6株奶牛乳房炎肺炎克雷伯菌的分离、鉴定及生物学特性
|
摘要
为研究陕西关中地区奶牛乳房炎肺炎克雷伯菌的流行情况,从某规模化奶牛场无菌采集乳样53份,进行细菌分离鉴定。通过细菌形态学观察、生化试验以及16S rRNA序列分析,共分离出6株肺炎克雷伯菌,并对其进行药物敏感性、生物被膜形成、random amplified polymorphic DNA(RAPD)分型以及小鼠致病性的试验。生化特性结果表明6株肺炎克雷伯菌对各种糖的分解能力无明显差异。药敏试验结果表明,6株菌均对恩诺沙星、阿米卡星、环丙沙星、链霉素、庆大霉素、四环素、美罗培南、头孢曲松和头孢他啶敏感,对青霉素和氨苄西林耐药。生物被膜表型检测结果表明,6株菌均可产生生物被膜,其中菌株KP4为中等黏附,其他5株为弱黏附,生物被膜成分中均含有胞外多糖,不含卷曲菌毛和纤维素。RAPD分型结果表明,分离的6株菌株共分3个基因型,其中C型占50.00%(3/6),B型占33.33%(2/6),A型占16.67%(1/6)。小鼠致病性结果表明,分离株对小鼠均具有较强的致病性,被感染小鼠的肝脏、脾脏和肺脏均有不同程度的出血。本试验为防控陕西省内肺炎克雷伯菌引起的奶牛乳房炎提供依据。
The purpose of this study was to detect the epidemiology and biological characteristics of Klebsiella pneumonia,which were isolated from dairy cows with mastitis in Guanzhong area,Shaanxi province.The six strains of pathogenic Klebsiella pneumoniaisolated from 53 milk samples in a large-scale dairy farm were identified by bacterial morphology observation,biochemical analysis,16 SrRNA sequence and PCR detection.WHO recommended by Kirby-Bauer(K-B)determination of 18 antimicrobial drug resistance,and isolated six Klebsiella pneumoniae biofilm formation and random amplified polymorphic DNA(RAPD)characteristics were further compared.Also,mouse virulence test of these isolates were tested.The results showed that the biochemical characteristics of six isolated strains have no significant difference in the ability to decomposition of various sugars;The results of drug sensitivity test showed that six isolated strains were highly sensitive to enrofloxacin,amikacin,ciprofloxacin,streptomycin,gentamicin,tetracycline,meropenem,ceftriaxone,ceftazidime,and resistant to ampicillin and penicillin.The biofilm phenotype test results showed that all 6 strains produced biofilms,the strain KP4 was moderately adherent,and the other 5 strains were weakly adherent.The composition of the biofilm contain extracellular polysaccharide,and free from curli and cellulose.And the 6 strains of Klebsiella pneumonia wouldalso be catergoried into three grops by RAPD grouping system,3 strains in type C(50.00%),2 strains in type B(33.33%),1 strain in type A(16.67%).The results of pathogenicity in mice showed that the isolates had strong pathogenicity to mice,and the lung,liver and spleen of infected mice all displayed different degrees of bleeding.This study provides useful reference materials for prevention and control of mastitis caused by Klebsiella pneumoniae in Shaanxi province.
The purpose of this study was to detect the epidemiology and biological characteristics of Klebsiella pneumonia,which were isolated from dairy cows with mastitis in Guanzhong area,Shaanxi province.The six strains of pathogenic Klebsiella pneumoniaisolated from 53 milk samples in a large-scale dairy farm were identified by bacterial morphology observation,biochemical analysis,16 SrRNA sequence and PCR detection.WHO recommended by Kirby-Bauer(K-B)determination of 18 antimicrobial drug resistance,and isolated six Klebsiella pneumoniae biofilm formation and random amplified polymorphic DNA(RAPD)characteristics were further compared.Also,mouse virulence test of these isolates were tested.The results showed that the biochemical characteristics of six isolated strains have no significant difference in the ability to decomposition of various sugars;The results of drug sensitivity test showed that six isolated strains were highly sensitive to enrofloxacin,amikacin,ciprofloxacin,streptomycin,gentamicin,tetracycline,meropenem,ceftriaxone,ceftazidime,and resistant to ampicillin and penicillin.The biofilm phenotype test results showed that all 6 strains produced biofilms,the strain KP4 was moderately adherent,and the other 5 strains were weakly adherent.The composition of the biofilm contain extracellular polysaccharide,and free from curli and cellulose.And the 6 strains of Klebsiella pneumonia wouldalso be catergoried into three grops by RAPD grouping system,3 strains in type C(50.00%),2 strains in type B(33.33%),1 strain in type A(16.67%).The results of pathogenicity in mice showed that the isolates had strong pathogenicity to mice,and the lung,liver and spleen of infected mice all displayed different degrees of bleeding.This study provides useful reference materials for prevention and control of mastitis caused by Klebsiella pneumoniae in Shaanxi province.
