H7 clinical, diagnosis, and epidemiological aspects of human, Infection.1995, 20(1): 9-10
21. CDC.University outbreak of calicivirus infection mistakenly attributed to Shiga toxin-producing Escherichia coli O157: H7--Virginia, 2000.MMWR-Morb-Mortal-Wkly-Rep. 2001, 50(23): 489-91
22. 徐建国,景怀奇.中国1997-1998年O157:H7大肠杆菌检测情况分析.疾病监测.1999,14(5):176-178
23. Michel,-P; Wilson,-J-B; Martin,-S-W.et al.Temporal and geographical distributions of reported cases of Escherichia coli O157:H7 infection in Ontario.Epidemiol-Infect. 1999 ,122(2): 193-200
24. PritchardGC,WillshawGA,BaileyJR et al. Verocytotoxin-producing Escherichia coli 0157 on a farm open to the public: outbreak investigation and longitudinal bacteriological study. Vet Rec 2000,147(10):259-264
BielaszewskaM,SchmidtH,LiesegangA et al . Cattle can be a reservoir of sorbitol-fermenting shiga toxin-producing Escherichia coli O157:H(-) strains and
25. a source of human diseases.J Clin Microbiol.2000,38(9):3470-3473
26. CerqueiraAM,GuthBE,JoaquimRM. High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil. Vet Mirobiol.1999,70(1-2):111-121
27. CobboldR, DesmarchelierP. A longitudinal study of Shiga-toxigenic Escherichia coli (STEC) prevalence in three Australian diary herds. Vet Microbiol .2000,71(1-2): 125-137
28. HancockD,BesserT,LejeaneJ et al . The control of VTEC in the animal reservoir.Int J Food Microbiol.2001,66(1-2):71-78
29. VersalovicJ .DNA-based identification and epidemiologic typing of bacteriol pathogens.Arch Pathol Lab Med.1993,117(11):1088
30. FrataminoPM,BhaduriS,BuchananRL et al. Studies on Escherichia coli serotype O157:H7 strains containing a 60MDa plasmid and on 60MDa plasmid-cured derivatives.J Med Microbiol.1993,39(5):371-381
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