羊腹泻样本中大肠杆菌的分离、毒力基因与耐药性分析

doi:10.11843/j.issn.0366-6964.2023.08.029

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (8): 3445-3454.doi: 10.11843/j.issn.0366-6964.2023.08.029

羊腹泻样本中大肠杆菌的分离、毒力基因与耐药性分析

刘鑫欢^1,2, 恽佳蕾^2,3, 毛立², 李基棕², 郝飞², 何苗锋^1,2, 杨蕾蕾², 张纹纹², 程子龙², 孙敏², 刘茂军^1,2,3,5, 王少辉⁴, 白娟¹, 李文良^1,2,3,4,5*

1. 南京农业大学动物医学院, 南京 210095;
2. 江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室, 南京 210014;
3. 江苏大学食品与生物工程学院, 镇江 212013;
4. 中国农业科学院上海兽医研究所, 上海 200241;
5. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2022-12-14 出版日期:2023-08-23 发布日期:2023-08-22
通讯作者: 李文良,主要从事动物重要病原感染致病机制与防控技术研究E-mail:kfliwenliang@163.com
作者简介:刘鑫欢(1999-),女,重庆人,硕士生,主要从事预防兽医学研究,E-mail:1922236149@qq.com;恽佳蕾(1997-),女,江苏无锡人,硕士,主要从事预防兽医学研究,E-mail:834462138@qq.com。
基金资助:
江苏省重点研发计划（现代农业）重点项目（BE2022394）；江苏省农业科技自主创新资金项目（CX（18）2003）

Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep

LIU Xinhuan^1,2, YUN Jialei^2,3, MAO Li², LI Jizong², HAO Fei², HE Miaofeng^1,2, YANG Leilei², ZHANG Wenwen², CHENG Zilong², SUN Min², LIU Maojun^1,2,3,5, WANG Shaohui⁴, BAI Juan¹, LI Wenliang^1,2,3,4,5*

1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Engineering and Technology for Veterinary Biological Products, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China;
3. School of Food and Bioengineering, Jiangsu University, Zhenjiang 212013, China;
4. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
5. Guotai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China

Received:2022-12-14 Online:2023-08-23 Published:2023-08-22

摘要/Abstract

摘要： 为确定华东地区发生腹泻的羊群中大肠杆菌感染、毒力基因携带及耐药性情况，本研究通过分离鉴定、进化分群、毒力基因检测、药敏试验等方法对发生腹泻的羊场病死羊肠道、粪便样品进行检测。结果显示，从187份样品中分离到163株大肠杆菌，其中B1群及A群的菌株占大多数，检出率分别为59.51%和28.83%；B2和D群的检出率分别为9.82%和1.84%。对分离菌株31个毒力基因进行检测，结果表明yijp、crlA、mat、ompA、fimC、ibeB 6种毒力基因的检出率均大于87%；所有菌株均未检出毒力基因afa/draB和LT。fimC、mat、crlA、ibeB、yijp及ompA在各进化群大肠杆菌均广泛存在。根据毒力基因检测结果，有72株菌为产志贺毒素大肠杆菌（STEC），主要携带毒力基因stx2。STEC另一标志性毒力基因eae检出率为61.11%。药敏试验检测的16种抗菌药中有4种的耐药率大于80%，有13种大于50%。属于多重耐药（MDR）的菌株有150株，占92.02%，且多为五重及以上耐药菌（84.05%）。华东地区临床流行的羊源大肠杆菌菌株毒力基因多样、耐药性普遍且耐药性强。该结果为本地区羊大肠杆菌病的防控提供了科学依据。

