Kerry A. Hamilton

Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks

A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella longbeachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an accidental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1×10(6) gene copies per L rainwater for Legionella spp., 9.6×10(5) gene copies per L for P. aeruginosa, 6.8×10(5) gene copies per L for M. intracellulare, 6.6×10(5) gene copies per L for Acanthamoeba spp., 1.1×10(5) gene copies per L for M. avium, and 9.8×10(3) gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau=0.151, P=0.009) and E. coli vs. M. intracellulare (tau=0.14, P=0.015). However, M. avium weakly correlated with both L. pneumophila (Kendalls tau=0.017, P=0.006) and M. intracellulare (tau=0.088, P=0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau=0.128, P=0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses documented from the current survey data.

Utility of Helicobacter spp. associated GFD markers for detecting avian fecal pollution in natural waters of two continents

Avian fecal droppings may negatively impact environmental water quality due to the presence of high concentrations of fecal indicator bacteria (FIB) and zoonotic pathogens. This study was aimed at evaluating the performance characteristics and utility of a Helicobacter spp. associated GFD marker by screening 265 fecal and wastewater samples from a range of avian and non-avian host groups from two continents (Brisbane, Australia and Florida, USA). The host-prevalence and -specificity of this marker among fecal and wastewater samples tested from Brisbane were 0.58 and 0.94 (maximum value of 1.00). These values for the Florida fecal samples were 0.30 (host-prevalence) and 1.00 (host-specificity). The concentrations of the GFD markers in avian and non-avian fecal nucleic acid samples were measured at a test concentration of 10 ng of nucleic acid at Brisbane and Florida laboratories using the quantitative PCR (qPCR) assay. The mean concentrations of the GFD marker in avian fecal nucleic acid samples (5.2 × 10(3) gene copies) were two orders of magnitude higher than non-avian fecal nucleic acid samples (8.6 × 10(1) gene copies). The utility of this marker was evaluated by testing water samples from the Brisbane River, Brisbane and a freshwater creek in Florida. Among the 18 water samples tested from the Brisbane River, 83% (n = 18) were positive for the GFD marker, and the concentrations ranged from 6.0 × 10(1)-3.2 × 10(2) gene copies per 100 mL water. In all, 92% (n = 25) water samples from the freshwater creek in Florida were also positive for the GFD marker with concentrations ranging from 2.8 × 10(1)-1.3 × 10(4) gene copies per 100 mL water. Based on the results, it can be concluded that the GFD marker is highly specific to avian host groups, and could be used as a reliable marker to detect the presence and amount of avian fecal pollution in environmental waters.

Evaluating the Potential for a Helicobacter pylori Drinking Water Guideline

Helicobacter pylori is a microaerophilic, gram-negative bacterium that is linked to adverse health effects including ulcers and gastrointestinal cancers. The goal of this analysis is to develop the necessary inputs for a quantitative microbial risk assessment (QMRA) needed to develop a potential guideline for drinking water at the point of ingestion (e.g., a maximum contaminant level, or MCL) that would be protective of human health to an acceptable level of risk while considering sources of uncertainty. Using infection and gastric cancer as two discrete endpoints, and calculating dose-response relationships from experimental data on humans and monkeys, we perform both a forward and reverse risk assessment to determine the risk from current reported surface water concentrations of H. pylori and an acceptable concentration of H. pylori at the point of ingestion. This approach represents a synthesis of available information on human exposure to H. pylori via drinking water. A lifetime risk of cancer model suggests that a MCL be set at <1 organism/L given a 5-log removal treatment because we cannot exclude the possibility that current levels of H. pylori in environmental source waters pose a potential public health risk. Research gaps include pathogen occurrence in source and finished water, treatment removal rates, and determination of H. pylori risks from other water sources such as groundwater and recreational water.

Human health risks for Legionella and Mycobacterium avium complex (MAC) from potable and non-potable uses of roof-harvested rainwater

A quantitative microbial risk assessment (QMRA) of opportunistic pathogens Legionella pneumophila (LP) and Mycobacterium avium complex (MAC) was undertaken for various uses of roof-harvested rainwater (RHRW) reported in Queensland, Australia to identify appropriate usages and guide risk management practices. Risks from inhalation of aerosols due to showering, swimming in pools topped up with RHRW, use of a garden hose, car washing, and toilet flushing with RHRW were considered for LP while both ingestion (drinking, produce consumption, and accidental ingestion from various activities) and inhalation risks were considered for MAC. The drinking water route of exposure presented the greatest risks due to cervical lymphadenitis and disseminated infection health endpoints for children and immune-compromised populations, respectively. It is therefore not recommended that these populations consume untreated rainwater. LP risks were up to 6 orders of magnitude higher than MAC risks for the inhalation route of exposure for all scenarios. Both inhalation and ingestion QMRA simulations support that while drinking, showering, and garden hosing with RHRW may present the highest risks, car washing and clothes washing could constitute appropriate uses of RHRW for all populations, and toilet flushing and consumption of lettuce irrigation with RHRW would be appropriate for non- immune-compromised populations.

