Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila , Mycobacterium avium , and Pseudomonas aeruginosa

1Because community water systems supply water to > 95% of the approximately 300 million people in the United States, there is a need for a firm understanding of the precise contribution of these water systems to the spread of waterborne disease. Community water systems deliver water to premise plumbing, which is the portion of the water distribution system beyond the property line and includes households, office buildings, and hospitals. Therefore, premise plumbing serves as the interface of exposure of people to the microbes inhabiting their water supply.

2Several unique features of premise plumbing can increase risk of microbial infection. High surface-to-volume ratio, intermittent stagnation, low disinfectant residual, and warming cycles can stimulate growth of waterborne pathogens. High surface-to-volume ratios of premise plumbing means that there is a large area conducive for biofilm formation. Biofilms can be attractive habitats for pathogens and offer protection from disinfectants. Opportunistic pathogens have been found to grow in shower heads, faucets, along pipe walls, and in water heaters. The long residence time of water in premise plumbing enhances biofilm formation, including growth of resident pathogens. Although greater water ages are thought to enhance attenuation of traditional enteric pathogens, opportunistic pathogens can adapt and grow at low oxygen levels characteristic of stagnation in premise plumbing.

3Herein, we review the epidemiology and ecology of opportunistic premise plumbing pathogens (OPPPs), focusing on three of the most commonly tracked bacterial agents: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. Infections by all three have been linked to human exposure via premise plumbing (Table 1). It has been estimated that the costs of the estimated 29,636 cases of OPPP disease per year is approximately $850 million.

4Until recently [e.g., 1976 (Legionella), 1980 (Mycobacterium), 1997 (Pseudomonas aeruginosa)], there was no consideration of pathogenic microorganisms that were natural inhabitants of water and drinking water systems. In the past, the term “waterborne pathogens” referred to those agents in human or animal waste that entered water as contaminants with ingestion of drinking water, the principle route of exposure and infection. Classically, these included polio virus, Shigella, Salmonella, and other enteric bacteria, all examples of pathogens with a fecal–oral route of infection. Those pathogens are not normal inhabitants of water but contaminants, and generally are not capable of reproduction in the water supply. Operationally, one can identify the point source for such “waterborne pathogens” by moving upstream as their numbers increase as they get closer to the point source. In contrast, the numbers of OPPPs increase as the distance from the treatment plant increases because they can multiply in pipes and plumbing systems. Further, OPPP numbers do not correlate with fecal coliform numbers.

5Increasingly, the role of biofilms within water distribution systems and premise plumbing is recognized as important for the establishment and maintenance of the chronic colonization associated with L. pneumophila, M. avium, and P. aeruginosa. Biofilms and amoebic host organisms can protect these pathogens from effective treatment with disinfectants, such as chlorine.

6On 25–27 March 2012, a Water Research Foundation–sponsored expert workshop, “Research Needs for Opportunistic Pathogens in Premise Plumbing,” was held at the Virginia Tech Northern Virginia Center, Falls Church, Virginia, to document the widespread prevalence and impact of OPPPs on humans and to review their epidemiology and ecology and identify common features contributing to the infective process. The 2-day workshop assembled > 50 experts in drinking water and waterborne pathogens to a) review the state of knowledge of OPPPs, b) identify gaps in knowledge of OPPPs, and c) identify and prioritize research objectives. This literature review serves to summarize the state of the knowledge with respect to epidemiology and ecology of key bacterial OPPPs, which may better inform the development of approaches to reduce human exposure and infection. Key knowledge gaps identified at the expert workshop that should be prioritized for future research are also described.

7There are three major presentations of Legionella spp. infection: a) Pontiac fever and b) either community-acquired or c) outbreak-associated Legionnaires’ disease. Pontiac fever is an influenza-like, mild illness that is spontaneously resolved without therapy and is caused by a variety of Legionella spp. from either water or soil. The focus here will be on L. pneumophila, the causative agent of serious, life-threatening pneumonia (“Legionnaires’ disease”), often requiring hospitalization. In the United States, the most recent estimated number of hospitalized cases, based on a population-based study in 2000–2009, is 8,000–18,000. Moreover, the number of reported legionellosis cases increased 3.5-fold between 2000 and 2011 (Table 1);. Legionellosis is an acute illness and, in its most severe form, is generally responsive to timely and appropriate antimicrobial therapy.

