奶牛牧场养殖环境中产ESBL耐药菌的流行特征

doi:10.11843/j.issn.0366-6964.2022.11.028

摘要/Abstract

摘要： 本研究旨在探索奶牛养殖环境中产超广谱β-内酰胺酶（extended-spectrum beta-lactamases，ESBL）耐药菌的流行特征。2020-2021年，采集6个内蒙古和宁夏牧场共160份牛奶、乳区皮肤拭子和环境来源的多种类型样本，分别进行了β-内酰胺酶检测、产ESBL耐药菌的分离鉴定、药敏试验以及菌株的全基因组测序。结果显示：24份奶样的检测结果显示，各牧场β-内酰胺酶阳性率为75%~100%。其中，5个牧场的153份样本共分离出6种产ESBL耐药菌，这些产ESBL耐药菌对阿莫西林（100%）、头孢噻肟（91.0%）和头孢他啶（78.2%）均具有较高耐药率；全基因组显示，14株产ESBL大肠杆菌携带多种耐药基因，以bla_CTX-M（100%）携带率最高。通过对内酰胺酶阳性奶样进行分析，发现造成牧场奶样β-内酰胺酶阳性的主要菌株是产ESBL鲍曼不动杆菌，并且该耐药菌携带有bla_CTX-M、bla_TEM、bla_OXA型β-内酰胺类耐药基因。本研究明确了内蒙古和宁夏牧场养殖环境中产ESBL耐药菌的流行特征，并对造成内蒙古1个牧场牛奶内酰胺酶阳性原因进行了追溯研究。

关键词: 产ESBL耐药菌, 牧场养殖环境, 奶牛乳房炎, 流行特征, β-内酰胺酶

Abstract: The purpose of this study was to explore the epidemiological characteristics of ESBL-producing resistant bacteria in the dairy farming environments. A total of 160 samples were collected from 6 farms in Inner Mongolia and Ningxia from 2020 to 2021, including milk, milk area skin swabs and environmental sources. β-lactamase rapid test strip, with the isolation and identification, drug sensitivity, and genome sequencing of ESBL-producing resistant bacteria, were conducted respectively. The positive rate of β-lactamase in all farms was 75%-100% in 24 milk samples. A total of 6 ESBL-producing resistant strains were isolated from 153 samples in 5 farms, and these ESBL-producing resistant strains had high resistance rates to amoxicillin (100%), cefotaxime (91.0%) and ceftazidime (78.2%). And 14 ESBL-producing Escherichia coli strains sequenced were found to carry multiple drug resistance gene types, with bla_CTX-M (100%) carrying rate being the highest. At the same time, the sixth special farm with "long-term positive lactamase test" was found that ESBL-producing Acinetobacter baumannii was the main strain of β-lactamase positive in the milk of the farm, and the resistant strain carried bla_CTX-M, bla_TEM and bla_OXA β-lactamase resistance genes. This study identified the epidemiological characteristics of ESBL-producing resistant bacteria in farm environments in Inner Mongolia and Ningxia, and the causes of lactamase positivity in the milk of one farm in Inner Mongolia were successfully traced back.

Key words: ESBL-producing resistant bacteria, dairy farming environment, bovine mastitis, epidemiological characteristics, β-lactamase

中图分类号:

S852.6

左扬, 李田, 胡秀花, 宋志强, 孙城涛, 吴聪明, 王少林. 奶牛牧场养殖环境中产ESBL耐药菌的流行特征[J]. 畜牧兽医学报, 2022, 53(11): 4027-4034.

ZUO Yang, LI Tian, HU Xiuhua, SONG Zhiqiang, SUN Chengtao, WU Congming, WANG Shaolin. Epidemiological Characteristics of ESBL-Producing Resistant Bacteria in Dairy Farming Environment[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4027-4034.

