山东地区乳房炎牛奶中大肠杆菌的分离鉴定及耐药性分析

doi:10.16431/j.cnki.1671-7236.2021.01.035

摘要/Abstract

摘要： 试验旨在了解山东地区乳房炎牛奶中大肠杆菌的污染状况及耐药情况。选择山东省3个地区的规模化奶牛场共采集227份牛奶样品，采用细菌学方法对大肠杆菌进行分离鉴定，用微量肉汤稀释法检测分离菌对11种常规抗菌药物的敏感性，采用PCR方法对常见的13种耐药基因、8种毒力基因和Ⅰ类整合子基因盒结构进行分析。结果显示，从227份牛奶样品中共分离出71株大肠杆菌；大肠杆菌对1种及1种以上抗菌药耐药的菌株达到77.5%，多重耐药率为15.5%，其中对多黏菌素耐药率为52.2%，对阿莫西林-克拉维酸耐药率为39.4%，而所有菌株均对新霉素表现为敏感。PCR检测耐药基因、毒力基因和Ⅰ类整合子结果显示，β-内酰胺类耐药基因中bla_TEM基因携带率为100%，其中全部为bla_TEM-1基因，bla_CTX-M基因携带率为32.4%，其中主要为bla_CTX-M-15基因，没有检测到bla_SHV、bla_OXA基因；多黏菌素的耐药基因mcr-1携带率为29.6%；喹诺酮类耐药基因中aac（6'）-Ⅰb-cr基因携带率为29.6%，qnrB基因携带率为20.8%，没有检测到qnrA和qnrC耐药基因；对8种毒力基因检测分析结果显示，仅Hly毒力基因没有被检出，Ecs3703、Irp2基因的检出率较高，分别为90.1%和63.4%，71株大肠杆菌中共有11株携带Ⅰ类整合子，检出率为15.5%，11株大肠杆菌携带6种耐药基因盒结构，最主要的耐药基因盒排列为dfr17-aadA5。本研究结果表明，山东地区乳房炎牛奶中大肠杆菌的耐药现象严重，携带毒力基因Ecs3703、Irp2的大肠杆菌可能是引起奶牛乳房炎的致病菌，Ⅰ类整合子的检测在细菌耐药性与基因携带率方面发挥着关键作用，可为临床预防和治疗奶牛乳房炎大肠杆菌病提供理论依据。

关键词: 大肠杆菌; 耐药性; 耐药基因; 毒力基因; Ⅰ类整合子

Abstract: This experiment was aimed to estimate the contamination and drug resistance of E.coli in mastitis milk in Shandong.The large-scale dairy farms located in three regions of Shandong province were selected as the sampling sites for this study.A total of 227 milk samples were collected,the bacteriological method was used to isolate and identify E.coli,the micro-broth dilution method was used to detect 11 conventional antibacterial drugs,the 13 common drug resistance genes,8 virulence genes and class Ⅰ integron were screened by PCR.The results showed that a total of 71 E.coli strains were isolated from 227 milk samples.Antibiotic susceptibility testing of E.coli strains showed that strains which were resistant to one or more antibacterial drugs reached 77.5%,the multi-drug resistance rate was 15.5%,of which the resistance rate to polymyxin was 52.2%,and the amoxicillin-clavulanic acid resistance rate was 39.4%.However,all strains were sensitive to neomyci.Of the 71 E.coli strains,all of them carried bla_TEM gene,and they were all bla_TEM-1 gene,32.4% of the strains carried bla_CTX-M gene,and most of them were bla_CTX-M-15 gene.In addition,bla_SHV and bla_OXA genes were not detected;29.6% of the isolates carried mobile colistin resistance genes mcr-1;29.6% of the strains carried aac (6')-Ⅰb-cr gene,and 20.8% carried qnrB gene,qnrA and qnrC genes were not detected.For the analysis of 8 virulence genes,only Hly gene were not detected,and the detection rates of Ecs3703 and Irp2 genes were higher,which were 90.1% and 63.4%,respectively.Class Ⅰ integrons were detected in 11 strains,with a rate of 15.5%,and carried 6 types of gene cassettes,the prevalence of the gene cassette was dfr17-aadA5.The results indicated that the drug resistance of E.coli in mastitis milk in Shandong was serious.E.coli that carrying virulence genes Ecs3703 and Irp2 might be the pathogenic bacteria causing mastitis in dairy cow.The detection of class Ⅰ integrin played a key role in bacterial resistance and gene carrying rate.This study could provide a theoretical basis for clinical prevention and treatment colibacillosis of dairy cow mastitis.

Key words: E.coli; drug resistance; drug resistance gene; virulence gene; class Ⅰ integron

中图分类号:

S852.61⁺2

苑晓萌, 赵效南, 李璐璐, 张庆, 胡明, 骆延波, 张印, 齐静, 刘玉庆. 山东地区乳房炎牛奶中大肠杆菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2021, 48(1): 312-323.

