Interactive effects of temperature, organic carbon, and pipe material on microbiota composition and Legionella pneumophila in hot water plumbing systems

Background: Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32-53 degrees C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 mu g/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing. Results: Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41 degrees C in the presence of PEX (1.58 x 10(5) gene copies/mL). At 53 degrees C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49 degrees C and 20% at 53 degrees C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log(10) lower) at temperatures <= 41 degrees C, but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures <= 45 degrees C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 degrees C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing. Conclusions: Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature increase. While copper pipe may also help shape the microbiota composition and limit L. pneumophila proliferation, its benefits might be constrained at higher temperatures. Influent assimilable organic carbon affected total bacterial numbers, but had minimal influence on opportunistic pathogen gene numbers or microbiota composition. These findings provide guidance among multiple control measures for the growth of opportunistic pathogens in hot water plumbing and insight into the mediating role of microbial ecological factors.