鸭源多杀性巴氏杆菌对磺胺类药物的耐药性检测及耐药基因分析
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摘要
为明确鸭源多杀性巴氏杆菌对磺胺类药物的耐药性及耐药基因的分布特征,试验采用琼脂二倍稀释法对分离自福建、广东和江西等地的89株鸭源多杀性巴氏杆菌进行甲氧苄啶/磺胺甲噁唑的耐受性检测,用PCR法检测各耐药菌株的磺胺类耐药基因(sul1、sul2和sul3),并利用多位点序列分型(MLST)分析耐药菌株间的亲缘关系。结果显示,33株鸭源多杀性巴氏杆菌对甲氧苄啶/磺胺甲噁唑耐受,耐药率为37.1%(33/89);在耐药菌株中,sul1、sul2和sul3基因检出率分别为100%(33/33)、97.0%(32/33)和21.2%(7/33),其中sul1和sul2基因同时检出率为97.0%(32/33),sul1、sul2和sul3基因同时检出率为21.2%(7/33);所有耐药菌株均属于ST129型。以上结果表明,鸭源多杀性巴氏杆菌对磺胺类药物具有一定的耐药性,其中的耐药菌株都属于同一序列型ST129,均携带有sul1耐药基因,且绝大多数耐药菌株同时携带sul1与sul2两种耐药基因,提示在福建、广东和江西等地需慎用磺胺类药物来防治该细菌的感染。
In order to understand the antimicrobial sensitibity to sulfonamides and distribution ofsulfonamides resistance genes of Pasteurella multocida strains isolated from ducks in Fujian,Guangdong,Jiangxi,etc,89 Pasteurella multocida strains were tested for antimicrobial susceptibility to trimethoprim-sulfamethoxazole by agar dilution method,PCR method and multi-locus sequence typing(MLST)were used for detection of sulfonamides resistance genes and to analyze the genetic relationship among the sulfonamides-resistant strains.The results showed that 33strains(37.1%)were resistant to trimethoprim-sulfamethoxazole.Among the 33 sulfonamidesresistant strains,the detection rates of sul1,sul2 and sul3genes were 100%(33/33),97.0%(32/33)and 21.2%(7/33).The simultaneous detection rate of sul1 and sul2genes were 97.0%(32/33).The simultaneous detection rate of sul1,sul2 and sul3genes were 21.2%(7/33).All of the33sulfonamides-resistant strains belonged to the ST129 sequence type.The results indicated that there was sulfonamides resistance to Pasteurella multocidaisolated from ducks,and all the sulfonamides-resistant strains,which belonged to the same sequence type ST129,carried sul1 resistant gene,most of sulfonamides-resistant strains carried sul1 and sul2resistant genes,suggesting that it should be rationally used sulfonamides to prevent and cure the infection of Pasteurella multocidaisolated from ducks in some areas of Fujian,Guangdong and Jiangxi,etc.
In order to understand the antimicrobial sensitibity to sulfonamides and distribution ofsulfonamides resistance genes of Pasteurella multocida strains isolated from ducks in Fujian,Guangdong,Jiangxi,etc,89 Pasteurella multocida strains were tested for antimicrobial susceptibility to trimethoprim-sulfamethoxazole by agar dilution method,PCR method and multi-locus sequence typing(MLST)were used for detection of sulfonamides resistance genes and to analyze the genetic relationship among the sulfonamides-resistant strains.The results showed that 33strains(37.1%)were resistant to trimethoprim-sulfamethoxazole.Among the 33 sulfonamidesresistant strains,the detection rates of sul1,sul2 and sul3genes were 100%(33/33),97.0%(32/33)and 21.2%(7/33).The simultaneous detection rate of sul1 and sul2genes were 97.0%(32/33).The simultaneous detection rate of sul1,sul2 and sul3genes were 21.2%(7/33).All of the33sulfonamides-resistant strains belonged to the ST129 sequence type.The results indicated that there was sulfonamides resistance to Pasteurella multocidaisolated from ducks,and all the sulfonamides-resistant strains,which belonged to the same sequence type ST129,carried sul1 resistant gene,most of sulfonamides-resistant strains carried sul1 and sul2resistant genes,suggesting that it should be rationally used sulfonamides to prevent and cure the infection of Pasteurella multocidaisolated from ducks in some areas of Fujian,Guangdong and Jiangxi,etc.
