Opportunistic Premise Plumbing Pathogens Research Articles

Photodynamic Inactivation of Opportunistic Premise Plumbing Pathogens and Their Biofilms

Opportunistic premise plumbing pathogens (OPPPs) form a group of microorganisms that normally live in water supply systems and have adapted especially well to the conditions in premise plumbing systems, and as such pose a threat to human health. Since the beginning of the 21st century, this threat has been escalating, and it is becoming increasingly evident that current water disinfection methods fall short in effectively controlling these pathogens. In researching new approaches to this emergency, phototherapy looks promising, especially one that combines photosensitizers, light, and oxygen, which is known as photodynamic inactivation (PDI). This review describes the main characteristics of the recognized (Pseudomonas aeruginosa, Legionella pneumophila, and Mycobacterium avium) and most important emerging OPPPs, and it offers a brief overview of current disinfection methods and their limitations in the fight against OPPPs. The principle and outcomes of PDI with endogenous and, in particular, exogenous photosensitizers are then explained and described through representative examples of PDI on recognized and emerging OPPPs and their biofilms. Finally, the prospects and future directions of PDI research in water disinfection and control of OPPPs are discussed.

Assessing residential activity in a home plumbing system simulator: monitoring the occurrence and relationship of major opportunistic pathogens and phagocytic amoebas.

Opportunistic premise plumbing pathogens (OPPPs) have been detected in buildings' plumbing systems causing waterborne disease outbreaks in the United States. In this study, we monitored the occurrence of OPPPs along with free-living amoeba (FLA) and investigated the effects of residential activities in a simulated home plumbing system (HPS). Water samples were collected from various locations in the HPS and analyzed for three major OPPPs: Legionella pneumophila, nontuberculous mycobacterial species (e.g., Mycobacterium avium, M. intracellulare, and M. abscessus), and Pseudomonas aeruginosa along with two groups of amoebas (Acanthamoeba and Vermamoeba vermiformis). A metagenomic approach was also used to further characterize the microbial communities. Results show that the microbial community is highly diverse with evidence of spatial and temporal structuring influenced by environmental conditions. L. pneumophila was the most prevalent pathogen (86% of samples), followed by M. intracellulare (66%) and P. aeruginosa (21%). Interestingly, M. avium and M. abscessus were not detected in any samples. The data revealed a relatively low prevalence of Acanthamoeba spp. (4%), while V. vermiformis was widely detected (81%) across all the sampling locations within the HPS. Locations with a high concentration of L. pneumophila and M. intracellulare coincided with the highest detection of V. vermiformis, suggesting the potential growth of both populations within FLA and additional protection in drinking water. After a period of stagnation lasting at least 2-weeks, the concentrations of OPPPs and amoeba immediately increased and then decreased gradually back to the baseline. Furthermore, monitoring the microbial population after drainage of the hot water tank and partial drainage of the entire HPS demonstrated no significant mitigation of the selected OPPPs. This study demonstrates that these organisms can adjust to their environment during such events and may survive in biofilms and/or grow within FLA, protecting them from stressors in the supplied water.

The composition of planktonic prokaryotic communities in a hospital building water system depends on both incoming water and flow dynamics.

In recent years, the frequency of nosocomial infections has increased. Hospital water systems support the growth of microbes, especially opportunistic premise plumbing pathogens. In this study, planktonic prokaryotic communities present in water samples taken from hospital showers and hand basins, collected over three different sampling phases, were characterized by 16S rRNA gene amplicon sequencing. Significant differences in the abundance of various prokaryotic taxa were found through univariate and multivariate analysis. Overall, the prokaryotic communities of hospital water were taxonomically diverse and dominated by biofilm forming, corrosion causing, and potentially pathogenic bacteria. The phyla Proteobacteria, Actinobacteriota, Bacteroidota, Planctomycetota, Firmicutes, and Cyanobacteria made up 96% of the relative abundance. The α-diversity measurements of prokaryotic communities showed no difference in taxa evenness and richness based on sampling sites (shower or hand basins), sampling phases (months), and presence or absence of Vermamoeba vermiformis. However, β-diversity measurements showed significant clustering of prokaryotic communities based on sampling phases, with the greatest difference observed between the samples collected in phase 1vs phase 2/3. Importantly, significant difference was observed in prokaryotic communities based on flow dynamics of the incoming water. The Pielou's evenness diversity index revealed a significant difference (Kruskal Wallis, p < 0.05) and showed higher species richness in low flow regime (< 13 minutes water flushing per week and ≤ 765 flushing events per six months). Similarly, Bray-Curtis dissimilarity index found significant differences (PERMANOVA, p < 0.05) in the prokaryotic communities of low vs medium/high flow regimes. Furthermore, linear discriminant analysis effect size showed that several biofilm forming (e.g., Pseudomonadales), corrosion causing (e.g., Desulfobacterales), extremely environmental stress resistant (e.g., Deinococcales), and potentially pathogenic (e.g., Pseudomonas) bacterial taxa were in higher amounts under low flow regime conditions. This study demonstrated that a hospital building water system consists of a complex microbiome that is shaped by incoming water quality and the building flow dynamics arising through usage.