YUAN Xiaomeng, ZHAO Xiaonan, LI Lulu, ZHANG Qing, HU Ming, LUO Yanbo, ZHANG Yin, QI Jing, LIU Yuqing. Isolation,Identification and Drug Resistance Analysis of E.coli from Mastitis Milk in Shandong Province[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 312-323.

参考文献

[1] LIPPOLIS J D,HOLMAN D B,BRUNELLE B W,et al.Genomic and transcriptomic analysis of Escherichia coli strains associated with persistent and transient bovine mastitis and the role of colanic acid[J].Infection and Immunity,2018,86(1):00566-17.
[2] JORGENSEN H J,MORK T,RORVIK L M.The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese[J].Journal of Dairy Science,2005,88(11):3810-3817.
[3] YANG F,ZHANG S,SHANG X,et al.Short communication:N-acetylcysteine-mediated augmenta-tion of beta-lactam antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from bovine mastitis cases[J].Journal of Dairy Science,2019,102(8):6920-6922.
[4] NAIR M K,JOY J,VASUDEVAN P,et al.Antibacterial effect of caprylic acid and monocaprylin on major bacterial mastitis pathogens[J].Journal of Dairy Science,2005,88(10):3488-3495.
[5] 刘风波,连亮亮.复方中药治疗奶牛乳房炎初探[J].国外畜牧学(猪与禽),2020,40(1):63-64. LIU F B,LIAN L L.Preliminary study on Chinese herbal medicine compound for dairy cow mastitis[J].Animal Science Abroad(Pigs and Poultry),2020,40(1):63-64.(in Chinese)
[6] 李松建,周雅坪,吕天星,等.IgG FcRn介导奶牛乳房炎无乳链球菌保护性抗原Sip亚单位疫苗的研究[J].中国预防兽医学报,2018,40(10):945-949. LI S J,ZHOU Y P,LYU T X,et al.Construction and clinical trial of the protective antigen Sip subunit vaccine against Streptococcus agalactiae in dairy cow mastitis mediated by IgG FcRn[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(10):945-949.(in Chinese)
[7] 张霖,薛盛东,周宗爱,等.铜绿假单胞菌Ⅰ类整合子遗传标记及耐药基因研究[J].中华医院感染学杂志,2007,5:496-498. ZHANG L,XUE S D,ZHOU Z A,et al.Genetic markers of class Ⅰ integron and drug-resistant genes in Pseudomonas aeruginosa[J].Chinese Journal of Nosocomiology,2007,5:496-498.(in Chinese)
[8] SINGH N S,SINGHAL N,VIRDI J S.Genetic environment of bla_TEM-1,bla_CTX-M-15,bla_CMY-42 and characterization of integrons of Escherichia coli isolated from an Indian urban aquatic environment[J].Frontiers in Microbiology,2018,9:382.
[9] HEGDE A,BALLAL M,SHENOY S.Detection of diarrheagenic Escherichia coli by multiplex PCR[J].Indian Journal of Medical Research,2012,30(3):279-284.
[10] CLINICAL AND LABORATORY STANDARDS INSTITUTE.Performance standards for antimi-crobial susceptibility testing[S].M100-S28.USA:CLSI,2018.
[11] 尹召华.奶牛乳腺炎的病原菌分离鉴定、毒力基因检测与基因治疗研究[D].扬州:扬州大学,2009. YIN Z H.Identification,virulent gene detection and gene therapy of mastitis-causing bacteria in dairy cows[D].Yangzhou:Yangzhou University,2009.(in Chinese)
[12] NUESCH-INDERBINEN M,KAPPELI N,MORACH M,et al.Molecular types,virulence profiles and antimicrobial resistance of Escherichia coli causing bovine mastitis[J].Veterinary Record Open,2019,6(1):e369.
[13] JOHNSON T J,WANNEMUEHLER Y,DOETKOTT C,et al.Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool[J].Journal of Clinical Microbio-logy,2008,46(12):3987-3996.
[14] 姚伟,高锋,曹明慧,等.辽宁地区奶牛乳房炎源大肠杆菌毒力基因及耐药基因检测分析[J].中国畜牧兽医,2017,44(6):1832-1839. YAO W,GAO F,CAO M H,et al.Detection and analysis of virulence genes and drug resistance gene in E.coli strains isolated from dairy cows with clinical mastitis in Liaoning region[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1832-1839.(in Chinese)
