121 research outputs found
Probiotic Lactobacillus reuteri effective in treating infantile colic and is associated with inflammatory marker reduction
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Growth of Opportunistic Pathogens in Domestic Plumbing: Building Standards, System Operation, and Design
No full textUnderstanding and limiting public health threats resulting from exposure to opportunistic pathogens (OPs) in domestic water (i.e., hot/cold water for human use) will be one of the grand challenges for water safety in the 21st century. This dissertation anticipates some of the complexities in balancing stakeholder goals and developing building standards to limit OP growth, and advances scientific understanding of OP survival and proliferation in domestic plumbing systems.
In a cross-sectional survey of water- and energy-efficient buildings, domestic water age ranged from 8 days to 6 months and resulted in pH and temperature fluctuations, rapid disinfectant residual decay up to 144 times faster than municipal water delivered to the buildings, and elevated levels of OP gene markers. This motivates future work to determine how to maintain high quality and safe water while preserving the sustainability goals of these cutting-edge buildings.
Head-to-head pilot-scale experiments examining OP growth in recirculating hot water systems revealed that elevated temperature had an overarching inhibitory effect on L. pneumophila growth where temperatures were maintained. However, control was undermined in distal branches, especially when density-driven convective mixing gradients maintained ideal growth temperatures and delivered nutrients to the otherwise stagnant branches. These results resolve discrepancies reported in the literature regarding the effects of flow, and identify important system design and operational conditions that facilitate OP growth.
Advancements were also made in understanding how corrosion can trigger OP growth. In Flint, MI, corrosive Flint River water damaged iron pipes, releasing iron nutrients, consuming chlorine residual, and supporting high levels of L. pneumophila in large building systems. This likely triggered two unprecedented clusters of Legionnaire's disease.
In pilot-scale systems, copper released from copper pipes, but not dosed as soluble cupric, triggered release of >1,100 times more H2 into the water due to deposition corrosion. The organic carbon fixed by autotrophic hydrogen oxidation has the potential to facilitate OP growth, but more work is needed to understand the limits of this mechanism.
Finally, well-controlled laboratory experiments confirmed past reports from field surveys that the use of chloramines trigger a trade-off between controlling Legionella and allowing non-tuberculous Mycobacteria to persist.Ph. D.Understanding and limiting public health threats resulting from exposure to opportunistic pathogens (OPs) in domestic water (i.e., hot and cold water intended for human use) will be one of the grand challenges for water safety in the 21st century. Unlike traditional fecal-based waterborne pathogens that have all but been eliminated through advanced treatment applied at water treatment facilities, OPs are native microbial members of drinking water and tend to proliferate in domestic plumbing. In addition to the complexity and technical nature of engineering controls applied in buildings to limit OP growth, there are many stakeholder groups with varied responsibilities and expertise in preventing, diagnosing, and/or remediating problems. Stakeholders sometimes present additional challenges when their goals have direct or indirect trade-offs with limiting OP growth in buildings. This dissertation anticipates some of the challenges to come, and advances scientific understanding of how OPs survive and proliferate in domestic plumbing systems.
Water- and energy-efficient buildings, while nobly seeking to preserve precious natural resources, potentially create unintended consequences with respect to water quality. In a cross-sectional survey of green building designs, water remained within domestic plumbing for over a week to months before being used by consumers, and resulted in water quality changes that facilitated the growth of OPs. While short-term solutions exist, such as flushing water to decrease water stagnation and introduce “fresh” water into the system, this work motivates future research for how to maintain high quality and safe water while preserving the sustainability goals of these cutting-edge buildings.
Systematic experiments were conducted on water heaters with a recirculating pump, which are marketed as a “green” technology for water and energy savings, to determine the effect of system design and operation on the growth of OPs. Elevated temperature was found to have an overarching inhibitory effect on growth of L. pneumophila, the most commonly reported OP. However, when the water heater temperature was not sufficient to completely eliminate L. pneumophila (51 °C), higher water temperatures actually supported high levels of L. pneumophila growth in infrequently used pipes by periodically disinfecting other microorganisms that are more susceptible to thermal disinfection and decreasing competition for nutrients.
