Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen.

ABSTRACTThe genusMycobacteriumcomprises a large number of well-characterized species, several of which are human and animal pathogens. Here, we report the whole-genome sequence ofMycobacteriumsp. strain 012931, a fish pathogen responsible for huge losses in aquaculture farms in Japan. The strain was isolated from a marine fish, yellowtail (Seriola quinqueradiata).

Febantel was developed as an oral anti-parasitic agent against monogenean Heterobothrium okamotoi for farmed tiger puffer Takifugu rubripes. In aquaculture, febantel might be used for the same fish repeatedly to obtain good anti-parasitic effects in fish farms. In this study, we investigated the safety of febantel for tiger puffer when used repeatedly. We also investigated the impact of febantel on the marine environment. Febantel was administered to fish at 25 mg and 50 mg per kg of body weight for five days and the process was repeated after a 14-day pause. External and internal clinical signs were investigated in tiger puffer as well as hematological parameters. During the examination period, tiger puffers showed no decrease in appetite or changes in growth pattern compared with the control group. In addition, we observed the effect of febantel on diatoms (Skeletonema costatum), crustaceans (Artemia saline), and fishes (Pagrus major) as proxies for marine organisms. The results confirmed no influence on the tested organisms. These results suggest that febantel is safe for tiger puffer and has no toxic effects in marine organisms.

Mycobacterium marinum is difficult to distinguish from other species of Mycobacterium isolated from fish using biochemical methods. Here, we used genetic and proteomic analyses to distinguish three Mycobacterium strains: M. marinum strains MB2 and Europe were isolated from tropical and marine fish in Thailand and Europe, and Mycobacterium sp. 012931 strain was isolated from yellowtail in Japan. In phylogenetic trees based on gyrB, rpoB, and Ag85B genes, Mycobacterium sp. 012931 clustered with M. marinum strains MB2 and Europe, but in trees based on 16S rRNA, hsp65, and Ag85A genes Mycobacterium sp. 012931 did not cluster with the other strains. In proteomic analyses using a Bruker matrix-assisted laser desorption ionization Biotyper, the mass profile of Mycobacterium sp. 012931 differed from the mass profiles of the other two fish M. marinum strains. Therefore, Mycobacterium sp. 012931 is similar to M. marinum but is not the same, suggesting that it could be a subspecies of M. marinum.

Mycobacterium marinum is a major causative agent of mycobacteriosis in fish that has a broad range of hosts, including in human isolates. So far, genomic analyses have focused on the human isolate. Here, we compared the draft genome sequences of two strains of M. marinum isolated from fish (MB2 and Europe) with the M. marinum M isolated from humans. M. marinum MB2 and Europe have single, circular chromosomes of 6,134,389 and 6,029,340 bp, and average G + C contents of 65.7 and 65.5 %, respectively. A total of 5,464 coding DNA sequences were annotated in both M. marinum MB2 and Europe genome. Dot plot analyses showed that M. marinum MB2 and Europe were closer to M. marinum M when compared to three other Mycobacterium species. The insertion/deletion gene analysis showed that M. marinum MB2 and Europe contained 342 and 487 genes that were not found in M. marinum M, and lacked 625 and 776 genes found in M. marinum M, respectively. Most of the inserted and deleted genes were classified in the fatty acid, lipid, and isoprenoid subsystem and the virulence, disease, and defense subsystem. Therefore, these results provide insights into the genomic diversity associated with variable hosts and pathogens.