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Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.
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DNA Bacteriano , Água Potável , Legionella , RNA Ribossômico 16S , Microbiologia da Água , Legionella/isolamento & purificação , Legionella/genética , Legionella/classificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genética , Água Potável/microbiologia , Reação em Cadeia da Polimerase/métodosRESUMO
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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BACKGROUND: Respiratory tract infections are major contributors to the global disease burden. Quantitative microbial risk assessment (QMRA) holds potential as a rapidly deployable framework to understand respiratory pathogen transmission and inform policy on infection control. OBJECTIVES: The goal of this paper was to evaluate, motivate, and inform further development of the use of QMRA as a rapid tool to understand the transmission of respiratory pathogens and improve the evidence base for infection control policies. METHODS: We conducted a literature review to identify peer-reviewed studies of complete QMRA frameworks on aerosol inhalation or contact transmission of respiratory pathogens. From each of the identified studies, we extracted and summarized information on the applied exposure model approaches, dose-response models, and parameter values, including risk characterization. Finally, we reviewed linkages between model outcomes and policy. RESULTS: We identified 93 studies conducted in 16 different countries with complete QMRA frameworks for diverse respiratory pathogens, including SARS-CoV-2, Legionella spp., Staphylococcus aureus, influenza, and Bacillus anthracis. Six distinct exposure models were identified across diverse and complex transmission pathways. In 57 studies, exposure model frameworks were informed by their ability to model the efficacy of potential interventions. Among interventions, masking, ventilation, social distancing, and other environmental source controls were commonly assessed. Pathogen concentration, aerosol concentration, and partitioning coefficient were influential exposure parameters as identified by sensitivity analysis. Most (84%, n=78) studies presented policy-relevant content including a) determining disease burden to call for policy intervention, b) determining risk-based threshold values for regulations, c) informing intervention and control strategies, and d) making recommendations and suggestions for QMRA application in policy. CONCLUSIONS: We identified needs to further the development of QMRA frameworks for respiratory pathogens that prioritize appropriate aerosol exposure modeling approaches, consider trade-offs between model validity and complexity, and incorporate research that strengthens confidence in QMRA results. https://doi.org/10.1289/EHP12695.
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Infecções Respiratórias , Medição de Risco/métodos , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Humanos , SARS-CoV-2 , COVID-19/transmissão , COVID-19/prevenção & controle , Staphylococcus aureus , Controle de Infecções/métodos , Legionella , AerossóisRESUMO
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.
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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 â¼1-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.
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Anti-Infecciosos , Legionella pneumophila , Legionella , Cobre , Abastecimento de Água , Alumínio , Magnésio , Água , Eletrodos , Microbiologia da ÁguaRESUMO
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.
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Legionella pneumophila , Legionella , Probióticos , Biofilmes , EcossistemaRESUMO
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.
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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.
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Água Potável , Legionella , Mycobacterium , Água Potável/análise , Escherichia coli/genética , Inundações , Humanos , Legionella/genética , Mycobacterium/genética , RNA Ribossômico 16S/análise , Inquéritos e Questionários , Microbiologia da Água , Abastecimento de ÁguaRESUMO
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 500â¯000 well users (â¼130â¯000 to 260â¯000 wells) may have been affected, but only around 15â¯000 well users (â¼3800 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.
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Tempestades Ciclônicas , Água Potável , Escherichia coli , Texas , Poços de ÁguaRESUMO
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.
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Tempestades Ciclônicas , Água Potável , Humanos , Porto Rico , População Rural , Qualidade da ÁguaRESUMO
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.
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Anti-Infecciosos , Água Potável , Legionella pneumophila , Legionella , Cobre , Concentração de Íons de Hidrogênio , Fosfatos , Microbiologia da Água , Abastecimento de ÁguaRESUMO
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.
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Planejamento em Desastres , Desastres , Estudos Transversais , Escherichia coli , Inundações , HumanosRESUMO
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.
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Antibacterianos , Tempestades Ciclônicas , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Hispânico ou Latino , Humanos , Águas ResiduáriasRESUMO
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.
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Água Potável/efeitos adversos , Água Potável/microbiologia , Engenharia Sanitária/instrumentação , Microbiologia da Água , Biofilmes/crescimento & desenvolvimento , Fenômenos Químicos , Cobre/química , Desenho de Equipamento , Humanos , Ferro/química , Microbiota/genética , Modelos Biológicos , Infecções Oportunistas/etiologia , Infecções Oportunistas/microbiologia , Cloreto de Polivinila/química , RNA Ribossômico 16S/genética , Temperatura , Abastecimento de ÁguaRESUMO
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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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.
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Desinfecção , Animais , Cloro , Criptosporidiose , Cryptosporidium , Desinfetantes , Estados Unidos , Purificação da ÁguaRESUMO
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 â¼42 °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.
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Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Água , Microbiologia da Água , Abastecimento de ÁguaRESUMO
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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Água Potável/microbiologia , Genoma Bacteriano , Legionella pneumophila/genética , Doença dos Legionários/epidemiologia , Doença dos Legionários/microbiologia , Microbiologia da Água , Sequenciamento Completo do Genoma , Humanos , Legionella pneumophila/classificação , Legionella pneumophila/isolamento & purificação , Michigan/epidemiologia , Filogenia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
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.
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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 â 60 â 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.