貉源肺炎克雷伯菌的分离鉴定及药敏试验
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摘要
为确定河北昌黎地区某养殖场貉呼吸道疾病引起死亡的病因,本研究从送检的貉病料肺脏中分离到1株革兰氏阴性短杆菌,并对分离株进行形态学观察、16S rRNA序列测序及全自动细菌生化鉴定仪鉴定,在此基础上完成了分离株的分型、人工感染小鼠试验及药敏试验等。结果显示,该分离株为非K1/K2型肺炎克雷伯菌,并将其命名为CLKp18;该分离株的16S rRNA基因序列与GenBank中登录的多株肺炎克雷伯菌参考株同源性均高达99%。Vitek全自动细菌鉴定仪结果表明,该菌株与肺炎克雷伯菌鉴定标准相似度为99%。人工感染小鼠试验结果显示,试验组小鼠在攻毒后6 h开始出现临床症状。无菌取出死亡小鼠的肺脏、肝脏、脾脏及肾脏,划线涂板,均可分离到该菌,表明该分离株感染小鼠后,能侵入到小鼠体内多个组织器官,引起不同程度的病变,尤以肝脏和肺脏病变较为明显,对小鼠的半数致死量(LD_(50))为2.17×10~7 CFU。药敏试验结果表明,该菌对单环内酰胺类、氯霉素类、氨基糖苷类、四环素类、磺胺类及大部分头孢类耐药,但对阿米卡星、阿莫西林/克拉维酸、头孢他啶和亚胺培南敏感。本研究结果确定了引起本次貉呼吸道症状疾病的致病菌为非K1/K2型肺炎克雷伯菌,且该分离株有较强的致病性和多重耐药性。本研究为临床治疗由该菌引起的貉肺炎提供了参考依据。
To determine the pathogen caused a serious mortality of racoon dog in Hebei province,and one Gram-negative bacterium was isolated from the the lung samples of diseased racoon dog.The isolate received morphological obsrvation,16 S rRNA sequence analysis and biochemical test.Basis on this,the serotype characteristics,pathogenicity tests,drug sensitivity test were completed.The isolated strain was confirmed as not K1/K2 Klebsiella pneumoniae,named as CLKp18.16 S rRNA gene sequence analysis results showed that the bacterium was 99% homologous with many strains of Klebsiella accessed in GenBank.The Vitek automatic bacterial identification results showed that the similarity between the strain and Klebsiella pneumoniae standard was 99%.Pathogenicity test results showed that the mice in the experimental group began to develop clinical symptoms 6 h after the challenge.The lung,liver,spleen and kidney of the dead mice were aseptically removed,and the bacteria were isolated by scribing and plating.It indicated that the infected strain could invade many tissues and organs in mice after infection,which caused different degrees of lesions,especially liver and lung lesions,the LD_(50) in mice was 2.17×10~7 CFU.Drug susceptibility test results showed that the strain resistant to monocyclic lactam,chloramphenicol,aminoglycosides,tetracyclines,sulfonamides and most cephalosporins,and sensitive to amikacin,amoxycillin/clavulanic acid,imipenem and ceftazidime.This study confirmed that K1/K2 Klebsiella pneumoniae didn't cause the pathogens,and the strain with strong pathogenicity in mice and multi-drug resistance.The results provided a reference for clinical treatment of pneumonia caused by the strain.
To determine the pathogen caused a serious mortality of racoon dog in Hebei province,and one Gram-negative bacterium was isolated from the the lung samples of diseased racoon dog.The isolate received morphological obsrvation,16 S rRNA sequence analysis and biochemical test.Basis on this,the serotype characteristics,pathogenicity tests,drug sensitivity test were completed.The isolated strain was confirmed as not K1/K2 Klebsiella pneumoniae,named as CLKp18.16 S rRNA gene sequence analysis results showed that the bacterium was 99% homologous with many strains of Klebsiella accessed in GenBank.The Vitek automatic bacterial identification results showed that the similarity between the strain and Klebsiella pneumoniae standard was 99%.Pathogenicity test results showed that the mice in the experimental group began to develop clinical symptoms 6 h after the challenge.The lung,liver,spleen and kidney of the dead mice were aseptically removed,and the bacteria were isolated by scribing and plating.It indicated that the infected strain could invade many tissues and organs in mice after infection,which caused different degrees of lesions,especially liver and lung lesions,the LD_(50) in mice was 2.17×10~7 CFU.Drug susceptibility test results showed that the strain resistant to monocyclic lactam,chloramphenicol,aminoglycosides,tetracyclines,sulfonamides and most cephalosporins,and sensitive to amikacin,amoxycillin/clavulanic acid,imipenem and ceftazidime.This study confirmed that K1/K2 Klebsiella pneumoniae didn't cause the pathogens,and the strain with strong pathogenicity in mice and multi-drug resistance.The results provided a reference for clinical treatment of pneumonia caused by the strain.