关键词: 大肠杆菌, 羊, 分离鉴定, 进化群, 毒力基因, 多重耐药

Abstract: In order to determine the infection situation of Escherichia coli (E.coli), virulence genes and drug resistance of the E.coli strains in goat and sheep farms with diarrhea in Eastern China, the intestinal tract and fecal samples of sick and dead goat and sheep with diarrhea were examined by means of E.coli isolation and identification, phylogenetic grouping, virulence gene detection and drug sensitivity test. The results showed that a total 163 E.coli strains were isolated from 187 samples, of which the majority were classified into group B1 and group A, with detection rates of 59.51% and 28.83%, respectively. The detection rates of B2 and D groups were 9.82% and 1.84%, respectively. The results from 31 virulence genes detection showed that the detection rates of yijp, crlA, mat, ompA, fimC and ibeB were all greater than 87%. Virulence genes afa/draB and LT were not detected in all strains. fimC, mat, crlA, ibeB, yijp and ompA were widely present in all evolutionary groups. According to the results of virulence gene test, 72 strains were Shiga toxin-producing E.coli (STEC), which mainly carried virulence gene stx2. The detection rate of the other marker virulence gene eae was 61.11%. Drug sensitivity test results showed that the resistance rate of 4 and 13 out of the 16 antibacterial drugs tested was greater than 80% and 50%, respectively. Among the 163 strains, 150 were multi-drug resistant (MDR) ones, accounting for 92.02%, and most of them were resistant to five or more drug categories (84.05%). In conclusion, the results of this study indicated that the clinically prevalent E.coli strains from goat and sheep in eastern China were highly virulent, common and strong drug resistance. It provided a scientific basis for the prevention and control of E.coli in the farms in this region.

Key words: E.coli, goat and sheep, isolation and identification, phylogenetic group, virulence gene, multi-drug resistance

中图分类号:

S852.61

刘鑫欢, 恽佳蕾, 毛立, 李基棕, 郝飞, 何苗锋, 杨蕾蕾, 张纹纹, 程子龙, 孙敏, 刘茂军, 王少辉, 白娟, 李文良. 羊腹泻样本中大肠杆菌的分离、毒力基因与耐药性分析[J]. 畜牧兽医学报, 2023, 54(8): 3445-3454.

LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.

