Browsing by Author "Rhoads, William J."

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    Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016
    Garner, Emily; Brown, Connor L.; Schwake, David Otto; Rhoads, William J.; Arango-Argoty, Gustavo; Zhang, Liqing; Jospin, Guillaume; Coil, David A.; Eisen, Jonathan A.; Edwards, Marc A.; Pruden, Amy (Centers for Disease Control and Prevention, 2019-11)
    During the water crisis in Flint, Michigan, USA (2014–2015), 2 outbreaks of Legionnaires’ disease occurred in Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella pneumophila isolates submitted to a laboratory in Genesee County during the second outbreak with 103 water isolates collected the following year. We documented a genetically diverse range of L. pneumophila strains across clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates, 3 water isolates from a Flint hospital, 1 water isolate from a Flint residence, and the reference Paris strain) had a high degree of similarity (2–1,062 single-nucleotide polymorphisms), all L. pneumophila sequence type 1, serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were widespread in Flint hospital water samples but bore no resemblance to available clinical isolates. L. pneumophila strains in Flint tap water after the outbreaks were diverse and similar to some disease-causing strains.
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    Considerations for large building water quality after extended stagnation
    Proctor, Caitlin R.; Rhoads, William J.; Keane, Tim; Salehi, Maryam; Hamilton, Kerry; Pieper, Kelsey J.; Cwiertny, David M.; Prévost, Michèle; Whelton, Andrew J. (Wiley, 2020-06-09)
    The unprecedented number of building closures related to the coronavirus disease (COVID-19) pandemic is concerning because water stagnation will occur in many buildings that do not have water management plans in place. Stagnant water can have chemical and microbiological contaminants that pose potential health risks to occupants. Health officials, building owners, utilities, and other entities are rapidly developing guidance to address this issue, but the scope, applicability, and details included in the guidance vary widely. To provide a primer of large building water system preventative and remedial strategies, peer-reviewed, government, industry, and nonprofit literature relevant to water stagnation and decontamination practices for plumbing was synthesized. Preventative practices to help avoid the need for recommissioning (e.g., routine flushing) and specific actions, challenges, and limitations associated with recommissioning were identified and characterized. Considerations for worker and occupant safety were also indicated. The intended audience of this work includes organizations developing guidance.
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    Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing
    Rhoads, William J.; Pruden, Amy; Edwards, Marc A. (MDPI, 2016-03-12)
    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.
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    Did Municipal Water Distribution System Deficiencies Contribute to a Legionnaires' Disease Outbreak in Quincy, IL?
    Rhoads, William J.; Keane, Tim; Spencer, M. Storme; Pruden, Amy; Edwards, Marc A. (2020-12-08)
    Fifty-eight people were sickened and 12 died from a Legionnaires' disease (LD) outbreak in Quincy, IL, in 2015. The initial outbreak investigation identified deficiencies at the Illinois Veteran's Home (IVHQ), but these did not account for four community-acquired cases that occurred concurrently with no IVHQ exposure. We broaden the investigation to evaluate seven lines of evidence and assess whether municipal drinking water supply deficiencies potentially contributed to a community-wide outbreak. Notably, 3-6 months prior to the outbreak, the primary disinfectant was changed and corrosion control was interrupted, causing a sustained decrease in disinfectant residuals throughout Quincy's distribution system. We hypothesize this created more favorable conditions for Legionella growth throughout the system and an increase in water lead levels. These municipal system deficiencies were not identified in prior investigations of the outbreak, but their impacts on public health outcomes are consistent with those of the 2014-2016 Flint Water Crisis. However, they occurred in Quincy without any legal violations in the municipal water system or public acknowledgment of community-wide health risks. This study supports the critical need for improved data collection during changes in municipal water treatment. Additional regulatory and communication requirements can better protect public health from both LD and lead.
