六十二株水貂源大肠杆菌分离株耐药表型与耐药基因及克隆菌群分析

doi:10.11843/j.issn.0366-6964.2019.12.014

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (12): 2498-2508.doi: 10.11843/j.issn.0366-6964.2019.12.014

六十二株水貂源大肠杆菌分离株耐药表型与耐药基因及克隆菌群分析

蒋智宇¹, 王凡¹, 杨溢², 衣首静¹, 杨杰¹, 鞠孜敬¹, 宋艳¹, 朱国强², 孙淑红^1,3,4*

1. 山东农业大学, 泰安 271000;
2. 扬州大学兽医学院, 扬州 225009;
3. 山东省动物生物工程与疾病防治重点实验室, 泰安 271000;
4. 山东省畜禽疫病防制工程技术研究中心, 泰安 271000

收稿日期:2019-04-15 出版日期:2019-12-23 发布日期:2019-12-20
通讯作者: 孙淑红,主要从事预防兽医学相关研究,E-mail:sunshuhong@sdau.edu.cn
作者简介:蒋智宇(1993-),男,安徽亳州人,硕士,主要从事动物疾病与防控研究,E-mail:380402876@qq.com;王凡(1995-),女,山东郓城人,硕士,主要从事分子生物学研究,E-mail:wf20170719@163.com
基金资助:
“十三五”国家重点研发计划（2017YFD0500105）；泰山学者计划（201511023）；山东省“双一流”计划

Analysis of Drug Resistance Phenotype, Drug Resistance Genes and Cloned Flora of 62 Mink-derived Escherichia coli Isolates

JIANG Zhiyu¹, WANG Fan¹, YANG Yi², YI Shoujing¹, YANG Jie¹, JU Zijing¹, SONG Yan¹, ZHU Guoqiang², SUN Shuhong^1,3,4*

1. Shandong Agricultural University, Tai'an 271000, China;
2. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
3. Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an 271000, China;
4. Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Tai'an 271000, China

Received:2019-04-15 Online:2019-12-23 Published:2019-12-20

摘要/Abstract

摘要： 为了调查诸城地区某水貂养殖场粪便源大肠杆菌的表观及其分子特征，采集某个水貂养殖场的水貂粪便进行大肠杆菌分离鉴定，对分离鉴定的大肠杆菌进行血清型鉴定和对14种常见抗菌药物的耐药表型鉴定；使用PCR检测耐药基因以及Ⅰ整合子基因盒的携带情况，利用多位点序列分型（MLST）来分析菌株的克隆关系并构建系统发育树来分析相同克隆群菌株的遗传相似性。结果显示，自82份水貂粪便样品分离到62株大肠杆菌，分离率75.61%；大肠杆菌分离株对AMP和TET的耐药率超过90%，多重耐药菌株（MDR）占比为85.48%。PCR检测到5类耐药基因的存在，qnrS检出率最高，为61.29%（38/62）；aaC2、aaC4、sul1和aac（6'）-Ib-cr耐药基因与菌株产生相应的耐药抗性存在一致性（P<0.01）。分离菌株中Ⅰ类整合子可变区域的优势结构为dfrA27-aadA2-qnrA。鉴定出致病性血清型的存在，且对应菌株都具有多重耐药性，优势血清型为O104：H4。分离株中存在33个STs，ST46为优势STs（16.13%），具有3个主要克隆群，依次为CC10、CC46和CC176；与致病性相关菌株的STs和人源大肠杆菌具有共同的遗传背景。本研究表明，养殖场的水貂受到致病性和多重耐药性大肠杆菌的污染，相同克隆群菌株的耐药基因分布具有多态性，表观特征差异明显。

