新疆地区腹泻仔猪源大肠杆菌的分群、血清型鉴定及耐药性分析

doi:10.11843/j.issn.0366-6964.2023.01.038

摘要/Abstract

摘要： 旨在了解新疆地区腹泻仔猪源大肠杆菌的系统进化分群、血清型及耐药性。本研究对154份腹泻仔猪粪便样品进行大肠杆菌的分离鉴定，采用多重PCR方法对分离株进行系统进化分群和O血清型鉴定，通过K-B纸片法对其进行药物敏感性检测并通过PCR方法进行耐药基因检测。结果显示：共分离到154株大肠杆菌，包括ETEC(n=24)、STEC(n=21)、EPEC(n=1)、EPEC/STEC(n=2)、ETEC/STEC(n=1)和ETEC/EPEC(n=1)，其他104株。系统进化分群显示，多数菌株属于B1(37%)和A群(31%)。定型菌株44株，分别属于10种血清型，以O154、O12、O8、O141和O175为主要流行血清型。151株(98%)为多重耐药菌，对复方新诺明、四环素、氨苄西林、链霉素和氯霉素的耐药率为81%~100%，对阿莫西林/克拉维酸、头孢噻肟、庆大霉素、头孢曲松、环丙沙星和阿米卡星的耐药率为31%~66%，对左氧氟沙星、多黏菌素B、头孢他啶、头孢吡肟、氨苄西林-舒巴坦、哌拉西林-他唑巴坦和亚胺培南的耐药率为1%~19%。耐药基因tetA(88%)、tetG(60%)和cmlA(45%)的携带率较高，而bla_CTX-M-1G、aadA1、sul1、aac和bla_CTX-M-9G均低于30%，未检测出bla_CTX-M-2G、bla_TEM、bla_SHV和tetE。研究结果表明，新疆腹泻仔猪源大肠杆菌类型复杂，多重耐药形势严峻，耐药基因多样化，且检测出人医临床重要的抗生素耐药表型，应加强对猪场大肠杆菌的耐药性监测。

关键词: 腹泻仔猪, 大肠杆菌, 系统进化分群, 血清型, 耐药性

Abstract: This study was conducted to examine the distribution of phylogenetic clustering, virulence genes, serotypes and drug resistance of Escherichia coli (E. coli) from diarrhea with piglets in Xinjiang. In this study, 154 fecal samples were collected from diarrhea with piglets, and isolation, identification of E. coli was conducted. Phylogenetic clustering, virulence genes, serotype were tested by multiple PCR method. Drug sensitivity of isolates was determined by using a Kirby-Bauer disk diffusion method and drug resistance gene was detected by PCR. The results showed that 154 E. coli were isolated, including ETEC (n=24), STEC (n=21), EPEC (n=1), EPEC/STEC (n=2), ETEC/STEC (n=1), ETEC/EPEC (n=1), and other 104 strains. Phylotyping assays showed that most strains largely belong to group A (37%) and B1 (31%). O serogroups were identified for 44 E. coli isolates, of which O154, O12, O8, O141 and O175 were dominant serogroups. One hundred and fifty-one strains were multiple drug resistance (MDR), and the drug resistance rates of 154 E. coli isolates to cotrimoxazole, tetracycline, ampicillin, streptomycin and chloramphenicol ranged from 81% to 100%, that to amoxicillin/clavulanate, cefotaxime, gentamicin, ceftriaxone, ciprofloxacin and amikacin ranged from 31% to 66%, that to levofloxacin, polymyxins B, ceftazidime, cefepime, ampicillin -sulbactam, piperacillin -tazobactam and imipenem ranged from 1% to 19%. The prevalence of drug resistance genes tetA (88%), tetG (60%) and cmlA (45%) was higher, while bla_CTX-M-2G, bla_TEM, bla_SHVand tetE were all lower than 30%. bla_CTX-M-2G, bla_TEM, bla_SHV and tetE were not detected. The results indicated that the types of E. coli from diarrhea with piglets in Xinjiang were complex and the situation of MDR was severe, the prevalence of drug resistance genes was diversified, and important antibiotic resistance phenotypes were detected in human and clinical, so the monitoring of drug resistance of E. coli in pig farms should be strengthened.

Key words: diarrhea with piglets, Escherichia coli, phylogenetic clustering, serum group, drug resistance

中图分类号:

佟盼盼, 黄顺敏, 王芋丹, 施旭辉, 陈文霞, 宋鑫龙, 张毅, 苏战强, 谢金鑫. 新疆地区腹泻仔猪源大肠杆菌的分群、血清型鉴定及耐药性分析[J]. 畜牧兽医学报, 2023, 54(1): 414-420.

TONG Panpan, HUANG Shunmin, WANG Yudan, SHI Xuhui, CHEN Wenxia, SONG Xinlong, ZHANG Yi, SU Zhanqiang, XIE Jinxin. Phylogenetic Clustering, Serotype and Drug Resistance Analysis of Escherichia coli from Diarrhea with Piglets in Xinjiang[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 414-420.

