狐狸源肺炎克雷伯菌的分离鉴定及毒力检测
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摘要
从濒死期蓝狐肺脏中分离到1株革兰阴性短杆菌,并对分离菌株进行形态学观察、16S rRNA鉴定、全自动细菌生化鉴定仪鉴定及khe基因鉴定,在此基础上,完成了分离菌株的分型、人工感染小鼠试验、药敏试验及毒力基因检测等。最终鉴定结果显示,该分离菌株为K20型肺炎克雷伯菌,命名为SWKp17;同时,Vitek细菌鉴定仪鉴定该菌为肺炎克雷伯菌。人工感染试验和药敏试验结果表明,该细菌有致病性和多重耐药性。其对小鼠的半数致死量(LD_(50))为2.9×10~7 CFU;对三代头孢菌素类、氯霉素类、喹诺酮类、氨基糖苷类、四环素类、磺胺类耐药,但对亚胺培南和多黏菌素敏感。该菌株具有fim、ureA、mrkD及wabG毒力基因,但不具有rmpA、kfu、alls、iroNB、ybtA、uge、iucB等毒力基因。结果表明,引起本次狐狸肺炎的致病菌为非K1、K2、K3、K5、K20、K54及K57型肺炎克雷伯菌,本研究为临床治疗由该菌引起的狐狸肺炎提供参考依据。
In order to identify the pathogen caused a serious mortality of blue fox in Shandong Province,the lung samples from the dying blue fox were collected and one gram-negative bacterium was isolated and purified.Then carried out the 16 S rRNA identification,automatic biochemical identification of bacteria identification and khe gene identification.On this basis,the serotype characteristics,pathogenicity tests,drug sensitivity test as well as genetic evolution analysis of main virulence genes were completed.16 S rRNA and khe gene sequence identification results showed that the isolated strain K54 was Klebsiella pneumoniae,named as SWKp17;the same time,Vitek bacterial identification instrument identified as Klebsiella pneumoniae.Pathogenicity tests and drug sensitivity test results showed that the strain was pathogenic and multi-drug resistance.The median lethal dose(LD_(50)) in mice was 2.9×10~7 CFU.The antimicrobial sensitivity test showed that the pathogen was resistant to third-generation cephalosporins,chloramphenicol,quinolones,aminoglycosides,tetracyclines and sulfonamides,sensitive to imipenem and polymyxin.It contained fim,ureA,mrkD and wabG genes,but did not contain virulence rmpA,kfu,alls,iroNB,ybtA,uge and iucB genes.The results of this study confirmed that the died fox did not caused by K1,K2,K3,K5,K20,K54 and K57 Klebsiella pneumoniae.These results provided a reference for clinical treatment of fox pneumonia caused by the bacteria.
In order to identify the pathogen caused a serious mortality of blue fox in Shandong Province,the lung samples from the dying blue fox were collected and one gram-negative bacterium was isolated and purified.Then carried out the 16 S rRNA identification,automatic biochemical identification of bacteria identification and khe gene identification.On this basis,the serotype characteristics,pathogenicity tests,drug sensitivity test as well as genetic evolution analysis of main virulence genes were completed.16 S rRNA and khe gene sequence identification results showed that the isolated strain K54 was Klebsiella pneumoniae,named as SWKp17;the same time,Vitek bacterial identification instrument identified as Klebsiella pneumoniae.Pathogenicity tests and drug sensitivity test results showed that the strain was pathogenic and multi-drug resistance.The median lethal dose(LD_(50)) in mice was 2.9×10~7 CFU.The antimicrobial sensitivity test showed that the pathogen was resistant to third-generation cephalosporins,chloramphenicol,quinolones,aminoglycosides,tetracyclines and sulfonamides,sensitive to imipenem and polymyxin.It contained fim,ureA,mrkD and wabG genes,but did not contain virulence rmpA,kfu,alls,iroNB,ybtA,uge and iucB genes.The results of this study confirmed that the died fox did not caused by K1,K2,K3,K5,K20,K54 and K57 Klebsiella pneumoniae.These results provided a reference for clinical treatment of fox pneumonia caused by the bacteria.
引文
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[18] 和晋渝,邱景富,刘梦颖,等.PCR鉴定肺炎克雷伯菌的强毒性血清型[J].生物技术通讯,2012,23(4):554-557.