System design also impacted Legionella growth. In pipes that slowly mixed with the recirculating line (simulating a pipe running upward to a shower head from a recirculating line in the floor, for instance), L. pneumophila were consistently elevated relative to pipes that did not convectively mix (simulating a pipe running downward to a kitchen tap from a recirculating line in the ceiling, for instance). The slow mixing maintained ideal Legionella growth temperature in the pipes with mixing, even when water heaters were maintained well above thermal disinfection levels for Legionella (i.e., at 60 °C). This is due to continuous delivery of nutrients to the upward pipes with mixing, but not the downward pipes without it. This result is significant because it outlines scenarios encountered in real buildings where even the most effective thermal disinfection strategy can be undermined in distal branches within the building.
This work also outlines the importance of corrosion in potentially triggering OP proliferation. In Flint, MI, when the water utility began distributing very corrosive Flint River water, the new water source damaged iron water pipes, releasing iron (which is a nutrient for Legionella) and eliminating disinfectant residual in the distribution system (which is needed to prevent Legionella regrowth). As a result, the corrosion supported increased Legionella levels and likely triggered two unprecedented clusters of Legionnaires’ disease.
In the pilot-scale systems, corrosion of the water heater anode rod caused trace nutrients to evolve into the water. However, this only occurred when low levels of copper released to the water from the natural corrosion of copper pipes were present. Ionic copper, which is sometimes used to disinfect Legionella, did not have the same effect when it was dosed to the experiment at similar concentrations. These trace nutrients generated from copper-enhanced corrosion of the water heater anode rod are a potential source of carbon for OP growth, and may help explain the variable effects of copper that have been reported in the literature. More work is needed to fully understand this potential growth mechanism.
Finally, this work confirmed past field observations that there is a trade-off between controlling Legionella and allowing Mycobacteria, another OP, to persist when using chloramines disinfectant residual. Reproducing this phenomenon in controlled laboratory settings is an important step in understanding, and ultimately preventing it
Impact of Flow Rate and Water Age on Opportunistic Pathogen Growth: Implications for Water Conservation, Fixture Design, and Policy
No full textWater conservation efforts have led to a decrease of flow rates in buildings, increasing water retention time (WRT) and sometimes opportunistic pathogens (OPs) growth. A novel experiment with replicated distal pipes operated at commonly used flow rates was designed to evaluate the effects of water age, flush frequency, flow rate, pipe diameter, water temperature, disinfectant residual presence, and microbial regrowth in hot and cold pipes. In cold water, total bacterial regrowth was a function of water age, plateauing after approximately 6 days at cell counts 20 times higher than influent water with minimal disinfectants. In warm (40 °C) water, most regrowth occurred in the heater tank, reducing the relative growth in the pipes. When cold water with ~1 mg/L chloramine was present, cold-water total bacteria regrowth plateaued after about 2 days WRT with cell counts 14 times higher than influent water, but regrowth still occurred in the heater tank. With 1 mg/L chloramine and elevated heater temperature (60 °C), regrowth in the tank was suppressed and cell counts in the pipes increased 82 times above cold-water influent levels at 7.5 days WRT. Legionella spp. and Mycobacterium spp. demonstrated opposite responses to flow rate with chloramine minimization. The highest levels of Legionella spp. (1.7 log higher than influent) were present when flow velocity was >2 feet per second (fps), but the highest levels of Mycobacterium spp. (1.5 log higher than influent) were observed at the lowest flow velocity (0.33 fps). This study highlights the tradeoffs between water conservation and water quality.Master of ScienceRegulations that decrease flow rates of faucets and showers have driven water conservation in buildings, increasing the time water sits in pipes and tanks (i.e., water retention time or WRT) elevating the likelihood of harmful bacterial growth. A novel faucet rig was designed to carry out a comprehensive experiment revealing the combined effects of WRT, flush frequency, flow rate, pipe diameter, water temperature, and disinfectant residual presence on water quality at the tap. In water without disinfectant, growth in cold water pipes increased with WRT, but in hot water the growth of bacteria occurred mostly in the warm water tank at 40 °C, which is a temperature known to leave a system vulnerable to bacterial growth. Cold pipes with a disinfectant residual saw a decrease in bacterial regrowth in comparison to cold pipes without disinfectant. However, if there was a disinfectant residual and an elevated water heater temperature set point in the tank, regrowth occurred when water was in the pipes at room temperature and there were lower disinfectant residuals. Potentially harmful bacteria, like Legionella spp. and Mycobacterium spp., which cause Legionnaires' disease and nontuberculous Mycobacteria (NTM) infections, grew more readily at higher flow rates, whereas others grew less readily, but all harmful bacteria were reduced by lowering WRT to less than ≈ 2 days and maintaining the water at 60 °C with a disinfectant. This study has important implications for regulations requiring minimum disinfectant levels to buildings, faucet flow rates regulations, and design and operation of building plumbing systems
How does metabolism of an “immuno acid” (tryptophan) by commensal Lactobacillus reuteri educate resident intestinal intraepithelial lymphocytes?
