扬子鳄源ST-25型肺炎克雷伯氏菌的分离鉴定及致病性分析

Isolation, identification, and pathogenicity analysis of ST25 Klebsiella pneumoniae from Alligator sinensis

摘要:

目的 探明一例起源于安徽省扬子鳄繁殖研究中心的成年鳄死因。方法 分别从该成年鳄的心、肝、脾、肺中进行病原分离培养，采用形态观察、生化特性及分子生物学方法进行鉴定；利用拉丝试验判定黏液表型；根据7个管家基因进行多位点序列分型(multilocus sequence typing, MLST)；通过毒力基因分析、致病性试验、耐药基因分析及药物敏感性试验，阐明分离菌株的致病性和耐药性。结果 从4种组织脏器中分离出的病原菌为形态一致的革兰阴性杆菌，结合生化试验、16S rRNA基因和khe基因鉴定结果，确定菌株均为肺炎克雷伯氏菌(命名为YZE01)，经荚膜血清型鉴定为K2型；拉丝试验呈阳性，表明该菌株为高黏液型；MLST分析表明，该菌为ST25型；毒力基因检测显示，其携带rmpA、rmpA2、entB、fimH、markD和wabG这6种毒力基因。致病性试验显示，部分受试小鼠在感染后24 h内死亡，剖检后在其心、肝、脾、肺中均可分离出该菌；且肝肺组织呈现不同程度的出血和淤血病变；此外，RT-qPCR结果显示，肺组织炎性因子IL-1β、IL-6、IL-8及TNF-α的表达水平在12 h时达到高峰后下降。在耐药性方面，该菌株同时携带blaSHV、armA和ermB这3种耐药基因，对头孢氨苄、头孢唑林、氨苄西林、链霉素、庆大霉素、红霉素、罗红霉素和克林霉素均不敏感。结论 所分离的肺炎克雷伯氏菌YZE01株携带多种毒力基因，具有较强的致病性和耐药性，对扬子鳄具有显著的损伤和致死作用，可为扬子鳄疾病的防控提供有效手段。

Abstract:

Objective To determine the cause of death of an adult crocodile in the Alligator sinensis Management Center in Anhui Province.Methods Bacteria were isolated from the heart, liver, lung, and spleen via the culture method, and the isolates were identified by morphological observation, biochemical tests, and molecular biological methods. Furthermore, the mucus phenotype was determined by means of the string test. Multilocus sequence typing (MLST) was conducted on the basis of seven housekeeping loci. Virulence gene analysis, pathogenicity test, drug resistance gene analysis, and antimicrobial susceptibility testing were conducted to clarify the pathogenicity and drug resistance of the isolates.Results The pathogenic bacteria isolated from the four organs were morphologically consistent Gram-negative bacilli. Through biochemical tests and 16S rRNA gene and khe sequencing, the isolates were identified as Klebsiella pneumoniae YZE01, capsular serotype K2. String test showed that the strain was hypermucinous K. pneumoniae, and MLST analysis showed that the strain belonged to sequence type 25 (ST25). The strain carried six virulence genes: fimH, entB, rmpA, rmpA2, mrkD, and wabG. Pathogenicity tests showed that some of the tested mice died within 24 h after infection with YZE01, and the same strain was isolated from the heart, liver, lung, and spleen. The lung tissue of infected mice showed hemorrhage and congestion lesions to different degrees. In addition, RT-qPCR revealed that the transcript levels of IL-1β, IL-6, IL-8, and TNF-α in the lung peaked at 12 h post-infection and then declined. The strain carried three drug resistance genes (blaSHV, armA, and ermB), and it was not sensitive to cephalexin, cefazolin, ampicillin, streptomycin, gentamicin, erythromycin, roxithromycin, and clindamycin.Conclusion The isolated strain K. pneumoniae YZE01 carries a variety of virulence genes and has strong pathogenicity and drug resistance. It is considered as a major cause of death in A. sinensis. The findings are conducive to the prevention and control of diseases in A. sinensis.