[19] MAGIORAKOS A P,SRINIVASAN A,CAREY R B,et al.Multidrug-resistant,extensively drug-resistant and pandrug-resistant bacteria:an international expert proposal for interim standard definitions for acquired resistance[J].Clin Microbiol Infect,2012,18(3):268-281.
[2] AYLING-SMITH B,PITT T L.State of the art in typing:Klebsiella spp[J].J Hosp Infect,1990,16(4):287-295.
[3] CORTES G,BORRELL N,DE A B,et al.Molecular analysis of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia[J].Infect Immun,2002,70(5):2583-2590.
[4] TURTON J F,PERRY C,ELGOHARI S,et al.PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific,variable number tandem repeat and virulence gene targets[J].J Med Microbiol,2010,59(5):541-547.
[5] CHOU H C,LEE C Z,MA L C,et al.Isolation of a chromosomal region of Klebsiella pneumoniae associated with allantoin metabolism and liver infection[J].Infect Immun,2004,72(7):3783-3792.
[6] 冯娜,高翔,肖敏,等.牛源肺炎克雷伯氏菌的分离鉴定及遗传进化分析[J].中国兽医科学,2016,46(11):1358-1364.
[7] 胡本刚,杜崇涛,雷连成,等.貂源肺炎克雷伯菌的分离与鉴定[J].中国预防兽医学报,2011,33(8):598-600.
[8] WANG J L,SHANG Y Y,GUO S Y,et al.Serotype and virulence genes of Klebsiella pneumoniae isolated from mink and its pathogenesis in mice and mink[J].Sci Rep,2017,7(1):17291.
[9] COLODNER R,ROCK W,CHAZAN B,et al.Risk factors for the development of extended-spectrum beta-lactamase-producing bacteriain nonhospitalized patients[J].Eur J Clin Microbiol Infect Dis,2004,23(3):163-167.
[10] FANG C T,LAI S Y,YI W C,et al.Klebsiella pneumoniae genotype K1:an emerging pathogen that causes septicocular or central nervous system complications from pyogenic liver abscess[J].Clin Infect Dis,2007,45(3):284-293.
[11] PAN Y J,FANG H C,YANG H C,et al.Capsular polysac charide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype[J].J Clin Microbiol,2008,46(7):2231-2240.
[12] LIN J C,KOH T H,LEE N,et al.Genotypes and virulence in serotype K2 Klebsiella pneumoniae from liver abscess and non-infectious carriers in Hong Kong,Singapore and Taiwan[J].Gut Pathogens,2014,6(21):1-7.
[13] HSIEH P F,LIN T L,LEE C Z,et al.Serum-induced iron-acquisition systems and TonB contribute to virulence in Klebsiella pneumoniae causing primary pyogenic liver abscess[J].J Infect Dis,2008,197(12):1717-1727.
[14] YU W L,KO W C,CHENG K C,et al.Association between rmpA and magA genes and clinical syndromes caused by Klebsiella pneumoniae in Taiwan[J].Clin Infect Dis,2006,42(10):1351-1358.
[15] DAS A,ACHARYA S,BEHERA B K,et al.Isolation,identification and characterization of Klebsiella pneumoniae from infected farmed Indian major carp Labeo rohita (Hamilton 1822) in West Bengal,India[J].Aquaculture,2018,482:111-116.
[16] CHEN I L,LEE C H,SU L H,et al.Antibiotic consumption and healthcare-associated infections caused by multidrug-resistant gram-negative bacilli at a large medical center in Taiwan from 2002 to 2009:implicating the importance of antibiotic stewardship[J].PLoS One,2013,8(5):1-6.
[17] CUBERO M,GRAU I,TUBAU F,et al.Hypervirulent Klebsiella pneumoniae clones causing bacteraemia in adults in a teaching hospital in Barcelona,Spain(2007-2013)[J].Clin Microbiol Infect,2016,22(2):154-160.
[18] 和晋渝,邱景富,刘梦颖,等.PCR鉴定肺炎克雷伯菌的强毒性血清型[J].生物技术通讯,2012,23(4):554-557.
[19] MAGIORAKOS A P,SRINIVASAN A,CAREY R B,et al.Multidrug-resistant,extensively drug-resistant and pandrug-resistant bacteria:an international expert proposal for interim standard definitions for acquired resistance[J].Clin Microbiol Infect,2012,18(3):268-281.