参考文献 34

[1]	GARCÍA A, Fox J G. A one health perspective for defining and deciphering Escherichia coli pathogenic potential in multiple hosts[J]. Comp Med, 2021, 71(1):3-45.
[2]	陈家露, 蔡重振, 次旦, 等. 西藏那曲市羊源大肠杆菌分离鉴定及耐药性研究[J]. 中国畜牧兽医, 2019, 46(12):3759-3767.CHEN J L, CAI C Z, CI D, et al. Isolation, identification and drug resistance analysis of sheep Escherichia coli in Naqu, Tibet[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(12):3759-3767. (in Chinese)
[3]	BÉLANGER L, GARENAUX A, HAREL J, et al. Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E.coli[J]. FEMS Immunol Med Microbiol, 2011, 62(1):1-10.
[4]	WIRIYAPROM R, NGASAMAN R, KAEWNOI D, et al. Prevalence and virulent gene profiles of sorbitol non-fermenting Shiga toxin-producing Escherichia coli isolated from goats in southern Thailand[J]. Trop Med Infect Dis, 2022, 7(11):357.
[5]	YANG X, SUN H, FAN R Y, et al. Genetic diversity of the intimin gene (eae) in non-O157 Shiga toxin-producing Escherichia coli strains in China[J]. Sci Rep, 2020, 10(1):3275.
[6]	郑晓风, 张妍, 刘英玉, 等. 新疆部分地区牛羊源产志贺毒素大肠埃希菌菌株检测与分析[J]. 畜牧兽医学报, 2020, 51(10):2518-2527.ZHENG X F, ZHANG Y, LIU Y Y, et al. Detection and analysis of Shiga toxin-producing Escherichia coli isolates from cattle and sheep sources in some regions of Xinjiang, China[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10):2518-2527. (in Chinese)
[7]	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, 2019, 40(4):299-304.YU J L, DONG C, WANG X J, et al. Isolation and identification of bovine-Derived non-O157 Escherichia coli strains[J]. Food Science, 2019, 40(4):299-304. (in Chinese)
[8]	YUN J L, MAO L, LI J Z, et al. Molecular characterization and antimicrobial resistance profile of pathogenic Escherichia coli from goats with respiratory disease in eastern China[J]. Microb Pathog, 2022, 166:105501.
[9]	ABDULKAREEM M H, ABOOD A I, DAKHEEL M M. Antimicrobial resistance of tannin extract against E.coli isolates from sheep[J]. Arch Razi Inst, 2022, 77(2):697-701.
[10]	KRIZMAN M, AVGUSTIN J A, ZDOVC I, et al. Antimicrobial resistance and molecular characterization of extended-spectrum β-lactamases and other Escherichia coli isolated from food of animal origin and human intestinal isolates[J]. J Food Prot, 2017, 80(1):113-120.
[11]	赵学亮, 王斌, 苗永强, 等. 陕西地区羊源致病性大肠杆菌耐药性分析与毒力基因检测[J]. 畜牧兽医学报, 2022, 53(5):1644-1648.ZHAO X L, WANG B, MIAO Y Q, et al. Detection of virulence genes and antimicrobial resistance analysis of Escherichia coli isolated from diarrhea sheep in Shanxi Province[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5):1644-1648. (in Chinese)
[12]	王显峰, 白金英. 羊源大肠杆菌血清型鉴定、毒力基因检测及耐药性分析[J]. 畜牧与兽医, 2020, 52(3):69-72.WANG X F, BAI J Y. Serotype identification, virulence gene and drug resistance detection of Escherichia from sheep[J]. Animal Husbandry & Veterinary Medicine, 2020, 52(3):69-72. (in Chinese)
[13]	宋晓莉, 成大荣, 刘晓明, 等. 临床腹泻肉羊源大肠杆菌流行病学调查及耐药性分析[J]. 黑龙江畜牧兽医, 2019(12):71-75, 80.SONG X L, CHENG D R, LIU X M, et al. Epidemiological investigation and drug resistance analysis of Escherichia coli from mutton sheep with clinical diarrhea[J]. Heilongjiang Animal Science and Veterinary Medicine, 2019(12):71-75, 80. (in Chinese)
[14]	FRANK J A, REICH C I, SHARMA S, et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes[J]. Appl Environ Microbiol, 2008, 74(8):2461-2470.
[15]	CLERMONT O, BONACORSI S, BINGEN E. Rapid and simple determination of the Escherichia coli phylogenetic group[J]. Appl Environ Microbiol, 2000, 66(10):4555-4558.
[16]	ZHU Y C, DONG W Y, MA J L, et al. Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China[J]. BMC Vet Res, 2017, 13(1):94.
[17]	JAKOBSEN L, SPANGHOLM D J, PEDERSEN K, et al. Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients[J]. Int J Food Microbiol, 2010, 142(1-2):264-272.
[18]	OBENG A S, RICKARD H, NDI O, et al. Antibiotic resistance, phylogenetic grouping and virulence potential of Escherichia coli isolated from the faeces of intensively farmed and free range poultry[J]. Vet Microbiol, 2012, 154(3-4):305-315.