引文
[1] LAM T J,BH V D B,JANSEN J,et al.Improving bovine udder health:a national mastitis control program in the Netherlands[J].J Dairy Sci,2013,96(2):1301-1311.
[2] 孙垚,贾坤,平晓坤,等.广东省部分地区奶牛乳房炎凝固酶阴性葡萄球菌的毒力基因和耐药性分析[J].中国兽医学报,2017,37(8):1495-1500.
[3] 王正兵,严作廷,罗金印,等.我国奶牛乳房炎常见病原菌的耐药性检测[J].贵州农业科学,2011,39(4)139-141.
[4] 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.
[5] SAISHU N,OZAKI H,MURASE T.CTX-M-type extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from cases of bovine mastitis in Japan[J].J Vet Med Sci,2014,76(8):1153-1156.
[6] 佟德炎.奶牛乳房炎源性肺炎克雷伯杆菌的耐药性和毒力基因检测[D].北京:中国农业大学,2017.
[7] QIN X,WEISSMAN S J,CHESNUT M F,et al.Kirby-Bauer disc approximation to detect inducible third-generation cephalosporin resistance in Enterobacteriaceae[J].Ann Clin Microbiol Antimicrob,2004,3(1):13.
[8] VUOTTO C,LONGO F,PASCOLINI C,et al.Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains[J].J Appl Microbiol,2017,123(4):1003-1018.
[9] JAIN S,CHEN J.Antibiotic resistance profiles and cell surface components of Salmonellae[J].J Food Prot,2006,69(5):1017-1023.
[10] 叶明亮,刁亚林,侯晓琦.随机引物PCR法基因分型肺炎克雷伯菌[J].中国误诊学杂志,2005,5(8):1403-1405.
[11] BOU G,CERVERO G,DOMINGUEZ M A,et al.Characterization of a nosocomial outbreak caused by a multiresistant Acinetobacte rbaumannii strain with a carbapenem-hydrolyzing enzyme:high-level carbapenem resistance in A.rbaumannii is not due solely to the presence of beta-lactamases[J].J Clin Microbiol,2000,38(9):3299-3305.
[12] 和晋渝.肺炎克雷伯菌的血清分型及毒力基因分布的研究[D].重庆:重庆医科大学,2012.
[13] 林雅,韩冠双,郭建华,等.肉牛上呼吸道肺炎克雷伯菌的分离鉴定及耐药分析[J].中国兽医学报,2015,35(11):1777-1781.
[14] HARAHAP N,SANTOSA D A,GOFAR N.The potential of exopolysaccharide-producing bacteria from rhizosphere of rubber plants for improving soil aggregate[J].2018,5(3):1275-1281.
[15] BRISSE S,EV D.Identification and antimicrobial susceptibility of 100 Klebsiella,animal clinical isolates[J].Vet Microbiol,2005,105(3):307-312.
[16] 徐丽,林迪斯,杨靖,等.肺炎克雷伯菌KbvR调控因子对细菌生物膜与荚膜形成能力的影响[J].南方医科大学学报,2016(10):1435-1439.
[17] DAVIES D.Understanding biofilm resistance to antibacterial agents[J].Nat Rev Drug Discov,2003,2(2):114-122.
