Tracking the reservoir of non-tuberculous mycobacteria – environmental and molecular studies

A total of 435 environmental samples were collected between 2024 and 2025 across five major cities in Poland. Samples were grouped into three main categories: (i) outdoor (ii) household, and (iii) hospital reservoirs.Outdoor samples included soil, air, and water sources, covering natural waters, fountains, and drinking fountains.Household samples comprised tap water, air, and biofilms collected from 50 households.Hospital samples included tap water (n = 25), air (n = 25), and biofilms (pipe sludge; tap limescale) collected from 25 sites across five hospitals, including ICUs (5), specialist clinics (5), patient rooms (5), scrub rooms (5), and public restrooms (5).Liquid (water) and solid (soil) samples were aseptically collected in quadruplicates following CDC standard procedures and immediately processed for: culture (2 samples), DNA isolation for metagenomic analysis, and physico-chemical testing. Biofilm swabs were collected aseptically in triplicates and analyzed as above, excluding physico-chemical testing. Active air sampling was conducted by collecting 1000 L of air directly onto Petri dishes using a Microflow Alfa 90/C air sampler (Aquaria).A detailed questionnaire documenting each sampling site was completed for every sample.Primary isolation, culturing, species identification, and DST were performed with standard mycobacteriological procedures.