[15] 南海辰,夏利宁,刘英玉,等.新疆塔城某规模化养殖场大肠埃希菌耐药性调查[J].动物医学进展,2014,35(9):118-123. NAN H C,XIA L N,LIU Y Y,et al.Investigation on antimicrobial drug resistance to Escherichia coli isolates from a farm in Tacheng,Xinjiang[J].Progress in Veterinary Medicine,2014,35(9):118-123.(in Chinese)
[16] 李孟,李金磊,申水莹,等.猪源大肠杆菌floR、CTX-M、mcr-1耐药基因多重PCR检测方法的建立[J].中国兽药杂志,2019,53(7):1-7. LI M,LI J L,SHEN S Y,et al.Development of multiplex PCR for detection of floR,CTX-M,mcr-1 in pig-derived Escherichia coli[J].Chinese Journal of Veterinary Drug,2019,53(7):1-7.(in Chinese)
[17] 嵇辛勤,李世静,文明,等.鸭源大肠杆菌β-内酰胺类耐药基因的检测[J].广东农业科学,2015,42(15):88-91. JI X Q,LI S J,WEN M,et al.Detection of β-lactamases-resistant genes in Escherichia coli isolated from ducks[J].Guangdong Agricultural Sciences,2015,42(15):88-91.(in Chinese)
[18] WANG P,HU L,HAO Z.Palmatine is a plasmid-mediated quinolone resistance (PMQR) inhibitor that restores the activity of ciprofloxacin against QnrS and AAC(6')-Ⅰb-cr-producing Escherichia coli[J].Infection and Drug Resistance,2020,13:749-759.
[19] DONG W L,ODAH K A,LIU L,et al.Multidrug resistance genes are associated with a 42-kb island TGI1 carrying a complex class 1 integron in Trueperella pyogenes[J].Journal of Global Antimi-crobial Resistance,2019,22:1-4.
[20] FLEMINGER G,RAGONES H,MERIN U,et al.Low molecular mass peptides generated by hydrolysis of casein impair rennet coagulation of milk[J].International Dairy Journal,2013,30(2):74-78.
[21] DALANEZI F M,JOAQUIM S F,GUIMARAES F F,et al.Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows[J].Journal of Dairy Science,2020,103(4):3648-3655.
[22] ASHRAF A,IMRAN M.Diagnosis of bovine mastitis:From laboratory to farm[J].Tropical Animal Health and Production,2018,50(6):1193-1202.
[23] 王晓,王新,郝丹,等.奶牛乳源大肠杆菌耐药性、毒素基因鉴定及PFGE分型研究[J].畜牧兽医学报,2014,45(3):463-470. WANG X,WANG X,HAO D,et al.Study on antibiotic resistance,virulence toxin genes and PFGE patterns of Escherichia coli strains isolated from dairy cattle milk[J].Chinese Journal of Animal and Veterinary Sciences,2014,45(3):463-470.(in Chinese)
[24] 黄梦夏,马光强,杨德凤,等.贵阳市散养奶牛牛奶中大肠杆菌的分离鉴定与耐药性分析[J].贵州畜牧兽医,2018,42(2):18-21. HUANG M X,MA G Q,YANG D F,et al.Isolation and identification and drug resistance analysis of Escherichia coli in milk from the free-range cows in Guiyang city[J].Guizhou Animal Science and Veterinary Medicine,2018,42(2):18-21.(in Chinese)
[25] 郭莉娟,孙丰慧,张艳娇,等.生牛奶中分离大肠杆菌对季铵盐类消毒剂耐药性研究[J].四川动物,2019,38(6):623-629. GUO L J,SUN F H,ZHANG Y J,et al.Research on ammonium compounds disinfectant-resistance of Escherichia coli in raw milk[J].Sichuan Journal of Zoology,2019,38(6):623-629.(in Chinese)
[26] LI W,XUE M,YU L,et al.QseBC is involved in the biofilm formation and antibiotic resistance in Escherichia coli isolated from bovine mastitis[J].PeerJ,2020,8:e8833.
[27] BROWN P,DAWSON M J.Development of new polymyxin derivatives for multi-drug resistant Gram-negative infections[J].Journal of Antibiotics,2017,70(4):386-394.
[28] 白凤佳,李欢,刘海泉,等.上海市某大型乳牛养殖环境中大肠杆菌对多粘菌素的耐药性分析[A].中国食品科学技术学会第十四届年会暨第九届中美食品业高层论坛[C].2017. BAI F J,LI H,LIU H Q,et al.Analysis of the resistance of Escherichia coli to polymyxin in a large dairy farm in Shanghai[A].The 14th Annual Meeting of the China Food Science and Technology Society and the 9th China-US Food Industry Senior Forum[C].2017.(in Chinese)
[29] 石润佳,韩荣伟,王军,等.华北地区乳房炎奶样中大肠杆菌的耐药性研究[J].现代食品科技,2019,35(5):274-280. SHI R J,HAN R W,WANG J,et al.Study on antibiotics resistance of Escherichia coli in milk samples from cows with mastitis in North China[J].Modern Food Science and Technology,2019,35(5):274-280.(in Chinese)