参考文献 31

[1]	张铁, 王春光, 王谦, 等. 猪源大肠杆菌的分离、鉴定及耐药性监测[J]. 中国农学通报, 2005, 21(12):23-25, 30.ZHANG T, WANG C G, WANG Q, et al. Serological identification and surveillance of susceptibility to antibiotics and isolate of Escherichia coli from swine[J]. Chinese Agricultural Science Bulletin, 2005, 21(12):23-25, 30. (in Chinese)
[2]	王秀梅, 蒋红霞, 廖晓萍, 等. 猪源致病性大肠杆菌的血清型、毒力基因及抗菌药耐药性的调查[J]. 中国农业科学, 2010, 43(19):4109-4115.WANG X M, JIANG H X, LIAO X P, et al. Prevalence of serotypes and virulence genes and antimicrobial susceptibility of pathogenic Escherichia coli isolates from swine[J]. Scientia Agricultura Sinica, 2010, 43(19):4109-4115. (in Chinese)
[3]	BRILHANTE M, PERRETEN V, DONÀ V. Multidrug resistance and multivirulence plasmids in enterotoxigenic and hybrid Shiga toxin-producing/enterotoxigenic Escherichia coli isolated from diarrheic pigs in Switzerland[J]. Vet J, 2019, 244:60-68.
[4]	BRAND P, GOBELI S, PERRETEN V. Pathotyping and antibiotic resistance of porcine enterovirulent Escherichia coli strains from Switzerland (2014-2015)[J]. Schweiz Arch Tierheilkd, 2017, 159(7):373-380.
[5]	LEONARD S R, MAMMEL M K, RASKO D A, et al. Hybrid Shiga Toxin-producing and Enterotoxigenic Escherichia sp. cryptic lineage 1 strain 7v harbors a hybrid plasmid[J]. Appl Environ Microbiol, 2016, 82(14):4309-4319.
[6]	葛海燕. 包头地区仔猪源致泻性大肠杆菌进化分群、生物被膜形成能力及耐药性检测[J]. 中国兽医杂志, 2020, 56(1):81-84.GE H Y. Evolutionary cluster and biofilm formation ability of laxative E. coli from piglets in Baotou area and detection of drug resistance[J]. Chinese Journal of Veterinary Medicine, 2020, 56(1):81-84. (in Chinese)
[7]	孟婷,崔平福,董亚青,等. 江苏地区仔猪腹泻性大肠杆菌的分离鉴定及其生物学特性研究[J]. 中国预防兽医学报, 2021, 43(2):202-206.MENG T, CUI P F, DONG Y Q, et al. Isolation, identification and biological characteristics of diarrheagenic E. coli from piglets in Jiangsu Province[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(2):202-206. (in Chinese)
[8]	TEICHMANN A, DE CUNHA AGRA H N, DE SOUZA NUNES L, et al. Antibiotic resistance and detection of the sul2 gene in urinary isolates of Escherichia coli in patients from Brazil[J]. J Infect Dev Ctries, 2014, 8(1):39-43.
[9]	杨德鸿, 张鹏, 于沛欣, 等. 苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估[J]. 微生物学报, 2020, 60(2):261-272.YANG D H, ZHANG P, YU P X, et al Isolation, identification and evaluation of inactivated vaccine of enterotoxigenic Escherichia coli (ETEC) in large-scale pig farms in northern Jiangsu[J]. Acta Microbiologica Sinica, 2020, 60(2):261-272. (in Chinese)
[10]	VORA G J, MEADOR C E, STENGER D A, et al. Nucleic acid amplification strategies for DNA microarray-based pathogen detection[J]. Appl Environ Microbiol, 2004, 70(5):3047-3054.
[11]	PATON A W, PATON J C. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157[J]. J Clin Microbiol, 1998, 36(2):598-602.
[12]	CLERMONT O, CHRISTENSON J K, DENAMUR E, et al. The Clermont Escherichia coli phylo-typing method revisited:improvement of specificity and detection of new phylo-groups[J]. Environ Microbiol Rep, 2013, 5(1):58-65.
[13]	IGUCHI A, IYODA S, SETO K, et al. Escherichia coli O-genotyping PCR:a comprehensive and practical platform for molecular O serogrouping[J]. J Clin Microbiol, 2015, 53(8):2427-2432.
[14]	PAGANI L, DELL'AMICO E, MIGLIAVACCA R, et al. Multiple CTX-M-type extended-spectrum β-lactamases in nosocomial isolates of Enterobacteriaceae from a hospital in northern Italy[J]. J Clin Microbiol, 2003, 41(9):4264-4269.
[15]	ECKERT C, GAUTIER V, ARLET G. DNA sequence analysis of the genetic environment of various blaCTX-M genes[J]. J Antimicrob Chemother, 2006, 57(1):14-23.