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    Effect of heat shock on hot water plumbing microbiota and Legionella pneumophila control
    Ji, Pan; Rhoads, William J.; Edwards, Marc A.; Pruden, Amy (2018-02-09)
    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.
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    Effects of Copper on Legionella pneumophila Revealed via Viability Assays and Proteomics
    Song, Yang; Mena-Aguilar, Didier; Brown, Connor L.; Rhoads, William J.; Helm, Richard F.; Pruden, Amy; Edwards, Marc A. (MDPI, 2024-07-03)
    Cu is an antimicrobial that is commonly applied to premise (i.e., building) plumbing systems for Legionella control, but the precise mechanisms of inactivation are not well defined. Here, we applied a suite of viability assays and mass spectrometry-based proteomics to assess the mechanistic effects of Cu on L. pneumophila. Although a five- to six-log reduction in culturability was observed with 5 mg/L Cu2+ exposure, cell membrane integrity only indicated a <50% reduction. Whole-cell proteomic analysis revealed that AhpD, a protein related to oxidative stress, was elevated in Cu-exposed Legionella relative to culturable cells. Other proteins related to cell membrane synthesis and motility were also higher for the Cu-exposed cells relative to controls without Cu. While the proteins related to primary metabolism decreased for the Cu-exposed cells, no significant differences in the abundance of proteins related to virulence or infectivity were found, which was consistent with the ability of VBNC cells to cause infections. Whereas the cell-membrane integrity assay provided an upper-bound measurement of viability, an amoebae co-culture assay provided a lower-bound limit. The findings have important implications for assessing Legionella risk following its exposure to copper in engineered water systems.
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    Growth of Opportunistic Pathogens in Domestic Plumbing: Building Standards, System Operation, and Design
    Rhoads, William J. (Virginia Tech, 2017-03-15)
    Understanding and limiting public health threats resulting from exposure to opportunistic pathogens (OPs) in domestic water (i.e., hot/cold water for human use) will be one of the grand challenges for water safety in the 21st century. This dissertation anticipates some of the complexities in balancing stakeholder goals and developing building standards to limit OP growth, and advances scientific understanding of OP survival and proliferation in domestic plumbing systems. In a cross-sectional survey of water- and energy-efficient buildings, domestic water age ranged from 8 days to 6 months and resulted in pH and temperature fluctuations, rapid disinfectant residual decay up to 144 times faster than municipal water delivered to the buildings, and elevated levels of OP gene markers. This motivates future work to determine how to maintain high quality and safe water while preserving the sustainability goals of these cutting-edge buildings. Head-to-head pilot-scale experiments examining OP growth in recirculating hot water systems revealed that elevated temperature had an overarching inhibitory effect on L. pneumophila growth where temperatures were maintained. However, control was undermined in distal branches, especially when density-driven convective mixing gradients maintained ideal growth temperatures and delivered nutrients to the otherwise stagnant branches. These results resolve discrepancies reported in the literature regarding the effects of flow, and identify important system design and operational conditions that facilitate OP growth. Advancements were also made in understanding how corrosion can trigger OP growth. In Flint, MI, corrosive Flint River water damaged iron pipes, releasing iron nutrients, consuming chlorine residual, and supporting high levels of L. pneumophila in large building systems. This likely triggered two unprecedented clusters of Legionnaire's disease. In pilot-scale systems, copper released from copper pipes, but not dosed as soluble cupric, triggered release of >1,100 times more H2 into the water due to deposition corrosion. The organic carbon fixed by autotrophic hydrogen oxidation has the potential to facilitate OP growth, but more work is needed to understand the limits of this mechanism. Finally, well-controlled laboratory experiments confirmed past reports from field surveys that the use of chloramines trigger a trade-off between controlling Legionella and allowing non-tuberculous Mycobacteria to persist.