关键词: 水貂源大肠杆菌, 致病性血清型, 耐药基因, 克隆群, 系统发育树

Abstract: This experiment was conducted to investigate the apparent characteristics and molecular characteristics of Escherichia coli isolates from the feces of a mink farm in Zhucheng. Mink feces from the mink farm were collected for isolating E. coli strains, then the isolates were identified, and their serotypes and drug resistance phenotypes to 14 common antibacterial drugs were determined. The drug resistance genes and the carrying status of the Ⅰ integron gene cassette of these E. coli isolates were detected by using PCR. Multi-locus sequence typing (MLST) was used to analyze the clonal relationship of these strains,and a phylogenetic tree was constructed to analyze the genetic similarity of the same clonal strains. The results showed that 62 strains of E. coli were isolated from 82 samples of mink feces, with a separation rate of 75.61%, and the resistance rate of E. coli isolates to AMP and TET exceeded 90%, and the multidrug-resistant strain (MDR) accounted for 85.48%. Five drug-resistant genes were detected by PCR, and the detection rate of qnrS was the highest (61.29%,38/62). The aaC2, aaC4, sul1 and aac(6')-Ib-cr resistance genes were consistent with the corresponding resistance of these strains (P<0.01). The dominant structure of the class Ⅰ integron variable region in the isolated strain was dfrA27-aadA2-qnrA. The pathogenic serotype was identified and the corresponding strains had multiple drug resistance. The dominant serotype was O104:H4. There were 33 STs in the isolates, and ST46 was the dominant ST (16.13%). There were 3 main clones, CC10, CC46 and CC176, respectively. The STs strains associated with pathogenicity share a common genetic background with human E. coli. The results showed that the mink of the farm were contaminated by pathogenic and multi-drug resistant E. coli. The distribution of drug resistance genes in the same clonal strains showed significant differences in polymorphism and epigenetic characteristics.

Key words: Escherichia coli from mink, pathogenic serotype, drug resistance gene, clonal complexes, phylogenetic tree

中图分类号:

S852.612

蒋智宇, 王凡, 杨溢, 衣首静, 杨杰, 鞠孜敬, 宋艳, 朱国强, 孙淑红. 六十二株水貂源大肠杆菌分离株耐药表型与耐药基因及克隆菌群分析[J]. 畜牧兽医学报, 2019, 50(12): 2498-2508.

JIANG Zhiyu, WANG Fan, YANG Yi, YI Shoujing, YANG Jie, JU Zijing, SONG Yan, ZHU Guoqiang, SUN Shuhong. Analysis of Drug Resistance Phenotype, Drug Resistance Genes and Cloned Flora of 62 Mink-derived Escherichia coli Isolates[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12): 2498-2508.

