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Copper Pipe, Lack of Corrosion Control, and Very Low pH May Have Influenced the Trajectory of the Flint Legionnaires’ Disease Outbreak
... We recently examined L. pneumophila growth in simulated glass water heaters with either cross-linked polyethylene (PEX) or copper pipe, Detroit tap water (sourced from Lake Huron) or treated Flint River water, and an initial influent pH 7.3 with continuous mixing representing hot water recirculation often present in large buildings [12]. After one year, very high levels of L. pneumophila (2.6-3.0 log CFU/mL) were observed in all treated Flint River water conditions with PEX pipe and with copper pipe when there was even low levels of phosphate corrosion control present (2.9 log 10 CFU/mL). ...
... After one year, very high levels of L. pneumophila (2.6-3.0 log CFU/mL) were observed in all treated Flint River water conditions with PEX pipe and with copper pipe when there was even low levels of phosphate corrosion control present (2.9 log 10 CFU/mL). By contrast, all treated Flint River water conditions with copper pipe and no phosphate corrosion control had 1-2 log lower levels of L. pneumophila, likely due to biotoxicity of copper, as directly evidence by an inverse correlation (R 2 = 0.85-0.95) between L. pneumophila and measured soluble copper or Cu +2 [12]. ...
... The presence of free chlorine has also been associated with lower levels of Legionella in general [21][22][23] and with LD incidence, in particular, during the Flint Water Crisis [1,3,5]. Thus, it is of interest to evaluate the extent to which recent phenomena observed to be at play in Flint's premise plumbing [12] hold true under a broader range of relevant conditions. Specifically, conditions with a higher influent pH (7.8-8.5), ...
Flint, MI experienced two outbreaks of Legionnaires’ Disease (LD) during the summers of 2014 and 2015, coinciding with use of Flint River as a drinking water source without corrosion control. Using simulated distribution systems (SDSs) followed by stagnant simulated premise (i.e., building) plumbing reactors (SPPRs) containing cross-linked polyethylene (PEX) or copper pipe, we reproduced trends in water chemistry and Legionella proliferation observed in the field when Flint River versus Detroit water were used before, during, and after the outbreak. Specifically, due to high chlorine demand in the SDSs, SPPRs with treated Flint River water were chlorine deficient and had elevated L. pneumophila numbers in the PEX condition. SPPRs with Detroit water, which had lower chlorine demand and higher residual chlorine, lost all culturable L. pneumophila within two months. L. pneumophila also diminished more rapidly with time in Flint River SPPRs with copper pipe, presumably due to the bacteriostatic properties of elevated copper concentrations caused by lack of corrosion control and stagnation. This study confirms hypothesized mechanisms by which the switch in water chemistry, pipe materials, and different flow patterns in Flint premise plumbing may have contributed to observed LD outbreak patterns.
In spring 2020, numerous buildings were closed or operated at reduced occupancies to slow the spread of COVID-19. An unintended consequence of these social distancing measures was a reduction in...
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.
Premise plumbing conditions can contribute to low chlorine or chloramine disinfectant residuals and reactions that encourage opportunistic pathogen growth and create risk of Legionnaires’ Disease outbreaks. This bench-scale study investigated the growth of Legionella spp. and Acanthamoeba in direct contact with premise plumbing materials—glass-only control, cross-linked polyethylene (PEX) pipe, magnesium anode rods, iron pipe, iron oxide, pH 10, or a combination of factors. Simulated glass water heaters (SGWHs) were colonized by Legionella pneumophila and exposed to a sequence of 0, 0.1, 0.25, and 0.5 mg/L chlorine or chloramine, at two levels of total organic carbon (TOC), over 8 weeks. Legionella pneumophila thrived in the presence of the magnesium anode by itself and or combination with other factors. In most cases, 0.5 mg/L Cl2 caused a significant rapid reduction of L. pneumophila, Legionella spp., or total bacteria (16S rRNA) gene copy numbers, but at higher TOC (>1.0 mg C/L), a chlorine residual of 0.5 mg/L Cl2 was not effective. Notably, Acanthamoeba was not significantly reduced by the 0.5 mg/L chlorine dose.
Flint, MI experienced two outbreaks of Legionnaires’ Disease (LD) during the summers of 2014 and 2015, coinciding with use of Flint River as a drinking water source without corrosion control. Using simulated distribution systems (SDSs) followed by stagnant simulated premise (i.e., building) plumbing reactors (SPPRs) containing cross-linked polyethylene (PEX) or copper pipe, we reproduced trends in water chemistry and Legionella proliferation observed in the field when Flint River versus Detroit water were used before, during, and after the outbreak. Specifically, due to high chlorine demand in the SDSs, SPPRs with treated Flint River water were chlorine deficient and had elevated L. pneumophila numbers in the PEX condition. SPPRs with Detroit water, which had lower chlorine demand and higher residual chlorine, lost all culturable L. pneumophila within two months. L. pneumophila also diminished more rapidly with time in Flint River SPPRs with copper pipe, presumably due to the bacteriostatic properties of elevated copper concentrations caused by lack of corrosion control and stagnation. This study confirms hypothesized mechanisms by which the switch in water chemistry, pipe materials, and different flow patterns in Flint premise plumbing may have contributed to observed LD outbreak patterns.
This review discusses Legionella, among the most prolific and publicly well-known waterborne pathogens, and advances in potential treatment technologies. The number of cases associated with Legionella continues to rise, as does its public awareness. Currently, cases associated with premise plumbing account for the largest number of legionellosis cases in the United States. So, while it is important to understand Legionella as such, it is also important to investigate how to treat drinking water in premise plumbing for Legionella and other waterborne pathogens. While there are currently several methods recognized as potential means of inactivating waterborne pathogens, several shortcomings continue to plague its implementation. These methods are generally of two types. Firstly, there are chemical treatments such as chlorine, chlorine dioxide, monochloramine, ozone, and copper-silver ionization. Secondly, there are physical treatments such as thermal inactivation and media filtration. Their shortcomings range from being labor-intensive and costly to having negative health effects if not properly operated. Recently developed technologies including ultraviolet (UV) irradiation using light emitting diodes (LEDs) and innovative carbon nanotube (CNT) filters can better control waterborne pathogens by allowing for the simultaneous use of different treatment measures in plumbing systems.