Critical review of mathematical approaches for quantitative microbial risk assessment (QMRA) of Legionella in engineered water systems: research gaps and a new framework

Legionella has been identified as the responsible agent for two-thirds of waterborne disease outbreaks in the United States from 2011–2012. The prevention of Legionella in engineered systems presents complex challenges for the drinking water industry due to its persistence, resistance to disinfection, and complex microbial ecology. Not all species of Legionella are of concern for human health, however, certain environmental conditions can cause human-virulent species such as L. pneumophila to proliferate, or modulate the distribution of virulence characteristics for relevant strains such that health risks are presented. Quantitative microbial risk assessment (QMRA) is a tool for integrating information on pathogen occurrence, infectivity, and exposure for guiding water quality management strategies. A standardized QMRA approach for Legionella has not been developed, and exposure models are highly varied based on scenario- and site-specific conditions. Detailed discussion of these varied mathematical approaches has been limited, but can aid in identifying research gaps for further QMRA development and public health risk management strategies. A summary of 18 studies that utilize Legionella exposure models for sewage treatment plants, cooling towers, drinking water distribution systems, whirlpool spas, showering, and recreational water scenarios are discussed here. Ten of these studies conducted a full QMRA, and provided human infection estimates. The summarized models utilized Gaussian dispersion, volumetric estimation, occupational hygiene, and aerosol science approaches. Parameters, implications, and limitations of each of these mathematical approaches are discussed, and a QMRA framework to address the identified limitations is proposed. This framework provides a comprehensive overview of key steps within an idealized Legionella QMRA model from exposure to risk characterization, including biofilm impacts, aerosol generation, survival and transport of bacteria within size-resolved water droplets, and interaction with a human receptor.

Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks

Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

Evidence of Avian and Possum Fecal Contamination in Rainwater Tanks as Determined by Microbial Source Tracking Approaches

ABSTRACT Avian and possum fecal droppings may negatively impact roof-harvested rainwater (RHRW) water quality due to the presence of zoonotic pathogens. This study was aimed at evaluating the performance characteristics of a possum feces-associated (PSM) marker by screening 210 fecal and wastewater samples from possums (n = 20) and a range of nonpossum hosts (n = 190) in Southeast Queensland, Australia. The host sensitivity and specificity of the PSM marker were 0.90 and 0.95 (maximum value, 1.00), respectively. The mean concentrations of the GFD marker in possum fecal DNA samples (8.8 × 107 gene copies per g of feces) were two orders of magnitude higher than those in the nonpossum fecal DNA samples (5.0 × 105 gene copies per g of feces). The host sensitivity, specificity, and concentrations of the avian feces-associated GFD marker were reported in our recent study (W. Ahmed, V. J. Harwood, K. Nguyen, S. Young, K. Hamilton, and S. Toze, Water Res 88:613–622, 2016, http://dx.doi.org/10.1016/j.watres.2015.10.050). The utility of the GFD and PSM markers was evaluated by testing a large number of tank water samples (n = 134) from the Brisbane and Currumbin areas. GFD and PSM markers were detected in 39 of 134 (29%) and 11 of 134 (8%) tank water samples, respectively. The GFD marker concentrations in PCR-positive samples ranged from 3.7 × 102 to 8.5 × 105 gene copies per liter, whereas the concentrations of the PSM marker ranged from 2.0 × 103 to 6.8 × 103 gene copies per liter of water. The results of this study suggest the presence of fecal contamination in tank water samples from avian and possum hosts. This study has established an association between the degradation of microbial tank water quality and avian and possum feces. Based on the results, we recommend disinfection of tank water, especially for tanks designated for potable use. IMPORTANCE The use of roof-harvested rainwater (RHRW) for domestic purposes is a globally accepted practice. The presence of pathogens in rainwater tanks has been reported by several studies, supporting the necessity for the management of potential health risks. The sources of fecal pollution in rainwater tanks are unknown. However, the application of microbial source tracking (MST) markers has the potential to identify the sources of fecal contamination in a rainwater tank. In this study, we provide evidence of avian and possum fecal contamination in tank water samples using molecular markers. This study established a potential link between the degradation of the microbial quality of tank water and avian and possum feces.

