Probiotic Approach to Pathogen Control in Premise Plumbing Systems? A Review

Critical Review pubs.acs.org/est

Probiotic Approach to Pathogen Control in Premise Plumbing Systems? A Review Hong Wang,† Marc A. Edwards,† Joseph O. Falkinham, III,‡ and Amy Pruden†,* †

Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States ‡ Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States ABSTRACT: Opportunistic pathogens occurring in premise (i.e., building) plumbing systems, including strains of Legionella, Mycobacterium, Acanthamoeba, and Pseudomonas, are now frequently cited agents of waterborne disease outbreaks. Unlike traditional fecal pathogens, opportunistic pathogens are part of the drinking water microbial ecology and therefore require new paradigms for their control. With the onset of the “microbiome era”, notions of eradicating all microbes in drinking water have proven unrealistic, making a probiotic concept worthy of consideration. Research is needed to better understand how engineering controls may individually, or in combination, select for a desirable microbiome, and how the microbiome itself may mediate proliferation of opportunistic pathogens. Ecological interactions such as competition, antagonism, and obligate parasite-host relationships offer potential targets for probiotic control of opportunistic pathogens. A probiotic approach may be defined as intentional inoculation of beneficial microbes or choosing conditions that select for a desirable microbiome. This critical review synthesizes the state of the knowledge of the factors governing opportunistic pathogen control in premise plumbing and potential opportunities for and barriers to implementation of a probiotic approach. Future effort is recommended to demonstrate the feasibility of the probiotic concept; to develop effective, practical, and safe protocols; and to engage relevant stakeholders in evaluating options and assessing corresponding risks.

1.0. INTRODUCTION Supply of safe potable water in buildings is paramount to public health. Although chemical contaminants in drinking water are also of concern, the vast majority of water-related health problems are caused by microbes (bacteria, viruses, protozoa, etc.).1 As early as the late 19th century, the importance of centralized water systems for controlling communicable diseases has been recognized.2 Disinfection of distributed drinking water, first implemented about 100 years ago, is credited with the control of several deadly waterborne diseases such as cholera, typhoid fever, and other diarrheal illnesses.2 For example, occurrence of typhoid fever per 100 000 people in the United States dropped from 100 cases in 1900 to 33.8 cases in 1920 and to 0.1 cases in 2006. Drinking water treatment and disinfection is considered to be one of the greatest public health achievements of the 20th century.3 However, in recent years the challenge of delivering safe, pathogen-free potable water has shifted. No longer is waterborne infection primarily due to fecal/gastrointestinal pathogens that escape from multiple drinking water treatment barriers, but instead to opportunistic pathogens who are indigenous drinking water microbes residing in premise (i.e., building) plumbing systems. Specifically, opportunistic pathogens; including Legionella spp., nontuberculosis mycobacteria (NTM), Pseudomonas aeruginosa, and Acanthamoeba spp., are © 2013 American Chemical Society

now recognized as the leading source of waterborne disease outbreak in developed countries in terms of increased prevalence4−8 and healthcare burden.9 These organisms are defined as “opportunistic” pathogens because they primarily cause disease in individuals with certain risk factors.10 In contrast to fecal-source pathogens, which are incapable of prolonged survival in drinking water distribution systems, opportunistic pathogens naturally colonize, persist, and multiply in potable water plumbing systems. Also noteworthy, inhalation of aerosols and direct contact are their primary transmission routes, instead of ingestion, which has traditionally been the primary exposure route of concern.11 The purpose of this critical review is to synthesize the state of the knowledge of opportunistic pathogens in premise plumbing systems and to provide a framework for opportunistic pathogen control that takes advantage of unique aspects of their microbial ecology. In particular, we explore the hypothesis that a “probiotic” approach may be appropriate for control of opportunistic pathogens in premise plumbing. Rather than continuing futile attempts to eradicate all microbes, a probiotic Received: Revised: Accepted: Published: 10117