[19]	孟庆美, 王少辉, 韩先干, 等. 禽致病性大肠杆菌毒力基因多重PCR方法的建立和应用[J]. 微生物学报, 2014, 54(6):696-702.MENG Q M, WANG S H, HAN X G, et al. Multiplex PCR assay for detection of virulence genes in avian pathogenic Escherichia coli[J]. Acta Microbiologica Sinica, 2014, 54(6):696-702. (in Chinese)
[20]	EWERS C, LI G W, WILKING H, et al. Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli:How closely related are they?[J]. Int J Med Microbiol, 2007, 297(3):163-176.
[21]	尚立宏, 张啸, 许伟, 等. 甘肃部分羊场羔羊腹泻病原菌分离鉴定及其耐药性研究[J]. 中兽医医药杂志, 2022, 41(4):7-11.SHANG L H, ZHANG X, XU W, et al. Isolation, identification and antimicrobial resistance of lamb diarrhea pathogens in some sheep farms in Gansu Province[J]. Journal of Traditional Chinese Veterinary Medicine, 2022, 41(4):7-11. (in Chinese)
[22]	刘晨阳. 羊源大肠杆菌的分离鉴定及耐药性与致病性分析[D]. 咸阳:西北农林科技大学, 2019.LIU C Y. Isolation and identification of Escherichia coli from goats and analysis of drug resistance and pathogenicity[D]. Xianyang:Northwest A&F University, 2019. (in Chinese)
[23]	NDEGWA E, O'BRIEN D, MATTHEW K, et al. Shiga toxin subtypes, serogroups, phylogroups, RAPD genotypic diversity, and select virulence markers of Shiga-toxigenic Escherichia coli strains from goats in mid-atlantic US[J]. Microorganisms, 2022, 10(9):1842.
[24]	NDEGWA E, ALAHMDE A, KIM C, et al. Age related differences in phylogenetic diversity, prevalence of Shiga toxins, intimin, hemolysin genes and select serogroups of Escherichia. coli from pastured meat goats detected in a longitudinal cohort study[J]. BMC Vet Res, 2020, 16(1):266.
[25]	ZHAO X L, LV Y Y, ADAM F E A, et al Comparison of antimicrobial resistance, virulence genes, phylogroups, and biofilm formation of Escherichia coli isolated from intensive farming and free-range sheep[J]. Front Microbiol, 2021, 12:699927.
[26]	BLUM S E, LEITNER G. Genotyping and virulence factors assessment of bovine mastitis Escherichia coli[J]. Vet Microbiol, 2013, 163(3-4):305-312.
[27]	张星星, 乔军, 孟庆玲, 等. 新疆犊牛源大肠杆菌系统进化及其耐药特性[J]. 家畜生态学报, 2016, 37(6):60-64.ZHANG X X, QIAO J, MENG Q L, et al. Phylogenetic analysis and resistance characteristics of Escherichia coli from calves in Xinjiang[J]. Acta Ecologae Animalis Domastici, 2016, 37(6):60-64. (in Chinese)
[28]	于伟伟, 乔军, 孟庆玲, 等. 奶牛乳房炎大肠杆菌新疆分离株系统分群及其耐药特性与毒力因子分布研究[J]. 中国奶牛, 2017(10):30-35.YU W W, QIAO J, MENG Q L, et al. Phylogenetic grouping, antimicrobial resistance and virulence factors of Escherichia coli isolated from bovine mastitis in Xinjiang[J]. China Dairy Cattle, 2017, (10):30-35. (in Chinese)
[29]	WANG S H, SHI Z Y, XIA Y J, et al. IbeB is involved in the invasion and pathogenicity of avian pathogenic Escherichia coli[J]. Vet Microbiol, 2012, 159(3-4):411-419.
[30]	TU J, XUE T, QI K Z, et al. The irp2 and fyuA genes in High Pathogenicity Islands are involved in the pathogenesis of infections caused by avian pathogenic Escherichia coli (APEC)[J]. Pol J Vet Sci, 2016, 19(1):21-29.
[31]	张凌, 佟盼盼, 张毅, 等. 羔羊STEC的耐药性、毒力基因和血清型分析[J]. 新疆农业科学, 2020, 57(10):1921-1930.ZHANG L, TONG P P, ZHANG Y, et al. Analysis of drug resistance, virulence genes and serotypes of STEC in a large-scale sheep farm in Xinjiang[J]. Xinjiang Agricultural Sciences, 2020, 57(10):1921-1930. (in Chinese)
[32]	冯杰, 马剑钢, 张新, 等. 宁夏地区羊源大肠埃希氏菌的分离鉴定及耐药性检测[J]. 动物医学进展, 2022, 43(5):126-130.FENG J, MA J G, ZHANG X, et al. Isolation, identification and antimicrobial resistance detection of Escherichia coli from sheep in Ningxia[J]. Progress in Veterinary Medicine, 2022, 43(5):126-130. (in Chinese)
[33]	PERSAD A K, RAJASHEKARA G, LEJEUNE J T. Shiga toxin (stx) encoding genes in sheep and goats reared in Trinidad and Tobago[J]. PLoS One, 2022, 17(11):e0277564.
[34]	杨跃飞. 临床动物源性大肠杆菌的分离鉴定、耐药基因检测及多位点序列分型分析[D]. 扬州:扬州大学, 2022.YANG Y F. Isolation, identification, antibiotic resistant gene detection and multi-locus sequence typing analysis of Escherichia coli isolates from animal origins in clinical[D]. Yangzhou:Yangzhou University, 2022. (in Chinese)