Mycobacterial contamination in tap and shower waters in Thailand.

Waterborne disease is increasingly becoming associated with opportunistic premise plumbing pathogens (OPPPs), which can resist residual chlorination, regrow throughout drinking water distribution systems, and colonize premise plumbing. Nontuberculous mycobacteria (NTM) include clinically important species and exert a high burden on healthcare systems. We briefly report a qPCR-based survey of Mycobacterium spp. numbers in tap, POU-treated, and shower waters from Bangkok, Thailand. Non-stagnant tap waters and non-stagnant shower waters had mean numbers of 1.3×103 and 2.4×103 copies/mL, respectively. Water stagnation resulted in mean numbers higher by up to 1.0 log. The lowest number, 25 copies/mL, was obtained from a POU-treated sample, while the highest number, 2.0×104 copies/mL, came from a stagnant tap. Comparing with international data, mean numbers in this study were greater than those in nine out of 11 (82%) comparable studies, and the maximum numbers in this study were also high. Our samples of Bangkok waters exhibited relatively high Mycobacterium spp. numbers, suggesting the need for appropriate POU treatment systems where NTM infection is a health concern. This survey data can be used to set inactivation performance targets in POU water disinfection system design and may also lead to quantitative microbial risk assessment (QMRA) studies.

A Critical Review on the Factors that Influence Opportunistic Premise Plumbing Pathogens: From Building Entry to Fixtures in Residences.

Residential buildings provide unique conditions for opportunistic premise plumbing pathogen (OPPP) exposure via aerosolized water droplets produced by showerheads, faucets, and tubs. The objective of this review was to critically evaluate the existing literature that assessed the impact of potentially enhancing conditions to OPPP occurrence associated with residential plumbing and to point out knowledge gaps. Comprehensive studies on the topic were found to be lacking. Major knowledge gaps identified include the assessment of OPPP growth in the residential plumbing, from building entry to fixtures, and evaluation of the extent of the impact of typical residential plumbing design (e.g., trunk and branch and manifold), components (e.g., valves and fixtures), water heater types and temperature setting of operation, and common pipe materials (copper, PEX, and PVC/CPVC). In addition, impacts of the current plumbing code requirements on OPPP responses have not been assessed by any study and a lack of guidelines for OPPP risk management in residences was identified. Finally, the research required to expand knowledge on OPPP amplification in residences was discussed.

Inactivation kinetics of 280 nm UV-LEDs against Mycobacterium abscessus in water

Nontuberculous mycobacteria (NTM) are opportunistic premise plumbing pathogens (OPPPs) that cause a burdensome waterborne respiratory disease. Due to their resistance to chemical disinfectants and regrowth in biofilms in drinking water distribution systems, treatment can be better performed using small ultraviolet disinfection units at the point-of-use (POU), such as at a tap or showerhead. Ultraviolet light-emitting diodes (UV-LEDs) are well suited for such applications, but fluence-response data are not available for one of the most important NTM, Mycobacterium abscessus. In this study, a bench-scale 280 nm UV-LED apparatus was used to irradiate M. abscessus in a water matrix. The fluence-response profile was sigmoidal, exhibiting both shoulder and tailing phenomena. Simple linear regression and the Geeraerd’s inactivation kinetics model yielded k values of 0.36 and 0.37 cm2/mJ, respectively, revealing that M. abscessus is more resistant to UV than Pseudomonas aeruginosa and Legionella pneumophila, which suggests that NTM are among the most UV-resistant OPPPs. Results of this study suggest that 280 nm UV-LED irradiation can be an effective and practical option to inactivate M. abscessus at the POU. Disinfection units that can deliver a fluence of 10 mJ/cm2 are expected to achieve nearly 2 log (99%) inactivation of M. abscessus.

