Standardised protocol for the detection and quantification of nontuberculous mycobacteria (NTM) in tap water and its application in investigating the source of NTM clinical infections

Nontuberculous mycobacteria (NTM) are emerging pathogens causing pulmonary and extrapulmonary diseases, including healthcare-related infections. Although water is one of the main infection sources, isolating NTM from water samples is rarely done due to the NTM fastidious growth and lack of a standardised method. We propose a standardised NTM detection protocol from tap water. We set a protocol for NTM cultivation, measured its repeatability (n = 20 experiments, 5 for each condition), reproducibility (n = 20, 5 for each condition), and performed an inter-laboratory comparison (n = 6) using sterile and tap water samples spiked with slowly growing ( M. avium ) and rapidly growing ( M. chelonae ) NTM, plus artificial contamination ( Pseudomonas aeruginosa ). We investigated water-related NTM infections using this protocol from 2014 to 2024. The protocol showed good repeatability, reproducibility, and recovery yield (range: 67–131 %) for quantifying NTM in artificial samples. The inter-laboratory agreement was 83 %. It was consistent with different growth media and temperatures. The addition of P. aeruginosa did not affect NTM recovery. We used the protocol in 12 investigations, involving 24 patients. NTM were detected in 11/12 investigations (92 %, 23 patients) with species being the same as patients’ isolates in 6/11 (55 %, 18 patients). Water was identified as a source of infection in 5/12 (42 %) investigations involving 15/24 patients (62 %) based on matching genotypes. This study provides a protocol for detecting and quantifying NTM colonies in tap water, which allows identifying the source of clinical infections. With an increase in NTM infections, using this method in mycobacteriology and in environmental laboratories could be beneficial. • We propose a standardised protocol for NTM detection and quantification from tap water. •Our protocol showed good repeatability, reproducibility and recovery yield for M. avium (slowly growing) and M. chelonae (rapidly growing) NTM •The protocol was efficient to identify the infection source in 86 % of NTM clinical infection investigations suspected of being water-related