Unveiling significant regrowth and potential risk of nontuberculous mycobacteria in hospital water supply system

• The complex hospital water supply system was the main reservoir for NTM. • Pathogenic NTM including M. abscessus were prevalent in building plumbing and POU. • Phylogenetic cluster ABS-GL4 of M. abscessus subsp. abscessus was dominant. • Potential transmission of pathogenic NTM via water use in the hospital was suspected. • Water quality control in the hospital water supply system should be revisited. The health burden of waterborne nontuberculous mycobacteria (NTM) is a rising concern. While the water supply systems can serve as a potential reservoir for NTM, their abundance, diversity, and transmission pathways remain unknown. This study aimed to characterize the prevalence and regrowth of NTM in building water supply system in a hospital where many M. abscessus were isolated from patients. The depletion of residual chlorine after stagnation and supply of warm water at the point of use promoted significant microbial regrowth, including NTM, in the hospital. The absolute abundance of Mycobacterium spp. 16S rRNA genes in tap water and shower water samples increased to approximately 10 4 copies/mL, while it was below the quantification limit in the finished water from a drinking water treatment plant. Amplicon sequencing of NTM-specific hsp 65 genes revealed that M. abscessus was prevalent in all samples, while the dominant NTM species varied depending on locations even in the same building. The presence of M. abscessus in water suggested the possibility of waterborne transmission in the hospital. M. abscessus was frequently isolated from tap water, shower water, and shower biofilms. These isolates demonstrated high clonality and were closely affiliated with the ABS-GL4 cluster of M. abscessus subsp. abscessus . Even though the automatic mixing equipment at the point of use was replaced with new one, the settlement and growth of NTM were reproducibly observed, suggesting mixing equipment as a hotspot for NTM proliferation. Additional interventions including water quality control are required as the hospital water supply system is a hot spot for NTM regrowth.