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    Impact of Flow Rate and Water Age on Opportunistic Pathogen Growth: Implications for Water Conservation, Fixture Design, and Policy
    Busch, Sarah Elizabeth (Virginia Tech, 2020-01-22)
    Water conservation efforts have led to a decrease of flow rates in buildings, increasing water retention time (WRT) and sometimes opportunistic pathogens (OPs) growth. A novel experiment with replicated distal pipes operated at commonly used flow rates was designed to evaluate the effects of water age, flush frequency, flow rate, pipe diameter, water temperature, disinfectant residual presence, and microbial regrowth in hot and cold pipes. In cold water, total bacterial regrowth was a function of water age, plateauing after approximately 6 days at cell counts 20 times higher than influent water with minimal disinfectants. In warm (40 °C) water, most regrowth occurred in the heater tank, reducing the relative growth in the pipes. When cold water with ~1 mg/L chloramine was present, cold-water total bacteria regrowth plateaued after about 2 days WRT with cell counts 14 times higher than influent water, but regrowth still occurred in the heater tank. With 1 mg/L chloramine and elevated heater temperature (60 °C), regrowth in the tank was suppressed and cell counts in the pipes increased 82 times above cold-water influent levels at 7.5 days WRT. Legionella spp. and Mycobacterium spp. demonstrated opposite responses to flow rate with chloramine minimization. The highest levels of Legionella spp. (1.7 log higher than influent) were present when flow velocity was >2 feet per second (fps), but the highest levels of Mycobacterium spp. (1.5 log higher than influent) were observed at the lowest flow velocity (0.33 fps). This study highlights the tradeoffs between water conservation and water quality.
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    Impact of water heater temperature setting and water use frequency on the building plumbing microbiome
    Ji, Pan; Rhoads, William J.; Edwards, Marc A.; Pruden, Amy (2017-06)
    Hot water plumbing is an important conduit of microbes into the indoor environment and can increase risk of opportunistic pathogens (for example, Legionella pneumophila). We examined the combined effects of water heater temperature (39, 42, 48, 51 and 58 degrees C), pipe orientation (upward/downward), and water use frequency (21, 3 and 1 flush per week) on the microbial composition at the tap using a pilot-scale pipe rig. 16S rRNA gene amplicon sequencing indicated that bulk water and corresponding biofilm typically had distinct taxonomic compositions (R-Adonis(2) = 0.246, P-Adonis = 0.001), yet similar predicted functions based on PICRUSt analysis (R-Adonis(2) = 0.087, P-Adonis = 0.001). Although a prior study had identified 51 degrees C under low water use frequency to enrich Legionella at the tap, here we reveal that 51 degrees C is also a threshold above which there are marked effects of the combined influences of temperature, pipe orientation, and use frequency on taxonomic and functional composition. A positive association was noted between relative abundances of Legionella and mitochondrial DNA of Vermamoeba, a genus of amoebae that can enhance virulence and facilitate replication of some pathogens. This study takes a step towards intentional control of the plumbing microbiome and highlights the importance of microbial ecology in governing pathogen proliferation.