8In addition to air-conditioning systems and cooling towers as sources, it is now understood that drinking water is an important source of L. pneumophila (; Boccia 2006;;;; ). L. pneumophila recently became the single most common cause of reported disease outbreaks involving drinking water, in part because of improved detection and diagnosis and also likely because it is the first OPPP-associated disease that must be reported to the National Notifiable Diseases Surveillance System (http://wwwn.cdc.gov/nndss/).

9Community-acquired L. pneumophila pneumonia. The prevalence of community-acquired and healthcare-associated legionellosis are both increasing. One-quarter (25%) of Legionella spp. infections are healthcare associated. Because L. pneumophila is present in drinking water distribution systems and household water, persons at risk for Legionnaires’ disease should take precautions. Risk factors for Legionnaires’ disease include reduced immune competence, smoking, alcoholism, and older age. Case reports are highest in summer and in the mid-Atlantic region of the United States.

10Outbreak-associated L. pneumophila pneumonia. Legionnaires’ disease was first described as an outbreak of pneumonia amongst attendees of an American Legion convention in Philadelphia, Pennsylvania, in 1976. In the absence of evidence of person-to-person transmission, it was hypothesized that the infective agent originated from the environment. Since that first report, outbreaks of L. pneumophila disease have been linked to water sources in hospitals, hotels, cruise ships, industrial facilities, and multiple and single family residences. The British Communicable Disease Surveillance Centre reported that 19 of 20 hospital outbreaks of Legionnaires’ disease in the United Kingdom from 1980 to 1992 were primarily attributed to hospital water systems. Legionella spp. in hospital drinking water samples have been linked to patient isolates by DNA-fingerprinting methods Consequently, Legionnaires’ disease should be considered for all pneumonia cases with prior hospital exposure, particularly the elderly, smokers, immunosuppressed, and those with chronic lung disease.

11The natural habitat for Legionella appears to be aquatic bodies including rivers, streams, and thermally polluted waters. Legionella bacteria have been detected in all segments of water distribution—from the source water (rivers and groundwater) to the tap. Natural aquatic bodies contain only small numbers of Legionella. The presence of Legionella in a water distribution system is not necessarily an indication that the system is poorly maintained because this bacterium may be a normal constituent of the microbial population of water distribution systems. It has been estimated that Legionella are found in approximately 50% of large building water systems and 10–30% of home water systems in the United States (; Stout and Yu 2011), and detection methods are becoming increasingly sensitive. Depending on the study and methods, a range of 12–70% of hospital water systems are estimated to be colonized with Legionella. A recent publication demonstrated the presence of “nonculturable” cells of L. pneumophila as well as methods for their resuscitation, to ensure that colony counts are not underestimated. In the first national study to use more sensitive molecular techniques, Legionella genetic material was detected in 50% of cold water samples.

12Cooling towers and, to a lesser degree, evaporative condensers were implicated in the earlier outbreaks prior to recognition of potable water as a reservoir. The emphasis of cooling towers in the dissemination of Legionella has been challenged. Reports of cooling towers as reservoirs for legionellosis have dwindled in comparison with those linked to building water distribution systems.

13Legionella are not completely eliminated from drinking water by standard water treatment practices. For example, Legionella are comparatively more resistant to chlorine than Escherichia coli. Legionella are also known to be sheltered within encysted amoebae; indeed, after phagocytosis by amoebae, whose cells are relatively chlorine-resistant, Legionella can survive up to 50 ppm chlorine. Legionella growth and proliferation occur in engineered habitats, especially water distribution systems, which provide favorable water temperatures (25–42°C), surfaces for biofilm formation, and nutrients. One important factor appears to be water temperature. Buildings with recirculating hot water distribution systems colonized with L. pneumophila were significantly more likely to have lower hot water heater temperatures (< 60°C) than systems that were not colonized. The microorganism is readily found in biofilm and detritus at the bottom of hot water tanks. Bacteria, protozoa, and amoebae also colonize water pipe surfaces, some of which have been shown to promote Legionella replication. Legionella and other microorganisms attach to surfaces and form biofilms on pipes throughout the water distribution system. Cold-water sources, such as ice from ice machines and water from fountains with stable biofilm-colonized surfaces, have also been implicated as a source of infection.

