鲮源嗜水气单胞菌的分离鉴定、生物学特性及防控药物筛选

Isolation,Identification,Biological Characterization,and Prevention and Control Drugs Screening of Aeromonas hydrophila from Cirrhinus molitorella

【目的】近年来,广东省多个规模化养殖场的鲮鱼出现体表出血、腹水和肠炎等症状,明确鲮(Cirrhinus molitorella)暴发性死亡的病原菌种类及其生物学特性,筛选能有效抑制病原菌的药物或微生态制剂,为鲮鱼气单胞菌败血症的科学防治提供指导。【方法】从患病鲮鱼的肝脏和肾脏等部位分离纯化细菌,通过16S rRNA、gyrB基因测序及生理生化特征分析进行细菌鉴定,通过毒力基因PCR检测和人工感染试验分析分离菌株的致病性,利用纸片扩散法评估分离菌株的耐药性,通过拮抗抑菌试验分析3株经实验室筛选、验证并保藏的贝莱斯芽孢杆菌(Bacillu velezensis)对分离菌株的抑菌效果,采用微量二倍稀释法评估12种中草药对分离菌株的抑菌和杀菌效果。【结果】根据分离菌株的形态特征、生物学特性,结合16S rRNA、gyrB基因序列分析结果,确定分离菌株为嗜水气单胞菌(Aeromonas hydrophila)。毒力基因PCR检测结果显示,分离菌株均携带act、aer、alt、ahyB、eprCAI、fl a、gcaT、ast、lip和exu毒力基因,属于广泛流行的毒力基因Ⅰ型菌株。回归感染试验结果表明分离菌株均具有较强致病力。药物敏感性试验结果显示,分离菌株对氨基糖苷类、四环素类、喹诺酮类、磺胺类和氯霉素类等抗生素敏感,对中草药单体姜黄素敏感度较高。贝莱斯芽孢杆菌WLYS23、CYS06和LF01菌株对分离菌株均具有较强的抑菌活性。【结论】引起鲮鱼死亡的病原菌为嗜水气单胞菌,含有10种毒力基因,具有高致病性,推荐选用多西环素、恩诺沙星或氟苯尼考进行科学防治。此外,3株贝莱斯芽孢杆菌对分离菌株具有较好的抑菌效果,可作为鲮鱼气单胞菌败血症潜在的生防益生菌,具有广泛的应用前景;中草药单体姜黄素对分离菌株的抑菌效果较好,推荐作为防控嗜水气单胞菌的绿色候选药物。

【Objective】In recent years,bacterial septicemia of mud carp(Cirrhinus molitorella)occurred in Guangdong Province,China.The clinical signs of the diseased fish show as hemorrhage on the body,ascites,and enteritis.The study aims to identify the pathogen causing explosive death in mud carp,and reveal the biological characteristics of the isolates.Furthermore,the drugs and probiotics that can effectively inhibit the pathogen were screened,which will provide guidance for the scientific control and prevention of Aeromonas septicemia in mud carp.【Method】The bacterial strains were isolated and purified from the liver and kidney of the diseased mud carp,and the isolates were identified based on 16S rRNA and gyrB gene sequencing and physiological and biological characteristics analysis.The pathogenicity of the isolates was evaluated by PCR detection of virulence-related genes and artificial infection test.The antimicrobial sensitivities of the isolates were determined using the disk diffusion method.The antibacterial effects of three Bacillus velezensis strains against the isolates were evaluated by antagonistic inhibition test,and the antibacterial and bactericidal effects of 12 Chinese herbal extracts on the isolated strains were evaluated by micro-double dilution method.【Result】A total of ten isolates were isolated from diseased mud carp of 4 breeding frams,and these isolates were identified as A.hydrophila based on its morphological and biological characteristics,16S rRNA and gyrB gene sequences analysis.Based on PCR detection results,these isolates harbored 10 virulence related genes,including act,aer,alt,ahyB,eprCAI,fl a,gcaT,ast,lip and exu,belonging to the main prevalent virulence genotype(genotypeⅠ).The results of artificial infection test showed that these isolates had high pathogenicity.The results of drug sensitivity test showed that the isolates were sensitive to aminoglycosides,tetracyclines,quinolones,sulfonamides and chloramphenicols,moreover,they were highly sensitive to the monomer curcumin.Strains B.velezensis WLYS23,CYS06 and LF01 showed strong antibacterial activity against the isolates.【Conclusion】The pathogen causing the bacterial outbreaks of mud carp is A.hydrophila,which contains 10 virulence related genes and has high pathogenicity.It is recommended to use doxycycline,enrofloxacin,or florfenicol for scientific prevention and control of bacterial diseases caused by A.hydrophila.In addition,several B.velezensis strains showed good antibacterial effects against the isolates,which indicate that they had broad application prospects as potential biocontrol probiotics.The Chinese herbal medicine curcumin has a good antibacterial activity against A.hydrophila,and curcumin is recommended as a green candidate drug for the prevention and control of Aeromonas septicemia in mud carp.