引文
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[2]朱玲,柳旭伟,剡文亮,等.牛源荚膜A型多杀性巴氏杆菌疫苗候选株的筛选及鉴定[J].中国畜牧兽医,2018,45(2):471-478.ZHU L,LIU X W,YAN W L,et al.Screening and identification of bovine capsular type A Pasteurella multicida vaccine candidate strains[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):471-478.(in Chinese)
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[13]金天明,高丰,佟庆彬,等.巴氏杆菌对链霉素和磺胺耐药机制的研究[J].畜牧与兽医,2007,39(2):5-7.JIN T M,GAO F,TONG Q B,et al.The mechanism of drug resistance of streptomycin and sulfonamides in Pastuerella multocida[J].Animal Husbandry&Veterinary Medicine,2007,39(2):5-7.(in Chinese)
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[15]ANTUNES P,MACHADO J,SOUSA J C,et al.Dissemination of sulfonamide resistance genes(sul1,sul2and sul3)in Portuguese Salmonella enteric strains and relation with integrons[J].Antimicrobial Agents and Chemotherapy,2005,49(2):836-839.
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[18]BARBOLLA R,CATALANO M,ORMAN B E,et al.Class1integrons increase trimethoprim-sulfamethoxazole MICs against epidemiologically unrelated Stenotrophomonas maltophiliaisolates[J].Antimicrobial Agents and Chemotherapy,2004,48(2):666-669.
[19]HUR J,KIMJ H,PARK J H,et al.Molecular and virulence characteristics of multi-drug resistant Salmonella enteritidis strains isolated from poultry[J].Veterinary Journal,2011,189(3):306-311.
[20]SANDVANG D,AARESTRUP F M,JENSEN L B.Characterization ofintegrons and antibiotic resistance genes in Danish multiresistant Salmonella enteric typhimurium DT104[J].FEMS Microbiology Letters,1997,157(1):177-181.
[21]金明兰,孟庆玲,陈松,等.大肠杆菌磺胺类药物抗性基因的检测[J].中国兽医学报,2014,37(12):2358-2363.JIN M L,MENG Q L,CHEN S,et al.Detection of sulfonamide sensitivity and resistant genes in E.coli[J].Chinese Journal of Veterinary Science,2014,37(12):2358-2363.(in Chinese)
[22]MAIDEN M C,BYQRAVES J A,FEIL E,et al.Multilocus sequence typing:A portable approach to the identification of clones within populations of pathogenic microorganisms[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(6):3140-3145.
[23]WANG Y,ZHU J,LU C,et al.Evidence of circulation of an epidemic strain of Pasteurella multocida in Jiangsu,China by multi-locus sequence typing(MLST)[J].Infection Genetics and Evolution,2013,20(12):34-38.
[24]王林柏,孙久鹤,郭东春,等.国内部分地区多杀性巴氏杆菌荚膜血清型和基因型的研究[J].中国预防兽医学报,2016,38(2):116-119.WANG L B,SUN J H,GUO D C,et al.Identification of capsule serotype and genotype of Pasteurella multocidain some areas of China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(2):116-119.(in Chinese)
[25]李伟杰,田野,岂晓鑫,等.禽源多杀性巴氏杆菌多位点序列分型研究[J].中国兽药杂志,2017,51(2):1-6.LI W J,TIAN Y,QI X X,et al.Multi-locus sequence typing of Pasteurella multocidaisolated from avian[J].Chinese Journal of Veterinary Drug,2017,51(2):1-6.(in Chinese)
[2]朱玲,柳旭伟,剡文亮,等.牛源荚膜A型多杀性巴氏杆菌疫苗候选株的筛选及鉴定[J].中国畜牧兽医,2018,45(2):471-478.ZHU L,LIU X W,YAN W L,et al.Screening and identification of bovine capsular type A Pasteurella multicida vaccine candidate strains[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):471-478.(in Chinese)
[3]SWEDBERG G,CASTENSSON S,SKLD O.Characterization ofmutationally altered dihydropteroate synthase and its ability to form a sulfonamide-containing dihydrofolate analog[J].Journal of Bateriology,1979,137(1):129-136.
[4]SWEDBERQ G,SKLD O.Characterization of different plasmid-borne dihydropteroate synthases mediating bacterial resistance to sulfonamides[J].Journal of Bateriology,1980,142(1):1-7.
[5]COURVALIN P.Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria[J].Antimicrobial Agents and Chemotherapy,1994,38(7):1447-1451.
[6]SELLYEI B,VARQA Z,SZENTESI-SAMU K,et al.Antimicrobial susceptibility of Pasteurella multocida isolated from swine and poultry[J].Acta Veterinaria Hungarica,2009,57(3):357-367.
[7]FURIAN T Q,BORGES K A,LAVINIKI V,et al.Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine[J].Brazilian Journal of Microbiology,2016,47(1):210-216.
[8]CLSI(CLINICAL AND LABORATORY STAN-DARDS INSTITUTE).Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals;Second informational supplement-document VET01-S2[S].Wayne,PA:Clinical and Laboratory Standards Institute,2013.