参考文献 39

[1]	MIAN G F, GODOY D T, LEAL C A G, et al.Aspects of the natural history and virulence of S.agalactiae infection in Nile tilapia[J].Vet Microbiol, 2009, 136(1-2):180-183.
[2]	高春生, 白翠, 马晓媛, 等.奶牛乳房炎的研究进展[J].吉林畜牧兽医, 2019, 40(11):58-59.GAO C S, BAI C, MA X Y, et al.Research progress of bovine mastitis[J].Jilin Animal Husbandry and Veterinary Medicine, 2019, 40(11):58-59.(in Chinese)
[3]	GRÖHN Y T, WILSON D J, GONZÁLEZ R N, et al.Effect of pathogen-specific clinical mastitis on milk yield in dairy cows[J].J Dairy Sci, 2004, 87(10):3358-3374.
[4]	薛晓聪.中国奶牛养殖生产布局演变及其增长效应研究[D].哈尔滨:东北农业大学, 2020.XUE X C.Research on the evolution and its growth effect of dairy farming production layout in China[D].Harbin:Northeast Agricultural University, 2020.(in Chinese)
[5]	IRAGUHA B, HAMUDIKUWANDA H, MUSHONGA B.Bovine mastitis prevalence and associated risk factors in dairy cows in Nyagatare District, Rwanda[J].J South Afr Vet Assoc, 2015, 86(1):a1228.
[6]	张行, 李新圃, 严勇, 等.我国奶牛主要养殖区乳房炎病原菌区系分布及对抗生素敏感性的最新调查研究[J].中兽医医药杂志, 2020, 39(4):5-9.ZHANG H, LI X P, YAN Y, et al.Latest investigation on the distribution and antimicrobial resistance of bovine mastitis pathogens from major dairy cow breeding areas in China[J].Journal of Traditional Chinese Veterinary Medicine, 2020, 39(4):5-9.(in Chinese)
[7]	吴海燕, 王纯结, 敖日格乐, 等.奶牛乳房炎常见致病菌的分离及药敏试验[J].养殖与饲料, 2019(9):10-13.WU H Y, WANG C J, AO R G L, et al.Isolation and drug sensitivity test of common pathogenic bacteria in bovine mastitis[J].Animals Breeding and Feed, 2019(9):10-13.(in Chinese)
[8]	郭日东, 吴晓亮, 宋丽华, 等.奶牛乳房炎病原菌的分离鉴定[J].养殖与饲料, 2015(8):13-14.GUO R D, WU X L, SONG L H, et al.Isolation and identification of pathogenic bacteria from bovine mastitis[J].Animals Breeding and Feed, 2015(8):13-14.(in Chinese)
[9]	赵祎云, 隋国燕, 唐峰, 等.兰州地区奶牛临床型乳房炎主要病原菌分离鉴定及耐药性分析[J].中国兽医杂志, 2020, 56(3):91-94.ZHAO Y Y, SUI G Y, TANG F, et al.Isolation, identification and drug resistance of pathogenic bacteria isolated from a cow with clinical mastitis in Lanzhou area[J].Chinese Journal of Veterinary Medicine, 2020, 56(3):91-94.(in Chinese)
[10]	陈杰, 魏勇, 王晨豫, 等.新疆部分地区9个奶牛场致奶牛乳房炎主要病原菌的检测与药敏试验[J].中国牛业科学, 2021, 47(6):9-15.CHEN J, WEI Y, WANG C Y, et al.Detection and drug sensitivity test of main pathogenic bacteria causing cow mastitis in 9 large-scale dairy farms in Xinjiang[J].China Cattle Science, 2021, 47(6):9-15.(in Chinese)
[11]	朱宁, 赵艳坤, 陈贺, 等.上海地区奶牛乳房炎病原菌的分离鉴定及耐药性分析[J].中国奶牛, 2020(9):31-35.ZHU N, ZHAO Y K, CHEN H, et al.Isolation and identification drug resistance analysis of pathogen of dairy cow mastitis[J].China Dairy Cattle, 2020(9):31-35.(in Chinese)
[12]	王慧, 刘伯承, 段洪峰, 等.湖南部分地区奶牛乳房炎主要病原菌的分离鉴定及药敏试验[J].中国奶牛, 2019(8):36-39.WANG H, LIU B C, DUAN H F, et al.Isolation, identification and drug susceptibility test for the main pathogenic bacteria of cow mastitis in some local areas of Hunan[J].China Dairy Cattle, 2019(8):36-39.(in Chinese)
[13]	陶晓丽.河南省临颍县奶牛乳房炎病原菌感染情况调查与药敏分析[J].中国兽医杂志, 2020, 56(3):107-108.TAO X L.Investigation on pathogenic bacteria infection and drug sensitivity analysis of bovine mastitis in Linying, Henan Province[J].Chinese Journal of Veterinary Medicine, 2020, 56(3):107-108.(in Chinese)
