西藏牦牛粪便大肠杆菌毒力特性、耐药性与Ⅰ类整合子分子特征相关性分析

doi:10.16431/j.cnki.1671-7236.2023.10.035

摘要/Abstract

摘要： 【目的】牦牛作为西藏自治区人畜共患及多重耐药病原体的重要宿主之一,在养殖过程中滥用抗菌药物是导致牦牛多重耐药发展及传播的主要原因。本研究旨在对西藏牦牛源大肠杆菌菌株毒力特性、耐药性、整合酶、生物被膜表型的相关性进行分析。【方法】在西藏拉萨、林芝、那曲养殖户采集200份牦牛腹泻样品,利用细菌学方法使用麦康凯培养基和伊红-美蓝培养基分离纯化大肠杆菌,用16S rDNA通用引物对疑似大肠杆菌进行PCR扩增及测序,所获序列用NCBI数据库进行BLAST比对。对大肠杆菌进行7类16种抗菌药物敏感性试验,选择致病性关系密切的4类10种毒力基因,以及20个常见耐药基因和2个Ⅰ类整合子进行PCR检测,采用改良半定量结晶紫染色法确定分离大肠杆菌生物被膜表型并进行相关性分析。【结果】共分离鉴定出91株牦牛源大肠杆菌,对7类16种抗菌药物的纸片扩散试验结果表明,大肠杆菌对克林霉素耐药性最强(87.91%),存在多重耐药现象且最多出现13耐。在毒力基因检测中,STEC、ETEC、EPEC、NTEC 4类毒力基因均有阳性存在,其中F17毒力基因占59.34%(54/91),stx1毒力基因占49.45%(45/91)。在22种耐药基因分析中,bla_TEM基因在分离菌株中占主导地位;其次为tetA和sul1基因;在Ⅰ类整合子中,intⅠ1和intⅠ2整合酶基因检出率分别为29.67%(27/91)和1.10%(1/91)。细菌在恶劣环境中生存的主要机制之一是生物被膜形成,在91株大肠杆菌中有48.35%(44/91)表现为弱黏附能力。当大肠杆菌STEC、ETEC检测率高时,耐药基因、Ⅰ类整合子生物被膜检测率也较高,二者呈正相关。【结论】91株西藏牦牛源大肠杆菌毒力基因丰富,耐药基因呈多样化并与Ⅰ类整合子和生物被膜的形成具有一定的相关性。试验结果为牦牛源大肠杆菌耐药机制研究和抗菌药物的合理使用提供了理论依据。

关键词: 西藏牦牛; 大肠杆菌; 毒力基因; 耐药性; 整合酶; 生物被膜

Abstract: 【Objective】 Yaks,as one of the important hosts of zoonotic and multi-drug resistant pathogens in Tibet Autonomous Region,the abuse of antibiotics during the breeding process is the main reason for the development and spread of multi-drug resistance in yaks.The aim of this study was to conduct correlation analysis on the virulence characteristics,drug resistance,integrase and biofilm-forming phenotype of Escherichia coli (E.coli) strains from Tibetan yaks.【Method】 200 samples of yaks diarrhea were collected from farmers in Lhasa,Linzhi,and Nagqu in Tibet.Use bacteriological methods to isolate and purify E.coli by MacConkey Agar medium and Eosin-Methylene Blue Agar medium.PCR amplification and sequencing of the suspected E.coli were performed with 16S rDNA universal primers.The obtained sequence were BLAST aligned in NCBI database.7 classes of 16 antimicrobial drug susceptibility tests were performed on E.coli.Using PCR amplification to detect 4 categories of 10 virulence genes,20 common drug resistance genes,and 2 class Ⅰ integrator that were closely related to pathogenicity.The modified semi-quantitative crystalline violet staining method was performed on the isolated E.coli to determine their biofilm-forming phenotype,and conducted correlation analysis.【Result】 91 strains of E.coli from yaks were isolated and identified.The antibiotic drug sensitivity test of 16 antibiotics in 7 categories showed that E.coli had the strongest drug resistance to clindamycin (87.91%).Meanwhile,there was a phenomenon of multiple drug resistance,with some strains being resistant to up to 13 antibiotics.4 types of virulence genes,STEC,ETEC,EPEC and NTEC,were found to be positive in virulence test,among these detected virulence genes,F17 gene accounted for 59.34% (54/91) and stx1 gene accounted for 49.45% (45/91).Drug resistance gene testing showed that the most common among the 22 drug resistance genes was bla_TEM gene,followed by tetracycline tetA and sul1 genes.Class Ⅰ integrator analysis detected intⅠ1 and intⅠ2 integrase genes,with separately rates of 29.67% (27/91) and 1.10% (1/91).The formation of biofilm was one of the main mechanisms for bacteria to survive in harsh environments.The detection of biofilm showed that 48.35% (44/91) of 91 E.coli strains showed weak adhesion ability,and the data analysis showed that the detection rate of STEC and ETEC of E.coli was positively correlated with the detection rate of drug resistant genes,class Ⅰ integrator and biofilm.【Conclusion】 A variety of virulence genes and drug resistance genes were detected from 91 strains of E.coli isolated from yaks,and the data showed that there was a certain correlation between the diversity of its drug resistance genes and class Ⅰ integrator and the ability of biofilm formation.The results provided a theoretical basis for the study of drug resistance mechanism of E.coli from yaks and the reasonable use of antibiotics.

Key words: Tibetan yaks; Escherichia coli; virulence genes; drug resistance; integrase; biofilm

中图分类号:

S852.61⁺2

辛娇娇, 索南顿珠, 娄永志, 阿旺克珠, 王新, 罡豪杰, 严佳辉, 常攀, 陈姝宇, 周洪婷, 益西措姆, 索朗斯珠, 贡嘎. 西藏牦牛粪便大肠杆菌毒力特性、耐药性与Ⅰ类整合子分子特征相关性分析[J]. 中国畜牧兽医, 2023, 50(10): 4210-4222.

XIN Jiaojiao, SUONAN Dunzhu, LOU Yongzhi, AWANG Kezhu, WANG Xin, GANG Haojie, YAN Jiahui, CHANG Pan, CHEN Shuyu, ZHOU Hongting, YIXI Cuomu, SUOLANG Sizhu, GONG Ga. Correlation Analysis on Virulence Characteristics,Drug Resistance and Molecular Features of Class Ⅰ Integrator of Escherichia coli Isolated from Feces in Tibetan Yak[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4210-4222.

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