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pathogens are of clinical importance with evident links between infections and premise plumbing sources. 2.2.1. Legionella. Legionella, in particular Legionella pneumophila, are the causative agent of a potentially fatal form of pneumonia called Legionnaires’ disease and a milder respiratory illness called Pontiac fever.28 It was estimated that Legionella infection hospitalized 8000−18 000 people29 and cost about $433.8 million in healthcare costs9 each year in the U.S. From 2005 to 2009, the death rate from Legionella infection was reported to be 8%.30 L. pneumophila was added to U.S. Environmental Protection Agency (U.S. EPA) drinking water candidate contaminant list (CCL) in 2009 due to increasing cases of Legionnaire’s disease and their association with drinking water systems, in particular premise plumbing.7,30,31 2.2.2. Mycobacterium avium complex (MAC). MAC contains a group of opportunistic pathogens in humans and animals whose source of infection is the environment. It is estimated that the prevalence of pulmonary NTM diseases was at least 8.6/100 000 persons in the United States, with MAC accounting for the majority of cases (i.e., about 87.5%).32 MAC pulmonary disease has increased in prevalence in recent years33 and was reported as the most expensive waterborne disease in terms of individual hospital visits (i.e., $1,649 per visit 9). Of particular concern is the fact that the majority of patients are slender, older women and men who lack classic risk factors for opportunistic infection.34 As the population of the United States continues to age, and older Americans live longer, it is expected that the number of individuals with MAC pulmonary disease will continue to increase. Several epidemiological studies have linked mycobacterial infection to tap water.35−37 2.2.3. Pseudomonas aeruginosa. P. aeruginosa is a leading cause of nosocomial dermal and disseminated infection in individuals with cancer, cystic fibrosis, and burns.38 P. aeruginosa infection can also cause pneumonia and bacteremia, which are associated with high mortality rates.39 Two studies reviewing recorded nosocomial P. aeruginosa infection from 1966 to 2001 and 1998−2005, respectively, revealed that hospital tap water is a source of disease-causing strains. It was estimated that 14.2−50% P. aeruginosa infection/colonization in patients were due to strains found in hospital water.5,40 P. aeruginosa infection were also linked to contamination of hot tubs41,42 and recreational water.43,44 However, P. aeruginosa infections outside the hospital environment were rarely reported, likely due to the facts that P. aeruginosa skin and ear infections are self-limiting44 and nonreportable. 2.2.4. Pathogenic Free-Living Amoebae (FLA). FLA are a group of eukaryotic microorganisms that are commonly found in the aquatic environment and soil. FLA have attracted recent attention in association with outbreaks of Acanthamoeba keratitis (AK)8,45 and occasional occurrences of fatal amoebic meningoencephalitis.46 Tap water was shown to be a source for pathogenic FLA, as evidenced by identical mtDNA profiles of clinical and water isolates6 and analysis of patients’ daily activity.46 As discussed in the following section, the greater public health concern regarding FLA in drinking water may be the role that they play in enhancing the growth of bacterial pathogens.47,48

approach would seek to foster establishment of a desirable microbiome, either by intentional inoculation or by choosing conditions that select for beneficial microbes and exclude pathogens.

2.0. THE DRINKING WATER MICROBIOME AND KEY OPPORTUNISTIC PATHOGENS OF CONCERN 2.1. New Insights into the Drinking Water Microbiome. From a historical engineering or consumer perspective, there exists a common misconception that drinking water is a sterile or nearly sterile environment. This myth is perpetuated by strict microbiological quality limits in main distribution systems (e.g., absence of total coliforms) and the assumption that disinfectants eradicate all microbes.12 Moreover, standard protocols by which samples are collected for microbial compliance testing typically employ extensive flushing of taps,13 which avoids detection of the microbes that naturally colonize premise plumbing. Premise plumbing bacterial levels are typically orders of magnitude higher than in the water main.11 In reality, drinking water systems actually harbor an astounding array of microorganisms, even in the presence of high disinfectant residuals. More than thirty heterotrophic bacterial genera have been isolated from drinking water, including: Acidovorax, Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Enterobacter, Flavobacterium, Klebsiella, Methylobacterium, Mycobacterium, Nocardia, Pseudomonas, Sphingomonas, Strenotrophomonas, and Xanthobacter,14−16 some of which contain pathogenic members. Over the past two decades, advances in culture-independent molecular techniques targeting DNA and RNA are shining new light into the profound depths of microbial diversity in drinking water. Poitelon17,18 reported an estimated richness of 173 to 333 bacterial and archael species (based on 16S rDNA cloning) and 24 eukaryotic species (e.g., protozoa, based on 18S rDNA cloning). αProteobacteria, β-Proteobacteria, γ-Proteobacteria, Actinobacteria, and Bacteroidetes were found to be the major classes of bacteria in finished drinking water by 16S rRNA or rDNA gene Sanger sequencing.19−21 Moreover, comparative RNA/DNA analyses indicate that drinking water microbes are not merely passive or dormant, but actively carrying out various functions within a stressed ecosystem.19,20 Over the past five years, nextgeneration DNA sequencing technologies, such as 454pyrosequencing and Illumina sequencing, have only served to crystallize the conclusion that the diversity of microbes in drinking water, and their corresponding metabolic capabilities, are indeed vast. Such methods provide access to millions of DNA sequences in a single reaction. Several recently published studies have applied next-generation DNA sequencing to drinking water samples, including associated biofilm from water meters,22 faucets,23 a pilot-scale microfiltration plant,24 and bulk water from different water treatment plants and distribution systems.25,26 Over 1000 species have been identified in drinking water-associated biofilm samples.23,24 Moreover, a recent metagenomic study of disinfected drinking water revealed a microbiome as functionally complex as that of the human gut.26 2.2. Emerging Premise Plumbing Opportunistic Pathogens. Drinking water opportunistic pathogens are of emerging public health concern. Although there are a variety of opportunistic pathogens drawing attention,27 this review will primarily focus on four groups of opportunistic pathogens including Legionella, M. avium complex (MAC), P. aeruginosa, and pathogenic free-living amoeba (FLA). All of these

