蛋鸡场中大肠杆菌对抗生素及消毒剂的耐药性
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摘要
为了解蛋鸡场中抗生素、消毒剂的耐药情况,从蛋鸡场的环境(粪便、土壤、水体等)和鸡蛋中分离大肠杆菌,测定7种抗生素和2种季铵盐消毒剂对大肠杆菌的最低抑制浓度(Minimum inhibitory concentration,MIC),检测18种抗生素耐药基因和5种季铵盐消毒剂耐药基因,并对大肠杆菌进行脉冲场凝胶电泳(Pulsed-field gel electrophoresis,PFGE)分析.共得到44株大肠杆菌;大肠杆菌对抗生素耐药率为6.82%-90.91%,对土霉素(OTC)耐药率最高,对庆大霉素(CN)耐药率最低,所有大肠杆菌对头孢噻呋钠(CFS)表现敏感,一共产生14种不同的耐药谱型,其中,AMLTMP-OTC是最主要的谱型(N=10,P=23.81%);抗生素耐药基因检出率为2.27%-84.09%,tet C检出率最高,qnr A检出率最低.消毒剂苯扎氯铵(Benzalkonium chloride,BC)、氯化十六烷基吡啶(Cetylpyridinium chloride,CPC)对大肠杆菌的MIC范围分别为8-64μg/m L、4-256μg/m L,仅检测出qac EΔ1耐药基因(N=18,P=40.91%),qac EΔ1阳性大肠杆菌与甲氧苄啶(TMP)耐药表型存在显著相关性(P<0.05);44株大肠杆菌PFGE基因型相似性在32.11%-96.62%之间.本研究表明蛋鸡场中大肠杆菌存在交叉传播的情况,对部分抗生素耐药性严重,同时对消毒剂出现耐药性,应引起足够重视.
The resistance to antibiotics and disinfectants was investigated by isolating 44 Escherichia coli strains from the surroundings of a chicken farm(including feces, soil, and water) and from eggs. The minimum inhibitory concentrations(MICs) of seven antibiotics and two quaternary ammonium compounds(QACs) in E. coli isolates were determined. Eighteen antibiotic-resistance genes and five QAC-resistance genes were amplified. The E. coli isolates were analyzed using pulsedfield gel electrophoresis(PFGE). The results showed that the resistance frequency of antibiotics in E. coli isolates ranged from 6.82% to 90.91%. The resistance frequency of oxytetracycline(OTC) was the highest and that of gentamicin was the lowest; all E. coli isolates were sensitive to ceftiofur sodium. Fourteen different antibiotic resistance profiles and the amoxicillintrimethoprim(TMP)-OTC profile were the most prevalent(N = 10, P = 23.81%). The prevalence of antibiotic resistance genes ranged from 2.27% to 84.09%. The tet C gene was dominant, and the qnr A gene was found to be the lowest. The MICs of benzalkonium chloride and cetylpyridinium chloride in E. coli isolates ranged from 8 to 64 μg/m L and from 4 to 256 μg/m L, respectively. The qac EΔ1 gene(N = 18, P = 40.91%) was the only QAC-resistance gene detected. A significant correlation was noted between qac EΔ1 gene and TMP resistance(P < 0.05). The similarity of PFGE genotypes ranged from 32.11% to 96.62%. Serious cross-contamination of E. coli was found in the chicken farm. The antibiotic and disinfectant resistances were strong in the E. coli having wide drug resistance profiles. Therefore, the study highlights the need for regularly monitoring the antibiotic and disinfectant resistance in bacteria from chicken farms and eggs.