Background:
A community-wide outbreak of Legionnaires' disease (LD) occurred in Genesee County, Michigan, in 2014 and 2015. Previous reports about the outbreak are conflicting and have associated the outbreak with a change of water source in the city of Flint and, alternatively, to a Flint hospital.
Objective:
The objective of this investigation was to independently identify relevant sources of Legionella pneumophila that likely resulted in the outbreak.
Methods:
An independent, retrospective investigation of the outbreak was conducted, making use of public health, health care, and environmental data and whole-genome multilocus sequence typing (wgMLST) of clinical and environmental isolates.
Results:
Strong evidence was found for a hospital-associated outbreak in both 2014 and 2015: a) 49% of cases had prior exposure to Flint hospital A, significantly higher than expected from Medicare admissions; b) hospital plumbing contained high levels of L. pneumophila; c) Legionella control measures in hospital plumbing aligned with subsidence of hospital A-associated cases; and d) wgMLST showed Legionella isolates from cases exposed to hospital A and from hospital plumbing to be highly similar. Multivariate analysis showed an increased risk of LD in 2014 for people residing in a home that received Flint water or was located in proximity to several Flint cooling towers.
Discussion:
This is the first LD outbreak in the United States with evidence for three sources (in 2014): a) exposure to hospital A, b) receiving Flint water at home, and c) residential proximity to cooling towers; however, for 2015, evidence points to hospital A only. Each source could be associated with only a proportion of cases. A focus on a single source may have delayed recognition and remediation of other significant sources of L. pneumophila. https://doi.org/10.1289/EHP5663.
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.
Biofilm heterogeneity has been characterized on various scales for both natural and engineered ecosystems. This heterogeneity has been attributed to spatial differences in environmental factors. Understanding their impact on localized biofilm heterogeneity in building plumbing systems is important for both management and representative sampling strategies. We assessed heterogeneity within the confined engineered ecosystem of a shower hose by high-resolution sampling (200 individual biofilm sections per hose) on varying scales (μm to m). We postulated that a biofilm grown on a single material under uniform conditions should be homogeneous in its structure, bacterial numbers, and community composition. A biofilm grown for 12 months under controlled laboratory conditions, showed homogeneity on large-scale. However, some small-scale heterogeneity was clearly observed. For example, biofilm thickness of cm-sections varied up to 4-fold, total cell concentrations (TCC) 3-fold, and relative abundance of dominant taxa up to 5-fold. A biofilm grown under real (i.e., uncontrolled) use conditions developed considerably more heterogeneity in all variables which was attributed to more discontinuity in environmental conditions. Interestingly, biofilm communities from both hoses showed comparably low diversity, with <400 taxa each, and only three taxa accounting for 57%, respectively, 73% of the community. This low diversity was attributed to a strong selective pressure, originating in migrating carbon from the flexible hoses as major carbon source. High-resolution sampling strategy enabled detailed analysis of spatial heterogeneity within an individual drinking water biofilm. This study gives insight into biofilm structure and community composition on cm-to m-scale and is useful for decision-making on sampling strategies in biofilm research and monitoring.
Microbial regrowth within building water systems are promoted by water stagnation, low disinfectant residual, high surface-to-volume ratio, amenable growth temperatures, and colonization of drinking water biofilms. Moreover, biofilms provide protection from environmental stresses, access to higher levels of nutrients, and opportunities for symbiotic interactions with other microbes. Disinfectant efficacy information is historically based on inactivation of pathogens in their planktonic, free-floating forms. However, due to the ecological importance of drinking water biofilms for pathogen survival, this study evaluated the efficacy of two common disinfectants, free chlorine and monochloramine, on Legionella pneumophila colonizing mature, drinking water biofilms established on copper and PVC surfaces. Results showed that inactivation was dependent on the disinfectant type and biofilm substratum. Overall, this, and other related research, will provide a better understanding of Legionella ecological stability and survival and aid policy makers in the management of exposure risks to water-based pathogens within building water systems.
In April 2014, the drinking water source in Flint, Michigan was switched from Lake Huron water with phosphate inhibitors to Flint River water without corrosion inhibitors. The absence of corrosion control and use of a more corrosive source increased lead leaching from plumbing. Our city-wide citizen science water lead results contradicted official claims that there was no problem– our 90th percentile was 26.8 µg/L, which was almost double the Lead and Copper Rule action level of 15 µg/L. Back calculations of a LCR sampling pool with 50% lead pipes indicated an estimated 90th percentile lead value of 31.7 µg/L (±4.3 µg/L). Four subsequent sampling efforts were conducted to track reductions in water lead after the switch back to Lake Huron water and enhanced corrosion control. The incidence of water lead varied by service line material. Between August 2015 and November 2016, median water lead reduced from 3.0 to <1 µg/L for homes with copper service lines, 7.2 to 1.9 µg/L with galvanized service lines, and 9.9 to 2.3 µg/L with lead service lines. As of summer 2017, our 90th percentile of 7.9 µg/L no longer differed from official results, which indicated Flint’s water lead levels were below the action level.