[16]	LIN C F, HSU S K, CHEN C H, et al. Genotypic detection and molecular epidemiology of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a regional hospital in central Taiwan[J]. J Med Microbiol, 2010, 59(6):665-671.
[17]	NG L K, MARTIN I, ALFA M, et al. Multiplex PCR for the detection of tetracycline resistant genes[J]. Mol Cell Probes, 2001, 15(4):209-215.
[18]	SENZ Y, BRIÑAS L, DOMÍNGUEZ E, et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins[J]. Antimicrob Agents Chemother, 2004, 48(10):3996-4001.
[19]	梁乔, 赵凤菊, 顾贵波, 等. 辽宁地区猪源大肠埃希菌氨基糖苷类抗生素耐药基因的检测与分析[J]. 河南农业科学, 2017, 46(6):134-137.LIANG Q, ZHAO F J, GU G B, et al. Detection and analysis of resistance genes to aminoglycoside antibiotics among swine Escherichia coli strains in Liaoning area[J]. Journal of Henan Agricultural Sciences, 2017, 46(6):134-137. (in Chinese)
[20]	KERRN M B, KLEMMENSEN T, FRIMODT-MØLLER N, et al. Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance[J]. J Antimicrob Chemother, 2002, 50(4):513-516.
[21]	KEYES K, HUDSON C, MAURER J J, et al. Detection of florfenicol resistance genes in Escherichia coli isolated from sick chickens[J]. Antimicrob Agents Chemother, 2000, 44(2):421-424.
[22]	Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Twenty-nine informational supplement[S]. PA:CLSI, 2019.
[23]	KIRATISIN P, APISARNTHANARAK A, LAESRIPA C, et al. Molecular characterization and epidemiology of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in thailand, where the CTX-M family is endemic[J]. Antimicrob Agents Chemother, 2008, 52(8):2818-2824.
[24]	VU-KHAC H, HOLODA E, PILIPCINEC E, et al. Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia[J]. Vet J, 2007, 174(1):176-187.
[25]	JOHNSON J R, KUSKOWSKI M A, O'BRYAN T T, et al. Epidemiological correlates of virulence genotype and phylogenetic background among Escherichia coli blood isolates from adults with diverse-source bacteremia[J]. J Infect Dis, 2002, 185(10):1439-1447.
[26]	李金朋, 温文彦, 靳曼玉, 等. 河南地区猪源大肠杆菌分子分群、耐药性及生物被膜表型分析[J]. 中国预防兽医学报, 2018, 40(3):190-194.LI J P, WEN W Y, JIN M Y, et al. Phylogenetic group, drug resistance and biofilm formation of Escherichia coli isolated from pigs in Henan regions[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(3):190-194. (in Chinese)
[27]	彭珂楠, 周雪珂, 殷鑫欢, 等. 四川地区猪源致病性大肠埃希菌分子分群、生物被膜形成能力及耐药性研究[J]. 浙江农业学报, 2019, 31(10):1599-1607.PENG K N, ZHOU X K, YIN X H, et al. Molecular typing, biofilm formation ability and drug resistance of pathogenic Escherichia coli isolated from pigs in Sichuan province[J]. Acta Agriculturae Zhejiangensis, 2019, 31(10):1599-1607. (in Chinese)
[28]	孙慧琴, 轩慧勇, 买占海, 等. 猪源大肠杆菌耐药性及相关耐药基因检测[J]. 新疆农业科学, 2021, 58(1):190-196.SUN H Q, XUAN H Y, MAI Z H, et al. Investigation on drug resistance and detection of related resistance genes of Escherichia coli from pigs[J]. Xinjiang Agricultural Sciences, 2021, 58(1):190-196. (in Chinese)
[29]	刘哲, 孙洋, 纪雪, 等. 仔猪腹泻大肠杆菌流行株鉴定及毒力与耐药性分析[J]. 中国畜牧兽医, 2017, 44(4):1226-1232.LIU Z, SUN Y, JI X, et al. Identification, virulence and drug resistance analysis of Escherichia coli from diarrhea piglets[J]. China Animal Husbandry & Veterinary Medicine, 2017, 44(4):1226-1232. (in Chinese)
[30]	KENNEDY C A, FANNING S, KARCZMARCZYK M et al. Characterizing the multidrug resistance of non-O157 Shiga toxin-producing Escherichia coli isolates from cattle farms and abattoirs[J]. Microb Drug Resist, 2017, 23(6):781-790.
[31]	王宏栋, 徐国锋, 矫薇薇, 等. 猪源氟喹诺酮耐药大肠杆菌通过接合水平传递耐药性的研究[J]. 畜牧兽医学报, 2016, 47(4):805-811.WANG H D, XU G F, JIAO W W, et al Investigation of antimicrobial agents resistance transferred horizontally in fluoroquinolone resistant Escherichia coli from swine[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(4):805-811. (in Chinese).