3.0. PREMISE PLUMBING AS AN ECOLOGICAL NICHE FOR OPPORTUNISTIC PATHOGENS Premise plumbing includes the portions of the potable water distribution system beyond the service pipe (i.e., pipeline connecting a building to a main pipe) and inside of buildings 10118

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Figure 1. Premise plumbing as the idealized ecological niche for opportunistic pathogens.

prerequisite for Legionella growth in oligotrophic environments.68,73 Biofilms are prevalent in drinking water systems and can support opportunistic pathogen growth. For example, M. avium gene copy numbers were enriched in showerhead biofilms as much as 100-fold above those numbers in bulk water,57 illustrating their preference for the biofilm environment, owing in part to the hydrophobicity of mycobacteria.61 Biofilms can provide nutrients and shelter against disinfection. Figure 1 provides a few examples of factors known to influence opportunistic pathogens in drinking water biofilms. Biofilmloving, FLA selectively graze on indigenous bacteria for food by phagocytosis. However, some amoeba-resisting bacteria, such as L. pneumophila and M. avium, are able to multiply within the phagosome by inhibiting phagosome acidification and lysosome fusion. Upon depletion of nutrients, Legionella are released into drinking water via phagosome rupture and amoeba cell lysis.64,74 In some cases, Legionella-containing vesicles can be expelled into the surrounding environment by their amoeba hosts. Live Legionella cells were found in Acanthamoeba castellanii vesicles even after 6 months, implying their importance in Legionella life cycle and transmission.75 Legionella was also located within amoeba cysts75 or between two layers of cyst walls during amoeba encystment.76 Encystment of amoeba is not only self-protective against harsh environments, but also facilitates survival of intracellular amoeba resisting bacteria (e.g., Legionella). For example, Acanthamoeba polyphaga cysts were able to protect Legionella from at least 50 mg/L free chlorine.77 Cell-to-cell interactions also widely exist between Legionella and other indigenous bacteria in the forms of competition,78 antagonism,79 and symbiosis.80 Legionella have also been reported to undergo extracellular growth by taking up nutrients from other dead cells (i.e., necrotrophic growth).81 Besides Legionella and mycobacteria, amoeba can interact with a variety of pathogenic bacteria (at least 102 species in EPA CCL3 Microbial Universe list), fungi, parasitic protozoa and viruses.82 The interplay between opportunistic pathogens and indigenous drinking water microbes is critical in shaping the phenotypic and genotypic compositions in both planktonic and biofilm microbes. Amoeba-resistant bacteria can also exchange genes, as