Coinciding with major changes to its municipal water system, Flint, MI, endured Legionnaires’ disease outbreaks in 2014 and 2015. By sampling premise plumbing in Flint in the fall of 2016, we found that 12% of homes harbored legionellae, a frequency similar to that in residences in neighboring areas. To evaluate the genetic diversity of Legionella pneumophila in Southeast Michigan, we determined the sequence type (ST) and serogroup (SG) of the 18 residential isolates from Flint and Detroit, MI, and the 33 clinical isolates submitted by hospitals in three area counties in 2013 to 2016. Common to one environmental and four clinical samples were strains of L. pneumophila SG1 and ST1, the most prevalent ST worldwide. Among the Flint premise plumbing isolates, 14 of 16 strains were of ST367 and ST461, two closely related SG6 strain types isolated previously from patients and corresponding environmental samples. Each of the representative SG1 clinical strains and SG6 environmental isolates from Southeast Michigan infected and survived within macrophage cultures at least as well as a virulent laboratory strain, as judged by microscopy and by enumerating CFU. Likewise, 72 h after infection, the yield of viable-cell counts increased >100-fold for each of the representative SG1 clinical isolates, Flint premise plumbing SG6 ST367 and -461 isolates, and two Detroit residential isolates. We verified by immunostaining that SG1-specific antibody does not cross-react with the SG6 L. pneumophila environmental strains. Because the widely used urinary antigen diagnostic test does not readily detect non-SG1 L. pneumophila, Legionnaires’ disease caused by SG6 L. pneumophila is likely underreported worldwide.
The 2014-2015 Legionnaires' disease (LD) outbreak in Genesee County, MI, and the outbreak resolution in 2016 coincided with changes in the source of drinking water to Flint's municipal water system. Following the switch in water supply from Detroit to Flint River water, the odds of a Flint resident presenting with LD increased 6.3-fold (95% CI: 2.5, 14.0). This risk subsided following boil water advisories, likely due to residents avoiding water, and returned to historically normal levels with the switch back in water supply. During the crisis, as the concentration of free chlorine in water delivered to Flint residents decreased, their risk of acquiring LD increased. When the average weekly chlorine level in a census tract was <0.5 mg/L or <0.2 mg/L, the odds of an LD case presenting from a Flint neighborhood increased by a factor of 2.9 (95% CI: 1.4, 6.3) or 3.9 (95% CI: 1.8, 8.7), respectively. During the switch, the risk of a Flint neighborhood having a case of LD increased by 80% per 1 mg/L decrease in free chlorine, as calculated from the extensive variation in chlorine observed. In communities adjacent to Flint, the probability of LD occurring increased with the flow of commuters into Flint. Together, the results support the hypothesis that a system-wide proliferation of legionellae was responsible for the LD outbreak in Genesee County, MI.
Background:
Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32-53 °C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 μg/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing.
Results:
Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41 °C in the presence of PEX (1.58 × 105 gene copies/mL). At 53 °C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49 °C and 20% at 53 °C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log10 lower) at temperatures ≤ 41 °C, but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures ≤ 45 °C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 °C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing.
Conclusions:
Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature increase. While copper pipe may also help shape the microbiota composition and limit L. pneumophila proliferation, its benefits might be constrained at higher temperatures. Influent assimilable organic carbon affected total bacterial numbers, but had minimal influence on opportunistic pathogen gene numbers or microbiota composition. These findings provide guidance among multiple control measures for the growth of opportunistic pathogens in hot water plumbing and insight into the mediating role of microbial ecological factors.
Driven by the development of water purification technologies and water quality regulations, the use of better source water and/or upgraded water treatment processes to improve drinking water quality have become common practices worldwide. However, even though these elements lead to improved water quality, the water quality may be impacted during its distribution through piped networks due to the processes such as pipe material release, biofilm formation and detachment, accumulation and resuspension of loose deposits. Irregular changes in supply-water quality may cause physiochemical and microbiological de-stabilization of pipe material, biofilms and loose deposits in the distribution system that have been established over decades and may harbor components that cause health or esthetical issues (brown water). Even though it is clearly relevant to customers' health (e.g., recent Flint water crisis), until now, switching of supply-water quality is done without any systematic evaluation. This article reviews the contaminants that develop in the water distribution system and their characteristics, as well as the possible transition effects during the switching of treated water quality by destabilization and the release of pipe material and contaminants into the water and the subsequent risks. At the end of this article, a framework is proposed for the evaluation of potential transition effects.
Flint, Michigan switched to the Flint River as a temporary drinking water source without implementing corrosion control in April 2014. Ten months later, water samples collected from a Flint residence revealed progressively rising water lead levels (104, 397, and 707 μg/L) coinciding with increasing water discoloration. An intensive follow-up monitoring event at this home investigated patterns of lead release by flow rate-all water samples contained lead above 15 μg/L and several exceeded hazardous waste levels (>5000 μg/L). Forensic evaluation of exhumed service line pipes compared to water contamination "fingerprint" analysis of trace elements, revealed that the immediate cause of the high water lead levels was the destabilization of lead-bearing corrosion rust layers that accumulated over decades on a galvanized iron pipe downstream of a lead pipe. After analysis of blood lead data revealed spiking lead in blood of Flint children in September 2015, a state of emergency was declared and public health interventions (distribution of filters and bottled water) likely averted an even worse exposure event due to rising water lead levels.
It is unclear how the water-based pathogen, Legionella pneumophila (Lp), and associated free-living amoeba (FLA) hosts change or are changed by the microbial composition of drinking water (DW) biofilm communities. Thus, this study characterized the bacterial community structure over a 7-month period within mature (> 600-day-old) copper DW biofilms in reactors simulating premise plumbing and assessed the impact of temperature and introduction of Lp and its FLA host, Vermamoeba vermiformis (Vv), co-cultures (LpVv). Sequence and quantitative PCR (qPCR) analyses indicated a correlation between LpVv introduction and increases in Legionella spp. levels at room temperature (RT), while at 37°C, Lp became the dominant Legionella spp. qPCR analysis suggested Vv presence may not be directly associated with Lp biofilm growth at RT and 37°C, but may contribute to or be associated with non-Lp legionellae persistence at RT. Two-way PERMANOVA and PCoA revealed that temperature was a major driver of microbiome diversity. Biofilm community composition also changed over the seven-month period and could be associated with significant shifts in dissolved oxygen, alkalinity and various metals in the influent DW. Hence, temperature, biofilm age, DW quality and transient intrusions/amplification of pathogens and FLA hosts may significantly impact biofilm microbiomes and modulate pathogen levels over extended periods.