[14]	刘景喜, 张进, 芦娜, 等.2018年天津市原料奶细菌总数、体细胞数及奶牛乳房炎病原菌、耐药基因调研报告[J].畜牧与兽医, 2020, 52(3):130-134.LIU J X, ZHANG J, LU N, et al.Report on investigation of total bacteria number, somatic cell count in raw milk and pathogens, resistance gene of mastitis in cows in Tianjin in 2018[J].Animal Husbandry & Veterinary Medicine, 2020, 52(3):130-134.(in Chinese)
[15]	吴利军, 李佳琳, 陈夏冰, 等.奶牛乳房炎主要病原菌的分离鉴定及耐药性研究[J].湖北农业科学, 2019, 58(21):145-149.WU L J, LI J L, CHEN X B, et al.Study on the isolation, identification and drug resistance of main pathogenic bacteria in dairy cow mastitis[J].Hubei Agricultural Sciences, 2019, 58(21):145-149.(in Chinese)
[16]	杜琳, 王丽芳, 冯小慧, 等.我国部分地区奶牛乳房炎主要病原菌的分离鉴定及药敏试验[J].动物医学进展, 2018, 39(3):124-128.DU L, WANG L F, FENG X H, et al.Isolation, identification and drug susceptibility test of main pathogens of dairy cow mastitis in some regions of China[J].Progress in Veterinary Medicine, 2018, 39(3):124-128.(in Chinese)
[17]	范蓉, 武智聪, 梁翠娟, 等.革兰阴性杆菌产超广谱β-内酰胺酶(ESBLs)的检出率及其与细菌耐药性的关系[J].河北医学, 2020, 26(3):522-525.FAN R, WU Z C, LIANG C J, et al.Relationship between the detection rate of extended spectrum β-lactamases (ESBLs) produced by gram negative bacilli and bacterial resistance[J].Hebei Medicine, 2020, 26(3):522-525.(in Chinese)
[18]	ALI T, RAHMAN S U, ZHANG L M, et al.Characteristics and genetic diversity of multi-drug resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from bovine mastitis[J].Oncotarget, 2017, 8(52):90144-90163.
[19]	BROLUND A.Overview of ESBL-producing Enterobacteriaceae from a Nordic perspective[J].Infect Ecol Epidemiol, 2014, 4(1):24555.
[20]	KLIBI A, JOUINI A, EL ANDOLSI R B, et al.Epidemiology of β-lactamase-producing staphylococci and gram negative bacteria as cause of clinical bovine mastitis in Tunisia[J].Biomed Res Int, 2019, 2019:2165316.
[21]	TIMOFTE D, MACIUCA I E, EVANS N J, et al.Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-lactamases from bovine mastitis isolates in the United Kingdom[J].Antimicrob Agents Chemother, 2014, 58(2):789-794.
[22]	TANIGUCHI T, LATT K M, TARIGAN E, et al.A 1-year investigation of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from bovine mastitis at a large-scale dairy farm in Japan[J].Microb Drug Resist, 2021, 27(10):1450-1454.
[23]	EISENBERGER D, CARL A, BALSLIEMKE J, et al.Molecular characterization of extended-spectrum β-lactamase-producing Escherichia coli isolates from milk samples of dairy cows with mastitis in Bavaria, Germany[J].Microb Drug Resist, 2018, 24(4):505-510.
[24]	KOOVAPRA S, BANDYOPADHYAY S, DAS G, et al.Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India[J].Infect Genet Evol, 2016, 44:395-402.
[25]	DAHMEN S, MÉTAYER V, GAY E, et al.Characterization of extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of Enterobacteriaceae causing cattle mastitis in France[J].Vet Microbiol, 2013, 162(2-4):793-799.
[26]	张颖欣, 陈友涵, 李树梅, 等.奶牛乳房炎源性肺炎克雷伯菌ESBLs基因型分布特征[J].中国兽医杂志, 2018, 54(11):10-12.ZHANG Y X, CHEN Y H, LI S M, et al.Genotypic characteristics of extended spectrum β-lactamases producing Klebsiella pneumoniae isolated from bovine mastitis[J].Chinese Journal of Veterinary Medicine, 2018, 54(11):10-12.(in Chinese)
[27]	SHAFIQ M, HUANG J, SHAH J M, et al.Characterization and virulence factors distribution of blaCTX-M and mcr-1 carrying Escherichia coli isolates from bovine mastitis[J].J Appl Microbiol, 2021, 131(2):634-646.