Drivers of Microbial Risk for Direct Potable Reuse and de Facto Reuse Treatment Schemes: The Impacts of Source Water Quality and Blending

Although reclaimed water for potable applications has many potential benefits, it poses concerns for chemical and microbial risks to consumers. We present a quantitative microbial risk assessment (QMRA) Monte Carlo framework to compare a de facto water reuse scenario (treated wastewater-impacted surface water) with four hypothetical Direct Potable Reuse (DPR) scenarios for Norovirus, Cryptosporidium, and Salmonella. Consumer microbial risks of surface source water quality (impacted by 0–100% treated wastewater effluent) were assessed. Additionally, we assessed risks for different blending ratios (0–100% surface water blended into advanced-treated DPR water) when source surface water consisted of 50% wastewater effluent. De facto reuse risks exceeded the yearly 10−4 infections risk benchmark while all modeled DPR risks were significantly lower. Contamination with 1% or more wastewater effluent in the source water, and blending 1% or more wastewater-impacted surface water into the advanced-treated DPR water drove the risk closer to the 10−4 benchmark. We demonstrate that de facto reuse by itself, or as an input into DPR, drives microbial risks more so than the advanced-treated DPR water. When applied using location-specific inputs, this framework can contribute to project design and public awareness campaigns to build legitimacy for DPR.

Seasonal Assessment of Opportunistic Premise Plumbing Pathogens in Roof-Harvested Rainwater Tanks

A seasonal study on the occurrence of six opportunistic premise plumbing pathogens (OPPPs) in 24 roof-harvested rainwater (RHRW) tanks repeatedly sampled over six monthly sampling events (n = 144) from August 2015 to March 2016 was conducted using quantitative qPCR. Fecal indicator bacteria (FIB) Escherichia coli (E. coli) and Enterococcus spp. were enumerated using culture-based methods. All tank water samples over the six events were positive for at least one OPPP (Legionella spp., Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Pseudmonas aeruginosa, or Acanthamoeba spp.) during the entire course of the study. FIB were positively but weakly correlated with P. aeruginosa (E. coli vs P. aeruginosa τ = 0.090, p = 0.027; Enterococcus spp. vs P. aeruginosa τ = 0.126, p = 0.002), but not the other OPPPs. FIBs were more prevalent during the wet season than the dry season, and L. pneumophila was only observed during the wet season. However, concentrations of Legionella spp., M. intracellulare, Acanthamoeba spp., and M. avium peaked during the dry season. Correlations were assessed between FIB and OPPPs with meteorological variables, and it was determined that P. aeruginosa was the only OPPP positively associated with an increased antecedent dry period, suggesting stagnation time may play a role for the occurrence of this OPPP in tank water. Infection risks may exceed commonly cited benchmarks for uses reported in the rainwater usage survey such as pool top-up, and warrant further exploration through quantitative microbial risk assessment (QMRA).

Quantitative microbial risk assessment of microbial source tracking markers in recreational water contaminated with fresh untreated and secondary treated sewage

Microbial source tracking (MST) methods have provided the means to identify sewage contamination in recreational waters, but the risk associated with elevated levels of MST targets such as sewage-associated Bacteroides HF183 and other markers is uncertain. Quantitative microbial risk assessment (QMRA) modeling allows interpretation of MST data in the context of the risk of gastrointestinal (GI) illness caused by exposure to pathogens. In this study, five sewage-associated, quantitative PCR (qPCR) MST markers [Bacteroides HF183 (HF183), Methanobrevibacter smithii nifH (nifH), human adenovirus (HAdV), human polyomavirus (HPyV) and pepper mild mottle virus (PMMoV)] were evaluated to determine at what concentration these nucleic acid markers reflected a significant health risk from exposure to fresh untreated or secondary treated sewage in beach water. The QMRA models were evaluated for a target probability of illness of 36 GI illnesses/1000 swimming events (i.e., risk benchmark 0.036) for the reference pathogens norovirus (NoV) and human adenovirus 40/41 (HAdV 40/41). Sewage markers at several dilutions exceeded the risk benchmark for reference pathogens NoV and HAdV 40/41. HF183 concentrations 3.22 × 103 (for both NoV and HAdV 40/41) gene copies (GC)/100 mL of water contaminated with fresh untreated sewage represented risk >0.036. Similarly, HF183 concentrations 3.66 × 103 (for NoV and HAdV 40/41) GC/100 mL of water contaminated with secondary treated sewage represented risk >0.036. HAdV concentration as low as 4.11 × 101 GC/100 mL of water represented risk >0.036 when water was contaminated with secondary treated sewage. Results of this study provide a valuable context for water quality managers to evaluate human health risks associated with contamination from fresh sewage. The approach described here may also be useful in the future for evaluating health risks from contamination with aged or treated sewage or feces from other animal sources as more data are made available.