Major waterborne (enteric) pathogens are relatively well understood and treatment controls are effective when well managed. However, water-based, saprozoic pathogens that grow within engineered water systems (primarily within biofilms/sediments) cannot be controlled by water treatment alone prior to entry into water distribution and other engineered water systems. Growth within biofilms or as in the case of Legionella pneumophila, primarily within free-living protozoa feeding on biofilms, results from competitive advantage. Meaning, to understand how to manage water-based pathogen diseases (a sub-set of saprozoses) we need to understand the microbial ecology of biofilms; with key factors including biofilm bacterial diversity that influence amoebae hosts and members antagonistic to water-based pathogens, along with impacts from biofilm substratum, water temperature, flow conditions and disinfectant residual—all control variables. Major saprozoic pathogens covering viruses, bacteria, fungi and free-living protozoa are listed, yet today most of the recognized health burden from drinking waters is driven by legionellae, non-tuberculous mycobacteria (NTM) and, to a lesser extent, Pseudomonas aeruginosa. In developing best management practices for engineered water systems based on hazard analysis critical control point (HACCP) or water safety plan (WSP) approaches, multi-factor control strategies, based on quantitative microbial risk assessments need to be developed, to reduce disease from largely opportunistic, water-based pathogens.
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.
We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.
Controlling organic carbon levels in municipal water has been hypothesized to limit downstream growth of bacteria and opportunistic pathogens in premise plumbing (OPPPs). Here, the relationships between influent organic carbon (0–15,000 µg ozonated fulvic acid /L) and the number of total bacteria [16S rRNA genes and heterotrophic plate counts (HPCs)] and a wide range of OPPPs (gene copy numbers of Acanthamoeba polyphaga, Vermamoeba vermiformis, Legionella pneumophila, and Mycobacterium avium) were examined in the bulk water of 120-mL simulated glass water heaters (SGWHs). The SGWHs were operated at 32–37 °C, which is representative of conditions encountered at the bottom of electric water heaters, with water changes of 80% three times per week to simulate low use. This design presented advantages of controlled and replicated (triplicate) conditions and avoided other potential limitations to OPPP growth in order to isolate the variable of organic carbon. Over seventeen months, strong correlations were observed between total organic carbon (TOC) and both 16S rRNA gene copy numbers and HPC counts (avg. R² > 0.89). Although M. avium gene copies were occasionally correlated with TOC (avg. R² = 0.82 to 0.97, for 2 out of 4 time points) and over a limited TOC range (0–1000 µg/L), no other correlations were identified between other OPPPs and added TOC. These results suggest that reducing organic carbon in distributed water is not adequate as a sole strategy for controlling OPPPs, although it may have promise in conjunction with other approaches.
Legionella pneumophila is a waterborne pathogen that can cause Legionnaires' disease, a fatal pneumonia, or Pontiac fever, a mild form of disease. Copper is an antimicrobial material used for thousands of years. Its incorporation in several surface materials to control the transmission of pathogens has been gaining importance in the past decade. In this work, the ability of copper to control the survival of L. pneumophila in biofilms was studied. For that, the incorporation of L. pneumophila in polymicrobial drinking water biofilms formed on copper, PVC and PEX, and L. pneumophila mono-species biofilms formed on copper and uPVC were studied by comparing cultivable and total numbers (quantified by peptide nucleic acid (PNA) hybridisation). L. pneumophila was never recovered by culture from heterotrophic biofilms; however, PNA-positive numbers were slightly higher in biofilms formed on copper (5.9 × 10(5) cells cm(-2)) than on PVC (2.8 × 10(5) cells cm(-2)) and PEX (1.7 × 10(5) cells cm(-2)). L. pneumophila mono-species biofilms grown on copper gave 6.9 × 10(5) cells cm(-2) for PNA-positive cells and 4.8 × 10(5) CFU cm(-2) for cultivable numbers, showing that copper is not directly effective in killing L. pneumophila. Therefore previous published studies showing inactivation of L. pneumophila by copper surfaces in potable water polymicrobial species biofilms must be carefully interpreted.
Abstract Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.
The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify
patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent
methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members.
Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water)
and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain
subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S
rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors
that may aid in controlling opportunistic pathogen proliferation in real-world water systems.
Two unchlorinated drinking water supplies were investigated to assess the potential of water treatment and distribution systems
to support the growth of Legionella spp. The treatment plant for supply A distributed treated groundwater with a low concentration (<0.5 ppm of C) of natural
organic matter (NOM), and the treatment plant for supply B distributed treated groundwater with a high NOM concentration (8
ppm of C). In both supplies, the water temperature ranged from about 10°C after treatment to 18°C during distribution. The
concentrations of Legionella spp. in distributed water, analyzed with quantitative PCR (Q-PCR), averaged 2.9 (± 1.9) × 102 cells liter−1 in supply A and 2.5 (± 1.6) × 103 cells liter−1 in supply B. No Legionella was observed with the culture method. A total of 346 clones (96 operational taxonomical units [OTUs] with ≥97% sequence similarity)
were retrieved from water and biofilms of supply A and 251 (43 OTUs) from supply B. The estimation of the average value of
total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77%
of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions
of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity
were observed in the supply with the low NOM concentration.
The role of bacteria in the occasional emergence of red water, which has been documented worldwide, has yet to be determined.
To better understand the mechanisms that drive occurrences of red water, the bacterial community composition and the relative
abundance of several functional bacterial groups in a water distribution system of Beijing during a large-scale red water
event were determined using several molecular methods. Individual clone libraries of the 16S rRNA gene were constructed for
three red water samples and one sample of normal water. Beta-, Alpha-, and Gammaproteobacteria comprised the major bacterial communities in both red water and normal water samples, in agreement with previous reports.
