河南省规模化猪场ESBLs大肠杆菌耐药基因检测及分析
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摘要
该研究目的是了解河南省规模化猪场大肠杆菌中ESBLs耐药基因的流行情况及耐药基因型分布情况,为猪场合理使用β-内酰胺类抗生素提供科学指导。从豫东、豫西、豫南和豫北10个规模化养猪场采样861份,对采样采用麦康凯平板初筛、ECC平板和16S rRNA测序鉴定相结合分离大肠杆菌。采用水煮法提取细菌基因组,PCR检测ESBLs耐药基因包括TEM、OXA、SHV和CTX-M。结果表明:861份样共分离出704株大肠杆菌,704株大肠杆菌中产ESBLs基因的检出580株,检出率为82.39%;ESBLs基因型以CTX-M、TEM和OXA为主,检出率分别为78.41%、32.81%和24.57%;SHV基因型检测率最低,检出率为2.56%;携带1种耐药基因ESBLs菌284株,占总检测菌株的40.34%;同时携带2种及以上耐药基因的ESBLs菌296株,占总检测菌株的42.05%;3种ESBLs基因共存菌株有88株。河南省规模化猪场广泛存在耐β-内酰胺类抗生素的大肠杆菌。
The objective of this study is to understand the prevalence of ESBLs drug-resistant genes and the distribution of drug-resistant genotypes in Escherichia coli from large-scale pig farms in Henan province, and to provide scientific guidance for the rational use of beta-lactam antibiotics. A total of 861 samples were collected from 10 large-scale pig farms in nine cities of Henan province. All E. coli isolates were initially screened by using Mac conkey plate, and then were identified by using ECC plate and 16 S rRNA sequencing. The genomic DNAs of E. coli isolates were extracted by using water-boiling method, and the ESBLs drug-resistant genes(TEM, OXA, SHV and CTX-M) in these isolates were detected by PCR amplification. A total of 704 E. coli isolates were identified from 861 samples; among these 704 isolates, 580 isolates contained ESBLs genes, and the detected rate reached 82.39%. The ESBLs genotypes mainly included CTX-M, TEM and OXA, and their detected rate was 78.41%, 32.81% and 24.57%, respectively; while the detected rate of genotype SHV was the lowest, only 2.56%. A total of 284 isolates carried a kind of ESBLs drug-resistant gene, accounting for 40.34% of all isolates. A total of 296 isolates carried 2 or over 2 kinds of ESBLs drug-resistant genes, accounting for 42.05% of all isolates. Three kinds of ESBLs genes co-existed in 88 isolates. The above results showed that the E. coli isolates which were resistant to beta-lactam antibiotics widely existed in large-scale pig farms of Henan province.
The objective of this study is to understand the prevalence of ESBLs drug-resistant genes and the distribution of drug-resistant genotypes in Escherichia coli from large-scale pig farms in Henan province, and to provide scientific guidance for the rational use of beta-lactam antibiotics. A total of 861 samples were collected from 10 large-scale pig farms in nine cities of Henan province. All E. coli isolates were initially screened by using Mac conkey plate, and then were identified by using ECC plate and 16 S rRNA sequencing. The genomic DNAs of E. coli isolates were extracted by using water-boiling method, and the ESBLs drug-resistant genes(TEM, OXA, SHV and CTX-M) in these isolates were detected by PCR amplification. A total of 704 E. coli isolates were identified from 861 samples; among these 704 isolates, 580 isolates contained ESBLs genes, and the detected rate reached 82.39%. The ESBLs genotypes mainly included CTX-M, TEM and OXA, and their detected rate was 78.41%, 32.81% and 24.57%, respectively; while the detected rate of genotype SHV was the lowest, only 2.56%. A total of 284 isolates carried a kind of ESBLs drug-resistant gene, accounting for 40.34% of all isolates. A total of 296 isolates carried 2 or over 2 kinds of ESBLs drug-resistant genes, accounting for 42.05% of all isolates. Three kinds of ESBLs genes co-existed in 88 isolates. The above results showed that the E. coli isolates which were resistant to beta-lactam antibiotics widely existed in large-scale pig farms of Henan province.
引文
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[5] 冯世文,李军,潘艳,等.广西猪源致病性大肠杆菌对抗菌药物的耐药性变化分析[J].南方农业学报,2017,48(2):350-356.
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[13] 郑新添,张孝辉,吴玲,等.猪源产ESBLs大肠杆菌三种耐药基因检测及分析[J].龙岩学院学报,2015,33(2):100-103.
