副猪嗜血杆菌临床分离菌的药物敏感性试验与ERIC-PCR基因分型
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摘要
从河南、湖北等7个省份的323份疑似副猪嗜血杆菌(HPS)典型病料中分离获得HPS菌104株。对这些分离菌株进行了16s RNA鉴定,用微量肉汤稀释法观察了这些菌株对12种抗菌药物的敏感性,并用ERIC-PCR方法对分离菌进行了基因分型。结果表明:所有菌株都对痢菌净和沃尼妙林敏感,对复方磺胺-5-甲氧嘧啶钠、四环素、环丙沙星、林可霉素、卡那霉素、甲砜霉素、红霉素、氨苄西林、头孢噻呋和头孢喹肟的耐药率分别为93.3%、51.9%、51.0%、26.0%、10.6%、13.5%、37.5%、39.4%、5.8%和1.9%;大多数菌株耐2~5种抗生素,所占比例达到69.2%;在104株分离株中有99株可以进行ERIC-PCR分型,按80%的相似性可分为18个ERIC-PCR谱型;包含菌株最多的谱型为型,有25株;其次依次为Ⅵ、Ⅷ、Ⅸ、Ⅶ型,分别有18、13、13、9株;除耐林可霉素的菌株较均匀地分布于12个ERIC-PCR型外,耐其它5种药物的菌株大多集中分布于2~3个ERIC-PCR型中。
One hundred and four strains of Haemophilus parasuis(HPS) were isolated from 323 suspected pathological pig materials in seven provinces(Henan,Hubei,etc.).These HPS isolates were identified through 16 s RNA PCR.The susceptibilities of these HPS isolates to 12 kinds of antibiotics were determined by broth microdilution method,and the genotyping of HPS isolates was carried out by using ERIC-PCR.The study showed that all strains were sensitive to mequindox and valnemulin,and their resistance rate to trimethoprim-sulfamethoxazole,tetracycline,ciprofloxacin,lincomycin,kanamycin,thiamphenicol,erythromycin,ampicillin,ceftiofur and cefquinome was 93.3%,51.9%,51.0%,26.0%,10.6%,13.5%,37.5%,39.4%,5.8% and 1.9%,respectively.Most strains were resistant to 2 ~ 5 kinds of antibiotics,and their proportion was 69.2%.Among 104 HPS isolates,99 isolates could be divided into 18 ERIC-PCR types according to 80% similarity.Among all ERIC-PCR types, type included the most strains(25 strains),which was followed by Ⅵ type(18 strains),Ⅷ type(13 strains),Ⅸ type(13 strains) and Ⅶ type(9 strains).Except the lincomycin-resistant isolates were almost equally distributed in 12 ERIC-PCR types,most of isolates resistant to other 5 kinds of antibiotics were concentratedly distributed in 2 ~ 3 ERIC-PCR types.
One hundred and four strains of Haemophilus parasuis(HPS) were isolated from 323 suspected pathological pig materials in seven provinces(Henan,Hubei,etc.).These HPS isolates were identified through 16 s RNA PCR.The susceptibilities of these HPS isolates to 12 kinds of antibiotics were determined by broth microdilution method,and the genotyping of HPS isolates was carried out by using ERIC-PCR.The study showed that all strains were sensitive to mequindox and valnemulin,and their resistance rate to trimethoprim-sulfamethoxazole,tetracycline,ciprofloxacin,lincomycin,kanamycin,thiamphenicol,erythromycin,ampicillin,ceftiofur and cefquinome was 93.3%,51.9%,51.0%,26.0%,10.6%,13.5%,37.5%,39.4%,5.8% and 1.9%,respectively.Most strains were resistant to 2 ~ 5 kinds of antibiotics,and their proportion was 69.2%.Among 104 HPS isolates,99 isolates could be divided into 18 ERIC-PCR types according to 80% similarity.Among all ERIC-PCR types, type included the most strains(25 strains),which was followed by Ⅵ type(18 strains),Ⅷ type(13 strains),Ⅸ type(13 strains) and Ⅶ type(9 strains).Except the lincomycin-resistant isolates were almost equally distributed in 12 ERIC-PCR types,most of isolates resistant to other 5 kinds of antibiotics were concentratedly distributed in 2 ~ 3 ERIC-PCR types.