引文
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[10] 杨锐,文继锋,龚永平,等. 小熊猫感染肺炎克雷伯氏菌的检查及药敏试验[J]. 中国人兽共患病学报,2017,33(3):271-275. YANG R,WEN J F,GONG Y P,et al.Detection and antibiotic sensitivity test of Klebsiella penumoniae in Ailurus fulgens[J].Chinese Journal of Zoonoses,2017,33(3):271-275.(in Chinese)
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[12] 李巧玲,肖丽荣,张文举,等. 蓝狐源肺炎克雷伯菌的鉴定与药敏试验[J]. 畜牧与兽医,2018,50(3):103-106. LI Q L,XIAO L R,ZHANG W J,et al.Isolation,identification and drug susceptibility of Klebsiella pneumoniae isolated from blue fox[J].Animal Husbandry & Veterinary Medicine,2018,50(3):103-106.(in Chinese)
[13] 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(1):111-116.
[14] PACZOSA M K,MECSAS J.Klebsiella pneumoniae:Going on the offense with a strong defense[J].Microbiology and Molecular Biology Reviews,2016,80(3):629-661.
[15] QUAN T P,FAWCETT N J,WRIGHTSON J M,et al.Increasing burden of community-acquired pneumonia leading to hospitalisation 1998-2014[J].Thorax,2016,71(6):535-542.
[16] TWENHAFEL N A,WHITEHOUSE C A,STEVENS E L,et al.Multisystemic abscesses in African green monkeys (Chlorocebus aethiops) with invasive Klebsiella pneumoniae—identification of the hypermucoviscosity phenotype[J].Veterinary Pathology,2008,45(2):226-231.
[17] JANG S,WHEELER L,CAREY R B,et al.Pleuritis and suppurative pneumonia associated with a hypermucoviscosity phenotype of Klebsiella pneumoniae in California sea lions (Zalophus californianus)[J].Veterinary Microbiology,2010,141(1-2):174-177.
[18] MANGES A R.Editorial commentary:Genomic epidemiology:Revealing hidden reservoirs for Klebsiella pneumoniae[J].Clinical Infectious Diseases,2015,61(6):900-902.
[19] 钟世勋,迟珊珊,王振,等. 山东规模化养殖场毛皮动物多重感染分析[J]. 中国兽医学报,2014,34(11):1770-1777. ZHONG S X,CHI S S,WANG Z,et al.The etiology analysis of multiple infections in Shandong scale fur frams[J].Chinese Journal of Veterinary Science,2014,34(11):1770-1777.(in Chinese)
[20] 韩坤,白雪,闫喜军,等. 貂源肺炎克雷伯氏菌的分离及毒力和耐药性的分析[J]. 中国兽医科学,2018,48(8):1019-1023. HAN K,BAI X,YAN X J,et al.Isolation,virulence and drug resistance of Klebsiella pneumoniae from mink[J].Chinese Veterinary Science,2018,48(8):1019-1023.(in Chinese)
[21] BRISSE S,GRIMONT F,GRIMONT P A D.The Genus Klebsiella,A Handbok on the Biology of Bacteria[M].3rd Edition.New York:Springer,2006.
[22] 黄海迪,郭卉,王贵升,等. 貂源肺炎克雷伯氏菌对小鼠的致病性及其药物防治研究[J].中国兽药杂志,2016,50(8):44-48. HUANG H D,GUO H,WANG G S,et al.Pathogenicity and medicine treatment of Klebsiella pneumoniae from mink in mice[J].Chinese Journal of Veterinary Drug,2016,50(8):44-48.(in Chinese)
[23] 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].Clinical Microbiology and Infection,2012,18(3):268-281.
[2] TRAUTMANN M,HELD T K,CROSS A S,et al.O antigen seroepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections[J].Vaccine,2004,22(7):818-821.
[3] GAYNES R,EDWARDS J R.Overview of nosocomial infections caused by Gram-negative bacilli[J].Clinical Infectious Diseases,2005,41(6):848-854.
[4] YU V L,HANSEN D S,KO W C,et al.Virulence characteristics of Klebsiella and clinical manifestations of K.pneumoniae bloodstream infections[J].Emerging Infectious Diseases,2007,13(7):986-993.