The resistance to antibiotics and disinfectants was investigated by isolating 44 Escherichia coli strains from the surroundings of a chicken farm(including feces, soil, and water) and from eggs. The minimum inhibitory concentrations(MICs) of seven antibiotics and two quaternary ammonium compounds(QACs) in E. coli isolates were determined. Eighteen antibiotic-resistance genes and five QAC-resistance genes were amplified. The E. coli isolates were analyzed using pulsedfield gel electrophoresis(PFGE). The results showed that the resistance frequency of antibiotics in E. coli isolates ranged from 6.82% to 90.91%. The resistance frequency of oxytetracycline(OTC) was the highest and that of gentamicin was the lowest; all E. coli isolates were sensitive to ceftiofur sodium. Fourteen different antibiotic resistance profiles and the amoxicillintrimethoprim(TMP)-OTC profile were the most prevalent(N = 10, P = 23.81%). The prevalence of antibiotic resistance genes ranged from 2.27% to 84.09%. The tet C gene was dominant, and the qnr A gene was found to be the lowest. The MICs of benzalkonium chloride and cetylpyridinium chloride in E. coli isolates ranged from 8 to 64 μg/m L and from 4 to 256 μg/m L, respectively. The qac EΔ1 gene(N = 18, P = 40.91%) was the only QAC-resistance gene detected. A significant correlation was noted between qac EΔ1 gene and TMP resistance(P < 0.05). The similarity of PFGE genotypes ranged from 32.11% to 96.62%. Serious cross-contamination of E. coli was found in the chicken farm. The antibiotic and disinfectant resistances were strong in the E. coli having wide drug resistance profiles. Therefore, the study highlights the need for regularly monitoring the antibiotic and disinfectant resistance in bacteria from chicken farms and eggs.
引文
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7 Kazama H,Hamashima H,Sasatsu M,Arai T.Distribution of the antiseptic-resistance genes qac E and qac EΔ1 in Gram-negative bacteria[J].FEMS Microbiol Lett,1998,159(2):173-178
8 Chaplin CE.Bacterial resistance to quaternary ammonium disinfectants[J].J Bacteriol,1952,63(4):453-458
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18宋立,宁宜宝,张秀英,沈青春,张广川,林树茂,吴好庭,赵晖,高光,冯忠武.中国不同地区家禽大肠杆菌血清型分布和耐药性比较研究[J].中国农业科学,2005,38(7):1466-1473[Song L,Ning YB,Zhang XY,Shen QC,Zhang GC,Lin SM,Wu HT,Zhao H,Gao G,Feng ZW.Comparative research on serotype distribution and antimicrobial resistance of Escherichia coli isolates from poultry in different areas of China[J].Sci Agric Sin,2005,38(7):1466-1473]
19李晓柳,林树乾,李桂明,赵增成,冯敏燕,傅剑,宋敏训,徐凌川.山东省部分地区鸡源大肠杆菌耐药性监测分析[J].中国家禽,2014,36(20):67-69[Li XL,Lin SQ,Li GM,Zhao ZC,Feng MY,Fu J,Song MX,Xu LC.Monitoring and analysis of antibiotic resistance of Escherichia coli from chickens in Shandong Province[J].Chin Poultry,2014,36(20):67-69]