14Multiple modes have been identified for transmission of Legionella to humans; there is evidence for aerosolization, aspiration, or even instillation into the lung during respiratory tract manipulation. Because one of the first environmental isolations of L. pneumophila was from a showerhead, it has been widely thought that aerosols from showers may be an important means for dissemination of this microorganism. However, as Legionella are prevalent in home water systems, any shower or faucet can be a source of infection, and detectable airborne Legionella aerosols have been detected in proximity to faucets.

15Aspiration of contaminated water or oropharyngeal secretions appears to be the major mode of transmission in the hospital setting. Colonization of oropharyngeal flora by L. pneumophila is a theoretical possibility, and the evidence for aspiration has accumulated. Nasogastric tube placement has been shown to be a significant risk factor for healthcare-associated legionellosis in intubated patients; microaspiration of contaminated water was the presumed mode of entry. It is possible that ingestion of water also can play a role. During the original 1976 outbreak, consumption of water and possible aspiration at the implicated hotel was associated with acquisition of disease—an association that has been generally overlooked.

16Healthcare personnel frequently use tap water to rinse respiratory apparatus and tubing used for ventilators. If the tap water contains L. pneumophila, the bacteria could possibly be instilled directly into the lung of a patient. In numerous studies, the risk of Legionnaires’ disease was significantly greater for patients who underwent endotracheal tube placement more often or had a significantly longer duration of intubation than for patients who had other causes of pneumonia. Use of sterile water for all nasogastric suspensions, for humidifiers in breathing circuits of mechanical ventilators, and for flushing tubes has been recommended to prevent Legionella infection.

17M. avium infections are known to originate from environmental sources. It is one among 175 species of the genus Mycobacterium that do not belong to the Mycobacterium tuberculosis complex and thus are called nontuberculous mycobacteria (NTM). Our focus is on M. avium as the causal agent of the majority of NTM infections in the United States, and it is the most prevalent Mycobacterium in drinking water.

18There are three major presentations of M. avium infection: a) bacteremia in HIV-infected individuals, b) cervical lymphadenitis in young children, and c) community-acquired M. avium infection in adults. There have been few reports of M. avium disease outbreaks, and these tend to be associated with contamination of solutions and instruments in hospitals. Reports have also linked isolation of M. avium from bronchoscopes, ice, whirlpool tubs, pools, footbaths, and prepared cleaning and irrigation solutions. M. avium bacteremia among HIV-infected and immunosuppressed persons emerged during the 1980s. At one time, approximately 50% of late-stage AIDS patients had M. avium bacteremia, but with the implementation of highly active antiretroviral therapy, the number of HIV-infected patients with M. avium infections has fallen dramatically. Although there are no national statistics documenting numbers of M. avium–associated cervical lymphadenitis in children, there has been no published evidence of this manifestation disappearing or increasing. In a comprehensive, prospective study of cervical lymphadenitis published in 1995 by a physician evaluating incident cases in the United States over the period of 1958–1990, only 105 cases were found. The age of the infected children (median age 3 years) suggests that exposure to water or soil containing M. avium, coupled with gum trauma due to erupting teeth, led to M. avium infection of the lymph nodes of the head and neck.

19Community-acquired M. avium infection. Currently, the majority of M. avium cases are community acquired. As disease caused by M. avium is not nationally notifiable in the United States, population-based studies are uncommon, and the public health burden of M. avium disease is difficult to measure. Two recent population-based studies in the United States have described rates of M. avium disease as increasing among older persons and women (Table 1). Estimated prevalence of M. avium lung disease varies by study, location, age, and susceptible population; prevalence increases with age and is highest among patients with AIDS, at 647 cases/100,000 persons.

20Among studies that evaluated numbers of M. avium isolates recovered from clinical specimens, rates of positive cultures appear to be increasing. These include reports from the United States, Japan, Canada, Taiwan, South Korea, and China. The increase in M. avium isolation and disease frequency has also coincided with a shift in the observed epidemiology, from disease occurring principally in older men with reduced lung function due to smoking or occupational dust exposure, to tall, slender, and older women. Among studies where age is reported, older age was associated with a higher prevalence of disease. However, factors (e.g., improved detection) other than age alone may be associated with increased rates reported per year in many studies, even in countries with aging populations.

