四株杂交鳢致病性舒伯特气单胞菌特性及致病性比较

Comparative on characteristics and pathogenicity of four strains of Aeromonas schubertii isolated from snakehead hybrid

摘要:

【背景】舒伯特气单胞菌为革兰氏阴性短杆菌，是一种人-畜-鱼共患的条件性致病菌，它能引起不同程度的人类疾病，近年来更是在水产养殖中被频繁报道。【目的】比较分析4株杂交鳢源舒伯特气单胞菌(WL-4、ZL-1、ZL-13、D075)菌株间生理生化特性、致病性以及对常用药物的敏感性差异，为水产舒伯特气单胞菌病的防治提供参考依据。【方法】使用全自动细菌生化鉴定仪检测菌株的生理生化特性；运用PCR扩增和测序比对菌株的gyrB、16S rRNA基因序列；采用改良平板法检测毒力因子表达，PCR技术检测菌株毒力基因和耐药基因的携带情况；通过人工注射感染分析4株舒伯特气单胞菌对杂交鳢的致病性差异；并采用微量二倍稀释法测试15种抗菌药物对菌株的最小抑菌浓度。【结果】以ATCC43700 (人源舒伯特气单胞菌)作为参考，4株菌株理化特性基本相同，gyrB和16S rRNA基因一致性分别为99.57%和100%，聚类分析聚为一簇；不同的舒伯特气单胞菌(ATCC43700、WL-4、ZL-1、ZL-13、D075)均具有溶血性、蛋白酶活性和脂肪酶活性；但人源菌株(ATCC43700)与杂交鳢源舒伯特气单胞菌携带的毒力基因存在差异，其中人源菌株携带了hlyA、ela、lip、ahal、act，而杂交鳢源菌株(WL-4、ZL-1、ZL-13、D075)携带hlyA、ela、lip、ahal、aer；4株杂交鳢源舒伯特气单胞菌对杂交鳢致病性存在差异，1.2×105 CFU/mL浓度腹腔注射感染健康杂交鳢，WL-4、ZL-1、ZL-13、D075对杂交鳢的感染致死率依次为30%、40%、70%和80%；不同菌株(ATCC43700、WL-4、ZL-1、ZL-13、D075)的药物敏感性以及携带的耐药基因也存在差异。【结论】4株杂交鳢源舒伯特气单胞菌在致病性、药物敏感性等方面存在差异，研究结果有助于进一步开展舒伯特气单胞菌病发病机制和防控技术研究。

关键词:舒伯特气单胞菌，毒力因子，致病性，药物敏感性

Abstract:

[Background] Aeromonas schubertii is a gram-negative short rod-shaped bacterium. It is a human-animal-fish co-infected conditional pathogen and can cause various human diseases, which has been reported frequently in recent years. [Objective] In order to provide references for the prevention and control of A. schubertii caused diseases in aquaculture , the physiological and biochemical characteristics, pathogenicity as well as the sensitivity to common antibiotics of the 4 strains of A. schubertii (WL-4, ZL-1, ZL-13, D075) isolated from hybrid snakehead have been analyzed and compared. [Methods] The physiological and biochemical characteristics of the strains were detected by automatic microorganism identification instrument, the gyrB and 16S rRNA gene sequences of the strains were compared by PCR and sequencing. The expression of virulence factor was tested by modified plate method and the carrying status of virulence gene and antibiotic resistance gene were detected by PCR. The differences of pathogenicity for 4 strains were analyzed by artificial injection infection. The minimum inhibitory concentration of 15 antimicrobial agents against the strains were measured by double microdilution method. [Results] As ATCC43700, a human origin strain, was used as a reference strain, the physiological and biochemical characteristics of these 5 A. schubertii were basically the same and the similarity of the gyrB and 16S rRNA genes of them were 99.57% and 100% respectively. five strains are clustered into one group though cluster analysis. All of them showed hemolytic activity, protease activity and lipase activity, but there are differences on virulence genes among 5 strain. ATCC43700 carried hlyA, ela, lip, ahal and act, while WL-4, ZL-1, ZL-13 and D075 carried hlyA, ela, lip, ahal and aer. four strains isolated from snakehead hybrid showed different pathogenicity. The mortality rate of hybrid snakehead by artificial injection of WL-4, ZL-1, ZL-13 and D075 at 1.2×105 CFU/mL solution was 30%, 40%, 70% and 80% respectively. The antibiotic sensitivity and antibiotic resistance gene are also different among them. [Conclusion] There are differences on pathogenicity and antibiotic sensitivity among the four strains of A. schubertii from hybrid snakehead. The results of this study are helpful to further research on the pathogenesis of A. schubertii and the prevention and control methods against it in aquaculture.

Key words:Aeromonas schubertii, Virulence factor, Pathogenicity, Antibiotic sensitivity