参考文献 33

[1]	陈晨,李朋,韩铠怿,等. 貂源绿脓杆菌的分离鉴定及其致病性[J]. 兽类学报, 2015, 35(2):196-202.CHEN C, LI P, HAN K Z, et al. Identification and Pathogenicity test of Pseudomonas aeruginosa from mink[J]. Acta Theriologica Sinica, 2015, 35(2):196-202. (in Chinese)
[2]	钟世勋, 迟珊珊,王振,等. 山东规模化养殖场毛皮动物多重感染病原学分析[J]. 中国兽医学报, 2014, 34(11):1770-1777.ZHONG S X, CHI S S, WANG Z, et al. The etiology analysis of multiple infections in Shandong scale fur farms[J]. Chinese Journal of Veterinary Science, 2014, 34(11):1770-1777. (in Chinese)
[3]	芮萍,刘曜综,马增军,等. 貂源伪狂犬病病毒的分离鉴定及gE基因分子特征[J]. 畜牧兽医学报, 2015, 46(7):1268-1272.RUI P, LIU Y Z, MA Z J, et al. Identification and glycoprotein e characteristic of pseudorabies virus from mink[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(7):1268-1272. (in Chinese)
[4]	王春梅, 何启盖,操继跃. 细菌多重耐药泵的研究进展[J]. 畜牧兽医学报, 2011, 42(4):455-467.WANG C M, HE Q G, CAO J Y. Multidrug efflux systems involved in resistance to antibacterial drugs[J]. Acta Veterinaria et Zootechnica Sinica, 2011, 42(4):455-467. (in Chinese)
[5]	冯敏燕, 林树乾,房玉波,等. 山东省鸡源致病性大肠杆菌O-抗原混合血清型分析[J]. 中国兽医学报, 2017, 37(9):1676-1679.FENG M Y, LIN S Q, FANG Y B, et al. Analysis of mixed O-antigen serotypes of E. coli isolated from chickens in Shandong province[J]. Chinese Journal of Veterinary Science, 2017, 37(9):1676-1679. (in Chinese)
[6]	金美伶, 白雪,王峰,等. 水貂大肠杆菌性腹泻的诊断与治疗[J]. 中国兽医杂志, 2015, 51(10):45-46.JIN M L, BAI X, WANG F, et al. Diagnosis and treatment of diarrhea caused by Escherichia coli in mink[J]. Chinese Journal of Veterinary Medicine, 2015, 51(10):45-46. (in Chinese)
[7]	田甜甜. 环境多重耐药细菌中分子遗传特征及耐药传播机制研究[D]. 新乡:河南师范大学, 2017.TIAN T T. Molecular characterization and transmission of antibiotic resistance in environmental multidrug resistant bacteria[D]. Xinxiang:Henan Normal University, 2017. (in Chinese)
[8]	KOCZURA R, MOKRACKA J, BARCZAK A, et al. Association between the presence of class 1 integrons, virulence genes, and phylogenetic groups of Escherichia coli isolates from river water[J]. Microb Ecol, 2013, 65(1):84-90.
[9]	CLERMONT O, OLIER M, HOEDE C, et al. Animal and human pathogenic Escherichia coli strains share common genetic backgrounds[J]. Infect, Genet Evol, 2011, 11(3):654-662.
[10]	王龙光, 姜雯,逄春华,等. 利用MLST技术探讨不同地区致病性耐药鸡源大肠杆菌的遗传进化关系[J]. 中国兽医学报, 2017, 37(9):1680-1686.WANG L G, JIANG W, PANG C H, et al. Genetic evolutionary relationship of drug-resistant and pathogenic chicken originated Escherichia coli strains from different regions with MLST[J]. Chinese Journal of Veterinary Science, 2017, 37(9):1680-1686. (in Chinese)
[11]	ZHAO X N, YANG J, JU Z J, et al. Molecular characterization of antimicrobial resistance in Escherichia coli from rabbit farms in Tai'an, China[J]. BioMed Res Int, 2018, 2018:8607647.
[12]	WIRTH T, FALUSH D, LAN R, et al. Sex and virulence in Escherichia coli:an evolutionary perspective[J]. Mol Microbiol, 2006, 60(5):1136-1151.
[13]	OOKA T, SETO K, KAWANO K, et al. Clinical significance of Escherichia albertii[J]. Emerg Infect Dis, 2012, 18(3):488-492.
[14]	EVANGELISTA D, THOUZÉ F, KOHLI M K, et al. Topological support and data quality can only be assessed through multiple tests in reviewing Blattodea phylogeny[J]. Mol Phylogenet Evol, 2018, 128:112-122.
[15]	TELLES G P, ARAÚJO G S, WALTER M E M T, et al. Live neighbor-joining[J]. BMC Bioinformatics, 2018, 19:172.