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    Limitations to Use Copper as an Antimicrobial Control of Legionella in Potable Water Plumbing Systems
    Song, Yang (Virginia Tech, 2022-01-28)
    The opportunistic pathogen Legionella is the leading cause of reported waterborne disease outbreaks in the United States. Legionella can thrive under the warm, stagnant, low-disinfectant conditions characteristic of premise (i.e., building) plumbing systems, making it challenging to identify effective interventions for its control. Copper (Cu) is a promising antimicrobial that can be dosed directly to water via copper-silver ionization systems or released naturally via corrosion of Cu pipes to help control growth of Legionella and other pathogens. However, prior research has shown that Cu does not always reliably control Legionella and sometimes seems to even stimulate its growth. A deeper understanding of the mechanistic effects of Cu on Legionella, at both pure-culture and real-world scales, is critical in order to inform effective controls for Legionella. The overarching objective of the research embodied by this dissertation was aimed at elucidating the chemical and microbial interactions in premise plumbing that govern efficacy of Cu for Legionella control through a series of complementary bench-, pilot-, and field-scale studies. A critical review and synthesis of the literature identified important knowledge gaps in relation to antimicrobial effects of Cu. In particular, changes in the pH, phosphate corrosion control, and rising levels of natural organic matter (NOM) in distributed water are predicted to be important controlling factors. The type of sacrificial anode rod material employed in water heaters was also identified as an underappreciated factor, which directly affects pH, evolution of hydrogen gas as a microbial nutrient, and release of metals (such as aluminum) that bind copper. Microbiological factors: including growth phase of Legionella (e.g., exponential or stationary), strain-specific Cu tolerance, background microbiome composition, and the possibility that viable but non-culturable (VBNC) Legionella might still cause human disease, were also identified as major confounding factors. These knowledge gaps are addressed from various dimensions across each chapter of the dissertation. The effects of pH, orthophosphate corrosion inhibitor concentration, and NOM were examined in bench-scale pure culture experiments over a range of conditions relevant to drinking water. Cupric ions and antimicrobial effects were drastically reduced at pH >7.5, especially in the presence of phosphate, which precipitates copper, or NOM, which complexes the Cu in a form that is less bioavailable. Chick-Watson disinfection models indicated that soluble Cu was the most robust correlate with observed Cu antimicrobial effects across a range of tested waters. This new knowledge suggests that measuring soluble rather than total Cu would be much more informative to guide practitioners in dosing. The research also demonstrated that changes in pH or orthophosphate that have been made to control corrosion over the last few decades, have significantly altered Cu chemistry in buildings, undermining antimicrobial capacity and increasing likelihood of Legionella growth. Pilot-scale experiments confirmed that soluble Cu is an effective indicator of Cu antimicrobial capacity, even in more complex environments represented by realistic hot water plumbing systems. In particular, dosing of orthophosphate, which is widely added by drinking water utilities to control corrosion, directly reduces soluble copper and overall antimicrobial capacity. In some cases, Cu added together with orthophosphate apparently promoted the growth of Legionella, providing an example of at least one circumstance where Cu addition can induce interactive effects that elevate Legionella compared to a control system with trace Cu. It was also demonstrated for the first time that different water heater sacrificial anode types are subject to different corrosion processes, which indirectly influence Cu antimicrobial capacity. Specifically, aluminum ions released from aluminum anode corrosion at 1 mg/L can form an Al(OH)3 gel, which can remove >80% of the soluble Cu from water and reduce Cu antimicrobial effects by >2-log at pH=7. Corrosion from magnesium anodes was found to dramatically increase the pH from 6.8 to >8, which correspondingly reduces Cu antimicrobial capacity. Cu deposition further increased the anode corrosion rate and promoted evolution of hydrogen gas, which is a potent electron donor that stimulates autotrophic microbial growth especially with a magnesium anode. Electric powered anodes did not release metals or alter pH and thus did not diminish Cu antimicrobial capacity. Still, across the pilot-scale experiments, even very high levels of Cu (>1.2 mg/L) at low pH (<7) failed to fully eradicate culturable Legionella. The much lower than expected antimicrobial efficacy of Cu in the pilot-scale hot water plumbing systems was found to be partially explained by the properties of the strain that colonized the systems. Based on fitting the data to a Chick-Watson disinfection model, the outbreak-associated strain that was inoculated into the systems was estimated to be 7 times more tolerant to Cu compared to the