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Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing.

ABSTRACT: Legionella pneumophila is known to proliferate in hot water plumbing systems, but little is known about the specific physicochemical factors that contribute to its regrowth. Here, L. pneumophila trends were examined in controlled, replicated pilot-scale hot water systems with continuous recirculation lines subject to two water heater settings (40 °C and 58 °C) and three distal tap water use frequencies (high, medium, and low) with two pipe configurations (oriented upward to promote convective mixing with the recirculating line and downward to prevent it). Water heater temperature setting determined where L. pneumophila regrowth occurred in each system, with an increase of up to 4.4 log gene copies/mL in the 40 °C system tank and recirculating line relative to influent water compared to only 2.5 log gene copies/mL regrowth in the 58 °C system. Distal pipes without convective mixing cooled to room temperature (23-24 °C) during periods of no water use, but pipes with convective mixing equilibrated to 30.5 °C in the 40 °C system and 38.8 °C in the 58 °C system. Corresponding with known temperature effects on L. pneumophila growth and enhanced delivery of nutrients, distal pipes with convective mixing had on average 0.2 log more gene copies/mL in the 40 °C system and 0.8 log more gene copies/mL in the 58 °C system. Importantly, this work demonstrated the potential for thermal control strategies to be undermined by distal taps in general, and convective mixing in particular.

SUBMITTER: Rhoads WJ

PROVIDER: S-EPMC4810150 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing.

Rhoads William J WJ Pruden Amy A Edwards Marc A MA

Pathogens (Basel, Switzerland) 20160312 1

Legionella pneumophila is known to proliferate in hot water plumbing systems, but little is known about the specific physicochemical factors that contribute to its regrowth. Here, L. pneumophila trends were examined in controlled, replicated pilot-scale hot water systems with continuous recirculation lines subject to two water heater settings (40 °C and 58 °C) and three distal tap water use frequencies (high, medium, and low) with two pipe configurations (oriented upward to promote convective mi ...[more]

PMID: 26985908

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Project description:BACKGROUND:Heat shock is a potential control strategy for Legionella pneumophila in hot water plumbing systems. However, it is not consistently effective, with little understanding of its influence on the broader plumbing microbiome. Here, we employed a lab-scale recirculating hot water plumbing rig to compare the pre- and post-"heat shock" (i.e., 40???60???40 °C) microbiota at distal taps. In addition, we used a second plumbing rig to represent a well-managed system at 60 °C and conducted a "control" sampling at 60 °C, subsequently reducing the temperature to 40 °C to observe the effects on Legionella and the microbiota under a simulated "thermal disruption" scenario. RESULTS:According to 16S rRNA gene amplicon sequencing, in the heat shock scenario, there was no significant difference or statistically significant, but small, difference in the microbial community composition at the distal taps pre- versus post-heat shock (both biofilm and water; weighted and unweighted UniFrac distance matrices). While heat shock did lead to decreased total bacteria numbers at distal taps, it did not measurably alter the richness or evenness of the microbiota. Quantitative PCR measurements demonstrated that L. pneumophila relative abundance at distal taps also was not significantly different at 2-month post-heat shock relative to the pre-heat shock condition, while relative abundance of Vermamoeba vermiformis, a known Legionella host, did increase. In the thermal disruption scenario, relative abundance of planktonic L. pneumophila (quantitative PCR data) increased to levels comparable to those observed in the heat shock scenario within 2 months of switching long-term operation at 60 to 40 °C. Overall, water use frequency and water heater temperature set point exhibited a stronger effect than one-time heat shock on the microbial composition and Legionella levels at distal taps. CONCLUSIONS:While heat shock may be effective for instantaneous Legionella control and reduction in total bacteria numbers, water heater temperature set point and water use frequency are more promising factors for long-term Legionella and microbial community control, illustrating the importance of maintaining consistent elevated temperatures in the system relative to short-term heat shock.

Project description: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 ?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.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.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.

Project description:Legionella pneumophila (Lp) is an opportunistic pathogen and its survival in water is critical for human infection. Therefore, identifying the genes of Lp that are required for survival in water may help devise strategies to prevent Legionella outbreaks. In this study, we exposed Lp in rich medium and in an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours to uncover the global transcriptomic changes of Lp in water. The repression of major metabolic pathways, such as division, transcription and translation, suggests that Lp enters a dormant state in water. The induction of the flagellar associated genes (flg, fli and mot), enhance entry genes (enh) and some Icm/Dot effectors suggests that Lp may be waiting to establish intracellular replication in suitable host. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to environmental stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than those cultured in rich medium. Apart from this, the gene bdhA involved in the degradation of the intracellular energy storage compound poly-hydroxybutyrate is highly expressed in water. Further characterization shows that bdhA is positively regulated by RpoS during short-term exposure to water. The deletion mutant of bdhA had a survival defect in water at 37°C, demonstrating that this gene is important for maintaining the long-term survivorship of Lp in water. Other identified genes highly induced upon exposure to water could also be necessary for Lp to survive in water. Legionella pneumophila Philadelphia-1 strain JR32 was grown in AYE broth at 25°C shaking to OD600 of 1 in triplicate. Samples were taken for analysis; this is the control. Then the cultures were washed three times in Fraquil and resuspended in Fraquil to an OD600 of 1 and transfered to vessels of bioreactior (Biostat Q-plus). Samples were taken after 2h, 6h and 24h.

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Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing.

Publications

Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing.

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