Legionella relative abundance in shower hose biofilms is associated with specific microbiome members.

Legionella are natural inhabitants of building plumbing biofilms, where interactions with other microorganisms influence their survival, proliferation, and death. Here, we investigated the associations of Legionella with bacterial and eukaryotic microbiomes in biofilm samples extracted from 85 shower hoses of a multiunit residential building. Legionella spp. relative abundance in the biofilms ranged between 0-7.8%, of which only 0-0.46% was L. pneumophila. Our data suggest that some microbiome members were associated with high (e.g. Chthonomonas, Vrihiamoeba) or low (e.g. Aquabacterium, Vannella) Legionella relative abundance. The correlations of the different Legionella variants (30 Zero-Radius OTUs detected) showed distinct patterns, suggesting separate ecological niches occupied by different Legionella species. This study provides insights into the ecology of Legionella with respect to: (i) the colonization of a high number of real shower hoses biofilm samples; (ii) the ecological meaning of associations between Legionella and co-occurring bacterial/eukaryotic organisms; (iii) critical points and future directions of microbial-interaction-based-ecological-investigations.

Shotgun Metagenomics Reveals Impacts of Copper and Water Heater Anodes on Pathogens and Microbiomes in Hot Water Plumbing Systems.

Hot water building plumbing systems are vulnerable to the proliferation of opportunistic pathogens (OPs), including Legionella pneumophila and Mycobacterium avium. Implementation of copper as a disinfectant could help reduce OPs, but a mechanistic understanding of the effects on the microbial community under real-world plumbing conditions is lacking. Here, we carried out a controlled pilot-scale study of hot water systems and applied shotgun metagenomic sequencing to examine the effects of copper dose (0-2 mg/L), orthophosphate corrosion control agent, and water heater anode materials (aluminum vs magnesium vs powered anode) on the bulk water and biofilm microbiome composition. Metagenomic analysis revealed that, even though a copper dose of 1.2 mg/L was required to reduce Legionella and Mycobacterium numbers, lower doses (e.g., ≤0.6 mg/L) measurably impacted the broader microbial community, indicating that the OP strains colonizing these systems were highly copper tolerant. Orthophosphate addition reduced bioavailability of copper, both to OPs and to the broader microbiome. Functional gene analysis indicated that both membrane damage and interruption of nucleic acid replication are likely at play in copper inactivation mechanisms. This study identifies key factors (e.g., orthophosphate, copper resistance, and anode materials) that can confound the efficacy of copper for controlling OPs in hot water plumbing.

Pilot-scale assessment reveals effects of anode type and orthophosphate in governing antimicrobial capacity of copper for Legionella pneumophila control.

Copper (Cu) is sometimes applied as an antimicrobial for controlling Legionella in hot water plumbing systems, but its efficacy is inconsistent. Here we examined the effects of Cu (0 - 2 mg/L), orthophosphate corrosion inhibitor (0 or 3 mg/L as phosphate), and water heater anodes (aluminum, magnesium, and powered anodes) on both bulk water and biofilm-associated L. pneumophila in pilot-scale water heater systems. Soluble, but not total, Cu was a good predictor of antimicrobial capacity of Cu. Even after months of exposure to very high Cu levels (>1.2 mg/L) and low pH (<7), which increases solubility and enhances bioavailability of Cu, culturable L. pneumophila was only reduced by ∼1-log. Cu antimicrobial capacity was shown to be limited by various factors, including binding of Cu ions by aluminum hydroxide precipitates released from corrosion of aluminum anodes, higher pH due to magnesium anode corrosion, and high Cu tolerance of the outbreak-associated L. pneumophila strain that was inoculated into the systems. L. pneumophila numbers were also higher in several instances when Cu was dosed together with orthophosphate (e.g., with an Al anode), revealing at least one scenario where high levels of total Cu appeared to stimulate Legionella. The controlled, pilot-scale nature of this study provides new understanding of the limitations of Cu as an antimicrobial in real-world plumbing systems.

Potential probiotic approaches to control Legionella in engineered aquatic ecosystems.