A high percentage of red water clones (25.2 to 57.1%) were affiliated with or closely related to a diverse array of iron-oxidizing
bacteria, including the neutrophilic microaerobic genera Gallionella and Sideroxydans, the acidophilic species Acidothiobacillus ferrooxidans, and the anaerobic denitrifying Thermomonas bacteria. The genus Gallionella comprised 18.7 to 28.6% of all clones in the three red water libraries. Quantitative real-time PCR analysis showed that the
16S rRNA gene copy concentration of Gallionella spp. was between (4.1 ± 0.9) × 107 (mean ± standard deviation) and (1.6 ± 0.3) × 108 per liter in red water, accounting for 13.1% ± 2.9% to 17.2% ± 3.6% of the total Bacteria spp. in these samples. By comparison, the percentages of Gallionella spp. in the normal water samples were 0.1% or lower (below the limit of detection), suggesting an important role of Gallionella spp. in the formation of red water.
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.
The maintenance of a chlorine or chloramine residual to suppress waterborne pathogens in drinking water distribution systems is common practice in the United States but less common in Europe. In this study, we investigated the occurrence of Bacteria and Legionella spp. in water-main biofilms and tap water from a chloraminated distribution system in the United States and a system in Norway with no residual using real-time quantitative polymerase chain reaction (qPCR). Despite generally higher temperatures and assimilable organic carbon levels in the chloraminated system, total Bacteria and Legionella spp. were significantly lower in water-main biofilms and tap water of that system (p < 0.05). Legionella spp. were not detected in the biofilms of the chloraminated system (0 of 35 samples) but were frequently detected in biofilms from the no-residual system (10 of 23 samples; maximum concentration = 7.8×10⁴ gene copies cm⁻²). This investigation suggests water-main biofilms may serve as a source of legionellae for tap water and premise plumbing systems, and residual chloramine may aid in reducing their abundance.
Shower hoses offer an excellent bacterial growth environment in close proximity to a critical end-user exposure route within building drinking water plumbing. However, the health risks associated with and processes underlying the development of biofilms in shower hoses are poorly studied. In a global survey, biofilms from 78 shower hoses from 11 countries were characterized in terms of cell concentration (4.1 × 104-5.8 × 108 cells/cm2), metal accumulation (including iron, lead, and copper), and microbiome composition (including presence of potential opportunistic pathogens). In countries using disinfectant, biofilms had on average lower cell concentrations and diversity. Metal accumulation (up to 5 μg-Fe/cm2, 75 ng-Pb/cm2, and 460 ng-Cu/cm2) seemed to be partially responsible for discoloration in biofilms, and likely originated from other pipes upstream in the building. While some genera that may contain potential opportunistic pathogens (Legionella, detected in 21/78 shower hoses) were positively correlated with biofilm cell concentration, others (Mycobacterium, Pseudomonas) had surprisingly non-existent or negative correlations with biofilm cell concentrations. In a controlled study, 15 identical shower hoses were installed for the same time period in the same country, and both stagnant and flowing water samples were collected. Ecological theory of dispersal and selection helped to explain microbiome composition and diversity of different sample types. Shower hose age was related to metal accumulation but not biofilm cell concentration, while frequency of use appeared to influence biofilm cell concentration. This study shows that shower hose biofilms are clearly a critical element of building drinking water plumbing, and a potential target for building drinking water plumbing monitoring.
We hypothesize that the increase in reported Legionnaires' disease from June 2014-November 2015 in Genesee County, MI (where Flint is located) was directly linked to the switch to corrosive Flint River water from non-corrosive Detroit water in April 2014-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 2010-2016 to evaluate characteristics of the Flint River water that were potentially conducive to Legionella growth. First, 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, Detroit = 63 versus Flint River = 157). The corrosive water also led to 1.3-2.2 times more water main breaks in 2014-2015 compared to 2010-2013, where such disruptions have been associated with outbreaks in other locales. Importantly, Legionella spp. and L. pneumophila decreased after switching back to Detroit water, both in terms of gene markers and culturablity, when comparing August/October 2015 to November 2016.
Complexities associated with drinking water plumbing systems can result in undesirable interactions among plumbing components, undermining 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 >1,100 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 re-colonizing biofilms, but M. avium gene numbers increased by 0.14-0.75 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.
Two clusters of Legionnaires' disease occurred in Flint, MI, subsequent to switching to a corrosive potable water source from April 2014 to October 2015. We hypothesized that the interrupted corrosion control and associated release of iron, nutrients, and depleted chlorine residual in the distribution system would lead to high levels of Legionella. A tap water survey conducted throughout Flint in August and October 2015 confirmed Legionella pneumophila in two hospitals (mean of 1890 ± 2220 gene copy numbers/mL, 48% positivity), but not small single-story buildings. The hospitals frequently had optimal Legionella growth temperatures and were located in high-water age zones of the distribution system (3 to >6 days). Relatively high concentrations of iron were present (mean of 51.0 ± 37.2 ppb), and Cl2 residual was sporadic (mean of 0.700 ± 0.775 mg/L) throughout the Flint distribution system. This study addresses knowledge gaps linking legionellosis outbreaks to changes in municipal water quality and distribution system operation.
Background:
The number of reported cases of Legionnaires' disease, a severe pneumonia caused by the bacterium Legionella, is increasing in the United States. During 2000-2014, the rate of reported legionellosis cases increased from 0.42 to 1.62 per 100,000 persons; 4% of reported cases were outbreak-associated. Legionella is transmitted through aerosolization of contaminated water. A new industry standard for prevention of Legionella growth and transmission in water systems in buildings was published in 2015. CDC investigated outbreaks of Legionnaires' disease to identify gaps in building water system maintenance and guide prevention efforts.
Methods:
Information from summaries of CDC Legionnaires' disease outbreak investigations during 2000-2014 was systematically abstracted, and water system maintenance deficiencies from land-based investigations were categorized as process failures, human errors, equipment failures, or unmanaged external changes.