[18] 石宗盛,薛世聪.产生物膜的肺炎克雷伯菌的体外药敏试验探讨[J].国际检验医学杂志,2017,38(4):490-492.
[19] ZOGAJ X,BOKRANZ W,NIMTZ M,et al.Production of cellulose and curli fimbriae by members of the family enterobacteriaceae isolated from the human gastrointestinal tract.[J].Infect Immun,2003,71(7):4151.
[20] 海岩,郝永清,张爱荣,等.奶牛乳房炎克雷伯氏菌的基因分型[J].中国兽药杂志,2007(5):21-23.
[2] 孙垚,贾坤,平晓坤,等.广东省部分地区奶牛乳房炎凝固酶阴性葡萄球菌的毒力基因和耐药性分析[J].中国兽医学报,2017,37(8):1495-1500.
[3] 王正兵,严作廷,罗金印,等.我国奶牛乳房炎常见病原菌的耐药性检测[J].贵州农业科学,2011,39(4)139-141.
[4] 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.
[5] SAISHU N,OZAKI H,MURASE T.CTX-M-type extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from cases of bovine mastitis in Japan[J].J Vet Med Sci,2014,76(8):1153-1156.
[6] 佟德炎.奶牛乳房炎源性肺炎克雷伯杆菌的耐药性和毒力基因检测[D].北京:中国农业大学,2017.
[7] QIN X,WEISSMAN S J,CHESNUT M F,et al.Kirby-Bauer disc approximation to detect inducible third-generation cephalosporin resistance in Enterobacteriaceae[J].Ann Clin Microbiol Antimicrob,2004,3(1):13.
[8] VUOTTO C,LONGO F,PASCOLINI C,et al.Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains[J].J Appl Microbiol,2017,123(4):1003-1018.
[9] JAIN S,CHEN J.Antibiotic resistance profiles and cell surface components of Salmonellae[J].J Food Prot,2006,69(5):1017-1023.
[10] 叶明亮,刁亚林,侯晓琦.随机引物PCR法基因分型肺炎克雷伯菌[J].中国误诊学杂志,2005,5(8):1403-1405.
[11] BOU G,CERVERO G,DOMINGUEZ M A,et al.Characterization of a nosocomial outbreak caused by a multiresistant Acinetobacte rbaumannii strain with a carbapenem-hydrolyzing enzyme:high-level carbapenem resistance in A.rbaumannii is not due solely to the presence of beta-lactamases[J].J Clin Microbiol,2000,38(9):3299-3305.
[12] 和晋渝.肺炎克雷伯菌的血清分型及毒力基因分布的研究[D].重庆:重庆医科大学,2012.
[13] 林雅,韩冠双,郭建华,等.肉牛上呼吸道肺炎克雷伯菌的分离鉴定及耐药分析[J].中国兽医学报,2015,35(11):1777-1781.
[14] HARAHAP N,SANTOSA D A,GOFAR N.The potential of exopolysaccharide-producing bacteria from rhizosphere of rubber plants for improving soil aggregate[J].2018,5(3):1275-1281.
[15] BRISSE S,EV D.Identification and antimicrobial susceptibility of 100 Klebsiella,animal clinical isolates[J].Vet Microbiol,2005,105(3):307-312.
[16] 徐丽,林迪斯,杨靖,等.肺炎克雷伯菌KbvR调控因子对细菌生物膜与荚膜形成能力的影响[J].南方医科大学学报,2016(10):1435-1439.
[17] DAVIES D.Understanding biofilm resistance to antibacterial agents[J].Nat Rev Drug Discov,2003,2(2):114-122.
[18] 石宗盛,薛世聪.产生物膜的肺炎克雷伯菌的体外药敏试验探讨[J].国际检验医学杂志,2017,38(4):490-492.
[19] ZOGAJ X,BOKRANZ W,NIMTZ M,et al.Production of cellulose and curli fimbriae by members of the family enterobacteriaceae isolated from the human gastrointestinal tract.[J].Infect Immun,2003,71(7):4151.
[20] 海岩,郝永清,张爱荣,等.奶牛乳房炎克雷伯氏菌的基因分型[J].中国兽药杂志,2007(5):21-23.