Opportunistic pathogens belonging to the genus Legionella are among the most reported waterborne-associated pathogens in industrialized countries. Legionella colonize a variety of engineered aquatic ecosystems and persist in biofilms where they interact with a multitude of other resident microorganisms. In this review, we assess how some of these interactions could be used to develop a biological-driven "probiotic" control approach against Legionella. We focus on: (i) mechanisms limiting the ability of Legionella to establish and replicate within some of their natural protozoan hosts; (ii) exploitative and interference competitive interactions between Legionella and other microorganisms; and (iii) the potential of predatory bacteria and phages against Legionella. This field is still emergent, and we therefore specifically highlight research for future investigations, and propose perspectives on the feasibility and public acceptance of a potential probiotic approach.

Variable Legionella Response to Building Occupancy Patterns and Precautionary Flushing.

When stay-at-home orders were issued to slow the spread of COVID-19, building occupancy (and water demand) was drastically decreased in many buildings. There was concern that widespread low water demand may cause unprecedented Legionella occurrence and Legionnaires' disease incidence. In lieu of evidenced-based guidance, many people flushed their water systems as a preventative measure, using highly variable practices. Here, we present field-scale research from a building before, during, and after periods of low occupancy, and controlled stagnation experiments. We document no change, a > 4-log increase, and a > 1.5-log decrease of L. pneumophila during 3- to 7-week periods of low water demand. L. pneumophila increased by > 1-log after precautionary flushing prior to reoccupancy, which was repeated in controlled boiler flushing experiments. These results demonstrate that the impact of low water demand (colloquially called stagnation) is not as straight forward as is generally assumed, and that some flushing practices have potential unintended consequences. In particular, stagnation must be considered in context with other Legionella growth factors like temperature and flow profiles. Boiler flushing practices that dramatically increase the flow rate and rapidly deplete boiler temperature may mobilize Legionella present in biofilms and sediment.

Occurrence of opportunistic pathogens in private wells after major flooding events: A four state molecular survey.

Private wells can become contaminated with waterborne pathogens during flooding events; however, testing efforts focus almost exclusively on fecal indicator bacteria. Opportunistic pathogens (OPs), which are the leading cause of identified waterborne disease in the United States, are understudied in private wells. We conducted a quantitative polymerase chain reaction survey of Legionella spp., L. pneumophila, Mycobacterium spp., M. avium, Naegleria fowleri, and shiga toxin-producing Escherichia coli gene markers and total coliform and E. coli in drinking water supplied by private wells following the Louisiana Floods (2016), Hurricane Harvey (2017), Hurricane Irma (2017), and Hurricane Florence (2018). Self-reported well characteristics and recovery status were collected via questionnaires. Of the 211 water samples collected, 40.3% and 5.2% were positive for total coliform and E. coli, which were slightly elevated positivity rates compared to prior work in coastal aquifers. DNA markers for Legionella and Mycobacterium were detected in 54.5% and 36.5% of samples, with L. pneumophila and M. avium detected in 15.6% and 17.1%, which was a similar positivity rate relative to municipal system surveys. Total bacterial 16S rRNA gene copies were positively associated with Legionella and Mycobacterium, indicating that conditions that favor occurrence of general bacteria can also favor OPs. N. fowleri DNA was detected in 6.6% of samples and was the only OP that was more prevalent in submerged wells compared to non-submerged wells. Self-reported well characteristics were not associated with OP occurrence. This study exposes the value of routine baseline monitoring and timely sampling after flooding events in order to effectively assess well water contamination risks.

Microbial Contamination of Drinking Water Supplied by Private Wells after Hurricane Harvey.