Results:
During 2000-2014, CDC participated in 38 field investigations of Legionnaires' disease. Among 27 land-based outbreaks, the median number of cases was 10 (range = 3-82) and median outbreak case fatality rate was 7% (range = 0%-80%). Sufficient information to evaluate maintenance deficiencies was available for 23 (85%) investigations. Of these, all had at least one deficiency; 11 (48%) had deficiencies in ≥2 categories. Fifteen cases (65%) were linked to process failures, 12 (52%) to human errors, eight (35%) to equipment failures, and eight (35%) to unmanaged external changes.
Conclusions and implications for public health practice:
Multiple common preventable maintenance deficiencies were identified in association with disease outbreaks, highlighting the importance of comprehensive water management programs for water systems in buildings. Properly implemented programs, as described in the new industry standard, could reduce Legionella growth and transmission, preventing Legionnaires' disease outbreaks and reducing disease.
Objectives:
We analyzed differences in pediatric elevated blood lead level incidence before and after Flint, Michigan, introduced a more corrosive water source into an aging water system without adequate corrosion control.
Methods:
We reviewed blood lead levels for children younger than 5 years before (2013) and after (2015) water source change in Greater Flint, Michigan. We assessed the percentage of elevated blood lead levels in both time periods, and identified geographical locations through spatial analysis.
Results:
Incidence of elevated blood lead levels increased from 2.4% to 4.9% (P < .05) after water source change, and neighborhoods with the highest water lead levels experienced a 6.6% increase. No significant change was seen outside the city. Geospatial analysis identified disadvantaged neighborhoods as having the greatest elevated blood lead level increases and informed response prioritization during the now-declared public health emergency.
Conclusions:
The percentage of children with elevated blood lead levels increased after water source change, particularly in socioeconomically disadvantaged neighborhoods. Water is a growing source of childhood lead exposure because of aging infrastructure. (Am J Public Health. Published online ahead of print December 21, 2015: e1-e8. doi:10.2105/AJPH.2015.303003).
Like other utilities in the United States, the water providers participating in this study had experienced lead problems as a result of seemingly innocuous changes to their drinking water treatment. Stock cures such as increasing pH or dosing orthophosphate corrosion inhibitor were not able to reduce lead in water in some cases. Bench-scale tests conducted in this research unambiguously demonstrated that raising the chloride-to-sulfate mass ratio (CSMR) increased lead leaching from lead plumbing materials, whereas lowering the CSMR decreased lead corrosion. The CSMR can be altered by any of the following: a change from a sulfate-based coagulant to a chloride-based coagulant, a switch from a freshwater source (surface water or groundwater) to seawater (i.e., desalinated water), implementation of anion exchange, and sodium chloride brine leaks from onsite hypochlorite generators. Before making treatment changes, utilities can use the simple bench-scale test protocol described in this article to screen for potential effects on lead release from solder.
Culture remains the gold-standard for the enumeration of environmental Legionella. However, it has several drawbacks including long incubation and poor sensitivity, causing delays in response times to outbreaks of Legionnaires' disease. This study aimed to validate real-time PCR assays to quantify Legionella species (ssrA gene), Legionella pneumophila (mip gene) and L. pneumophila serogroup-1 (wzm gene) to support culture-based detection in a frontline public health laboratory.
Each qPCR assay had 100% specificity, excellent sensitivity (5 GU/reaction) and reproducibility. Comparison of the assays to culture-based enumeration of Legionella from 200 environmental samples showed that they had a negative predictive value of 100%. Thirty eight samples were positive for Legionella species by culture and qPCR. One hundred samples were negative by both methods whereas 62 samples were negative by culture but positive by qPCR. The average log10 increase between culture and qPCR for Legionella spp. and L. pneumophila was 0.72 (p = 0.0002) and 0.51 (p = 0.006) respectively.
The qPCR assays can be conducted on the same 1L water sample as culture thus can be used as a supplementary technique to screen out negative samples and allow more rapid indication of positive samples.
The assay could prove informative in public health investigations to identify or rule out sources of Legionella as well as to specifically identify L. pneumophila serogroup 1 in a timely manner not possible with culture. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Cast-iron pipes have been used to transport potable water for more than 500 years (Gedge, 1992), and iron pipe corrosion has been a problem for just as long. AWWA estimates that it will cost US water utilities 77.2 billion for service and replacement of transmission and distribution system lines over the next 20 years (Davies et al, 1997). The majority of distribution system pipes are composed of iron material: cast iron (38%), ductile iron (22%), and steel (5%) (AWWA, 1996). Moreover, a 1997 survey of the 100 largest AWWA Research Foundation (AWWARF) member utilities found that "the most common distribution system problem is corrosion of cast-iron pipe" (Bray, 1997). Although the body of literature on iron corrosion is quite large, most studies were conducted at experimental conditions inconsistent with the potable water distribution system environment (e.g., brine solutions, oil and gas pipelines, highly acidic conditions, or very high temperatures). The goal of this literature review is to summarize the results of several hundred peer-reviewed articles relevant to the drinking water industry. Corrosion of iron pipes in a distribution system can cause three distinct but related problems. First, pipe mass is lost through oxidation to soluble iron species or iron-bearing scale. Second, the scale can accumulate as large tubercles that increase head loss and decrease water capacity. Finally, the release of soluble or particulate iron corrosion by-products to the water decreases its aesthetic quality and often leads to consumer complaints of "red water" at thr tap. The water industry must be concerned with all three of these aspects of corrosion.