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[15] 白雪.猪源大肠杆菌耐药性分析及ESBLs类基因的质粒传播[D].杨凌:西北农林科技大学,2018.
[16] 沈莉萍,徐锋,张维谊,等.1997～2010年上海市猪源大肠杆菌耐药性和产ESBLs菌的基因型检测[J].中国动物检疫,2017,34(9):26-30,45.
[17] 曹敏,谭艾娟,吕世明,等.贵州部分地区猪源大肠杆菌耐药性分析及ESBLs基因型检测[J].中国畜牧兽医,2016,43(4):1098-1104.
[18] 矫薇薇.猪源致病大肠杆菌耐药性及质粒介导ESBLs耐药基因检测[D].哈尔滨:东北农业大学,2015.
[19] 刘日昂.大肠杆菌超广谱β-内酰胺酶种类及相关基因检测[D].贵阳:贵州大学,2017.
[20] Bush K, Jacoby G A, Medeiros A A. A functional classification scheme for beta-lactamases and its correlation with molecular structure[J]. Antimicrob Agents Chemother, 1995, 39(6): 1211-1233.
[2] Ae V D B, Stobberingh E E. Epidemiology of resistance to antibiotics. Links between animals and humans[J]. International Journal of Antimicrobial Agents, 2000, 14(4): 327-335.
[3] Paterson D L, Bonomo R A. Extended-spectrum β-lactamases: a clinical update[J]. Clinical Microbiology Reviews, 2005, 18(4): 657.
[4] Zheng H, Zeng Z, Chen S, et al. Prevalence and characterisation of CTX-M β-lactamases amongst Escherichia coli isolates from healthy food animals in China[J]. International Journal of Antimicrobial Agents, 2012, 39(4): 305-310.
[5] 冯世文,李军,潘艳,等.广西猪源致病性大肠杆菌对抗菌药物的耐药性变化分析[J].南方农业学报,2017,48(2):350-356.
[6] 裴亚玲.鸡源大肠杆菌ESBLs基因型检测[D].郑州:河南农业大学,2009.
[7] 李凤娟.鸭源大肠杆菌ESBLs的基因型检测[D]. 郑州:河南农业大学,2009.
[8] 杜向党,焦显芹,莫娟,等.鸡猪源大肠杆菌CTX-M型ESBLs的分子检测[J].华北农学报,2009,24(2):90-93.
[9] 苑丽.鸡源分离菌超广谱β-内酰胺酶基因分型和整合子分子特征[D]. 郑州:河南农业大学,2010.
[10] 潘玉善.禽源大肠杆菌、奇异变形杆菌多重耐药的分子特征及CTX-M基因传播扩散机制[D]. 郑州:河南农业大学,2012.
[11] 冯建昆.鸭源大肠杆菌CTX-M型ESBLs的基因亚型检测及基因环境研究[D]. 郑州:河南农业大学,2013.
[12] 匡秀华.鸡大肠杆菌超广谱β-内酰胺酶(ESBLs)的基因检测及其感染的化学治疗[D]. 郑州:河南农业大学,2006.
[13] 郑新添,张孝辉,吴玲,等.猪源产ESBLs大肠杆菌三种耐药基因检测及分析[J].龙岩学院学报,2015,33(2):100-103.
[14] 刘河冰,李新生,莫娟,等.ESBLs在产16S rRNA甲基化酶鸡大肠杆菌中的流行[J].江西农业大学学报,2009(4):621-624.
[15] 白雪.猪源大肠杆菌耐药性分析及ESBLs类基因的质粒传播[D].杨凌:西北农林科技大学,2018.
[16] 沈莉萍,徐锋,张维谊,等.1997～2010年上海市猪源大肠杆菌耐药性和产ESBLs菌的基因型检测[J].中国动物检疫,2017,34(9):26-30,45.
[17] 曹敏,谭艾娟,吕世明,等.贵州部分地区猪源大肠杆菌耐药性分析及ESBLs基因型检测[J].中国畜牧兽医,2016,43(4):1098-1104.
[18] 矫薇薇.猪源致病大肠杆菌耐药性及质粒介导ESBLs耐药基因检测[D].哈尔滨:东北农业大学,2015.
[19] 刘日昂.大肠杆菌超广谱β-内酰胺酶种类及相关基因检测[D].贵阳:贵州大学,2017.
[20] Bush K, Jacoby G A, Medeiros A A. A functional classification scheme for beta-lactamases and its correlation with molecular structure[J]. Antimicrob Agents Chemother, 1995, 39(6): 1211-1233.