Hurricane Hurricane Harvey made landfall on the Texas Gulf Coast on August 25, 2017, as a Category 4 hurricane and caused widespread flooding. We explored spatial and temporal distributions of well testing and contamination rates; relationships between contamination and system characteristics and recovery behaviors; and efficacy of mitigation strategies. We estimated that over 500 000 well users (∼130 000 to 260 000 wells) may have been affected, but only around 15 000 well users (∼3800 to 7500 wells) had inundated systems based on inundation maps. Local health departments and our team sampled 8822 wells in 44 counties in the 10 months that followed. Total coliform occurrence was 1.5 times and Escherichia coli was 2.8 times higher after Hurricane Harvey compared to baseline levels. Microbial contamination was more likely (1.7-2.5 times higher) when wells were inundated and/or residents felt their water was unsafe. Although more wells in urban counties were affected, E. coli rates were higher in wells in rural counties. Disinfection did not always eliminate contamination, highlighting concerns about the implementation and efficacy of chlorination procedures. Despite this extensive well testing conducted after Hurricane Harvey, we estimate that only 4.1% of potentially affected wells were tested, underscoring the magnitude of recovery assistance needed to assist well users after flooding events.

Source-to-Tap Assessment of Microbiological Water Quality in Small Rural Drinking Water Systems in Puerto Rico Six Months After Hurricane Maria.

Maria made a landfall in Puerto Rico on September 20, 2017 as a category 4 hurricane, causing severe flooding, widespread electricity outages, damage to infrastructure, and interruptions in water and wastewater treatment. Small rural community water systems face unique challenges in providing drinking water, which intensify after natural disasters. The purpose of this study was to evaluate the functionality of six very small rural public water systems and one large regulated system in Puerto Rico six months after Maria and survey a broad sweep of fecal, zoonotic, and opportunistic pathogens from the source to tap. Samples were collected from surface and groundwater sources, after water treatment and after distribution to households. Genes indicative of pathogenic Leptospira spp. were detected by polymerase chain reaction (PCR) in all systems reliant on surface water sources. Salmonella spp. was detected in surface and groundwater sources and some distribution system water both by culture and PCR. Legionella spp. and Mycobacteria spp. gene numbers measured by quantitative PCR were similar to nonoutbreak conditions in the continental U.S. Amplicon sequencing provided a nontarget screen for other potential pathogens of concern. This study aids in improving future preparedness, assessment, and recovery operations for small rural water systems after natural disasters.

Natural Organic Matter, Orthophosphate, pH, and Growth Phase Can Limit Copper Antimicrobial Efficacy for Legionella in Drinking Water.

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 2.5× 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 (>10×) than did the total or soluble Cu (2×) over recent decades. The overall findings aid in improving the efficacy of Cu as an actively dosed or passively released antimicrobial against L. pneumophila.

Evaluation of Preparedness and Recovery Needs of Private Well Users After the Great Louisiana Flood of 2016.

CONTEXT: The August 2016 Louisiana flood marked the second 500-year flood in the state in 1 year. OBJECTIVE: The aim of this study was to identify private well user needs in the aftermath of the flood and to develop disaster planning and recovery recommendations for flood-prone well-reliant communities. DESIGN: A descriptive cross-sectional study was conducted to collect information from a convenience sample of flood-impacted well users via surveys and water sampling kits, which were distributed to well users 9 to 11 weeks after floodwaters receded (n = 106). SETTING: Surveys and kits were distributed at roadside flood response and recovery stations set up by local churches in French Settlement, Livingston Parish, Louisiana, an area at the epicenter of the flood-impacted area. PARTICIPANTS: Subjects were included if they self-reported having a flood-impacted well. MAIN OUTCOME MEASURES: Surveys collected information to characterize knowledge gaps, risk perceptions, flood impacts, resource accessibility, and well maintenance barriers. Well water tests evaluated total coliform and Escherichia coli. RESULTS: Among those in low-risk flood zones (n = 22), 27% were in areas designated as having flooded. Among flood-impacted wells that were shock chlorinated after the flood (n = 16), 31.3% tested positive for total coliform and 12.5% for E coli. Only 26% of respondents received well-related information after the disaster. CONCLUSIONS: Results highlight critical needs for disaster planning and well user education in flood-prone areas, changes to flood risk maps, and concerns with the efficacy of disinfection strategies. Information and resources needs for flood-impacted well users are presented and recommendations on how to improve flood preparedness and recovery are made.

Demonstrating an Integrated Antibiotic Resistance Gene Surveillance Approach in Puerto Rican Watersheds Post-Hurricane Maria.