common lab strain applied in the bench-scale tests. Further, exponential growth phase L. pneumophila were found to be 2.5 times more susceptible to Cu relative to early stationary phase cultures. It is important to also recognize that, in the pilot-scale systems, drinking water biofilms and the amoeba hosts that colonize them can further shield Legionella from the antimicrobial effects of Cu. Application of shotgun metagenomic sequencing offered the opportunity to more deeply examine the response of Legionella and other pathogens to Cu dosed to the pilot-scale hot water systems in the context of the broader microbiome. It was found that metagenomic analysis provided a sensitive indication of the bioavailability of Cu to the broader microbial community inhabiting the hot water systems, further confirming that the outbreak-associated strain of Legionella that colonized the rigs was relatively tolerant of Cu. Functional gene analysis provided further insight into the mechanistic action of Cu, suggesting multi-modal action of both membrane damage and interruption of nucleic acid replication. The metagenomic analysis further revealed that protozoan host numbers tended to increase in the pilot-scale systems with time, and this could also increase the potential for Legionella proliferation with time. Additional pure culture studies aiming to further assess the mechanistic action of Cu provided strong evidence that Cu can induce a VBNC state for Legionella. This is a concern, given that other studies have indicated that VBNC Legionella are still capable of causing legionellosis. However, VBNC cells are not detected by conventional culturing. Multiple lines of evidence supported the conclusion that Cu induced a VBNC state for Legionella, including membrane integrity, enzyme activity, ATP generation, and Amoebae resuscitation assays applied to two different strains of L. pneumophila. After exposure to Cu, up to a 5-log (99.999%) reduction in culturable Legionella was observed, whereas corresponding reductions in the various viability measures were only by <1-log (90%). In other words, conventional culturing may miss up to 99.99% of the Legionella that is still capable of causing disease. To our knowledge, this is the first study that has assessed the potential for Cu-induced VBNC Legionella. Additional research is needed to further quantify the contribution of VBNC status to challenges in effective Cu-based control of Legionella in premise plumbing. This research further examines, for the first time, the proteomic response of Legionella to Cu, comparing both presumably VBNC and culturable cells. Functional annotation of proteins that were differentially produced by the cells in response to Cu addition revealed that VBNC L. pneumophila modulated its proteome to favor cell membrane- and motility-related proteins, while reducing production of other proteins related to primary metabolism compared to culturable cells. These observations are consistent with the metagenomic-based observations and support the hypothesis that Cu inactivates cells by damaging the cell membrane. The findings also confirmed reduced general cell metabolism that is characteristic of a VBNC state. This dissertation highlights the important and complex effects of Cu on Legionella growth in potable water systems as modified by water chemistry, water heater anode type, characteristics of the surrounding microbiome, and Legionella strain characteristics and growth status. The findings raise important questions about how to measure disinfectant efficacy and provide fundamental new knowledge that can help to better optimize the application of Cu as an antimicrobial to drinking water systems and better protect public health.
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    Natural Organic Matter, Orthophosphate, pH, and Growth Phase Can Limit Copper Antimicrobial Efficacy for Legionella in Drinking Water
    Song, Yang; Pruden, Amy; Edwards, Marc A.; Rhoads, William J. (2021-02-02)
    Copper (Cu) is a promising antimicrobial for premise plumbing, where ions can be dosed directly via copper silver ionization or released naturally via corrosion of Cu pipes, but Cu sometimes inhibits and other times stimulates Legionella growth. Our overarching hypothesis was that water chemistry and growth phase control the net effect of Cu on Legionella. The combined effects of pH, phosphate concentration, and natural organic matter (NOM) were comprehensively examined over a range of conditions relevant to drinking water in bench-scale pure culture experiments, illuminating the effects of Cu speciation and precipitation. It was found that cupric ions (Cu2+) were drastically reduced at pH > 7.0 or in the presence of ligand-forming phosphates or NOM. Further, exponential phase L. pneumophila were 2.5x more susceptible to Cu toxicity relative to early stationary phase cultures. While Cu2+ ion was the most effective biocidal form of Cu, other inorganic ligands also had some biocidal impacts. A comparison of 33 large drinking water utilities' field-data from 1990 and 2018 showed that Cu2+ levels likely decreased more dramatically (>10x) than did the total or soluble Cu (2x) over recent decades. The overall findings aid in improving the efficacy of Cu as an actively dosed or passively released antimicrobial against L. pneumophila.