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21 Marilyn CR.Update on acquired tetracycline resistance genes[J].FEMS Microbiol Lett,2005,245(2):195-203
22何雪梅,郭莉娟,吴国艳,程琳,李蓓,罗燕,邹立扣,卿玲杉.猪肉源大肠杆菌对抗生素及消毒剂的耐药性[J].食品科学,2014,35(7):132-137[He XM,Guo LJ,Wu GY,Cheng L,Li B,Luo Y,Zou LK,Qing LS.Antibiotic and disinfectant resistance of Escherichia coli isolated from pork in Sichuan province[J].Food Sci,2014,35(7):132-137]
23张立平,张忠,曾瑾,包少文,邓光存.宁夏地区162株大肠杆菌临床分离株对四环素类药物的耐药性分析[J].广东医学,2013,34(23):3580-3583[Zhang LP,Zhang Z,Zeng J,Bao SW,Deng GC.Tetracycline resistance of 162 clinical isolates of Escherichia coli in Ningxia[J].Guangdong Med J,2013,34(23):3580-3583]
24许瑞,刘建华,胡功政,吴华,陈玉霞,赵金凤,孟春萍.鸭源大肠杆菌3种tet基因的多重PCR检测[J].江西农业学报,2011,23(3):150-152[Xu R,Liu JH,Hu GZ,Wu H,Chen YX,Zhao JF,Meng CP.Multiplex PCR for detection of three tetracycline-resistant genes in Escherichia coli isolated from duck[J].Acta Agric Jiangxi,2011,23(3):150-152]
25张纯萍,宁宜宝,宋立.健康鸡猪体内大肠杆菌对四环素的耐药性及耐药基因分布[J].中国农业科学,2010,43(12):2578-2583[Zhang CP,Ning YB,Song L.Resistance to tetracycline and distribution of tetracycline resistance determinants in commensal Escherichia coli isolated from clinically healthy chickens and pigs[J].Sci Agric Sin,2010,43(12):2578-2583]
26 Toleman MA,Bennett PM,Walsh TR.ISCR Elements:novel genecapturing systems of the 21st century?[J].Microbiol Mol Biol Rev,2006,70(2):296-316
27郭莉娟,何雪梅,邓雯文,吴国艳,龙梅,李蓓,张安云,王红宁,邹立扣.大熊猫源大肠杆菌及肺炎克雷伯氏菌对消毒剂耐药性研究[J].四川动物,2014,33(6):801-807[Guo LJ,He XM,Deng WW,Wu GY,Long M,Li B,Zhang AY,Wang HN,Zou LK.Disinfectant resistance of Escherichia coli and Klebsiella pneumonia isolated from giant panda[J].Sichuan J Zool,2014,33(6):801-807]
28 Guerra B,Junker E,Schroeter A,Malorny B,Lehmann S,Helmuth R.Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle,swine,and poultry[J].J Antimicrob Chemother,2003,52(3):489-492
29 Carattoli A.Importance of integrons in the diffusion of resistance[J].Vet Res,2001,32(3-4):243-259
30 Zhao WH,Chen G,Ito R,Kimura S,Hu ZQ.Identification of a plasmidbor ne bla IMP-11 gene in clinical isolates of Escherichia coli and Klebsiella pneumonia[J].J Med Microbiol,2012,61(Pt 2):246-251
31 Sandt CH,Krouse DA,Cook CR,Hackman AL,Chmielecki WA,Warren NG.The key role of pulsed-field gel electrophoresis in investigation of a large multiserotype and multistate food-borne outbreak of Salmonella infections centered in Pennsylvania[J].J Clin Microbiol,2006,44(9):3208-3212
32杨玲丽,陈杖榴,刘健华,曾振灵,马俊英,吴聪明.动物、环境及饲养员多重耐药大肠杆菌的PFGE分型研究[J].中国农业科学,2005,39(12):2597-2602[Yang LL,Chen ZL,Liu JH,Zeng ZL,Ma JY,Wu CM.Molecular typing of multidrug-resistant Escherichia coli isolated from farm animals,farmers and environment by pulsed-field gel electrophoresis[J].Sci Agric Sin,2005,39(12):2597-2602]
2 Tao CW,Hsu BM,Ji WT,Hsu TK,Kao PM,Hsu CP,Shen SM,Shen TY,Wan TJ,Huang YL.Evaluation of five antibiotic resistance genes in wastewater treatment systems of swine farms by real-time PCR[J].Sci Total Environ,2014,496:116-121