21Both pulmonary and extrapulmonary M. avium infections have been described. Extrapulmonary sites of M. avium infection include skin and soft tissue, catheter-related bacteremia, exit-site infection, surgical site infections, and infections such as peritonitis, lymphadenitis, keratitis, and osteomyelitis. M. avium disease typically involves subacute to chronic infections that are difficult to treat and are frequently resistant to antimicrobials. Susceptibility to M. avium disease is poorly characterized; however, multiple host risk factors have been associated with M. avium infection. Competent cell-mediated (innate) immunity is an essential first line of defense against mycobacterial infections; defects in immune response pathways or in T-cell function may be a risk factor for infection. Chronic lung diseases, such as chronic obstructive pulmonary disease, bronchiectasis, silicosis, cystic fibrosis, alveolar proteinosis, pneumoconiosis, and previous tuberculosis, have all been identified as risk factors for NTM-associated pulmonary disease. Other risk factors for NTM pulmonary disease include connective tissue disorders and abnormal cystic fibrosis genotypes.

22Multiple reports have linked M. avium disease with exposure to drinking water, using molecular typing methods. Closely related M. avium isolates have been recovered from hospitalized patients and water in the hospitals, from residents and their household water, and from a resident and his/her municipal water. Strains of M. avium isolated from humans have been shown to be competent biofilm formers. An in vitro study suggested that a M. avium strain’s ability to form biofilm is associated with pathogenicity and invasion of bronchial epithelial cells.

23Many factors appear to interact in complex and poorly characterized ways to support or inhibit M. avium growth in water pipes. Factors include temperature, water flow, nutrients, pipe material and condition, residual disinfectant, free-living phagocytic amoebae, mycobacteriophages, and other bacteria. Reports implicate some of these risk factors, but none alone predict M. avium concentration at the point of use. Concentrations of M. avium in water were significantly correlated with organic carbon concentrations, hot water plumbing lines, and plastic pipe material, although reported significant M. avium concentrations in water independent of pipe material.

24M. avium has been isolated from multiple environmental sources, including from water and biofilm. Reports of M. avium in water distribution and treatment plants suggest that biofilms that form in the distribution system act as an important niche for the survival of M. avium. Further, M. avium, like L. pneumophila and P. aeruginosa, is an amoeba-resisting microorganism, able to grow and survive in amoebae. M. avium is approximately 500 times more resistant to chlorine than Escherichia coli and 40 times more tolerant to chlorine than P. aeruginosa. Further, M. avium survives and multiplies in distribution systems despite ambient chlorine residual concentrations. M. avium grown in water is more chlorine resistant than the same strains grown in culture medium, and most strains are more resistant to chloramine than to free chlorine.

25Drinking water is a known environmental source of NTM and has been extensively studied in an attempt to characterize the risk of human exposure to NTM. Quantitative interpretation of the results of these studies is problematic because isolation methods vary and because decontamination steps to prevent the growth of more rapidly growing microorganisms are known to reduce concentrations of M. avium in water samples. Unfortunately, there are no selective or differential media for the cultivation of NTM from samples containing other microorganisms. Therefore, observed and reported occurrence in water samples should be interpreted as conservative estimates of true occurrence and abundance. M. avium isolation from drinking water has been documented at points of use within both public and private buildings.

26Water is a well-documented source of M. avium exposure. M. avium isolation from hospital water supplies is of particular concern due to the potential for exposure of immunosuppressed patients. The persistence of a single clone of M. avium (up to 18 months) in hospital and distributed municipal drinking water as a potential chronic source of human exposure is a major challenge. It is important to point out that identification of identical M. avium clones in patients and their household plumbing does not necessarily indicate that the tap water is the original source, especially if the patients collected the samples. It could be that patients continually reinfect their own taps.

27Filters have been recommended as a means to reduce M. avium exposure from water. However, some types of filters, that is, granular activated charcoal (GAC) filters, have been shown to be colonized by M. avium and support their growth. Consequently, the GAC filter becomes a source of M. avium and likely the other OPPPs.

28There are four major presentations of P. aeruginosa infection (Table 1): a) bacteremia in immunocompromised individuals, b) pneumonia in cystic fibrosis (CF) patients, c) community-acquired ear and pneumonia infections, and d) hospital-acquired outbreaks, principally associated with contaminated solutions or medical devices used in general patients or those in intensive care units (ICUs). In all four presentations, water containing P. aeruginosa is the source of infection.

