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

doi:10.11843/j.issn.0366-6964.2020.12.029

Abstract

Abstract: To detect the drug resistance genes, the multiplex PCR method for drug resistance genes of β-lactams, tetracyclines, aminoglycosides, amphenicols, and sulfonamides were developed. Based on the sequences of drug resistance genes from GenBank, 17 pairs of specific primers were designed. Then, 4 multiple PCR assays were established through the optimization of PCR reaction conditions and primers concentrations (cat+floR+tetB+tetC; dfrA12+sul2+sul1+bla_CTX-M+bal_TEM-1; aac3+aph3+aadA1+strB; tetA+cmlA+strA+sul3). The sensitivity and specificity of these assays were determined. The multiplex PCR assays were used to detect the drug resistance genes of 42 avian pathogenic Escherichia coli (APEC). The drug sensitivity of these APEC strains were also determined, which were compared to the distributions of drug resistance genes in these strains. The results showed that the 17 drug resistance genes were effectively and specifically amplified in these 4 optimized multiplex PCR assays. The detection limits of the 4 multiplex PCR were 10³, 10⁴, 10⁴ and 10⁵CFU of bacteria, respectively. The established multiplex PCR assays are specific and rapid for the detection of drug resistance genes in APEC strains, which showed 92.86% coincident with the drug resistance for these strains. The developed 4 multiplex PCR are simple and rapid assays for drug resistance genes detection, which can be used for the epidemiologic study for drug resistance genes.

Key words: drug resistance gene, multiplex PCR, detection

CLC Number:

S852.612

ZHANG Yaodong, ZHU Hong, AFAYIBO Dossêh Jean Apôtre, WANG Yao, YI Zhengfei, XIN Suhua, TAO Chenglin, LI Tao, QI Jingjing, TIAN Mingxing, DING Chan, YU Shengqing, WANG Shaohui. Development and Application of Multiplex PCR Method for the Drug Resistance Genes Detection in Avian Pathogenic Escherichia coli[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3193-3198.

References 18

[1]	NGUYEN F, STAROSTA A L, ARENZ S, et al. Tetracycline antibiotics and resistance mechanisms[J]. Biol Chem, 2014, 395(5):559-575.
[2]	BABAKHANI S, OLOOMI M. Transposons:the agents of antibiotic resistance in bacteria[J]. J Basic Microbiol, 2018, 58(11):905-917.
[3]	方结红. 生猪屠宰厂大肠杆菌磺胺耐药基因sul1、sul2和sul3的传播与适应性机制研究[D]. 杭州:浙江工商大学, 2018.FANG J H. Research on the dissemination and fitness mechanism of Escherichia coli sulfonamide-resistant genes sul1, sul2 and sul3 in a pig slaughterhouse[D]. Hangzhou:Zhejiang Gongshang University, 2018. (in Chinese)
[4]	叶满玉. 动物源细菌对β-内酰胺类药物耐药基因三重PCR检测方法建立与应用[D]. 成都:四川农业大学, 2009.YE M Y. The establishment and application on multiplex PCR detection of animal original bacteria β-Lactamase resistance genes[D]. Chengdu:Sichuan Agricultural University, 2009. (in Chinese)
[5]	MARTíNEZ J L. Antibiotics and antibiotic resistance genes in natural environments[J]. Science, 2008, 321(5887):365-367.
[6]	BOHNERT J A, KERN W V. Selected arylpiperazines are capable of reversing multidrug resistance in Escherichia coli overexpressing RND efflux pumps[J]. Antimicrob Agents Chemother, 2005, 49(2):849-852.
[7]	蒋智宇, 王凡, 杨溢, 等. 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 Z Y, WANG F, YANG Y, et al. Analysis of drug resistance phenotype, drug resistance genes and cloned flora of 62 mink-derived Escherichia coli isolates[J]. Acta Veterinaria et Zootechnica Sinica, 2011, 50(12):2498-2508. (in Chinese)
[8]	FALAGAS M E, KARAGEORGOPOULOS D E. Pandrug resistance (PDR), extensive drug resistance (XDR), and multidrug resistance (MDR) among Gram-Negative Bacilli:need for international harmonization in terminology[J]. Clin Infect Dis, 2008, 46(7):1121-1122.
[9]	KAPIL A. The challenge of antibiotic resistance:need to contemplate[J]. Indian J Med Res, 2005, 121(2):83-91.
[10]	张扬, 陈丽鹏, 吴华, 等. 鸡源大肠杆菌四环素耐药基因检测[J]. 江西农业学报, 2014, 26(1):94-96, 106.ZHANG Y, CHEN L P, WU H, et al. Detection of tetracycline-tolerant genes in Escherichia coli isolated from chickens[J]. Acta Agriculturae Jiangxi, 2014, 26(1):94-96, 106. (in Chinese)
[11]	杨鑫, 王红宁, 张安云, 等. 大肠杆菌氯霉素类耐药基因三重PCR检测试剂盒的研究与应用[J]. 中国兽医杂志, 2009, 45(2):11-13.YANG X, WANG H N, ZHANG A Y, et al. Development of multiplex PCR detecting kit and its application to the detection of chloramphenicols resistant genes in Escherichia coli[J]. Chinese Journal of Veterinary Medicine, 2009, 45(2):11-13. (in Chinese)
[12]	GRAPE M, SUNDSTR?M L, KRONVALL G. Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources[J]. J Antimicrob Chemother, 2003, 52(6):1022-1024.
[13]	ABDELGADER S A, SHI D L, CHEN M M, et al. Antibiotics resistance genes screening and comparative genomics analysis of commensal Escherichia coli isolated from poultry farms between China and Sudan[J]. BioMed Res Int, 2018, 2018:5327450.
[14]	牛建宁, 崔恩慧, 唐攀, 等. 鸡源致病性大肠杆菌对四环素类抗生素耐药性及耐药基因检测[J]. 西北农业学报, 2014, 23(11):35-39.NIU J N, CUI E H, TANG P, et al. The resistance of pathogenic Escherichia coli isolates from chickens to tetracycline antibiotics and the detection of resistance genes[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2014, 23(11):35-39. (in Chinese)
[15]	GHARAIBEH M H, ABUTARBUSH S M, MUSTAFA F G, et al. Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates[J]. Infect Genet Evol, 2020, 83:104317.
[16]	SHAFIQ M, HUANG J H, RAHMAN S U, et al. High incidence of multidrug-resistant Escherichia coli coharboring mcr-1 and blaCTX-M-15 recovered from pigs[J]. Infect Drug Resist, 2019, 12:2135-2149.
[17]	李帆, 魏单单, 刘建华, 等. 247株猪源沙门菌对四环素类药物耐药性和耐药基因的检测分析[J]. 中国兽医学报, 2019, 39(10):1927-1933.LI F, WEI D D, LIU J H, et al. Detection of tetracycline resistance and resistance genes in 247 Salmonella isolates from swines[J]. Chinese Journal of Veterinary Science, 2019, 39(10):1927-1933. (in Chinese)
[18]	谭耀荣, 李迪, 郑瑶瑶, 等. 一株猪源肺炎克雷伯氏菌的分离鉴定及耐药性、耐药基因的检测[J]. 黑龙江畜牧兽医, 2020(6):57-62.TAN Y R, LI D, ZHENG Y Y, et al. Isolation, identification, drug resistance and antibiotic resistance genes detection of a Klebsiella pneumoniae strain from swine[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(6):57-62.(in Chinese)

Development and Application of Multiplex PCR Method for the Drug Resistance Genes Detection in Avian Pathogenic Escherichia coli

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