Susceptibility of Non‐Tuberculous Mycobacteria Biofilm to Common Disinfectants in Aquaculture Systems

Mycobacteriosis is a common and persistent bacterial disease affecting cultured, wild and pet fish. The disease can be caused by various Mycobacterium spp. Currently, depopulation and disinfection are the main recommended measures for containing disease outbreaks, as no vaccines are commercially available, and only a few reports of successful antimicrobial therapies have been made. While disinfectant susceptibility studies have been conducted on planktonic forms of some non-tuberculous mycobacteria (NTM) affecting fish, biofilm-related research remains limited. In this study, biofilm formation of Mycobacterium chelonae, Mycobacterium salmoniphilum, Mycobacterium arcueilense and Mycobacterium marinum isolates recovered from diseased fish were initially evaluated using the minimal biofilm eradication concentration (MBEC) assay system. All Mycobacterium spp. were able to form biofilms within a 2-week period when incubated at 25°C, but M. chelonae, M. salmoniphilum and M. arcueilense produced a faster and greater biofilm than M. marinum. To investigate the susceptibility of mycobacterial biofilms to common disinfectants, mature biofilms were divided into six treatment groups based on disinfectant type and exposure time: povidone-iodine (50 mg L^-1 free iodine) and bleach (200 mg L^-1 free chlorine) were applied for 30 min each; hydrogen peroxide (3% H₂O₂) and Virkon Aquatic (10 g L^-1) were applied for 15 min each; and 70% ethanol was tested at both 15 and 30 min. Results demonstrated variable susceptibility among species. M. marinum was susceptible to all disinfectants, while M. arcueilense was susceptible to bleach, povidone-iodine and 70% ethanol (30-min exposure). None of the tested disinfectants at recommended doses effectively eradicated M. chelonae or M. salmoniphilum biofilms. These findings underscore species-specific differences in biofilm formation and resistance, emphasising the critical need for robust disinfection protocols in aquaculture settings to prevent mycobacteriosis transmission. Further research is essential to understand the mechanisms underlying disinfectant resistance among NTMs, optimise disinfection strategies and ensure the health and biosecurity of aquaculture facilities.