29Community-acquired pneumonia. Very few data are available about the occurrence of P. aeruginosa disease outside of the healthcare setting. Much of what is known about infections in the community setting is from reports in the published literature (e.g., case reports, outbreak investigations). Infections produced by P. aeruginosa are not nationally notifiable, so the burden of disease is difficult to assess. The primary infections (ear and skin) acquired in the community involve the use of swimming pools, hot tubs, and whirlpools where there has been a failure to maintain the equipment or to maintain sufficient residual disinfectant. P. aeruginosa is rarely carried by healthy individuals (2–10% of individuals, likely in the ear) but can be recovered from 50–60% of hospitalized patients. P. aeruginosa is a major cause of otitis externa (“swimmer’s ear”), with a magnitude of 2.4 million cases per year and an estimated outpatient cost of approximately $500 million.

30Hospital-acquired infections. In a meta-analysis of 43 water-associated outbreaks in hospitals covering 35 years (1966–2001), it was estimated that there were approximately 1,400 nosocomial pneumonia deaths per year in the United States caused by P. aeruginosa. Among gram-negative healthcare-associated pathogens, P. aeruginosa is the second most frequent pathogen causing ventilator-associated pneumonia, and the third or fourth most frequent pathogen causing septicemia, urinary tract infections, and surgical wound infections. A 10-year molecular epidemiological study attempted to determine the respective roles of exogenous and endogenous flora and time on infection and the effect of P. aeruginosa infection in ICU patients. Isolates fell into three types: 1) identical patient and faucet isolates; 2) identical patient isolates, but none in faucets; and 3) unrelated patient and faucet isolates. Higher levels of faucet contamination with P. aeruginosa were correlated with higher numbers of cases in type 1; namely 34/1,000 patient admissions. The number of type 3 or “endogenous” cases was considerably lower and stable over time. Two studies of P. aeruginosa transmission among ICU patients, using either pulsed-field gel electrophoresis (PFGE) or arbitrary-primed polymerase chain reaction (AP-PCR), established that patient isolates contaminated faucets. This information should be considered when hypothesizing transmission pathways involving other OPPPs; for example, are M. avium–infected patients the source of identical clones of M. avium in household plumbing?

31P. aeruginosa in cystic fibrosis. Cystic fibrosis (CF) patients become colonized early in life with P. aeruginosa, and the prevalence of colonization increases with age. There are 30,000 cystic fibrosis patients in the United States, and 1,000 new patients are diagnosed annually. P. aeruginosa is a major pathogen of CF patients; 60–80% of CF patients are infected. Although a proportion of CF patients are infected with P. aeruginosa from other patients (cohabitating hospital wards), the major source of infection is water.

32P. aeruginosa is a ubiquitous gram-negative bacillus commonly associated with soil and water. It has minimal nutritional requirements, which enables it to survive in many different environments; it is especially known for possessing a characteristically large genome with vast metabolic capabilities, and it is a model biofilm-forming organism. In addition to the ability of pseudomonads to grow on a wide variety of organic compounds, P. aeruginosa is resistant to chlorine and other disinfectants used in water treatment. P. aeruginosa has been isolated from water, antimicrobial soaps, as well as disinfectant solutions and chlorine-based sanitizing solutions. P. aeruginosa is widely detected in a variety of aquatic environments, including tap water; it is notoriously subject to multiple antibiotic resistance, and therefore P. aeruginosa infections can be very difficult to treat.

33The role of tap water as a source of P. aeruginosa disease has been established in a number of published studies. The modes of transmission have included direct contact with water and aerosols, aspiration, indirect transfer from moist environmental surfaces, and via healthcare worker hands. Several studies used molecular markers to demonstrate relatedness of tap water and patient isolates. Tap water samples from sinks within ICUs have been found to contain P. aeruginosa strains that were identical by molecular typing to those obtained from infected and colonized patients. Tap water faucets were colonized with the same P. aeruginosa strain for > 2 years, even though P. aeruginosa was not recovered from the mains supplying the sinks. Tap water and outlets appear to be the reservoir for P. aeruginosa within healthcare facilities. However, in a surveillance study of hospitalized patients, most were colonized before admission. Based on the identity of isolates from both water and patients, P. aeruginosa in tap water was found to be the source of infection in 1 of 14 patients.