Effect of UV Light and Sodium Hypochlorite on Formation and Destruction of Pseudomonas fluorescens Biofilm In Vitro

Pseudomonas fluorescens is one of the first colonizers of bacterial biofilm in water systems and a member of opportunistic premise plumbing pathogens (OPPPs). The aim of this study was to examine the effect of UV light and sodium hypochlorite on the formation and destruction of mature P. fluorescens biofilm on ceramic tiles. Planktonic bacteria or bacteria in mature biofilm were exposed to UV light (254 nm) for 5, 20 s. and to 0.4 mg/L sodium hypochlorite for 1 min. Mature biofilm was also exposed to increased concentration of sodium hypochlorite of 2 mg/L for 0.5, 1 and 2 h and combined with UV. Prolonged action of sodium hypochlorite and an increase in its concentration in combination with UV gave the best results in the inhibition of biofilm formation after the pre-treatment and destruction of mature biofilm. The effect of hyperchlorination in combination with UV radiation shows better results after a long exposure time, although even after 120 min there was no completely destroyed biofilm. Furthermore, the mechanism of the effect of combined methods should be explored as well as the importance of mechanical cleaning that is crucial in combating bacterial biofilm in swimming pools.

Impact of pipe material, operation, and disinfectant on cold water plumbing microbiology

AbstractThis study examined the effects of pipe materials, pipe diameters, frequency of use, and disinfectant types on the water quality microbiology in experimental pipe rack systems. Two pipe rack systems were installed in buildings in Colorado (CO) and Pennsylvania (PA) and sampled for 8–9 months of operation, respectively, under simulated high and low use conditions. Opportunistic premise plumbing pathogens including Legionella pneumophila, nontuberculous mycobacteria, Pseudomonas aeruginosa, and free‐living amoebae (FLA) were the target organisms. Disinfectant type (free chlorine vs. chloramine) and use frequency (flushed every 12 h vs. weekly) had the greatest impact on bulk water microbiology. Under low use conditions, neither system had detectable disinfectant residual but under high use, residual was detected. Heterotrophic plate count bacteria predominated in the free chlorine system while nontuberculous mycobacteria were predominant in the chloraminated system. P. aeruginosa and FLA were not detected in the free chlorine system and legionellae were not detected in either system.

Hospital water as the source of healthcare-associated infection and antimicrobial-resistant organisms.

Drinking water is considered one of the most overlooked and underestimated sources of healthcare-associated infections (HAIs). Recently, the prevention and control of opportunistic premise plumbing pathogens (OPPPs) in healthcare water systems has been receiving increasing attention in infection control guidelines. However, these fail to address colonization of pathogens that do not originate from source water. Subsequently, this review explores the role of water and premise plumbing biofilm in HAIs. The potential mechanisms of contamination and transmission of antimicrobial-resistant (AMR) pathogens originating both from supply water and human microbiota are discussed. OPPPs, such as Legionella pneumophila, Pseudomonas aeruginosa and Mycobacterium avium have been described as native to the plumbing environment. However, other pathogens, not found in the source water, have been found to proliferate in biofilms formed on outlets devices and cause HAI outbreaks. Biofilms formed on outlet devices, such as tap faucets, showers and drains provide an ideal niche for the dissemination of antimicrobial resistance. Thus, comprehensive surveillance guidelines are required to understand the role that drinking water and water-related devices play in the transmission of AMR HAIs and to improve infection control guidelines.

The Presence of Opportunistic Premise Plumbing Pathogens in Residential Buildings: A Literature Review

Opportunistic premise plumbing pathogens (OPPP) are microorganisms that are native to the plumbing environment and that present an emerging infectious disease problem. They share characteristics, such as disinfectant resistance, thermal tolerance, and biofilm formation. The colonisation of domestic water systems presents an elevated health risk for immune-compromised individuals who receive healthcare at home. The literature that has identified the previously described OPPPs (Aeromonas spp., Acinetobacter spp., Helicobacter spp., Legionella spp., Methylobacterium spp., Mycobacteria spp., Pseudomonas spp., and Stenotrophomonas spp.) in residential drinking water systems were systematically reviewed. By applying the Preferred reporting items for systematic reviews and meta-analyses guidelines, 214 studies were identified from the Scopus and Web of Science databases, which included 30 clinical case investigations. Tap components and showerheads were the most frequently identified sources of OPPPs. Sixty-four of these studies detected additional clinically relevant pathogens that are not classified as OPPPs in these reservoirs. There was considerable variation in the detection methods, which included traditional culturing and molecular approaches. These identified studies demonstrate that the current drinking water treatment methods are ineffective against many waterborne pathogens. It is critical that, as at-home healthcare services continue to be promoted, we understand the emergent risks that are posed by OPPPs in residential drinking water. Future research is needed in order to provide consistent data on the prevalence of OPPPs in residential water, and on the incidence of waterborne homecare-associated infections. This will enable the identification of the contributing risk factors, and the development of effective controls.