羊腹泻样本中大肠杆菌的分离、毒力基因与耐药性分析

Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 34

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王海波, 占今舜, 谷志勇, 陈新锋, 潘月, 贾浩滨, 钟小军, 李开嵘, 赵生国, 霍俊宏. 湖羊与三元杂交绵羊夏杜湖、夏澳湖肉质特性比较研究[J]. 畜牧兽医学报, 2024, 55(1): 110-119.
[2]	严晓春, 习海娇, 李金泉, 王志英, 苏蕊. 内蒙古绒山羊绒毛性状基因组育种值估计准确性研究[J]. 畜牧兽医学报, 2024, 55(1): 120-128.
[3]	张晨俭, 李隐侠, 丁强, 刘伟佳, 王慧利, 何南, 吴家顺, 曹少先. CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J]. 畜牧兽医学报, 2024, 55(1): 129-141.
[4]	段香茹, 康佳, 杨若晨, 单新雨, 李太春, 赵雯, 张英杰, 刘月琴. L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响[J]. 畜牧兽医学报, 2024, 55(1): 179-191.
[5]	张德安, 杨若渚, 刘杰, 刘德武, 邓铭, 柳广斌, 孙宝丽, 郭勇庆, 李耀坤. 饲喂青贮黄梁木代替青贮玉米川中黑山羊肝转录组的表达分析[J]. 畜牧兽医学报, 2024, 55(1): 232-244.
[6]	陈玥彤, 刘晓涵, 王芷洋, 赵宇馨, 周铁忠, 胡增金, 朱悦, 王少辉, 田明星, 丁思羽, 祁晶晶, 于圣青. 广东规模化鸡场死鸡胚中鸡毒支原体的分离鉴定、致病性及药物敏感性[J]. 畜牧兽医学报, 2024, 55(1): 290-299.
[7]	唐崟梅, 李琪, 李海洋, 林亚秋, 王永, 向华, 黄炼, 朱江江. 山羊FATP2基因的克隆及对前体脂肪细胞脂质沉积的影响[J]. 畜牧兽医学报, 2023, 54(9): 3642-3652.
[8]	邵鹏, 唐崟梅, 林亚秋, 王永, 向华, 黄炼, 朱江江. PSMD9对山羊前体脂肪细胞脂质沉积的调控作用研究[J]. 畜牧兽医学报, 2023, 54(9): 3653-3663.
[9]	张寒月, 赵丹, 梁煜, 赵弼时, 范蒙丹, 乔利英, 刘建华, 杨凯捷, 潘洋洋, 刘文忠. miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2023, 54(8): 3262-3274.
[10]	刘林丽, 彭雪兰, 李博, 程乐凡, 次仁拉姆, 张恩平. 过表达UCP3基因对萨湖羊前体脂肪细胞分化的影响[J]. 畜牧兽医学报, 2023, 54(8): 3275-3285.
[11]	贺名扬, 马钰静, 王泳, 杨若晨, 刘月琴, 张英杰, 段春辉. 褪黑激素对绵羊卵巢颗粒细胞增殖、凋亡、类固醇激素分泌的影响[J]. 畜牧兽医学报, 2023, 54(8): 3313-3324.
[12]	宋美君, 郝科兴, 海思妤, 陈岩, 王静, 胡广东. SRIF-14对绵羊子宫内膜上皮细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2023, 54(8): 3325-3334.
[13]	李悦欣, 刘爱菊, 马晓菲, 郑忠, 胡伯欣, 智云霞, 田树军. TGFβR1介导TGF-β/Smad信号通路对绵羊颗粒细胞功能的影响[J]. 畜牧兽医学报, 2023, 54(8): 3335-3347.
[14]	马友记, 陈鹏飞, 马青, 吴怡. 不同运动量对滩羊瘤胃菌群多样性的影响[J]. 畜牧兽医学报, 2023, 54(8): 3393-3405.
[15]	张颖, 金华, 韩杨, 眭丹, 肖鑫, 郝秀静, 李敏. 患呼吸道疾病羊鼻腔及生活环境微生物多样性分析[J]. 畜牧兽医学报, 2023, 54(8): 3455-3465.