3 Lee JH.Antimicrobial resistance of Escherichia coli O26 and O111isolates from cattle and their characteristics[J].Vet Microbiol,2009,135(3-4):401-405
4杨汉春,陈声,Meng JH,吴清明,查振林,郭鑫.鸡源大肠杆菌对氟喹诺酮类药物的多重耐药性[J].畜牧兽医学报,2003,34(4):398-404[Yang HC,Chen S,Meng JH,Wu QM,Cha ZL,Guo X.Multiple fluoroquinolone resistance in clinical isolates of Escherichia coli from chickens[J].Acta Vet Zootech Sin,2003,34(4):398-404]
5 Langsrud S,Sundheim G.Factors contributing to the survival of poultry associated Pseudomonas spp.exposed to a quaternary ammonium compound[J].J Appl Microbiol,1997,82(6):705-712
6 Sidhu MS,S?rum H,Holck A.Resistance to quaternary ammonium compounds in food-related bacteria[J].Microb Drug Resist,2002,8(4):393-399
7 Kazama H,Hamashima H,Sasatsu M,Arai T.Distribution of the antiseptic-resistance genes qac E and qac EΔ1 in Gram-negative bacteria[J].FEMS Microbiol Lett,1998,159(2):173-178
8 Chaplin CE.Bacterial resistance to quaternary ammonium disinfectants[J].J Bacteriol,1952,63(4):453-458
9 M100-S24 Performance standards for antimicrobial susceptibility testing:twenty-fourth informational supplement[S].USA:Clinical and Laboratory Standards Institute,2014
10 Guo LJ,Long M,Huang Y,Wu GY,Deng WW,Yang XJ,Li B,Meng Y,Cheng L,Fan LQ,Zhang HM,Zou LK.Antimicrobial and disinfectant resistance of Escherichia coli isolated from giant pandas[J].J Appl Microbiol,2015,119(1):55-64
11 Zou LK,Meng JH,Mc Dermott PF,Wang F,Yang QR,Cao GJ,Hoffmann M,Zhao SH.Presence of disinfectant resistance genes in Escherichia coli isolated from retail meats in the United States[J].JAntimicrob Chemother,2014,69(10):2644-2649
12夏青青,王红宁,张安云.三重PCR方法对猪、鸡源细菌中四环素类药物耐药基因的检测[J].四川大学学报(自然科学版),2013,50(1):171-176[Xia QQ,Wang HN,Zhang AY.Detection of tetracycline resistant genes in bacterial by using a trip-PCR method[J].J Sichuan Univ:Nat Sci Ed,2013,50(1):171-176]
13田国宝,王红宁,张安云,张毅,杨鑫,徐昌文.大肠杆菌β-内酰胺酶耐药基因bla(TEM)、bla(SHV)、bla(CTX-M)三重PCR检测方法建立[J].中国兽医杂志,2013,49(1):3-5[Tian GB,Wang HN,Zhang AY,Zhang Y,Yang X,Xu CW.Development of multiplex PCR for detection ofβ-lactamases blaTEM,blaSHV,blaCTX-M in Escherichia coli[J].Chin JVet Med,2013,49(1):3-5]
14周万蓉.细菌对磺胺类药物耐药基因三重PCR检测试剂盒研制与应用[D].雅安:四川农业大学,2007
15 Zhao S,Blickenstaff K,Glenn A,Ayers SL,Friedman SL,Abbott JW,Mc Dermott PF.Beta-Lactam resistance in Salmonella strains isolated from retail meats in the United States by the National Antimicrobial Resistance Monitoring System between 2002 and 2006[J].Appl Environ Microbiol,2009,75(24):7624-7630
16杜向党,连明香,李德喜,张素梅,刘建华,潘玉善,李新生.质粒介导的喹诺酮类耐药基因在猪源大肠杆菌中的检测[J].江西农业学报,2009,21(8):9-11[Du XD,Lian MX,Li DX,Zhang SM,Liu JH,Pan YS,Li XS.Detection of plasmid-mediated quinolone resistant genes among Escherichia coli strains isolated from healthy pigs[J].Acta Agric Jiangxi,2009,21(8):9-11]