Illustrative distribution system operation and management practices shaped the occurrence and persistence of Legionella spp., nontuberculous mycobacteria (NTM), Pseudomonas aeruginosa and two amoebae host (Acanthamoeba spp., Vermamoeba vermiformis) gene markers in the effluent of standardized simulated household water heaters (SWHs). The interplay between disinfectant type (chlorine or chloramine), water age (2.3-5.7 d) and materials (polyvinyl chloride (PVC), cement or iron) in upstream simulated distribution systems (SDSs) profoundly influenced levels of pathogen gene markers in corresponding SWH bulk waters. For example, Legionella spp. were 3-4 log higher in SWHs receiving water from chloraminated vs. chlorinated SDSs, because of disinfectant decay from nitrification. By contrast, SWHs fed with chlorinated PVC SDS water not only harbored the lowest levels of all pathogen markers, but effluent from the chlorinated SWHs were even lower than influent levels in several instances (e.g., 2 log less Legionella spp. and NTM for PVC and 3-5 log less P. aeruginosa for cement). However, pathogen gene marker influent levels correlated positively to effluent levels in the SWHs (P<0.05). Likewise, microbial community structures were similar between SWHs and the corresponding SDS feed waters. This study highlights the importance and challenges of distribution system management/operation to help control opportunistic pathogens.
Biofilms in drinking water distribution systems (DWDS) could exacerbate the persistence and associated risks of pathogenic Legionella pneumophila, thus raising human health concerns. However, mechanisms controlling adhesion and subsequent detachment of L. pneumophila associated with biofilms remain unclear. We determined the connection between L. pneumophila adhesion and subsequent detachment with biofilm physical structure characterization using optical coherence tomography (OCT) imaging technique. Analysis of the OCT images of multi-species biofilms grown under low nutrient condition up to 29 weeks revealed the lack of biofilm deformation even when these biofilms were exposed to flow velocity of 0.7 m/s, typical flow for DWDS. L. pneumophila adhesion on these biofilm under low flow velocity (0.007 m/s) positively correlated with biofilm roughness due to enlarged biofilm surface area and local flow conditions created by roughness asperities. The pre-adhered L. pneumophila on selected rough and smooth biofilms were found to detach when these biofilms were subjected to higher flow velocity. At the flow velocity of 0.1 and 0.3 m/s, the ratio of detached cell from the smooth biofilm surface was from 1.3 to 1.4 times higher than that from the rough biofilm surface, presumably because of the low shear stress zones near roughness asperities. This study determined that physical structure and local hydrodynamics control adhesion and detachment from simulated drinking water biofilm, thus it is the first step toward reducing the risk of L. pneumophila exposure and subsequent infections.
In the United States, 3,522 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and recovered from a variety of natural freshwater environments. Human exposure to L. pneumophila Sg1 may occur from aerosolation and subsequent inhalation of household and facility water. In this study, two primer/probe sets (one able to detect L. pneumophila and the other L. pneumophila Sg1) were determined to be highly sensitive and selective for their respective targets. Over 274 water samples, collected in 2009 and 2010 from 68 public and private water taps across the United States, were analyzed using the two qPCR assays to evaluate the incidence of L. pneumophila Sg1. Nearly half of the taps showed the presence of L. pneumophila Sg1 in one sampling event and 16% of taps showed it in more than one sampling event. This study is the first national survey in documenting the occurrence and colonization of L. pneumophila Sg1 in cold water from point of use taps.
Legionella is ubiquitous in freshwater systems worldwide and can also be found in soil. Legionellosis may be caused by inhalation of aerosolized water or soil particles containing Legionella. Isolation of Legionella from the environment is an essential step in outbreak investigation and may also be performed within the context of a hazard analysis and control risk management plan. Culture remains the gold standard for detection of Legionella in environmental samples. Specific properties of environmental sites that could be a source of Legionella contamination, collection of samples from such sites, and procedures for culture of these samples for Legionella are described in this chapter.
The aim of this study was to compare biofilm accumulation and heterotrophic bacterial diversity on three pipe materials-cast iron, medium density polyethylene (MDPE), and unplasticised polyvinyl chloride (uPVC) - using a laboratory model system run over a short period (21 d) and a longer period (7 months). Newly Modified Robbins Devices (nMRD) were run in parallel, each containing 25 discs of one material with cold tap water flowing through the devices at 3 ml min−1 (Reynolds Number 9.05) for 21 d. The numbers of bacteria on each material increased exponentially between 0 and 11 d when the biofilm viable count remained constant. The mean doubling times of the heterotrophic population on the materials during the exponential phase was 13.2 h for cast iron and 15.6 h for MDPE and uPVC. The same experiment was repeated under different environmental conditions with a lower temperature, higher free chlorine and lower number of organisms ml−1 of incoming water. The exponential phase lengthened to 16 d but the steady state count remained the same. The mean viable count after 21 d and after 7 months was on average 97% higher on cast iron than on the other materials. Very few different colony types were isolated from each material with the largest number (nine) recovered from cast iron. The numbers of planktonic bacteria in the effluent water leaving each of the nMRDs directly correlated with the numbers in the biofilm phase on each of the materials. In addition the distribution and thickness of the biofilms on the MDPE and uPVC were observed using confocal scanning laser microscopy. In conclusion, MDPE and uPVC support the lowest numbers of bacteria in a steady state biofilm in the short term (21 d) and over a longer term (7 months). The diversity of heterotrophic bacteria was greatest on cast iron.
Copper/silver ionization is a new disinfection method that is being used to eradicate Legionella pneumophila from hospital hot water recirculating systems. The objective of this study was to determine the susceptibility of L. pneumophila serogroup 1 to copper and silver ions alone and in combination. L. pneumophila serogroup 1 (L. p. sg-1) was completely inactivated (6-log reduction) at copper concentration of 0.1 mg/l within 2.5 h, whereas more than 24 h was required to achieve a similar reduction at the highest silver ion concentration tested (0.08 mg/l). Checkerboard method and Gard additive model prediction demonstrated that copper and silver ions in combination could result in additive or synergistic effect depending on the concentration of copper and silver ions. Under the experimental conditions used in this study, synergism of copper/silver ions in eradicating L. p. sg-1 was observed at higher concentration combinations of copper/silver ions (e.g. 0.04/0.04 mg/l) while only an additive effect was observed at lower concentration combinations (e.g. 0.02/0.02 mg/l). This study suggested that both copper and silver ions are effective in inactivating L. pneumophila and the combined effect is greater than that seen with either ion alone.
