Species identification of aquatic mycobacterium isolates by sequencing and PCR-RFLP of the 16S–23S rDNA internal transcribed spacer (ITS) region

Abstract

Fast and reliable identification of clinically significant Mycobacterium species strains attempted to identify on the basis of amplification, RFLP and sequencing of the internal transcribed spacer 1 (ITS1) 16S–23S Mycobacteria in Apparently Healthy Freshwater Aquarium Fish in Uttar Pradesh, India. Sixty apparently healthy freshwater fish aquariums belonging to eight different species have been harvested from six outlets in several aquarium cities and processed for the isolation of Mycobacterium species. Using the initial decontamination protocol tissue homogenates (with 1N HCl and NaOH 2N) and incubating at 30°C for two months, Mycobacterium species was isolated of 25% of them. These isolates have been identified by standard biochemical tests. All the fragments of genes of the ITS region of the 23S rRNA were amplified by 16S fragment performance of different dimensions between 230bp and 350bp digested by two fast digest restriction enzymes; HaeIII and Sau96I. The digested products were analyzed using gel electrophoresis with agarose. There has also been sequencing amplified fragments of the ITS gene. The isolates were identified as five strains of M. abscessus, M. gordonae as three, two M. fortuitum, M. Parascrofulaceum, M. arupense, one isolate as M. senegalense, M. aubagnense and M. conceptionense. The incidence of mycobacteria in freshwater fish, seemingly healthy is terrible and the study is relevant due to the variety of Mycobacterium related to aquarium fish and zoonotic importance. All Mycobacterium species isolated during this study are well known pathogens both to fish and humans.