34Information available for disease incidence for each bacterial OPPP that we focus on in this review are listed in Table 1, with two amoebal OPPPs also indicated for comparison as emerging and serious health threats. In particular, Naegleria fowleri, a brain-eating amoeba that prefers warm aquatic environments, was recently detected in premise plumbing and linked to high-profile deaths in the southern United States, with investigations ongoing. Overall, it is expected that the number of people susceptible to infection with OPPPs will grow in the United States. Cystic fibrosis patients, transplant recipients, and immunosuppressed patients are living longer lives, resulting in more time to become colonized or infected. For example, the U.S. population is aging, and it is estimated that the proportion of individuals > 60 years of age will increase from 16.1% in 2000 to 24.8% in 2025. Such an increase in individuals > 60 years of age, coupled with higher rates of infection by OPPPs in that age group, strongly suggests that the prevalence of OPPP-related disease will rise. Our public water systems are deteriorating and in desperate need of maintenance and replacement; thus, there are increased opportunities for intrusion of soil-associated OPPPs and for biofilm formation, which favors their growth. At the same time, exposure occurs via premise plumbing, and knowledge of the interaction between the chemistry and microbiology of municipal water and premise plumbing is needed to inform risk management strategies.

35There are several traits shared by the three bacterial OPPPs that select for their presence and persistence in premise plumbing. Those traits include disinfectant resistance, biofilm formation, survival at high temperatures, and growth in free-living phagocytic amoebae. The concentrations of water treatment disinfectants (e.g., chlorine, chloramine) required to kill 99.9% of the bacterial OPPPs are higher than those needed to obtain a 3-log reduction in Giardia lamblia cysts, the standard used for water disinfection. The presence of residual disinfectant provides these resistant OPPPs a competitive advantage. One factor likely leading to the occurrence and persistence of the bacterial OPPPs in premise plumbing and distributions systems is their ability to adhere to surfaces and form biofilms. Bacteria in biofilms are also more resistant to disinfectants. Residence in biofilms also makes bacterial OPPPs more accessible to free-living, phagocytic amoebae [e.g., Acanthamoeba and Vermamoeba (formerly called Hartmanella], which can actually enhance their proliferation in drinking water. All three bacterial OPPPs belong to the category of amoeba-resisting microorganisms: they are not necessarily killed by amoebae following phagocytosis but can actually survive and grow. Finally, because these OPPS have relative resistance to high temperatures that are encountered in hot water pipes (e.g., 35–45°C), their numbers actually increase in hot water heaters and premise plumbing.

36Currently, there are no documented broadly effective community-level engineering control strategies for OPPPs in municipal drinking water or premise plumbing. Identification and use of methods to effectively and economically control OPPPs is in its infancy. Preventative measures could be imposed by water utilities, healthcare operators, and homeowners (Table 2). Although a simple increase in disinfectant concentrations is possible, it would likely be counterproductive in that it would create taste and odor problems, and additional chlorination may result in potentially carcinogenic disinfection by-products. In addition, increasing the disinfection concentrations might be ineffective because the OPPPs are very resistant to disinfectants, particularly those encased within amoebae cysts.

37Participants of the workshop, “Research Needs for Opportunistic Pathogens in Premise Plumbing,” took the view that novel approaches are needed: for example, manipulation of the chemistry or microbiome of drinking water distribution systems and premise plumbing. A recent review identified specific challenges for in-building control of Legionella, much of which is driven by limitations of water chemistry, scaling, and corrosion control. Even if possible measures are identified, there may be wide variance in outcomes because of differences in types of disinfectant, organic carbon levels, pipe composition, system design, water chemistry, and even bacterial species and strains. At the level (and responsibility) of the water provider, reduction of turbidity and organic carbon levels and the employment of biofilm-discouraging pipes (e.g., antimicrobial coated or impregnated) might prove useful (Table 2). For the hydrophobic M. avium and other mycobacteria, turbidity reductions do reduce numbers, most likely as the cells enter the treatment plant adhered to soil particulates. Reducing organic carbon might also be expected to reduce OPPP concentrations because all three of the species discussed herein are heterotrophs, requiring organic carbon for growth. Although speculative, it might be possible to reduce OPPP biofilm numbers by pretreating distribution and plumbing pipes with agents (e.g., antimicrobials or surfactants) that reduce adherence and biofilm growth.