No full textLimitations to Use Copper as an Antimicrobial Control of Legionella in Potable Water Plumbing Systems
No full textThe opportunistic pathogen Legionella is the leading cause of reported waterborne disease outbreaks in the United States. Legionella can thrive under the warm, stagnant, low-disinfectant conditions characteristic of premise (i.e., building) plumbing systems, making it challenging to identify effective interventions for its control. Copper (Cu) is a promising antimicrobial that can be dosed directly to water via copper-silver ionization systems or released naturally via corrosion of Cu pipes to help control growth of Legionella and other pathogens. However, prior research has shown that Cu does not always reliably control Legionella and sometimes seems to even stimulate its growth. A deeper understanding of the mechanistic effects of Cu on Legionella, at both pure-culture and real-world scales, is critical in order to inform effective controls for Legionella. The overarching objective of the research embodied by this dissertation was aimed at elucidating the chemical and microbial interactions in premise plumbing that govern efficacy of Cu for Legionella control through a series of complementary bench-, pilot-, and field-scale studies.
A critical review and synthesis of the literature identified important knowledge gaps in relation to antimicrobial effects of Cu. In particular, changes in the pH, phosphate corrosion control, and rising levels of natural organic matter (NOM) in distributed water are predicted to be important controlling factors. The type of sacrificial anode rod material employed in water heaters was also identified as an underappreciated factor, which directly affects pH, evolution of hydrogen gas as a microbial nutrient, and release of metals (such as aluminum) that bind copper. Microbiological factors: including growth phase of Legionella (e.g., exponential or stationary), strain-specific Cu tolerance, background microbiome composition, and the possibility that viable but non-culturable (VBNC) Legionella might still cause human disease, were also identified as major confounding factors. These knowledge gaps are addressed from various dimensions across each chapter of the dissertation.
The effects of pH, orthophosphate corrosion inhibitor concentration, and NOM were examined in bench-scale pure culture experiments over a range of conditions relevant to drinking water. Cupric ions and antimicrobial effects were drastically reduced at pH >7.5, especially in the presence of phosphate, which precipitates copper, or NOM, which complexes the Cu in a form that is less bioavailable. Chick-Watson disinfection models indicated that soluble Cu was the most robust correlate with observed Cu antimicrobial effects across a range of tested waters. This new knowledge suggests that measuring soluble rather than total Cu would be much more informative to guide practitioners in dosing. The research also demonstrated that changes in pH or orthophosphate that have been made to control corrosion over the last few decades, have significantly altered Cu chemistry in buildings, undermining antimicrobial capacity and increasing likelihood of Legionella growth.
Pilot-scale experiments confirmed that soluble Cu is an effective indicator of Cu antimicrobial capacity, even in more complex environments represented by realistic hot water plumbing systems. In particular, dosing of orthophosphate, which is widely added by drinking water utilities to control corrosion, directly reduces soluble copper and overall antimicrobial capacity. In some cases, Cu added together with orthophosphate apparently promoted the growth of Legionella, providing an example of at least one circumstance where Cu addition can induce interactive effects that elevate Legionella compared to a control system with trace Cu.
