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

Abstract

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 °C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 μg/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 °C in the presence of PEX (1.58 × 10⁵ gene copies/mL). At 53 °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 °C and 20% at 53 °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 log₁₀ lower) at temperatures ≤ 41 °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 °C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 °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.

Keywords: Hot water; Legionella; Opportunistic pathogen; Pipe material; Premise plumbing; Temperature control.

Fig. 1

Effect of temperature and pipe material on gene copy numbers of total bacteria (a), L. pneumophila (b), V. vermiformis (c), and Acanthamoeba (d) in SWHs dosed with 700 μg/L AOC in the influent. All microbial numbers are log₁₀-transformed gene copies/mL. SWHs with copper (circle) or PEX (triangle) pipe section were incubated constantly at 32 °C (black) or at increasing temperatures from 32 to 53 °C (red for copper, green for PEX) from day 1 to day 316. Error bars are standard deviations of triplicate SWHs

Fig. 2

Effect of dosed assimilable organic carbon (AOC) on gene copy numbers. a Correlations between the numbers of total bacteria and dosed AOC level. Linear regression lines are color-coded according to temperature as indicated. Means and standard errors from replicated SWHs are shown (n = 6 for combined pipe material data at 32–49 °C, n = 3 for 53 °C, with each pipe material plotted individually). b Distribution of gene copy numbers of specific microbes of interest under temperature/pipe conditions where a significant effect of AOC supplementation was noted. For each boxplot, n = 9 for 32 °C and n = 3 for 37 °C. Asterisk indicates significant difference compared to lower AOC levels according to post hoc ANOVA test

Fig. 3

Effect of various factors on relative abundances of potential opportunistic pathogens. Changes in the relative abundance of a Mycobacterium spp. and b Legionella spp. with temperature based on Illumina sequencing. c Relative abundance of L. pneumophila based on qPCR (mip gene copies/16S rRNA gene copies) for PEX condition only. Water supplemented with 0–700 μg/L AOC were fed to SWHs and incubated at increasing temperatures (32–53 °C). Circles and triangles represent SWHs with copper and PEX pipe sections, respectively. Error bars represent standard errors of triplicate SWHs

Fig. 4

Effect of copper ion dosing on total bacteria and L. pneumophila. Total bacteria (16S rRNA gene copy numbers—circles) and L. pneumophila (mip gene copy numbers—triangles or CFU—diamonds) were measured in SWHs with PEX pipe at 32 °C. No AOC was supplemented. An increasing amount of Cu²⁺ was dosed over the course of the experiment, from 5 to 1000 ppb as shown in the top panel, to the SWHs indicated in red. Standard errors were determined from triplicate SWHs. Asterisk indicates significant difference in comparison to the no Cu²⁺ control

Fig. 5

NMDS plot illustrating the overall dissimilarities among hot water microbiota. Circles represent SWHs with copper (solid) or PEX (open) pipe incubated at varying temperatures (blue, 32 °C; cyan, 37 °C; green, 41 °C; brown, 45 °C; magenta, 49 °C; red, 53 °C) in comparison the control set maintained at 32 °C (triangles, Ctl32°C). Cross symbols represent influent water (after GAC filtration) at the time of 37 °C incubation. Labels of “7” above a symbol indicate 700 μg/L dosed AOC, while non-labeled conditions represent 0 and 30 μg/L dosed AOC. The trajectory lines (solid—copper; dashed—PEX) indicate the trend of microbiota changing with increasing temperatures

Fig. 6

Effects of temperature and pipe material on microbiota composition and diversity. a Phylum composition, (b) Shannon diversity, (c) 14 persistent OTUs at all temperatures for both pipe materials, and (d) OTUs selectively enriched with temperature. OTUs being undetected in any temperature or pipe material are grouped as “Transient OTUs”. Standard errors were determined from triplicate SWHs (b and d panels). Each plotted line in panel d represents three to five OTUs enriched at each temperature (see Additional file 1: Table S2 for details)

Fig. 7

Impact of pipe material on hot water microbiota illustrated using PCoA plot of SWHs with copper (circles) or PEX (triangles) pipe sections supplemented with 0 (white), 20 (light gray), or 700 (dark gray) microgram per liter AOC and incubated at 32 °C. The most abundant OTUs significantly correlated with both axes are shown as the arrows and labeled with their best-known taxonomy identification. In bold are OTUs significantly enriched in one pipe material at all lower temperatures (32–41 °C). Embedded figures show the relative abundance of copper (lower) and PEX (upper) enriched OTUs in SWHs at 32 °C

Fig. 8

Absolute number of L. pneumophila quantified by qPCR in comparison to the number of Legionella spp. determined by Illumina sequencing. To determine absolute number of Legionella spp. using Illumina sequencing information, the relative abundance (%) of OTUs belonging to the genera Legionella spp. was multiplied with the number of 16S gene copies quantified qPCR