Isolation and molecular identification of non-tuberculous mycobacteria from diseased farmed rainbow trout in western Iran

Non-tuberculous mycobacteria (NTM) are important pathogens affecting marine, brackish, and freshwater fish and may persist in aquatic environments. Identification of NTM species is essential for understanding their epidemiology and implications for aquaculture. Moreover biofilm-forming capacity is also recognized as an important survival strategy that facilitates the long-term persistence of NTM in aquatic environments. This study aimed to isolate and identify NTM species from dead/diseased farmed rainbow trout ( Oncorhynchus mykiss ) using phenotypic and molecular methods and to evaluate their biofilm-forming capacity. A total of 123 dead/diseased fish samples were collected from different fish farming ponds. Following initial sample processing, samples were inoculated into two Lowenstein-Jensen media and were incubated at 25 °C and 37 °C for 8 weeks. The positive colonies were identified using conventional phenotypic tests and molecular identification using the hsp65 and rpoB genes. Biofilm-forming capacity was assessed using the crystal violet microtiter plate assay. Associations between NTM species, growth rate category, and clinical signs were evaluated using Fisher’s exact test. Out of 123 samples, 19 samples (15.45%) were positive for NTM. The identified species included M. fortuitum ( n = 9, 47.37%), followed by M. chelonae ( n = 5, 26.32%) and M. marinum ( n = 5, 26.32%). No statistically significant association was found between NTM species and clinical manifestations ( p = 0.090), whereas a statistically significant association was observed between growth rate category and clinical signs ( p = 0.01). All positive isolates were able to produce biofilm, with 15 (78.95%) isolates classified as strong biofilm producers and 4 (21.05%) isolates classified as moderate biofilm producers. In conclusion, this study indicates the presence of NTM in dead/diseased farmed rainbow trout, with M. fortuitum being the predominant species among the recovered isolates. The high biofilm-forming capacity of fish-associated NTM isolates may contribute to their persistence in aquatic environments. These findings highlight the necessity of regular monitoring and reliable identification of NTM in aquatic environments.