Abstract
ABSTRACTNontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTM rpoB genes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance of Mycobacterium avium subsp. avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, including Mycobacterium abscessus and Mycobacterium chelonae. Overall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.
Highlights
Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease
A few of the Ͼ150 nontuberculous mycobacteria (NTM) species that have been described [11] cause infections in humans [10, 12, 13]. Among these are NTM frequently detected in DW and DW distribution systems (DS), including species commonly associated with human infections (e.g., Mycobacterium avium and Mycobacterium abscessus) [7, 10, 13], as well as species that rarely cause infections (e.g., Mycobacterium frederiksbergense and Mycobacterium aurum) [14, 15]
Methods are needed that yield high NTM DNA levels during extraction from low-biomass samples and recover relatively large, phylogenetically informative segments of NTM DNA to advance our understanding of the potential health risks posed by NTM in DW systems
Summary
Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits This method, combined with highthroughput, single-molecule real-time sequencing of NTM rpoB genes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. IMPORTANCE An extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTM rpoB genes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). While studies to date have relied on Sanger sequencing of clone libraries of such genes [22,23,24], this approach is labor intensive and not well suited to high-throughput screening to detect NTM in environmental samples such as DW
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