凡纳滨对虾肠源美人鱼发光杆菌美人鱼亚种的分离鉴定、耐药性及其潜在致病性

Isolation, identification, and drug resistance and potential pathogenicity evaluation of Photobacterium damselae subsp. damselae isolated from the gut of Litopenaeus vannamei

摘要:

背景 美人鱼发光杆菌(Photobacterium damselae)是对虾肠道中的常驻细菌，同时也可能引发多种对虾疾病。然而，目前关于对虾肠源美人鱼发光杆菌的生物学特性及潜在致病性仍缺乏系统研究。目的 分离鉴定和表征凡纳滨对虾(Litopenaeus vannamei)肠源美人鱼发光杆菌，揭示其耐药性和潜在致病性，为对虾养殖中该菌的防控和利用提供依据。方法 从广东省湛江市6个养殖区采集对虾，从其肠道分离并遴选发光杆菌属细菌，结合形态、生理生化和ureC基因扩增对代表菌株进行鉴定；利用平板法测试其溶血性和产胞外酶能力；采用纸片扩散法分析其对18种抗菌药物的耐药性；通过典型毒力基因检测和卤虫(Artemia franciscana)幼体试验评价其毒力与潜在致病性。结果 从不同养殖区的对虾肠道中均分离到美人鱼发光杆菌美人鱼亚种(P. damselae subsp. damselae, Pdd)。除对蔗糖、甘露醇、纤维二糖和半乳糖利用存在差异外，16株代表菌的其他生理生化特征一致。16株中，7株呈现α溶血，9株为β溶血；所有菌株均产生胞外脂肪酶和卵磷脂酶，其中11株还能分泌几丁质酶，但在酶活性上呈现差异。16株Pdd的多重耐药率高达87.5%，对氨苄西林、阿莫西林、链霉素和呋喃唑酮的耐药率均≥50.0%。所有Pdd均携带磷脂酶基因plpV，β溶血型菌株均携带溶血素基因hlyAch，另有4株具有胶原酶基因colP，但毒性质粒pPHDD1及其相关溶血素基因dly和hlyApl均未检出。除2株Pdd对卤虫幼体无明显毒性外，其余14株均具有显著致死作用(P<0.05)。结论 Pdd在凡纳滨对虾肠道中较普遍存在，绝大多数菌株表现出明显的胞外酶活性和多重耐药性，并对卤虫幼体具有显著致死作用，可能对凡纳滨对虾构成较高的致病风险。

Abstract:

Background Photobacterium damselae is a resident bacterium in the shrimp gut and can cause various shrimp diseases. However, systematic research on the biological characteristics and potential pathogenicity of gut-derived P. damselae in shrimps remains lacking.Objective To provide a basis for prevention and utilization of this bacterium in shrimp aquaculture, we isolated, identified, and characterized P. damselae from the gut of the Pacific white shrimp (Litopenaeus vannamei).Methods Photobacterium strains were isolated and screened from the gut of L. vannamei sampled across six farming areas in Zhanjiang, Guangdong. Representative strains were identified based on morphological, physiological, and biochemical characteristics and ureC sequence. Their hemolytic activity and extracellular enzyme production were tested via the plate method. The resistance of the strains to 18 antimicrobial agents was evaluated via the disk diffusion method, and their virulence and potential pathogenicity were assessed by detecting typical virulence genes and conducting toxicity assays in Artemia franciscana larvae.Results P. damselae subsp. damselae (Pdd) was isolated from the guts of shrimps across different farming areas. Except differences in the utilization of sucrose, mannitol, cellobiose, and galactose, the physiological and biochemical characteristics of the 16 representative strains were consistent. Among the 16 strains, 7 showed α-hemolysis, while 9 exhibited β-hemolysis. All the strains produced extracellular lipase and lecithinase, with 11 of them also secreting chitinase. However, differences were observed in their enzymatic activities. The multidrug resistance rate of the 16 strains was as high as 87.5%. Moreover, at least 50.0% of the strains were resistant to ampicillin, amoxicillin, streptomycin, and furazolidone. All Pdd strains carried the phospholipase gene plpV, and the β-hemolytic strains all carried the hemolysin gene hlyAch. Additionally, four strains possessed the collagenase gene colP, while the virulence plasmid pPHDD1 and its related hemolysin genes dly and hlyApl were not detected in any of the strains. Except two Pdd strains that showed no obvious toxicity to A. franciscana larvae, the remaining 14 strains exhibited lethal effects (P<0.05).Conclusion Pdd is commonly found in the gut of L. vannamei, with the vast majority of strains exhibiting visible extracellular enzyme activity and multidrug resistance, as well as significant lethality to A. franciscana larvae, posing a potentially high pathogenic risk to L. vannamei.