It was also demonstrated for the first time that different water heater sacrificial anode types are subject to different corrosion processes, which indirectly influence Cu antimicrobial capacity. Specifically, aluminum ions released from aluminum anode corrosion at 1 mg/L can form an Al(OH)3 gel, which can remove >80% of the soluble Cu from water and reduce Cu antimicrobial effects by >2-log at pH=7. Corrosion from magnesium anodes was found to dramatically increase the pH from 6.8 to >8, which correspondingly reduces Cu antimicrobial capacity. Cu deposition further increased the anode corrosion rate and promoted evolution of hydrogen gas, which is a potent electron donor that stimulates autotrophic microbial growth especially with a magnesium anode. Electric powered anodes did not release metals or alter pH and thus did not diminish Cu antimicrobial capacity. Still, across the pilot-scale experiments, even very high levels of Cu (>1.2 mg/L) at low pH (<7) failed to fully eradicate culturable Legionella.
The much lower than expected antimicrobial efficacy of Cu in the pilot-scale hot water plumbing systems was found to be partially explained by the properties of the strain that colonized the systems. Based on fitting the data to a Chick-Watson disinfection model, the outbreak-associated strain that was inoculated into the systems was estimated to be 7 times more tolerant to Cu compared to the common lab strain applied in the bench-scale tests. Further, exponential growth phase L. pneumophila were found to be 2.5 times more susceptible to Cu relative to early stationary phase cultures. It is important to also recognize that, in the pilot-scale systems, drinking water biofilms and the amoeba hosts that colonize them can further shield Legionella from the antimicrobial effects of Cu.
Application of shotgun metagenomic sequencing offered the opportunity to more deeply examine the response of Legionella and other pathogens to Cu dosed to the pilot-scale hot water systems in the context of the broader microbiome. It was found that metagenomic analysis provided a sensitive indication of the bioavailability of Cu to the broader microbial community inhabiting the hot water systems, further confirming that the outbreak-associated strain of Legionella that colonized the rigs was relatively tolerant of Cu. Functional gene analysis provided further insight into the mechanistic action of Cu, suggesting multi-modal action of both membrane damage and interruption of nucleic acid replication. The metagenomic analysis further revealed that protozoan host numbers tended to increase in the pilot-scale systems with time, and this could also increase the potential for Legionella proliferation with time.
Additional pure culture studies aiming to further assess the mechanistic action of Cu provided strong evidence that Cu can induce a VBNC state for Legionella. This is a concern, given that other studies have indicated that VBNC Legionella are still capable of causing legionellosis. However, VBNC cells are not detected by conventional culturing. Multiple lines of evidence supported the conclusion that Cu induced a VBNC state for Legionella, including membrane integrity, enzyme activity, ATP generation, and Amoebae resuscitation assays applied to two different strains of L. pneumophila. After exposure to Cu, up to a 5-log (99.999%) reduction in culturable Legionella was observed, whereas corresponding reductions in the various viability measures were only by <1-log (90%). In other words, conventional culturing may miss up to 99.99% of the Legionella that is still capable of causing disease. To our knowledge, this is the first study that has assessed the potential for Cu-induced VBNC Legionella. Additional research is needed to further quantify the contribution of VBNC status to challenges in effective Cu-based control of Legionella in premise plumbing.
This research further examines, for the first time, the proteomic response of Legionella to Cu, comparing both presumably VBNC and culturable cells. Functional annotation of proteins that were differentially produced by the cells in response to Cu addition revealed that VBNC L. pneumophila modulated its proteome to favor cell membrane- and motility-related proteins, while reducing production of other proteins related to primary metabolism compared to culturable cells. These observations are consistent with the metagenomic-based observations and support the hypothesis that Cu inactivates cells by damaging the cell membrane. The findings also confirmed reduced general cell metabolism that is characteristic of a VBNC state.