[16]	TRUCCO M I, BURATTI C C. Taxonomic review of Argentine mackerel Scomber japonicus (Houttuyn, 1782) by phylogenetic analysis[J]. Mol Biol Res Commun, 2017, 6(3):141-152.
[17]	JI X W, LIAO Y L, ZHU Y F, et al. Multilocus sequence typing and virulence factors analysis of Escherichia coli O157 strains in China[J]. J Microbiol, 2010, 48(6):849-855.
[18]	LEWIS II J S, HERRERA M, WICKES B, et al. First report of the emergence of CTX-M-type extended-spectrum β-lactamases (ESBLs) as the predominant ESBL isolated in a U. S. health care system[J]. Antimicrob Agents Chemother, 2007, 51(11):4015-4021.
[19]	OLIVEIRA M, FREIRE D, PEDROSO N M. Escherichia coli is not a suitable fecal indicator to assess water fecal contamination by otters[J]. Braz J Biol, 2017, 78(1):155-159.
[20]	李莎莎. 山东省禽源大肠杆菌血清型鉴定及喹诺酮类药物耐药性研究[D]. 泰安:山东农业大学, 2010.LI S S. Fluoroquinolones resistance and serogroups of Escherichia coli from Shandong province of China[D]. Tai'an:Shandong Agricultural University, 2010. (in Chinese)
[21]	潘鑫. 山东省高密地区鸭源大肠杆菌的分离鉴定及耐药基因检测[D]. 泰安:山东农业大学, 2012.PAN X. Isolation, identification and detection of drug resistance genes of duck E. coli isolates from Gaomi in Shandong province[D]. Tai'an:Shandong Agricultural University, 2012. (in Chinese)
[22]	SAFADI R A, ABU-ALI G S, SLOUP R E, et al. Correlation between in vivo biofilm formation and virulence gene expression in Escherichia coli O104:H4[J]. PLoS One, 2012, 7(7):e41628.
[23]	DEVAUX I, VARELA-SANTOS C, PAYNE-HALLSTRÖM L, et al. Investigation of travel-related cases in a multinational outbreak:example of the Shiga-toxin producing E. coli outbreak in Germany, May-June 2011[J]. Epidemiol Infect, 2015, 143(16):3468-3474.
[24]	KUMARASAMY K K, TOLEMAN M A, WALSH T R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK:a molecular, biological, and epidemiological study[J]. Lancet Infect Dis, 2010, 10(9):597-602.
[25]	PEDERSEN K, HAMMER A S, SØRENSEN C M, et al. Usage of antimicrobials and occurrence of antimicrobial resistance among bacteria from mink[J]. Vet Microbiol, 2009, 133(1-2):115-122.
[26]	RADHOUANI H, IGREJAS G, GONÇALVES A, et al. Antimicrobial resistance and virulence genes in Escherichia coli and enterococci from red foxes (Vulpes vulpes)[J]. Anaerobe, 2013, 23:82-86.
[27]	ROTIMI V O, JAMAL W, PAL T, et al. Emergence of CTX-M-15 type extended-spectrum β-lactamase-producing Salmonella spp. in Kuwait and the United Arab Emirates[J]. J Med Microbiol, 2008, 57(7):881-886.
[28]	BRYAN A, SHAPIR N, SADOWSKY M J. Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli Strains isolated from diverse human and animal sources[J]. Appl Environ Microbiol, 2004, 70(4):2503-2507.
[29]	GILLINGS M R. Class 1 integrons as invasive species[J]. Curr Opin Microbiol, 2017, 38:10-15.
[30]	NICOLAS-CHANOINE M H, BERTRAND X, MADEC J Y. Escherichia coli ST131, an intriguing clonal group[J]. Clin Microbiol Rev, 2014, 27(3):543-574.
[31]	LARSEN M V, COSENTINO S, RASMUSSEN S, et al. Multilocus sequence typing of total-genome-sequenced bacteria[J]. J Clin Microbiol, 2012, 50(4):1355-1361.
[32]	MANGES A R, JOHNSON J R. Food-borne origins of Escherichia coli causing extraintestinal infections[J]. Clin Infect Dis, 2012, 55(5):712-719.
[33]	CHEN Y, CHEN X, ZHENG S, et al. Serotypes, genotypes and antimicrobial resistance patterns of human diarrhoeagenic Escherichia coli isolates circulating in southeastern China[J]. Clin Microbiol Infect, 2014, 20(1):52-58.

