Isolation and identification of non-tuberculous mycobacteria from dead edible fish in Iran

Problem Statement
Non-tuberculous mycobacteria (NTM) are environmental opportunistic pathogens widely present in aquatic and terrestrial environments, posing risks to both fish and humans. Fish mycobacteriosis (fish tuberculosis) is a granulomatous disease affecting over 200 fish species, often leading to lethargy, poor growth, organomegaly, and mortality. While Mycobacterium marinum, M. chelonae, and M. fortuitum are known common causes, traditional phenotypic identification methods are time-consuming, complex, and frequently fail to provide precise species-level identification. Furthermore, the zoonotic potential of these pathogens highlights the need for rapid and accurate detection in edible fish, particularly in aquaculture systems where they may serve as a source of infection for humans.

Methodology
The study investigated the presence and identification of NTM in 123 dead edible fish samples collected from various fish farming ponds in Ilam, western Iran, between January 2024 and February 2025. Three healthy fish per pond served as controls.

• Sample Processing: Tissue samples were homogenized, stained with Ziehl-Neelsen (ZN) for acid-fast bacilli, and decontaminated using NaOH and HCl.
• Culture: Sediments were inoculated onto Lowenstein-Jensen (LJ) media and incubated at both 25°C and 37°C for 8 weeks.
• Phenotypic Characterization: Positive colonies were evaluated based on morphological features, growth rate, pigment production, and biochemical tests (niacin production, nitrate reduction, catalase, urease, and arylsulfatase activity) following CDC protocols.
• Molecular Identification: DNA was extracted using a commercial kit. Species identification was performed via PCR amplification and sequencing of two housekeeping genes: a 441-bp fragment of the hsp65 gene and a 750-bp fragment of the rpoB gene.
• Analysis: Sequencing results were analyzed using BLASTn against NCBI databases. Phylogenetic trees were constructed using the Neighbor-Joining (NJ) approach with the Kimura two-parameter (K2P) distance model and 1000 bootstrap replications in MEGA 7 software.

Key Results

• Prevalence: NTM were isolated from 19 of the 123 samples (15.44%). All control samples were negative.
• Clinical Signs: Among the infected fish, 47.36% (9/19) presented with skin ulcerations, 26.32% (5/19) with scale loss, 5.26% (1/19) with severe emaciation, and 21.05% (4/19) showed no clinical signs.
• Growth Characteristics: 73.68% (14/19) of isolates were Rapidly Growing Mycobacteria (RGM), while 26.32% (5/19) were Slowly Growing Mycobacteria (SGM).
• Identification Discrepancies: Phenotypic methods identified 14 isolates as the M. fortuitum complex but failed to distinguish species in 7 cases (53.8% of the complex).
• Molecular Findings: Sequencing of hsp65 and rpoB genes allowed for precise species discrimination. The hsp65 gene demonstrated higher similarity values than rpoB. The final species distribution was:
  • Mycobacterium fortuitum: 9 isolates (47.36%)
  • Mycobacterium chelonae: 5 isolates (26.32%)
  • Mycobacterium marinum: 5 isolates (26.32%)
    (Note: The abstract text contains a typographical error listing "M. fortuitum" twice in the summary; the detailed results and Table 1 clarify the presence of M. chelonae).

Significance and Claims
The authors claim that this study provides more comprehensive quantitative data on the frequency and distribution of specific clinical signs associated with fish tuberculosis compared to previous qualitative descriptions. The research underscores the limitations of phenotypic methods, which could not achieve species-level identification for a significant portion of the isolates, whereas molecular methods proved significantly more accurate.

The study concludes that edible fish in the region harbor NTM species (M. fortuitum, M. chelonae, and M. marinum) that are pathogenic to both fish and humans. Consequently, the authors emphasize the importance of implementing rapid and accurate molecular detection methods in aquaculture systems to mitigate potential health threats to human consumers and improve disease management in fish farming. The paper calls for further molecular studies to better understand the epidemiology of these pathogens.