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    Replicable simulation of distal hot water premise plumbing using convectively-mixed pipe reactors
    Spencer, M. Storme; Cullom, Abraham C.; Rhoads, William J.; Pruden, Amy; Edwards, Marc A. (2020-09-16)
    A lack of replicable test systems that realistically simulate hot water premise plumbing conditions at the laboratory-scale is an obstacle to identifying key factors that support growth of opportunistic pathogens (OPs) and opportunities to stem disease transmission. Here we developed the convectively-mixed pipe reactor (CMPR) as a simple reproducible system, consisting of off-the-shelf plumbing materials, that self-mixes through natural convective currents and enables testing of multiple, replicated, and realistic premise plumbing conditions in parallel. A 10-week validation study was conducted, comparing three pipe materials (PVC, PVC-copper, and PVC-iron; n = 18 each) to stagnant control pipes without convective mixing (n = 3 each). Replicate CMPRs were found to yield consistent water chemistry as a function of pipe material, with differences becoming less discernable by week 9. Temperature, an overarching factor known to control OP growth, was consistently maintained across all 54 CMPRs, with a coefficient of variation <2%. Dissolved oxygen (DO) remained lower in PVC-iron (1.96 +/- 0.29 mg/L) than in PVC (5.71 +/- 0.22 mg/L) or PVC-copper (5.90 +/- 0.38 mg/L) CMPRs as expected due to corrosion. Further, DO in PVC-iron CMPRs was 33% of that observed in corresponding stagnant pipes (6.03 +/- 0.33 mg/L), demonstrating the important role of internal convective mixing in stimulating corrosion and microbiological respiration. 16S rRNA gene amplicon sequencing indicated that both bulk water (P-adonis= 0.001, R-2= 0.222, P-betadis= 0.785) and biofilm (P-adonis= 0.001, R-2= 0.119, P-betadis= 0.827) microbial communities differed between CMPR versus stagnant pipes, consistent with creation of a distinct ecological niche. Overall, CMPRs can provide a more realistic simulation of certain aspects of premise plumbing than reactors commonly applied in prior research, at a fraction of the cost, space, and water demand of large pilot-scale rigs.
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    Shotgun Metagenomics Reveals Taxonomic and Functional Shifts in Hot Water Microbiome Due to Temperature Setting and Stagnation
    Dai, Dongjuan; Rhoads, William J.; Edwards, Marc A.; Pruden, Amy (Frontiers, 2018-11-13)
    Hot water premise plumbing has emerged as a critical nexus of energy, water, and public health. The composition of hot water microbiomes is of special interest given daily human exposure to resident flora, especially opportunistic pathogens (OPs), which rely on complex microbial ecological interactions for their proliferation. Here, we applied shotgun metagenomic sequencing to characterize taxonomic and functional shifts in microbiomes as a function of water heater temperature setting, stagnation in distal pipes, and associated shifts in water chemistry. A cross-section of samples from controlled, replicated, pilot-scale hot water plumbing rigs representing different temperature settings (39, 42, and 51 degrees C), stagnation periods (8 h vs. 7 days), and time-points, were analyzed. Temperature setting exhibited an overarching impact on taxonomic and functional gene composition. Further, distinct taxa were selectively enriched by specific temperature settings (e.g., Legionella at 39 degrees C vs. Deinococcus at 51 degrees C), while relative abundances of genes encoding corresponding cellular functions were highly consistent with expectations based on the taxa driving these shifts. Stagnation in distal taps diminished taxonomic and functional differences induced by heating the cold influent water to hot water in recirculating line. In distal taps relative to recirculating hot water, reads annotated as being involved in metabolism and growth decreased, while annotations corresponding to stress response (e.g., virulence disease and defense, and specifically antibiotic resistance) increased. Reads corresponding to OPs were readily identified by metagenomic analysis, with L. pneumophlla reads in particular correlating remarkably well with gene copy numbers measured by quantitative polymerase chain reaction. Positive correlations between L. pneumophila reads and those of known protozoan hosts were also identified. Elevated proportions of genes encoding metal resistance and hydrogen metabolism were noted, which was consistent with elevated corrosion-induced metal concentrations and hydrogen generation. This study provided new insights into real-world factors influencing taxonomic and functional compositions of hot water microbiomes. Here metagenomics is demonstrated as an effective tool for screening for potential presence, and even quantities, of pathogens, while also providing diagnostic capabilities for assessing functional responses of microbiomes to various operational conditions. These findings can aid in informing future monitoring and intentional control of hot water microbiomes.