六十二株水貂源大肠杆菌分离株耐药表型与耐药基因及克隆菌群分析

Analysis of Drug Resistance Phenotype, Drug Resistance Genes and Cloned Flora of 62 Mink-derived Escherichia coli Isolates

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 33

相关文章 15

编辑推荐

Metrics

本文评价

[1]	赵菲菲, 李杰, 韩宁, 谢仕廷, 曾振灵. 分离自屠宰场的肺炎克雷伯菌的耐药性分析[J]. 畜牧兽医学报, 2023, 54(7): 3044-3053.
[2]	蒋增海, 滕霖, 贺安文, 刘言言, 乐敏, 何启盖. 猪产业链中鼠伤寒沙门菌及沙门菌血清型4,[5],12:i:-基因组学分析[J]. 畜牧兽医学报, 2023, 54(3): 1199-1209.
[3]	陶洁, 李本强, 程靖华, 石迎, 刘佩红, 刘惠莉. 肉鸽肠道微生物菌群差异分析和抗生素耐药基因预测[J]. 畜牧兽医学报, 2023, 54(12): 5293-5300.
[4]	杨梦林, 郑世奇, 彭凯, 王玮, 黄燕华, 彭杰. 鸽源鼠伤寒沙门菌的分离鉴定及致病性分析[J]. 畜牧兽医学报, 2023, 54(11): 4880-4888.
[5]	赵学亮, 王斌, 苗永强, 赵浩宇, 谢青芳, 王娟, 杨增岐. 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.
[6]	余蕴, 向勇, 李庆钵, 刘鹏, 黎丽珍, 廖明, 曹伟胜. 种蛋孵化死胚及孵化厅环境样本中铜绿假单胞菌的分离鉴定及其耐药性分析[J]. 畜牧兽医学报, 2022, 53(2): 548-555.
[7]	刘健, 白艺兰, 李鑫, 桂亚萍, 鞠厚斌, 龚国华, 夏炉明, 陈伟锋, 朱晓英, 常晓静, 李增强, 唐聪圣, 王建, 赵洪进. 猫杯状病毒分离株VP1基因序列与致病性分析[J]. 畜牧兽医学报, 2022, 53(10): 3530-3539.
[8]	张海霞, 张振彪, 代禾根, 宋昱, 李蕾, 夏兆飞. 中国部分地区犬源伪中间葡萄球菌的流行特征分析[J]. 畜牧兽医学报, 2021, 52(9): 2561-2568.
[9]	章婧, 蔡萍, 贺奕卓, 陈伟涛, 黄宇晨, 蒋红霞. 广东省养禽场肠球菌耐药性及毒力因子流行分布特征[J]. 畜牧兽医学报, 2021, 52(9): 2650-2659.
[10]	张耀东, 朱红, AFAYIBO Dossêh Jean Apôtre, 王瑶, 易正飞, 信素华, 陶程琳, 李涛, 祁晶晶, 田明星, 丁铲, 于圣青, 王少辉. 常见耐药基因多重PCR方法的建立及用于禽致病性大肠杆菌耐药性监测[J]. 畜牧兽医学报, 2020, 51(12): 3193-3198.
[11]	邓雯文, 李才武, 晋蕾, 李果, 吴虹林, 张贵权, 魏荣平, 陈锐, 王明磊, 何永果, 李倜, 李德生, 张和民, 黄炎, 邹立扣. 大熊猫粪便中微生物与寄生虫的宏转录组学分析[J]. 畜牧兽医学报, 2020, 51(11): 2812-2824.
[12]	龚永平, 陈珍容, 阴文奇, 文继峰, 易可可, 颜其贵. 林麝源屎肠球菌LS170308株多重耐药基因岛PRI1分析[J]. 畜牧兽医学报, 2019, 50(6): 1275-1283.
[13]	王少君, 孙康泰, 熊智颖, 瞿孝云, 马叶本, 陈政权, 张红霞, 温俊平, 白洁, 詹泽强, 高远, 韦懿桓, 廖明, 张建民. 广东省零售市场鸡肉中肯塔基沙门菌的流行情况及耐药性分析[J]. 畜牧兽医学报, 2019, 50(12): 2509-2517.
[14]	徐耀辉, 邓同炜, 齐亚如, 韩文龙, 卢建洲, 蒋增海. 三黄种鸡场孵化死胚中沙门菌的分离鉴定与耐药性分析[J]. 畜牧兽医学报, 2018, 49(5): 1081-1088.
[15]	孙朋浩, 邱荣超, 李书宁, 郑双双, 杨伟聪, 张丽娜, 符颖, 詹泽强, 张建民, 廖明, 瞿孝云. 广州市生鲜鸡中肠炎沙门菌的监测及耐药基因的分子鉴定[J]. 畜牧兽医学报, 2018, 49(2): 388-395.