38Individuals or building managers can also employ measures to reduce OPPP numbers in premise plumbing (Table 2). First, temporary high disinfectant concentrations or high-temperature water can be applied to a building plumbing system to kill viable OPPPs. Although that has been performed successfully, the OPPPs occasionally return, sometimes in higher numbers than were present before the temporary treatment—likely due to necrophilic growth. Point-of-use microbiological filters have been used in some healthcare settings to prevent exposure of patients to OPPPs or to prevent contamination of solutions that may come in contact with patients (e.g., dye, disinfecting solutions). However, those filters can, over time, become sources of OPPPs.

39One outcome of the 2012 expert workshop sponsored by the Water Research Foundation was a listing of research gaps in our understanding and knowledge of the bacterial OPPPs. Those research gaps specifically focused on epidemiology and ecology of OPPPs are listed in Appendix 1 and are ordered by priority as judged by the experts participating in the workshop. Of importance above all research needs identified at the workshop was the determination of the prevalence, incidence, and trends of disease caused by OPPPs. Because disease caused by M. avium and P. aeruginosa is not required to be reported, the full impact of disease caused by these OPPPs can only be estimated—and those are underestimates. In addition, it will be important to determine the relationship between OPPP densities (infectious dose) and disease. Two factors make such risk–analysis calculation difficult: The OPPPs vary widely in virulence (even within single species) and individuals vary widely in susceptibility.

40Although the focus of the workshop and this review is on three OPPPs, there are other opportunistic pathogens in premise plumbing. As mentioned above, amoebal OPPPs, such as N. fowleri and Acanthamoeba, are of growing concern both as pathogens and as hosts for bacterial OPPPs. Other important bacterial OPPPs include Acinetobacter spp., Stentrophomonas spp., Brevundimonas spp., Sphingomonas spp., and Chryseobacterium spp.. In addition, the slow-growing, lipid-rich, mycobacterial-like Segniliparus spp. isolated from cystic fibrosis patients are also waterborne OPPPs.

41One novel research concept that was identified by the workshop was the possibility that the microbiome of a water distribution system or premise plumbing could be manipulated to influence the presence of OPPPs. Studies of that type might provide understanding of the distribution system microbiome to aid in predicting when colonization by OPPPs would be favored. This idea was reviewed recently by. Putting such an idea into practice would not only require study of the roles of other bacteria but also better understanding the role of bacterial viruses (bacteriophage) and amoebae. Evidence that a switch from chlorine disinfection to chloramine resulted in an absence of Legionella but an increase in M. avium illustrates the complexity of the challenge presented by OPPPs and deserves further exploration and research. Studies of the impact of disinfection methods should include the use of ultraviolet irradiation, an agent that not only results in killing but also in mutations. Some studies also indicate that overuse of chlorination can also enhance selection of antibiotic-resistant pathogens. Because the OPPPs belong to the category of amoeba-resisting microorganisms, a thorough investigation of the role of free-living, phagocytic amoebae in supporting the persistence of OPPPs in premise plumbing is warranted. There is a dynamic, changing relationship between OPPPs and amoebae over time. Thus, the interaction between OPPPs and free-living phagocytic amoebae and disinfectant regulation needs careful examination to provide a guide for remedial measures. As discussed above concerning remediation and control of OPPPs, it will be important to determine whether microbial ecologic controls could effectively reduce human exposure to OPPPs.

42Determine the prevalence, incidence, and trends of disease caused by OPPPs

43Determine the dose–response relationship between OPPP numbers and disease

44Determine whether the microbiome of a distribution system is a determinant of the premise plumbing microbiome

45Determine the role of free-living phagocytic amoebae species on the prevalence, persistence, growth, and survival of OPPPs

46Determine whether microbial ecologic controls could reduce exposure to OPPPs

47Determine the contributions of bacterial, viral, and eukaryotic microorganisms to the persistence of OPPPs

48Determine the impact of disinfection methods, including ultraviolet irradiation, on the emergence of antibiotic-resistant OPPPs.