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    Survey of green building water systems reveals elevated water age and water quality concerns
    Rhoads, William J.; Pruden, Amy; Edwards, Marc A. (The Royal Society of Chemistry, 2015-11-05)
    Widespread adoption of innovative water conservation strategies has potential unintended consequences for aesthetics and public health. A cross-section of green buildings were surveyed and compared to typical conventional buildings in terms of water retention time (i.e., water age), water chemistry, and levels of opportunistic pathogen genetic markers. Water age was estimated to be 2-6.7 months in an off-grid office, an average of 8 days in a Leadership in Environmental Engineering Design certified healthcare suite, and was increased to 2.7 days from 1 day due to installation of a solar "pre-heat" water tank in a net-zero energy house. Chlorine and chloramine residuals were often completely absent in the green building systems, decaying up to 144 times faster in premise plumbing with high water age when compared to distribution system water. Concentration of 16S rRNA and opportunistic pathogen genus level genetic markers were 1-4 orders of magnitude higher in green versus conventional buildings. This study raises concerns with respect to current green water system practices and the importance of considering potential public health impacts in the design of sustainable water systems.
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    Water heater temperature set point and water use patterns influence Legionella pneumophila and associated microorganisms at the tap
    Rhoads, William J.; Ji, Pan; Pruden, Amy; Edwards, Marc A. (Biomed Central, 2015-12-01)
    Background Lowering water heater temperature set points and using less drinking water are common approaches to conserving water and energy; yet, there are discrepancies in past literature regarding the effects of water heater temperature and water use patterns on the occurrence of opportunistic pathogens, in particular Legionella pneumophila. Our objective was to conduct a controlled, replicated pilot-scale investigation to address this knowledge gap using continuously recirculating water heaters to examine five water heater set points (39–58 °C) under three water use conditions. We hypothesized that L. pneumophila levels at the tap depend on the collective influence of water heater temperature, flow frequency, and the resident plumbing ecology. Results We confirmed temperature setting to be a critical factor in suppressing L. pneumophila growth both in continuously recirculating hot water lines and at distal taps. For example, at 51 °C, planktonic L. pneumophila in recirculating lines was reduced by a factor of 28.7 compared to 39 °C and was prevented from re-colonizing biofilm. However, L. pneumophila still persisted up to 58 °C, with evidence that it was growing under the conditions of this study. Further, exposure to 51 °C water in a low-use tap appeared to optimally select for L. pneumophila (e.g., 125 times greater numbers than in high-use taps). We subsequently explored relationships among L. pneumophila and other ecologically relevant microbes, noting that elevated temperature did not have a general disinfecting effect in terms of total bacterial numbers. We documented the relationship between L. pneumophila and Legionella spp., and noted several instances of correlations with Vermamoeba vermiformis, and generally found that there is a dynamic relationship with this amoeba host over the range of temperatures and water use frequencies examined. Conclusions Our study provides a new window of understanding into the microbial ecology of potable hot water systems and helps to resolve past discrepancies in the literature regarding the influence of water temperature and stagnation on L. pneumophila, which is the cause of a growing number of outbreaks. This work is especially timely, given society’s movement towards “green” buildings and the need to reconcile innovations in building design with public health.