[5] 王羽,董文龙,王巍,等. 猪源肺炎克雷伯菌的分离鉴定及其耐药性分析[J]. 中国兽医科学,2017,47(12):1570-1576. WANG Y,DONG W L,WANG W,et al.Isolation,identification and drug resistance analysis of Klebsiella pneumoniae from swine[J].Chinese Veterinary Science,2017,47(12):1570-1576.(in Chinese)
[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. FENG N,GAO X,XIAO M,et al.Isolation,identification and genetic evolution analysis of Klebsiella pneumon strain from calves[J].Chinese Veterinary Science,2016,46(11):1358-1364.(in Chinese)
[7] 杨帆,李淑梅,魏纪东,等. 动物源肺炎克雷伯菌毒力基因及生物膜研究[J]. 中国预防兽医学报,2017,39(9):764-766. YANG F,LI S M,WEI J D,et al.Virulence genes and biofilm of Klebsiella pneumonia isolated from ani-mals[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(9):764-766.(in Chinese)
[8] 卢玉婷,郑丹丹,胡扬扬,等. 鲤鱼肺炎克雷伯氏菌分离与鉴定[J]. 中国兽药杂志,2014,48(9):9-13. LU Y T,ZHENG D D,HU Y Y,et al.Isolation and identification of Klebsiella pneumoniae from carp[J].Chinese Journal of Veterinary Drug,2014,48(9):9-13.(in Chinese)
[9] 马磊,颜其贵,万莉,等. 竹鼠肺炎克雷伯氏菌分离鉴定[J]. 中国人兽共患病学报,2011,27(9):825-827. MA L,YAN Q G,WAN L,et al.Isolation and identification on K.pneumoniae in Rhizomys[J].Chinese Journal of Zoonoses,2011,27(9):825-827.(in Chinese)
[10] 杨锐,文继锋,龚永平,等. 小熊猫感染肺炎克雷伯氏菌的检查及药敏试验[J]. 中国人兽共患病学报,2017,33(3):271-275. YANG R,WEN J F,GONG Y P,et al.Detection and antibiotic sensitivity test of Klebsiella penumoniae in Ailurus fulgens[J].Chinese Journal of Zoonoses,2017,33(3):271-275.(in Chinese)
[11] 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].Scientific Reports,2017,7(1):17291-17297.
[12] 李巧玲,肖丽荣,张文举,等. 蓝狐源肺炎克雷伯菌的鉴定与药敏试验[J]. 畜牧与兽医,2018,50(3):103-106. LI Q L,XIAO L R,ZHANG W J,et al.Isolation,identification and drug susceptibility of Klebsiella pneumoniae isolated from blue fox[J].Animal Husbandry & Veterinary Medicine,2018,50(3):103-106.(in Chinese)
[13] 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(1):111-116.
[14] PACZOSA M K,MECSAS J.Klebsiella pneumoniae:Going on the offense with a strong defense[J].Microbiology and Molecular Biology Reviews,2016,80(3):629-661.
[15] QUAN T P,FAWCETT N J,WRIGHTSON J M,et al.Increasing burden of community-acquired pneumonia leading to hospitalisation 1998-2014[J].Thorax,2016,71(6):535-542.
[16] TWENHAFEL N A,WHITEHOUSE C A,STEVENS E L,et al.Multisystemic abscesses in African green monkeys (Chlorocebus aethiops) with invasive Klebsiella pneumoniae—identification of the hypermucoviscosity phenotype[J].Veterinary Pathology,2008,45(2):226-231.
[17] JANG S,WHEELER L,CAREY R B,et al.Pleuritis and suppurative pneumonia associated with a hypermucoviscosity phenotype of Klebsiella pneumoniae in California sea lions (Zalophus californianus)[J].Veterinary Microbiology,2010,141(1-2):174-177.
[18] MANGES A R.Editorial commentary:Genomic epidemiology:Revealing hidden reservoirs for Klebsiella pneumoniae[J].Clinical Infectious Diseases,2015,61(6):900-902.
[19] 钟世勋,迟珊珊,王振,等. 山东规模化养殖场毛皮动物多重感染分析[J]. 中国兽医学报,2014,34(11):1770-1777. ZHONG S X,CHI S S,WANG Z,et al.The etiology analysis of multiple infections in Shandong scale fur frams[J].Chinese Journal of Veterinary Science,2014,34(11):1770-1777.(in Chinese)
[20] 韩坤,白雪,闫喜军,等. 貂源肺炎克雷伯氏菌的分离及毒力和耐药性的分析[J]. 中国兽医科学,2018,48(8):1019-1023. HAN K,BAI X,YAN X J,et al.Isolation,virulence and drug resistance of Klebsiella pneumoniae from mink[J].Chinese Veterinary Science,2018,48(8):1019-1023.(in Chinese)
[21] BRISSE S,GRIMONT F,GRIMONT P A D.The Genus Klebsiella,A Handbok on the Biology of Bacteria[M].3rd Edition.New York:Springer,2006.
[22] 黄海迪,郭卉,王贵升,等. 貂源肺炎克雷伯氏菌对小鼠的致病性及其药物防治研究[J].中国兽药杂志,2016,50(8):44-48. HUANG H D,GUO H,WANG G S,et al.Pathogenicity and medicine treatment of Klebsiella pneumoniae from mink in mice[J].Chinese Journal of Veterinary Drug,2016,50(8):44-48.(in Chinese)
[23] 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].Clinical Microbiology and Infection,2012,18(3):268-281.