(Figure 1), where low disinfectant residual is more likely to occur as a result of high surface area to volume ratio, long retention times, warmer temperature, and reactive pipe surfaces.11,49 Premise plumbing is an ideal ecological niche for opportunistic pathogens and also represents a front line of exposure to humans. Colonization of premise plumbing by opportunistic pathogens is well-documented, especially in hospital buildings48,50−52 and hotels.53−55 Overnight stagnation can significantly increase Legionella, mycobacteria, and total bacterial (16S rRNA) gene copy numbers in premise plumbing.56 Opportunistic pathogens were widely found in cold and hot water,37 biofilms from taps or shower heads,57 shower aerosols,58 eye wash stations,59 and occasionally water filters.60 The drinking water environment is a selective ecosystem as the presence of disinfectant, low available carbon, and competing microorganisms all demand adaptation to a constrained set of conditions. Opportunistic pathogens, like other drinking water microbes, generally possess several adaptive features to aid their survival under the extreme conditions of the drinking water environment, including oligotrophy, resistance to disinfection and heat, slow growth rates, and tendency to form biofilms.61−64 Given the low organic carbon levels, establishment of autotrophic microbes, such as nitrifiers, may act to fix carbon in the system and indirectly lead to proliferation of heterotrophic microbes.65 Nitrifiers can also act to decay disinfectant residuals,66 making the environment more hospitable for other microbes. An association of opportunistic pathogens and nitrification was recently observed in simulated distribution systems disinfected with chloramine.67 Opportunistic pathogens can establish as a part of the premise plumbing microbial ecology, and certain interactions with other microbes can stimulate their proliferation. Reproduction of L. pneumophila and M. avium in drinking water systems involves a critical relationship with FLA68,69 and biofilms.70 Co-culturing with an amoeba host has been confirmed to increase the virulence of L. pneumophila71 and M. avium.72 In particular, intracellular replication within an amoeba host, such as Acanthamoeba, is now thought to be a 10119

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observed by comparing the genome sequences of Legionella drancourtii and Parachlamydia acanthamoebae.83

Disinfectants can also be applied at the building-level (e.g., hospitals), including copper−silver ionization, chlorine dioxide, UV light, and hyperchlorination. A recent review of hospital drinking water disinfection strategies suggested that copper− silver ionization may be the only method that satisfies the “4step standardized evaluation criteria” which consider long-term control of Legionella.101 Chlorine dioxide is an alternative disinfectant that has proved effective in reducing Legionella contamination in several studies.102−107 However, challenges of chlorine dioxide disinfection include rapid residual loss in presence of corrosion scale,108 and possibly development of pipe leaks.109 Expertise and maintenance required for buildinglevel disinfection are serious barriers for building owners 4.2. Pipe Material. Pipe materials affect biofilm attachment and growth due to variations in surface roughness, chemical activity, and influence on water chemistry. Metal-based materials can form corrosion products on pipe surfaces and release metals into water as a result of chemical or biological reactions.110−112 Potential links between pipe materials and opportunistic pathogens in the drinking water environment have been explored in field surveys and laboratory experiments. Unfortunately, the effects of pipe material on the existing microbial biofilm have not been fully assessed in relation to the changes in numbers of opportunistic plumbing pathogens; for example, a decline in levels of a particular pathogen could be due to changes in a nonpathogenic member of the normal microbial biofilm microbiota that interacts or competes with the pathogen. An inhibitory effect of copper pipe toward Legionella growth and adherence was observed in a biofilm reactor113 and a model warm water system during the first two years of operation.114 A negative association between copper levels >50 μg/L and Legionella colonization was also observed in real-world hot water systems.115,116 However, the opposite effect was observed in one Legionella survey of German residences, where plumbing with copper pipes was more frequently colonized with Legionella than those made of synthetic materials or galvanized steel, and a positive correlation was observed between copper concentration and Legionella growth in hot water.117 Though the reason for the discrepancy is unknown, it is reasonable to speculate that levels of bioavailable copper concentration near the pipe surface and bulk water would differ markedly in these studies, and that copper can also increase rates of disinfectant loss,49,110 which would also be influential on Legionella growth. In our previous study of simulated distribution systems it was observed that the effect of pipe material (PVC, iron, or cement) on mycobacteria, P. aeruginosa, and Acanthamoeba was only significant at low disinfectant residual.67 Interactions between pipe material and disinfectant were observed for M. avium, Acanthamoeba, and P. aeruginosa at some water ages.67 In another study, lower M. avium concentrations were found on copper pipe surfaces when disinfected with free chlorine as compared to monochloramine, while chloramination of iron pipe surfaces controlled M. avium levels better than free chlorine.118 Therefore, effects of disinfectants and pipe materials should be considered along with other factors reviewed below 4.3. Assimilable Organic Carbon (AOC). Since opportunistic pathogens are heterotrophic microbes, it has been speculated that assimilable organic carbon (AOC) is a limiting factor for their proliferation. AOC refers to the fraction of organic carbon that can be utilized by specific strains or defined mixtures of bacteria, resulting in a quantifiable increase in