Cupriavidus spp. and other waterborne organisms in healthcare water systems across the UK

Cupriavidus pauculus is a rare clinical pathogen, cases of which have been linked to contaminated hospital water systems. An outbreak of three cases of C.pauculus and other waterborne organisms was reported in a Glasgow hospital in 2018. To determine whether Cupriavidus spp. are present in hospital water systems elsewhere in Scotland and the UK, and to ascertain the optimal laboratory methodology for detection. This study also sought to establish where in the water system these organisms are detected, and whether a selective media could be developed for isolation. In addition, water samples were tested for the presence of other Gram-negative waterborne organisms. Water samples were received from 10 UK National Health Service hospitals and from various parts of the water system. Isolates were plated on to tryptone soya agar (TSA) and Pseudomonas isolation agar, and were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S polymerase chain reaction. Cupriavidus spp. were detected in four of 10 hospitals tested, and all five isolates were from the periphery of the water system. All hospitals had evidence of other opportunistic premise plumbing pathogens. Cupriavidus spp. were identified using TSA, with some isolates growing on Pseudomonas isolation agar; as such, they may be detected inadvertently when testing water specifically for Pseudomonas aeruginosa. Isolation of Cupriavidus spp. was not unique to the Glasgow incident, and these bacteria are present in hospital water systems elsewhere in the UK. Water testing in response to clinical cases is recommended. Consideration should also be given to water testing following bacteraemias due to other rare and unusual water-borne pathogens.

Causes, Factors, and Control Measures of Opportunistic Premise Plumbing Pathogens—A Critical Review

This review critically analyses the chemical and physical parameters that influence the occurrence of opportunistic pathogens in the drinking water distribution system, specifically in premise plumbing. A comprehensive literature review reveals significant impacts of water age, disinfectant residual (type and concentration), temperature, pH, and pipe materials. Evidence suggests that there is substantial interplay between these parameters; however, the dynamics of such relationships is yet to be elucidated. There is a correlation between premise plumbing system characteristics, including those featuring water and energy conservation measures, and increased water quality issues and public health concerns. Other interconnected issues exacerbated by high water age, such as disinfectant decay and reduced corrosion control efficiency, deserve closer attention. Some common features and trends in the occurrence of opportunistic pathogens have been identified through a thorough analysis of the available literature. It is proposed that the efforts to reduce or eliminate their incidence might best focus on these common features.

Opportunistic Premise Plumbing Pathogens. A Potential Health Risk in Water Mist Systems Used as a Cooling Intervention.

Water mist systems (WMS) are used for evaporative cooling in public areas. The health risks associated with their colonization by opportunistic premise plumbing pathogens (OPPPs) is not well understood. To advance the understanding of the potential health risk of OPPPs in WMS, biofilm, water and bioaerosol samples (n = 90) from ten (10) WMS in Australia were collected and analyzed by culture and polymerase chain reaction (PCR) methods to detect the occurrence of five representative OPPPs: Legionella pneumophila, Pseudomonas aeruginosa, Mycobacterium avium, Naegleria fowleri and Acanthamoeba. P. aeruginosa (44%, n = 90) occurred more frequently in samples, followed by L. pneumophila serogroup (Sg) 2–14 (18%, n = 90) and L. pneumophila Sg 1 (6%, n = 90). A negative correlation between OPPP occurrence and residual free chlorine was observed except with Acanthamoeba, rs (30) = 0.067, p > 0.05. All detected OPPPs were positively correlated with total dissolved solids (TDS) except with Acanthamoeba. Biofilms contained higher concentrations of L. pneumophila Sg 2–14 (1000–3000 CFU/mL) than water samples (0–100 CFU/mL). This study suggests that WMS can be colonized by OPPPs and are a potential health risk if OPPP contaminated aerosols get released into ambient atmospheres.