This study describes the influence of different corrosion control programs on corrosion rates, and the short-term effects of various levels of corrosiveness on bacterial water quality in model drinking water distribution systems. Corrosion rates were strongly related to seasons and water temperature. This suggests that slightly higher corrosion inhibitor concentrations would be required during summer and possibly lower dosages during winter months. Over a one-year period, bacterial water quality data were similar in new pipes receiving waters with different corrosiveness and phosphate levels, suggesting that corrosion affects bacterial water quality over longer periods of time.
Copper corrosion by-product release to potable water is a complex function of pipe age, water quality, stagnation time, and type of phosphate inhibitor. Moderate (≈1 mg/l as P) doses of polyphosphate or orthophosphate generally decreased copper release in pipe rig tests of >3 years duration. The exception was pH 7.2 and alkalinity 300 mg/l as CaCO3, in which phosphate dosing significantly increased copper release over long time periods by hindering the formation of relatively insoluble malachite scale. Dosing of polyphosphates, which is invariably a blend of orthophosphate and polyphosphate due to reversion, was not as beneficial as orthophosphate. These relative effects are consistent with expectations based on simplistic solubility models, in which copper complexation by polyphosphate tends to increase soluble copper release, whereas higher doses of orthophosphate tend to decrease copper solubility.
Taste and odor issues occurring in new buildings were attributed to rapid loss of chloramine residual, high levels of microbes in the potable water system, and high water age due to use of advanced water conservation devices. Laboratory experiments confirmed that chloramine could decay rapidly in the presence of new copper pipe, providing a possible explanation for the rapid disinfectant loss in the new buildings. Higher temperature and lower pH also accelerated the rate of chloramine decay in copper pipes. The reaction was slowed by the addition of phosphate inhibitor or aluminum, which presumably formed barriers between the pipe wall and the chloramine in the bulk water. Additional research is needed to better understand how to maintain high quality water in buildings while also conserving water.
Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative
agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously
we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide
biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS
gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other
five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR
method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA
extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the
newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid
detection of high-risk L. pneumophila Sg1 in water and clinical samples.
Drinking water biofilms were grown on coupons of plumbing materials, including ethylene-propylene-diene-monomer (EPDM) rubber, silane cross-linked polyethylene (PE-X b), electron-ray cross-linked PE (PE-X c) and copper under constant flow-through of cold tap water. After 14 days, the biofilms were spiked with Pseudomonas aeruginosa, Legionella pneumophila and Enterobacter nimipressuralis (10(6)cells/mL each). The test bacteria were environmental isolates from contamination events in drinking water systems. After static incubation for 24h, water flow was resumed and continued for 4 weeks. Total cell count and heterotrophic plate count (HPC) of biofilms were monitored, and P. aeruginosa, L. pneumophila and E. nimipressuralis were quantified, using standard culture-based methods or culture-independent fluorescence in situ hybridization (FISH). After 14 days total cell counts and HPC values were highest on EPDM followed by the plastic materials and copper. P. aeruginosa and L. pneumophila became incorporated into drinking water biofilms and were capable to persist in biofilms on EPDM and PE-X materials for several weeks, while copper biofilms were colonized only by L. pneumophila in low culturable numbers.E. nimipressuralis was not detected in any of the biofilms. Application of the FISH method often yielded orders of magnitude higher levels of P. aeruginosa and L. pneumophila than culture methods. These observations indicate that drinking water biofilms grown under cold water conditions on domestic plumbing materials, especially EPDM and PE-X in the present study, can be a reservoir for P. aeruginosa and L. pneumophila that persist in these habitats mostly in a viable but non-culturable state.
Yolk sac suspensions infected with the Legionnaires disease bacterium (LDB) were plated onto 17 different bacteriological agar media. The LDB grew only on Mueller-Hinton agar supplemented with 1% Iso Vitale X and 1% hemoglobin (MH-IH). This medium was subsequently analyzed to determine the components required to support growth of the LDB. L-Cysteine hydrochloride can replace the Iso Vitale X reagent, and soluble ferric pyrophosphate can replace hemoglobin. A new medium, F-G agar, was formulated incorporating these chemicals. Different cultures conditions (oxygen tension, temperature, and pH) were also evaluated. The LDB grew optimally at 35 degrees C under 2.5% CO2 on the F-G agar adjusted to pH 6.9. When infected tissues were inoculated onto both F-G agar and MH-IH, the F-G agar produced colonies of the LDB more rapidly and in greater numbers than did MH-IH.
A chemically defined medium containing 21 amino acids and inorganic salts was developed which supported the growth of four isolates of Legionnaires disease bacterium (Legionella pneumophila). Growth in liquid defined medium at 37 degrees C with shaking approximated the generation time and growth kinetics observed for growth in complex media. After a 3-h lag, the culture grew exponentially with a generation time of 6 h and reached a maximum optical density of 230 Klett units (170 Klett units corrected for pigment). A soluble brown pigment was first observed as the culture entered late exponential to early stationary phase of growth. Morphologically, L. pneumophila grew in the liquid defined medium with extensive filamentation and numerous intracellular lipid granuoles. L-Serine, L-methionine, and L-cysteine were required for optimum growth. The latter amino acid could be replaced by L-cystine or reduced glutathione but not by D-cysteine, thiomalate, thioglycollate, or 2-mercaptoethanol. Ferric iron was needed for maximum growth, but supplemental iron was not an essential growth requirement. Carbohydrates (i.e., glucose) or organic acids did not stimulate growth. In fact, pyruvate, acetate, and citrate all gave varying degrees of inhibition (69, 37, and 0% of control growth, respectively).