[28]	王桂琴, 李继东, 许立华, 等.宁夏地区奶牛乳房炎主要病原菌毒力基因检测及多重耐药机理研究[Z/OL].国家科技成果数据库, 2021.[2022-07-10].http://cnki.scstl.org/KCMS/detail/detail.aspx?filename=SNAD000001914190&dbcode=SNAD& dbname=SNAD.WANG G Q, LI J D, XU L H, et al.Study on virulence gene detection and multiple drug resistance mechanism of main pathogenic bacteria of bovine mastitis in Ningxia[Z/OL].National scientific and technological achievements database, 2021.[2022-07-10].http://cnki.scstl.org/KCMS/detail/detail.aspx?filename=SNAD000001914190&dbco-de=SNAD&dbname=SNAD (in Chinese)
[29]	袁昕慧.NGS技术用于引起奶牛乳房炎的液化沙雷菌的耐药基因的筛选[D].延吉:延边大学, 2018.YUAN X H.Screening of drug-resistance genes of Serratia Liquefaciens causing cow mastitis by NGS technology[D].Yanji:Yanbian University, 2018.(in Chinese)
[30]	吴文斌.奶牛乳房炎的病因及综合防治措施[J].兽医导刊, 2021(1):119.WU W B.Etiology and comprehensive prevention and treatment of bovine mastitis[J].Veterinary Orientation, 2020(1):119.(in Chinese)
[31]	中华人民共和国卫生部卫生部.关于印发全国打击违法添加非食用物质和滥用食品添加剂专项整治抽检工作指导原则和方案的通知(食品整治办
	[2009]29号)[A].2009-03-23.Minister of Health of the People's Republic of China.Notice on printing and distributing the guiding principles and programs of special rectification and sampling inspection of national crackdown on illegal addition of non-edible substances and abuse of food additives (Food Control Office
	[2009] No.29)[A].2009-03-23.(in Chinese)
[32]	谢磊, 吉鸿武, 刘建华, 等.北京市生鲜牛乳中内源性β-内酰胺酶残留现状研究[J].中国乳业, 2015(2):51-56.XIE L, JI H W, LIU J H, et al.Study on endogenous β-lactamase residues in fresh milk in Beijing[J].China Dairy, 2015(2):51-56.(in Chinese)
[33]	侯海燕, 刘纯成, 刘靓.2014年淮安市市售牛乳中β-内酰胺酶残留状况调查[J].中国校医, 2015, 29(8):567-568, 571.HOU H Y, LIU C C, LIU L.Investigation on residues of β-lactamase in commercial milk in Huaian City, 2014[J].Chinese Journal of School Doctor, 2015, 29(8):567-568, 571.(in Chinese)
[34]	帕提古丽·托乎提.新疆巴州地区生鲜牛乳中β-内酰胺酶残留状况调查[J].中国动物检疫, 2015, 32(10):19-20.PATIGULI·TUOHUTI.Investigation of β-lactamase residue in fresh milk in Bazhou, Xinjiang[J].China Animal Health Inspection, 2015, 32(10):19-20.(in Chinese)
[35]	石润佳, 韩荣伟, 王军, 等.乳房炎牛乳中产β-内酰胺酶肠杆菌耐药性研究进展[J].动物医学进展, 2020, 41(4):86-90.SHI R J, HAN R W, WANG J, et al.Advances in drug resistance of Enterobacteria-producing β-lactamases from milk[J].Progress in Veterinary Medicine, 2020, 41(4):86-90.(in Chinese)
[36]	AHMED W, NEUBAUER H, TOMASO H, et al.Characterization of enterococci- and ESBL-producing Escherichia coli isolated from milk of bovides with Mastitis in Egypt[J].Pathogens, 2021, 10(2):97.
[37]	AMARUZZAMAN E A, ABDUL AZIZ S, BITRUS A A, et al.Occurrence and characteristics of extended-spectrum β-lactamase-producing Escherichia coli from dairy cattle, milk, and farm environments in Peninsular Malaysia[J].Pathogens, 2020, 9(12):1007.
[38]	宗辉, 孙钰芳, 陈思敏, 等.β-内酰胺酶抑制剂复方制剂研究进展[J].中国新药杂志, 2022, 31(4):343-351.ZONG H, SUN Y F, CHEN S M, et al.An update on the combinations with β-lactamase inhibitors[J].Chinese Journal of New Drugs, 2022, 31(4):343-351.(in Chinese)
[39]	苏晓东.兽用β-内酰胺类抗生素的规范使用[J].中国畜牧业, 2018(17):84-85.SU X D.Standard use of veterinary β-lactam antibiotics[J].China Animal Industry, 2018(17):84-85.(in Chinese)