[9]MATTER D,ROSSANO A,LIMAT S,et al.Antimicrobial resistance profile of Actinobacillus pleuropneumoniae and Actinobacillus porcitonsillarum[J].Veterinary Microbiology,2007,122(1):146-156.
[10]BOERLIN P,TRAVIS R,GYLES C L,et al.Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario[J].Applied and Environmental Microbiology,2005,71(11):6753-6761.
[11]SUBAAHARAN S,BLACKALL L L,BLACKALL P J.Development of a multi-locussequence typing scheme for avian isolates of Pasteurella multocida[J].Veterinary Microbiology,2010,141(3):354-361.
[12]SAN MILLAN A,ESCUDERO J A,GUTIERREZ B,et al.Multiresistance in Pasteurella multocida is mediated by coexistence of small plasmids[J].Antimicrobial Agents and Chemotherapy,2009,53(8):3399-3404.
[13]金天明,高丰,佟庆彬,等.巴氏杆菌对链霉素和磺胺耐药机制的研究[J].畜牧与兽医,2007,39(2):5-7.JIN T M,GAO F,TONG Q B,et al.The mechanism of drug resistance of streptomycin and sulfonamides in Pastuerella multocida[J].Animal Husbandry&Veterinary Medicine,2007,39(2):5-7.(in Chinese)
[14]TANG X,ZHAO Z,HU J,et al.Isolation,antimicrobial resistance,and virulence genes of Pasteurella multocida strains from swine in china[J].Journal of Clinical Microbobilolgy,2009,47(4):951-958.
[15]ANTUNES P,MACHADO J,SOUSA J C,et al.Dissemination of sulfonamide resistance genes(sul1,sul2and sul3)in Portuguese Salmonella enteric strains and relation with integrons[J].Antimicrobial Agents and Chemotherapy,2005,49(2):836-839.
[16]ENNE V I,LIVERMORE D M,STEPHENS P,et al.Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction[J].Lancet,2001,357(9265):1325-1328.
[17]HAMMERUM A M,SANDVANG D,ANDERSEN S R,et al.Detection of sul1,sul2and sul3in sulphonamide resistant Escherichia coli isolates obtained from healthy humans,pork and pigs in Denmark[J].International Journal of Food Microbiology,2006,106(2):235-237.
[18]BARBOLLA R,CATALANO M,ORMAN B E,et al.Class1integrons increase trimethoprim-sulfamethoxazole MICs against epidemiologically unrelated Stenotrophomonas maltophiliaisolates[J].Antimicrobial Agents and Chemotherapy,2004,48(2):666-669.
[19]HUR J,KIMJ H,PARK J H,et al.Molecular and virulence characteristics of multi-drug resistant Salmonella enteritidis strains isolated from poultry[J].Veterinary Journal,2011,189(3):306-311.
[20]SANDVANG D,AARESTRUP F M,JENSEN L B.Characterization ofintegrons and antibiotic resistance genes in Danish multiresistant Salmonella enteric typhimurium DT104[J].FEMS Microbiology Letters,1997,157(1):177-181.
[21]金明兰,孟庆玲,陈松,等.大肠杆菌磺胺类药物抗性基因的检测[J].中国兽医学报,2014,37(12):2358-2363.JIN M L,MENG Q L,CHEN S,et al.Detection of sulfonamide sensitivity and resistant genes in E.coli[J].Chinese Journal of Veterinary Science,2014,37(12):2358-2363.(in Chinese)
[22]MAIDEN M C,BYQRAVES J A,FEIL E,et al.Multilocus sequence typing:A portable approach to the identification of clones within populations of pathogenic microorganisms[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(6):3140-3145.
[23]WANG Y,ZHU J,LU C,et al.Evidence of circulation of an epidemic strain of Pasteurella multocida in Jiangsu,China by multi-locus sequence typing(MLST)[J].Infection Genetics and Evolution,2013,20(12):34-38.
[24]王林柏,孙久鹤,郭东春,等.国内部分地区多杀性巴氏杆菌荚膜血清型和基因型的研究[J].中国预防兽医学报,2016,38(2):116-119.WANG L B,SUN J H,GUO D C,et al.Identification of capsule serotype and genotype of Pasteurella multocidain some areas of China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(2):116-119.(in Chinese)
[25]李伟杰,田野,岂晓鑫,等.禽源多杀性巴氏杆菌多位点序列分型研究[J].中国兽药杂志,2017,51(2):1-6.LI W J,TIAN Y,QI X X,et al.Multi-locus sequence typing of Pasteurella multocidaisolated from avian[J].Chinese Journal of Veterinary Drug,2017,51(2):1-6.(in Chinese)