Vibrio vulnificus is the major pathogenic bacterium causing mass mortality in groupers (Epinephelus spp.) culture with symptoms as skin and fins hemorrhages and ulcerative necrosis. Microbiological and biochemical characterizations were used to identify Vibrio spp., but it is ambiguous to distinguish V. vulnificus and others. Therefore, molecular genotypic analysis was applied to identify and typing Vibrio spp. isolated from the diseased grouper. Firstly, the identification of Vibrio spp. was assessed by 16 s rRNA before generating the phylogenetic tree. It revealed that 40 samples from 63 samples were V. vulnificus biotype 1. After that, all V. vulnificus samples were classified into subgroup by molecular biotyping based on the 22 virulence genes identification. Accordingly, V. vulnificus can be categorized as subgroup 1, 2, and 3 (marked as G1, G2, and G3, respectively) based on the presence of those studied virulence genes. V. vulnificus isolates containing 16 virulence genes of vvpE, gloB, tlh, Mann Hemo, ManIIA, rimO, vllY, OmpU, vvhA, rtx, hlyIII, pilF, Mann TRAP, vcgC, 16S rRNA type B and nanA has been classified as subgroup 1, whereas subgroup 2 has shown the additional 4 genes including YJ-like nab 1, YJ-like nab 2, cps and sodA, as well as subgroup 3 harboring CM-like nab 1, CM-like nab2, and sodA. Surprisingly, different identification method using 10 house-keeping genes in multi-locus sequence typing (MLST) represented the new three different sequence type (ST) profile. Additionally, phenotypic analysis of isolated V. vulnificus was also performed through the disk diffusion method. This result demonstrated that G1 and G2 were susceptible to tetracycline at 44.4% and 53.8%, respectively, while G3 was susceptible to oxytetracycline (46.2%). Interestingly, G2 which lacking CM-like nab1 and CM-like nab2 alleles exhibited the highest virulent pathogenicity, despite showing a slower growth rate than G1 and G3. Meanwhile, G3, lacking cps gene but containing CM-like nab1 and CM-like nab2 alleles, did not cause mortality in experimental challenge fish. In the case of inactivated vaccine efficiency trial, the FKC-Mixed vaccine revealed the best efficacy when compared with other vaccinated groups (P < 0.05). Altogether, our findings suggested that the molecular genotyping analysis is useful for identifying Vibrio spp. and may apply as the appropriate platform for further characterization of other bacterial species.

创伤弧菌是导致石斑鱼（Epinephelus spp.）培养中大量死亡的主要病原菌，症状为皮肤和鳍出血和溃疡性坏死。微生物学和生化特征用于鉴定弧菌属，但很难区分创伤弧菌和其他弧菌。因此，分子基因型分析被用于鉴定和分型弧菌属。从患病的石斑鱼中分离出来。首先，弧菌属的鉴定。在生成系统发育树之前通过 16 s rRNA 进行评估。结果显示，63个样本中的40个样本为创伤弧菌1型。基于22个毒力基因鉴定，通过分子生物分型将创伤弧菌样品分为亚群。因此，根据所研究的毒力基因的存在，创伤弧菌可分为亚组 1、2 和 3（分别标记为 G1、G2 和 G3）。创伤弧菌含有16个毒力基因株vvpE，GLOB，TLH，曼出血天赋，ManIIA，RIMO，vllY，OmpU，vvhA，RTX，hlyIII，PILF，曼TRAP，VCG C，的16S rRNA B型和nanA被归类为亚组 1，而亚组 2 显示了额外的 4 个基因，包括 YJ-like nab 1、YJ-like nab 2、cps和sodA，以及包含 CM-like nab 1、CM-like nab2 的亚组 3 ，和苏打水。令人惊讶的是，在多位点序列分型 (MLST) 中使用 10 个看家基因的不同识别方法代表了新的三种不同序列类型 (ST) 谱。此外，分离的创伤弧菌的表型分析也通过盘扩散法进行。该结果表明，G1 和 G2 对四环素的敏感性分别为 44.4% 和 53.8%，而 G3 对土霉素的敏感性为 46.2%。有趣的是，尽管生长速度比 G1 和 G3 慢，但缺乏 CM 样nab 1 和 CM 样nab 2 等位基因的G2表现出最高的毒性致病性。同时，缺乏cps基因但含有 CM 样nab 1 和 CM 样nab 2 等位基因的G3不会导致实验挑战鱼的死亡。在灭活疫苗效率试验的情况下，FKC-Mixed 疫苗显示出与其他疫苗接种组相比的最佳疗效（P < 0.05)。总而言之，我们的研究结果表明分子基因分型分析可用于识别弧菌属。并可用作进一步表征其他细菌物种的适当平台。