[30] 高建鹏,张子荣,张奇波,等.致奶牛乳房炎大肠杆菌的分离鉴定及药敏试验[J].中国牛业科学,2018,44(1):26-29. GAO J P,ZHANG Z R,ZHANG Q B,et al.Isolation,identification and drug resistance test of Escherichia coli in dairy cow mastitis[J].China Cattle Science,2018,44(1):26-29.(in Chinese)
[31] 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 areas of Hunan[J].China Dairy Cattle,2019,8:36-39.(in Chinese)
[32] PERILLAUD-DUBOIS C,PILMIS B,DIEP J,et al.Performance of rapid antimicrobial susceptibility testing by disk diffusion on MHR-SIR agar directly on urine specimens[J].European Journal of Clinical Microbiology & Infectious Diseases,2019,38(1):185-189.
[33] FERRER M D,QUILES-PUCHALT N,HARWICH M D,et al.RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria[J].Nucleic Acids Research,2011,39(14):5866-5878.
[34] 张力国,卢婷,赵明礼,等.牛源大肠杆菌毒力基因的检测及其对小鼠致病性的研究[J].中国兽医科学,2014,44(8):817-822. ZHANG L G,LU T,ZHAO M L,et al.Detection of virulence genes of Escherichia coli from cattle and pathogenicity of the isolate in mice[J].China Veterinary Science,2014,44(8):817-822.(in Chinese)
[35] 张震,宋美英,只勇,等.黑龙江省部分牛场致病性大肠杆菌毒力因子的调查与分析[J].黑龙江畜牧兽医,2017,3:160-162. ZHANG Z,SONG M Y,ZHI Y,et al.Investigation and analysis of the virulence factors of pathogenic Escherichia coli from some cattle ranches in Heilongjiang province[J].Heilongjiang Animal Science and Veterinary Medicine,2017,3:160-162.(in Chinese)
[36] LIU G,ALI T,GAO J,et al.Co-occurrence of plasmid-mediated colistin resistance (mcr-1) and extended-spectrum beta-lactamase encoding genes in Escherichia coli from bovine mastitic milk in China[J].Microbial Drug Resistance,2020,26(6):685-696.
[37] 杨艳丽,张星星,黄新,等.牛源大肠杆菌质粒介导喹诺酮类耐药基因的检测分析[J].中国畜牧兽医,2019,46(8):2462-2469. YANG Y L,ZHANG X X,HUANG X,et al.Analysis and detection of plasmid-mediated quinolone resistance genes in bovine Escherichia coli[J].China Animal Husbandry & Veterinary Medicine,2019,46(8):2462-2469.(in Chinese)
[38] 卢婷.牛源大肠杆菌耐药性和耐药基因检测及对喹诺酮类药物防突变浓度的测定[D].哈尔滨:东北农业大学,2015. LU T.Study on antimicrobial resistance,resistance genes and mutant prevention concentration of Escherichia coli from cattle[D].Harbin:Northeast Agricultural University,2015.(in Chinese)
[39] GIAKKOUPI P,TZOUVELEKIS L S,TSAKRIS A,et al.IBC-1,a novel integron-associated class a beta-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain[J].Antimicrobial Agents and Chemotherapy,2000,44(9):2247-2253.
[40] 周亚玲,陈红,朱丽华,等.临床革兰阴性杆菌整合子携带情况及其与耐药性的相关性分析[J].临床检验杂志,2019,37(1):59-61. ZHOU Y L,CHEN H,ZHU L H,et al.Analysis of clinical Gram-negative bacilli integronts and their correlation with drug resistance[J].Chinese Journal of Clinical Laboratory Science,2019,37(1):59-61.(in Chinese)
[41] 邓阳,肖亦莎,李昕,等.氨基糖苷类抗生素治疗药物监测及其毒理机制研究进展[J].中国药理学与毒理学杂志,2019,33(12):1085-1092. DENG Y,XIAO Y S,LI X,et al.Research advances in therapeutic drug monitoring and toxicological mechanism of aminoglycoside antibiotics[J].Chinese Journal of Pharmacology and Toxicology,2019,33(12):1085-1092.(in Chinese)
[42] 王海生,扈瑞平,高玉敏,等.牛源大肠杆菌Ⅰ类整合子分子特征及其与耐药性关系的研究[J].中国畜牧兽医,2014,41(9):63-68. WANG H S,HU R P,GAO Y M,et al.Study on molecular characterization of class Ⅰ integron and integron-associated antimicrobial resistance in Escherichia coli from beef cattle[J].China Animal Husbandry & Veterinary Medicine,2014,41(9):63-68.(in Chinese)