Molecular genotyping and phenotyping of Vibrio vulnificus isolated from diseased, brown-marbled grouper (Epinephelus fuscoguttatus) in Thailand with preliminary vaccine efficacy analysis

Abstract

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.