4.0. PHYSICAL/CHEMICAL DISINFECTION MEASURES AND EFFECT ON OPPORTUNISTIC PATHOGENS Drinking water microbiology is driven by a combination of factors including water source and chemistry, disinfection type and concentration, pipe material and conditions, temperature, and seasons. Although such factors have been applied with the aim of reducing microbial numbers and limiting their growth, the effect on the overall microbiome has not generally been considered. It is our hypothesis that such engineered controls could be more overtly manipulated to select for a “desirable microbiome,” that is, a microbiome that restricts the establishment and growth of opportunistic pathogens in premise plumbing. 4.1. Role of Disinfectants. In the U.S. and other countries, maintaining disinfectant residual is a common strategy for limiting microbial proliferation in the distribution system. In a survey of hot water systems in public buildings in Japan, L. pneumophila-positive samples were also found to have very low chlorine concentrations.84 P. aeruginosa-positive hotels in Italy were also characterized with low chlorine concentrations54 and M. avium was found to be more prevalent in undisinfected hot water compared to chlorine dioxide disinfected systems.85 Negative associations between gene copy numbers of Legionella, Mycobacterium, P. aeruginosa, Acanthamoeba, and disinfectant (chloramine or chlorine) concentrations were observed in six simulated drinking water systems.67 Such studies illustrate that maintaining a sufficient disinfectant residual is one method for limiting opportunistic pathogen numbers. However, disinfectants add significant cost to treatment, can trigger corrosion, and form harmful disinfection byproducts.86 Further, disinfectant residual is particularly challenging to maintain in premise plumbing, where opportunistic pathogens tend to reside. Disinfectants alone are not guaranteed to control opportunistic pathogens. L. pneumophila serogroup 1 colonization was found to be positively associated with free chlorine concentration in some hot water systems.54 Many opportunistic pathogens are disinfectant-resistant.63,87−89 For example, M. avium is 100−600 times more resistant to chlorine than Escherichia coli.87,90 Encysted amoeba are disinfectant-resistant and L. pneumophila and M. avium present within cysts are protected from a wide range of environmental stressors (e.g., disinfectant, heat, acid, alkali).63,91,92 Shifting disinfectant type has been implemented as a water system-level strategy for opportunistic pathogen control.93 Changing from chlorine to chloramine residual in the distribution system was found to be effective for control of Legionella colonization in building plumbing served by two realworld drinking water distribution systems.93,94 However, there is evidence that chloramines may enhance mycobacterial prevalence.93,95 Recent studies also indicate that amoeba hosts vary in their sensitivity to chlorine versus chloramine.96 Different disinfection kinetics, effective dosage, and cellular responses toward chlorine and chloramine were observed for L. pneumophila, M. avium, and Acanthamoeba castellanii using labcultivated cells,97−100 implying that there may be trade-offs in the selection of chlorine versus chloramine as residual disinfectant. Such observations suggest that interventions should consider the wider context of effects on total microbiome. 10120

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biomass concentration.119 Generally, a guideline level of AOC < 50 μg/L is considered to be effective for limiting heterotrophic bacteria and total coliform regrowth in drinking water distribution systems.120 However, the relationship between AOC and opportunistic pathogens under typical drinking water conditions is largely unknown. Previous studies demonstrated a positive relationship between mycobacterial abundance and AOC concentration in real-world drinking water distribution systems in the U.S. and Finland.121,122 In a pilot study, though M. avium was found to be recovered at an AOC level as low as 50 μg/L, no significant quantitative relationship could be established between M. avium and AOC levels of 50−228 μg/L.118 No clear effect of AOC was found on NTM numbers among eight Dutch drinking water plants and distribution systems.123 Such observed differences may be due to the composition of the microbial biofilms, which hypothetically could exacerbate or buffer direct influence of AOC. L. pneumophila was found to be more prevalent in water with AOC concentrations above 10 μg/L than in water with AOC levels below 5 μg/L.123 Lower levels of Legionella and H. vermiformis were observed in unchlorinated drinking water supplied with low natural organic matter (NOM) concentration, implying a carbon source effect on proliferation.124,125 One lab-controlled study noted that extremely low levels of organic carbon produced by distillation and UV carbon destruction did seem to hinder growth of L. pneumophila if antimicrobial copper surfaces were present instead of PEX pipe. However, high levels of total bacteria (as indicated by 16S rRNA gene copies and HPCs) were still detected in the presence of copper surfaces at low organic carbon (