This dissertation highlights the important and complex effects of Cu on Legionella growth in potable water systems as modified by water chemistry, water heater anode type, characteristics of the surrounding microbiome, and Legionella strain characteristics and growth status. The findings raise important questions about how to measure disinfectant efficacy and provide fundamental new knowledge that can help to better optimize the application of Cu as an antimicrobial to drinking water systems and better protect public health.Doctor of PhilosophyThe opportunistic pathogen Legionella is the leading cause of reportable waterborne disease outbreaks in the United States. Legionella is capable of growing in drinking water plumbing systems in homes, hospitals, hotels, and other buildings. Legionella is spread by inhaling tiny droplets of water that are suspended in the air when using the water, for example when showering, resulting in a severe and deadly form of pneumonia called Legionnaires' Disease. Copper is a promising antimicrobial that can be dosed directly into a building's water system by installing a copper-silver ionization system. There is also interest in understanding whether copper released naturally from copper pipes could help control Legionella. However, prior research indicates that copper sometimes inhibits, sometimes has no effect, and sometimes even seems to stimulate Legionella growth. The purpose of this dissertation was to better understand how the chemical properties of the drinking water, such as pH, presence of corrosion inhibitors that are commonly added to the water by utilities, and natural organic matter impact the ability of copper to kill Legionella. Impacts of the design of the drinking water system were also examined, for example, the material used in the anodes of water heaters to prevent corrosive damage to other system components was hypothesized to change the water chemistry in such a way that could also interfere with copper disinfection. Finally, the effect of the strain of Legionella, its growth phase (exponential or stationary), and culturability status (culturable versus viable but non-culturable) was also examined. Experiments were conducted over a wide range of conditions, from bench-scale pure culture experiments of a few days to full-scale plumbing systems over a period of 3.5 years. The complementary approaches maximize the strength of scientific conclusions about approaches that can more effectively control Legionella.
Several discoveries were made as a result of this research that can help to improve the use of copper for controlling Legionella in drinking water systems. In particular, it was found that it is best to keep the pH less than 7.5, because above pH 7.5 copper reacts with orthophosphate corrosion inhibitor or natural organic matter in the water in a manner that makes it less potent to microbes. Through disinfection modeling it was found that soluble copper was the best predictor of the ability to kill Legionella. Therefore, it is recommended from this research that practitioners should monitor soluble copper instead of total copper for the purpose of assessing Legionella control.
From the pilot-scale experiments, it was further found that the type of anode installed in the water heater can affect the ability of copper to kill Legionella. Magnesium anodes performed the worst, likely because they raised the pH above the recommended level of 7.5. Aluminum anodes were also a problem because aluminum ions released form an aluminum hydroxide gel that can remove more than 80% of the soluble copper from water. Electric powered anodes did not reduce copper antimicrobial effects by raising pH or forming a gel, but they are much less commonly used.
A surprising finding throughout this study was that very high levels of copper (>1.2 mg/L) were required to measurably reduce Legionella in the pilot-scale systems. In the pure culture experiments, it was found that the outbreak-associated strain from Quincy, IL, that was inoculated into the system was highly copper tolerant. This demonstrated that the strain of Legionella that colonizes a particular drinking water system could be the reason why copper is sometimes less effective. Pure culture experiments also found that stationary phase Legionella are more difficult to kill than exponential phase Legionella, which could explain some discrepancies among lab studies reported in the literature. A particularly noteworthy discovery of this research was that copper can make it appear as if Legionella have been killed, because the traditional culture media indicate that there is no growth on the Petri dish; however, they are in fact still alive and capable of causing human disease. This is referred to as a "viable but non-culturable (VBNC)" state. The VBNC state of Legionella was confirmed using an array of techniques (membrane integrity, enzyme activity, ATP generation, and amoebae resuscitation) for two strains of L. pneumophila. We also examined how VBNC Legionella cellular functions were impacted by copper using whole cell proteome, i.e., analysis of all of the proteins extracted from Legionella. Copper induced VBNC Legionella modulated its proteome to favor cell membrane and motility related proteins, and reduced others related to primary metabolism compared with culturable cells. These results were consistent with those obtained via shotgun metagenomic analysis of the microbial community DNA in the pilot-scale water systems. Given the potential for VBNC organisms to prevail in systems disinfected with copper, it is recommended to supplement culture-based monitoring with molecular-based monitoring, e.g., with quantitative polymerase chain reaction.
This dissertation highlights the important and complex effects of copper on Legionella growth in potable water systems. The findings help to inform guidance on how to improve the antimicrobial effect of copper, through adjusting the water chemistry, selecting appropriate water heater anodes, and optimizing the overall hot water system design. The dissertation also helps to inform improved strategies for monitoring the efficacy of copper for killing Legionella in real-world systems. Overall, the findings can help to improve policy and practice aimed at reducing the incidence of Legionnaires' Disease and protecting public health
Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing
No full textLegionella 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
Alanine Enhances Jejunal Sodium Absorption in the Presence of Glucose Studies in Piglet Viral Diarrhea
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