Biofilm Degradation of Nontuberculous Mycobacteria Formed on Stainless Steel Following Treatment with Immortelle (Helichrysum italicum) and Common Juniper (Juniperus communis) Essential Oils

Nontuberculous mycobacteria, like other opportunistic premise plumbing pathogens, produce resistant biofilms on various surfaces in the plumbing system including pipes, tanks, and fittings. Since standard methods of water disinfection are ineffective in eradicating biofilms, research into new agents is necessary. Essential oils (EOs) have great potential as anti-biofilm agents. Therefore, the purpose of this research was to investigate the potential anti-biofilm effect of common juniper (Juniperus communis) and immortelle (Helichrysum italicum) EOs. Minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and minimum effective concentrations of EOs on Mycobacterium avium, M. intracellulare, and M. gordonae were tested. Additionally, biofilms on the surface of a stainless steel disc were treated with single or mixed concentration of EOs, in order to investigate their degeneration via the bacterial count and confocal laser scanning microscopy (CLSM). H. italicum EO showed the strongest biofilm degradation ability against all Mycobacteria strains that were tested. The strongest effect in the biofilm degradation after the single or mixed applications of EOs was observed against M. gordonae, followed by M. avium. The most resistant was the M. intracellulare biofilm. Synergistic combinations of J. communis and H. italicum EOs therefore seem to be an effective substance in biofilm degradation for use in small water systems such as baths or hot tubs.

Living with Legionella and Other Waterborne Pathogens.

Legionella spp. and other opportunistic premise plumbing pathogens (OPPPs), including Pseudomonas aeruginosa, Mycobacterium avium, Stenotrophomonas maltophilia, and Acinetobacter baumannii, are normal inhabitants of natural waters, drinking water distribution systems and premise plumbing. Thus, humans are regularly exposed to these pathogens. Unfortunately, Legionella spp. and the other OPPPs share a number of features that allow them to grow and persist in premise plumbing. They form biofilms and are also relatively disinfectant-resistant, able to grow at low organic matter concentrations, and able to grow under stagnant conditions. Infections have been traced to exposure to premise plumbing or aerosols generated in showers. A number of measures can lead to reduction in OPPP numbers in premise plumbing, including elevation of water heater temperatures.

Methylobacterium spp. as Emerging Opportunistic Premise Plumbing Pathogens

Methylobacterium spp. are emerging opportunistic premise plumbing pathogens. Human infections linked to premise plumbing provide evidence of their routes of infection. Cells of a collection of representative strains of different Methylobacterium species were tested for hydrophobicity by contact angle, adherence and biofilm formation on different plumbing materials, and temperature tolerance (50–60 °C); characteristics shared by OPPPs. Methylobacterium spp. strains were shown to grow in drinking water, have high cell-surface hydrophobicity, adhere to pipe surface materials, form biofilms, and survive exposure to high (60° C) temperatures. It can be concluded that Methylobacterium spp. strains share traits in common with other opportunistic premise plumbing pathogens (OPPPs).

Biofilms in premise plumbing systems as a double-edged sword: microbial community composition and functional profiling of biofilms in a tropical region.

To understand distributions of opportunistic premise plumbing pathogens (OPPPs) and microbial community structures governed by sample location, pipe materials, water temperature, age of property and type of house, 29 biofilm samples obtained from faucets, pipes, and shower heads in different households in Singapore were examined using next-generation sequencing technology. Predictive functional profiling of the biofilm communities was also performed to understand the potential of uncultivated microorganisms in premise plumbing systems and their involvement in various metabolic pathways. Microbial community analysis showed Proteobacteria, Bacteroidetes, Acidobacteria, Nitrospira, and Actinobacteria to be the most abundant phyla across the samples which was found to be significantly different when grouped by age of the properties, location, and the type of house. Meanwhile, opportunistic premise plumbing pathogens such as Mycobacterium, Citrobacter, Pseudomonas, Stenotrophomonas, and Methylobacterium were observed from the samples at 0.5% of the total reads. Functional prediction using 16S gene markers revealed the involvement of the biofilm communities in different metabolic pathways like nitrogen metabolism, biodegradation of xenobiotics, and bacterial secretion implying diverse functionalities that are yet to be studied in this environment. This study serves as a preliminary survey on the microbial communities harboring premise plumbing systems in a tropical region like Singapore.