引文
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[2]中国兽医学会.2010年执业兽医资格考试应试指南(上)[M].北京:中国农业出版社,2010:644-646.
[3]张悦,李蓓蓓,魏建超,等.上海副猪嗜血杆菌分离鉴定及其耐药性分析[J].中国动物传染病学报,2015(4):25-30.
[4]张悦.副猪嗜血杆菌耐药性调查及其对氟苯尼考耐药分子机制的研究[D].北京:中国农业科学院,2015.
[5]Guo L,Zhang J,Xu C,et al.Molecular characterization of fluoroquinolone resistance in Haemophilus parasuis isolated from pigs in South China[J].Journal of Antimicrobial Chemotherapy,2011,66(3):539-542.
[6]Clinical Laboratory Standards Institute.Performance standards for antimicrobial disk and dilution susceptibility test for bacteria isolated from animals:approved standard M100-S23[M].Wayne:Clinical Laboratory Standards Institute,2013.
[7]Clinical Laboratory Standards Institute.Performance standards for antimicrobial disk and dilution susceptibility test for bacteria isolated from animals:approved standard M100S[M}.Wayne:Clinical Laboratory Standards Institute,2016.
[8]The European Committee on Antimicrobial Susceptibility Testing(EUCAST).Breakpoint tables for interpretations of MICs and zone diameters,version 7.1[M].2017.
[9]Garch F E,Jong A D,Simjee S,et al.Monitoring of antimicrobial susceptibility of respiratory tract pathogens isolated from diseased cattle and pigs across Europe,2009~2012:Vet Path results[J].Veterinary Microbiology,2016,194:11-22.
[10]Appuhamy S,Parton R,Coote J G,et al.Genomic fingerprinting of Haemophilus somnus by a combination of PCR methods[J].Journal of Clinical Microbiology,1997,35(1):288-291.
[11]Versalovic J,Koeuth T,Lupski J R.Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes[J].Nucleic Acids Research,1991,19(24):6823.
[12]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.
[13]张立宪,游一.副猪嗜血杆菌的分离鉴定与药敏试验[J].河南农业科学,2015,44(9):105-107,111.
[14]Xu C,Zhang J,Zhao Z,et al.Antimicrobial susceptibility and PFGE genotyping of Haemophilus parasuis isolates from pigs in South China(2008~2010)[J].Journal of Veterinary Medical Science,2011,73(8):1061.
[15]忽占利,李军星,胡松华,等.副猪嗜血杆菌分离株的耐药性及耐药基因分析[J].华北农学报,2013(4):228-233.
[16]De la Fuente A J,Tucker A W,Nacas J.Antimicrobial susceptibility patterns of Haemophilus parasuis from pigs in the United Kingdom and Spain[J].Veterinary Microbiology,2007,120:184-191.
[17]周学利.副猪嗜血杆菌分离株的血清分型及其耐药性研究[D].武汉:华中农业大学,2009.
[18]万世平,王建,葛菲菲,等.副猪嗜血杆菌上海株的分离鉴定及耐药特性的研究[J].广东农业科学,2010,37(8):201-203.
[19]NedbalcováK,KucerováZ.Antimicrobial susceptibility of Pasteurella multocida and Haemophilus parasuis isolates associated with porcine pneumonia[J].Acta Veterinaria Brno,2013,82(1):3-7.
[20]San Millan A,GiufréM,Escudero J A,et al.Contribution of ROB-1 and PBP3 mutations to the resistance phenotype of aβ-lactamase-positive amoxicillin/clavulanic acid-resistant Haemophilus influenzae carrying plasmid p B1000 in Italy[J].Journal of Antimicrobial Chemotherapy,2011,66(1):96.
[21]Souza A V,Moreira C R,Pasternak J,et al.Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit[J].J Med Microb,2004,53(10):999-1005.
[22]Pettigrew M M,Foxman B,Ecevit Z,et al.Use of pulsedfield gel electrophoresis enterobacterial repetitive intergenic consensus typing,and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae[J].Journal of Clinical Microbiology,2002,40(2):660-662.
[23]Rafiee M,Bara M,Stephens C P,et al.Application of ERIC-PCR for the comparison of isolates of Haemophilus parasuis[J].Australian Veterinary Journal,2000,78(12):846.