Comprehensive surveillance approaches are needed to assess sources, clinical relevance, and mobility of antibiotic resistance genes (ARGs) in watersheds. Here, we examined metrics derived from shotgun metagenomic sequencing and relationship to human fecal markers (HFMs; crAssphage, enterococci) and anthropogenic antibiotic resistance markers (AARMs; intI1, sul1) in three distinct Puerto Rican watersheds as a function of adjacent land use and wastewater treatment plant (WWTP) input 6 months after Hurricane Maria, a category V storm. Relative abundance and diversity of total ARGs increased markedly downstream of WWTP inputs, with ARGs unique to WWTP and WWTP-impacted river samples predominantly belonging to the aminoglycoside and ß-lactam resistance classes. WWTP and other anthropogenic inputs were similarly associated with elevated resistome risk scores and mobility incidence (M%). Contig analysis indicated a wide variety of mobile ß-lactam ARGs associated with pathogens downstream of WWTP discharge that were consistent with regional clinical concern, e.g., Klebsiella pneumoniae contigs containing KPC-2 within an ISKpn6-like transposase. HFMs and AARMs correlated strongly with the absolute abundance of total ARGs, but AARMs better predicted the majority of ARGs in general (85.4 versus <2%) and ß-lactam ARGs in particular. This study reveals sensitive, quantitative, mobile, clinically relevant, and comprehensive targets for antibiotic resistance surveillance in watersheds.

Replicable simulation of distal hot water premise plumbing using convectively-mixed pipe reactors.

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 (Padonis = 0.001, R2 = 0.222, Pbetadis = 0.785) and biofilm (Padonis = 0.001, R2 = 0.119, Pbetadis = 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.

Considerations for large building water quality after extended stagnation.

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.

Improving state-level emergency well disinfection strategies in the United States.

After flooding events, well users are encouraged to disinfect their private wells. However, well disinfection strategies are not consistently applied or proven effective. This study examines the science-based evidence that disinfection procedures reduce microbial loading in well water; reviews inclusion of disinfection principles in state-level emergency protocols; and explores research gaps potentially hindering disinfection efficacy. Emergency well disinfection protocols from 34 states were reviewed based on instructions for creating chlorine solutions; circulating chlorine solutions throughout the distribution system; achieving effective CT disinfection (chlorine dose*contact time); and post-disinfection guidance. Many protocols were missing key information about fundamentals of disinfection. Only two protocols instructed well users to verify chlorine residuals and three protocols instructed users to measure water pH. Most protocols recommended that high chlorine doses be introduced into the well, circulated throughout the system, and stagnated for several hours. A CT value estimated to inactivate at least 99.9% (3-log removal) of Cryptosporidium (255 mg-hr/L) was predicted to be achieved by 72.7% of protocols, and estimated CT values ranged from 35 to 16,327 mg-hr/L. Two research gaps identified were determining whether chlorine doses should differ based on well water chemistries and evaluating the appropriate chlorine dose that should be recommended for inactivating pathogens. This effort underscores a need for consistent, evidence-based messaging in emergency well disinfection protocols.

Residential water heater cleaning and occurrence of Legionella in Flint, MI.

After the Federal emergency in Flint, MI was declared in early 2016 in response to elevated lead-in-water and incidence of Legionnaires' disease, concerns arose that contaminants in residential water heaters could continue to contribute to poor quality tap water. Here, a comprehensive field survey of residential water heaters (n = 30) and associated water quality was conducted and the subsequent effects of an aggressive manual water heater clean-out was determined, including draining the tank and removing sediments via brushing and flushing. Before cleaning, inorganics accumulated in the tank sediments did not serve as a source of metals measured at hot water outlets. After cleaning, hardness- (calcium, magnesium, silica) and corrosion-associated inorganics (lead, iron, copper, aluminum, zinc) decreased by 64% in samples from sediment cleanout drain valves. Culturable L. pneumophila was only detected in 1 home (3.3%) prior to cleaning and 2 homes (6.7%) after cleaning, thus quantitative polymerase chain reaction was used to quantify potential effects on unculturable strains despite the limitation of differentiating live and dead cells. After the cleaning protocol, Legionella spp. and L. pneumophila gene numbers decreased or remained non-detectable in 83% and 98% of samples, respectively. Homes with less than 0.4 mg/L influent free chlorine tended to have quantifiable Legionella spp. gene numbers in water entering the home and had elevated L. pneumophila and Legionella spp. gene numbers throughout the home plumbing. Also, Legionella spp. and L. pneumophila gene numbers were highest for water heaters set at or below ∼42 °C and significantly decreased >51 °C, consistent with Legionella's preferred temperature range. Examination of the only home that had culturable L. pneumophila both before and after the cleaning protocol revealed that the organism was culturable from several sample locations throughout the home, including in water representative of the water main. Notably, the home was located in close proximity to McLaren Hospital, where an outbreak of Legionnaires disease was reported, and the water heater had a setpoint within the Legionella growth range of 44.2 °C. Considering that other factors were more strongly associated with Legionella occurrence and water heater sediment was not detectably mobilizing to tap water, it was concluded that water heater cleaning had some benefits, but was not an overarching factor contributing to possible human health risks.

Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016.

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.

Shotgun Metagenomics Reveals Taxonomic and Functional Shifts in Hot Water Microbiome Due to Temperature Setting and Stagnation.

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°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°C vs. Deinococcus at 51°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. pneumophila 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.

Effect of heat shock on hot water plumbing microbiota and Legionella pneumophila control.

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.

Distribution System Operational Deficiencies Coincide with Reported Legionnaires' Disease Clusters in Flint, Michigan.

We hypothesize that the increase in reported Legionnaires' disease from June 2014 to November 2015 in Genesee County, MI (where Flint is located) was directly linked to the switch to corrosive Flint River water from noncorrosive Detroit water from April 2014 to October 2015. To address the lack of epidemiological data linking the drinking water supplies to disease incidence, we gathered physiochemical and biological water quality data from 2010 to 2016 to evaluate characteristics of the Flint River water that were potentially conducive to Legionella growth. The treated Flint River water was 8.6 times more corrosive than Detroit water in short-term testing, releasing more iron, which is a key Legionella nutrient, while also directly causing disinfectant to decay more rapidly. The Flint River water source was also 0.8-6.7 °C warmer in summer months than Detroit water and exceeded the minimum Legionella growth temperature of 20 °C more frequently (average number of days per year for Detroit was 63 versus that for the Flint River, which was 157). The corrosive water also led to 1.3-2.2 times more water main breaks in 2014-2015 compared to 2010-2013; such disruptions have been associated with outbreaks in other locales. Importantly, Legionella spp. and Legionella pneumophila decreased after switching back to Detroit water, in terms of both gene markers and culturability, when August and October 2015 were compared to November 2016.

Interactive Effects of Corrosion, Copper, and Chloramines on Legionella and Mycobacteria in Hot Water Plumbing.

Complexities associated with drinking water plumbing systems can result in undesirable interactions among plumbing components that undermine engineering controls for opportunistic pathogens (OPs). In this study, we examine the effects of plumbing system materials and two commonly applied disinfectants, copper and chloramines, on water chemistry and the growth of Legionella and mycobacteria across a transect of bench- and pilot-scale hot water experiments carried out with the same municipal water supply. We discovered that copper released from corrosion of plumbing materials can initiate evolution of >1100 times more hydrogen (H2) from water heater sacrificial anode rods than does presence of copper dosed as soluble cupric ions. H2 is a favorable electron donor for autotrophs and causes fixation of organic carbon that could serve as a nutrient for OPs. Dosed cupric ions acted as a disinfectant in stratified stagnant pipes, inhibiting culturable Legionella and biofilm formation, but promoted Legionella growth in pipes subject to convective mixing. This difference was presumably due to continuous delivery of nutrients to biofilm on the pipes under convective mixing conditions. Chloramines eliminated culturable Legionella and prevented L. pneumophila from recolonizing biofilms, but M. avium gene numbers increased by 0.14-0.76 logs in the bulk water and were unaffected in the biofilm. This study provides practical confirmation of past discrepancies in the literature regarding the variable effects of copper on Legionella growth, and confirms prior reports of trade-offs between Legionella and mycobacteria if chloramines are applied as secondary disinfectant residual.