Molecular detection of opportunistic premise plumbing pathogens in rural Louisiana's drinking water distribution system

Opportunistic premise plumbing pathogens (OPPPs) in drinking water distribution systems are responsible for causing numerous infections such as Legionnaires’ disease and pneumonia through the consumption of contaminated drinking water. The incidence of opportunistic pathogens and the number of individuals at risk of contracting infections caused by these OPPPs in drinking water has risen drastically in the past decade. Preflush and postflush water samples were collected from 64 houses in a rural town in northeast Louisiana to determine drinking water quality in terms of understanding abiotic and biotic factors on potential proliferation of OPPPs. Physical and chemical water quality parameters, such as pH, temperature, dissolved oxygen, salinity, and specific conductance were also measured. The quantitative polymerase chain reaction (qPCR) results indicated that Legionella spp. had the highest prevalence and was found in 46/64 samples (72%), followed by Mycobacterium spp. which was found in 43/64 samples (67%), E. coli in 31/64 samples (48%) and, Naegleria fowleri in 4/64 samples (6%) respectively. The results indicate the persistence of Legionella spp. DNA marker in these water samples.

Public Health Risks Associated with Heavy Metal and Microbial Contamination of Drinking Water in Australia.

Recently in Australia concerns have been raised regarding the contamination of municipal drinking water supplies with lead. This is of particular concern to children due to the impact of lead exposure on cognitive development and as such these findings have received much media attention. The response from legislators has been swift, and The Victorian School Building Authority has announced that all new schools and school upgrade works will only use lead-free tapware and piping systems. However, while the immediate replacement of lead-containing brass fittings may seem a logical and obvious response, it does not consider the potential implications on microbial contamination. This is particularly concerning given the increasing public health threat posed by opportunistic premise plumbing pathogens (OPPPs). This commentary explores this public health risk of lead exposure from plumbing materials compared to the potential public health risks from OPPPs. Non-tuberculous mycobacterium was chosen as the example OPPP, and the influence on plumbing material and its public health burden in Australia is explored. This commentary highlights the need for future research into the influence of plumbing material on OPPPs prior to any changes in legislation regarding plumbing material.

Reverse QMRA as a Decision Support Tool: Setting Acceptable Concentration Limits for Pseudomonas aeruginosa and Naegleria fowleri

Opportunistic premise plumbing pathogens such as Pseudomonas aeruginosa and Naegleria fowleri are a growing concern in building water systems because of their potential risks to human health. The aim of this study was to determine the critical concentrations of P. aeruginosa and N. fowleri in water that are associated with meaningful public health risks. To determine these concentrations, a reverse quantitative microbial risk assessment (QMRA) was conducted. Environmental concentrations of P. aeruginosa and N. fowleri corresponding to the risk target of one micro-disability-adjusted life year (DALY) per person per year and 10−4 annual risks of illness were calculated for several applicable exposure scenarios. To calculate the concentration of P. aeruginosa, cleaning contact lenses with potentially contaminated tap water in the absence of an appropriate cleaning solution was considered. For N. fowleri, two exposure scenarios, recreational exposure (swimming) and nasal cleansing (via the use of a neti pot™ or similar device) were considered. The highest critical concentration for P. aeruginosa was found to be 33 CFU/L with a 95% confidence interval of (2.0, 118) for the drop exposure scenario using the 10−4 annual risk target. For N. fowleri, based on the DALY approach, critical concentrations were 0.000030 N. fowleri/L for swimming and 0.00000060 N. fowleri/L for neti pot™ use scenario. Considering heat inactivation, the critical concentration limits for P. aeruginosa using the DALY approach and the 10−4 annual risk target approach were found to be 0.55 CFU/L and 55 CFU/L, respectively. For N. fowleri, the 10−4 annual risk target approach resulted in 0.022 N. fowleri/L and the DALY approach resulted in 0.00000064 N. fowleri/L for the neti pot™ scenario. For P. aeruginosa, N50 (the median infective dose) and alpha (α) contributed the most and contact rates the least to the variability and uncertainty of the estimates for all the scenarios. For N. fowleri, N50 and contact rates contributed the most and α the least to the variability and uncertainty to calculate the concentrations for all the scenarios. The QMRA framework implemented in this research can be used to incorporate more information regarding opportunistic pathogens to inform management decisions, and to prioritize the best interventions regarding estimated reduction in infections caused by opportunistic pathogens.