17于金玲,刘孝刚,张汉成.锦州地区鸡源大肠杆菌的分离鉴定[J].畜牧与兽医,2011,43(2):65-67[Yu JL,Liu XG,Zhang HC.Isolation and identification of Escherichia coli from chickens in Jinzhou[J].Anim Husb&Vet Med,2011,43(2):65-67]
18宋立,宁宜宝,张秀英,沈青春,张广川,林树茂,吴好庭,赵晖,高光,冯忠武.中国不同地区家禽大肠杆菌血清型分布和耐药性比较研究[J].中国农业科学,2005,38(7):1466-1473[Song L,Ning YB,Zhang XY,Shen QC,Zhang GC,Lin SM,Wu HT,Zhao H,Gao G,Feng ZW.Comparative research on serotype distribution and antimicrobial resistance of Escherichia coli isolates from poultry in different areas of China[J].Sci Agric Sin,2005,38(7):1466-1473]
19李晓柳,林树乾,李桂明,赵增成,冯敏燕,傅剑,宋敏训,徐凌川.山东省部分地区鸡源大肠杆菌耐药性监测分析[J].中国家禽,2014,36(20):67-69[Li XL,Lin SQ,Li GM,Zhao ZC,Feng MY,Fu J,Song MX,Xu LC.Monitoring and analysis of antibiotic resistance of Escherichia coli from chickens in Shandong Province[J].Chin Poultry,2014,36(20):67-69]
20张扬,陈丽鹏,吴华,赵金凤,许瑞,贺志沛,胡功政.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[Zhang Y,Chen LP,Wu H,Zhao JF,Xu R,He ZP,Hu GZ.Detection of tetracycline-tolerant genes in Escherichia coli isolated from chickens[J].Acta Agric Jiangxi,2014,26(1):94-96]
21 Marilyn CR.Update on acquired tetracycline resistance genes[J].FEMS Microbiol Lett,2005,245(2):195-203
22何雪梅,郭莉娟,吴国艳,程琳,李蓓,罗燕,邹立扣,卿玲杉.猪肉源大肠杆菌对抗生素及消毒剂的耐药性[J].食品科学,2014,35(7):132-137[He XM,Guo LJ,Wu GY,Cheng L,Li B,Luo Y,Zou LK,Qing LS.Antibiotic and disinfectant resistance of Escherichia coli isolated from pork in Sichuan province[J].Food Sci,2014,35(7):132-137]
23张立平,张忠,曾瑾,包少文,邓光存.宁夏地区162株大肠杆菌临床分离株对四环素类药物的耐药性分析[J].广东医学,2013,34(23):3580-3583[Zhang LP,Zhang Z,Zeng J,Bao SW,Deng GC.Tetracycline resistance of 162 clinical isolates of Escherichia coli in Ningxia[J].Guangdong Med J,2013,34(23):3580-3583]
24许瑞,刘建华,胡功政,吴华,陈玉霞,赵金凤,孟春萍.鸭源大肠杆菌3种tet基因的多重PCR检测[J].江西农业学报,2011,23(3):150-152[Xu R,Liu JH,Hu GZ,Wu H,Chen YX,Zhao JF,Meng CP.Multiplex PCR for detection of three tetracycline-resistant genes in Escherichia coli isolated from duck[J].Acta Agric Jiangxi,2011,23(3):150-152]
25张纯萍,宁宜宝,宋立.健康鸡猪体内大肠杆菌对四环素的耐药性及耐药基因分布[J].中国农业科学,2010,43(12):2578-2583[Zhang CP,Ning YB,Song L.Resistance to tetracycline and distribution of tetracycline resistance determinants in commensal Escherichia coli isolated from clinically healthy chickens and pigs[J].Sci Agric Sin,2010,43(12):2578-2583]
26 Toleman MA,Bennett PM,Walsh TR.ISCR Elements:novel genecapturing systems of the 21st century?[J].Microbiol Mol Biol Rev,2006,70(2):296-316
27郭莉娟,何雪梅,邓雯文,吴国艳,龙梅,李蓓,张安云,王红宁,邹立扣.大熊猫源大肠杆菌及肺炎克雷伯氏菌对消毒剂耐药性研究[J].四川动物,2014,33(6):801-807[Guo LJ,He XM,Deng WW,Wu GY,Long M,Li B,Zhang AY,Wang HN,Zou LK.Disinfectant resistance of Escherichia coli and Klebsiella pneumonia isolated from giant panda[J].Sichuan J Zool,2014,33(6):801-807]
28 Guerra B,Junker E,Schroeter A,Malorny B,Lehmann S,Helmuth R.Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle,swine,and poultry[J].J Antimicrob Chemother,2003,52(3):489-492
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