Opportunistic Premise Plumbing Pathogens: Increasingly Important Pathogens in Drinking Water

Opportunistic premise plumbing pathogens are responsible for a significant number of infections whose origin has been traced to drinking water. These opportunistic pathogens represent an emerging water borne disease problem with a major economic cost of at least $1 billion annually. The common features of this group of waterborne pathogens include: disinfectant-resistance, pipe surface adherence and biofilm formation, growth in amoebae, growth on low organic concentrations, and growth at low oxygen levels. Their emergence is due to the fact that conditions resulting from drinking water treatment select for them. As such, there is a need for novel approaches to reduce exposure to these pathogens. In addition to much-needed research, controls to reduce numbers and human exposure can be instituted independently by utilities and homeowners and hospital- and building-operators.

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title: Direct healthcare costs of selected diseases primarily or partially transmitted by water
abstract: Despite US sanitation advancements, millions of waterborne disease cases occur annually, although the precise burden of disease is not well quantified. Estimating the direct healthcare cost of specific infections would be useful in prioritizing waterborne disease prevention activities. Hospitalization and outpatient visit costs per case and total US hospitalization costs for ten waterborne diseases were calculated using large healthcare claims and hospital discharge databases. The five primarily waterborne diseases in this analysis (giardiasis, cryptosporidiosis, Legionnaires' disease, otitis externa, and non-tuberculous mycobacterial infection) were responsible for over 40 000 hospitalizations at a cost of $970 million per year, including at least $430 million in hospitalization costs for Medicaid and Medicare patients. An additional 50 000 hospitalizations for campylobacteriosis, salmonellosis, shigellosis, haemolytic uraemic syndrome, and toxoplasmosis cost $860 million annually ($390 million in payments for Medicaid and Medicare patients), a portion of which can be assumed to be due to waterborne transmission.

title: Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains
abstract: Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.

title: Chlorine, Chloramine, Chlorine Dioxide, and Ozone Susceptibility of Mycobacterium avium
abstract: Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking water.

title: Capability of mucoid Pseudomonas aeruginosa to survive in chlorinated water.
abstract: Mucoid strains of Pseudomonas aeruginosa are characterized by an overproduction of the extracellular polysaccharide alginate. When suspended into chlorinated swimming-pool water or drinking water samples, mucoid bacteria revealed enhanced survival compared with isogenic nonmucoid cells. Removal of slime from mucoid bacteria abolished chlorine resistance, addition of purified alginate to washed bacteria again enhanced survival. Thus, alginate-containing slime confers protection on P. aeruginosa against chlorine and may contribute to survival of these bacteria in chlorinated water systems.

title: Colonization of components of a model hot water system by Legionella pneumophila.
abstract: A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.

title: Direct measurement of chlorine penetration into biofilms during disinfection
abstract: Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.

title: Resistance of Legionella pneumophila serotype 1 biofilms to chlorine-based disinfection.
abstract: The presence of Legionella spp. in potable water systems is a major concern to municipal water providers and consumers alike. Despite the inclusion of chlorine in potable supplies and frequent chlorination cycles, the bacterium is a recalcitrant human pathogen capable of causing incidents of Legionnaires' disease, Pontiac fever and community-acquired pneumonia in humans. Using two materials routinely employed for the delivery of potable water as a substratum, copper and stainless steel, the development of Legionella pneumophila biofilms and their response to chlorination was monitored over a three-day and a three-month period, respectively. Preliminary in vitro studies using broth and sterile tap water as culture media indicated that the bacterium was capable of surviving in low numbers for 28 days in the presence of chlorine. Subsequently, biofilms were grown for three days, one month and two months, respectively, on stainless steel and copper sections, which are widely used for the conveyance of potable water. Immediately after exposure to 50mg/L chlorine for 1h, the biofilms yielded no recoverable colonies, but colonies did reappear in low numbers over the following days. Despite chlorination at 50mg/L for 1h, both one- and two-month-old L. pneumophila biofilms were able to survive this treatment and to continue to grow, ultimately exceeding 1x10(6)cfu per disc. This research provides an insight into the resistance afforded to L. pneumophila against high levels of chlorine by the formation of biofilms and has implications for the delivery of potable water.

title: Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality.
abstract: An increasing number of microorganisms, including bacteria but also viruses and eukaryotes, have been described as benefiting from interaction with free-living amoebae (FLA). Beneficial interaction can be due to resistance to predation conferring ecological advantage, intracellular survival and/or intracellular proliferation. This review highlights the potential risk associated with amoebae by listing all known pathogenic microbial species for which growth and/or survival promotion by FLA (mainly Acanthamoeba spp.) has been demonstrated. It focuses on the susceptibility of amoebal and intra-amoebal bacteria to various categories of biocides, the known mechanisms of action of these biocides against trophozoites and cysts and the various methods used to demonstrate efficacy of treatments against FLA. Brief descriptions of FLA ecology and prevalence in domestic/institutional water systems and their intrinsic pathogenicity are also presented. The intention is to provide an informed opinion on the environmental risks associated with the presence of FLA and on the survival of cysts following biocidal treatments, while also highlighting the need to conduct research on the roles of amoebae in aquatic ecosystems.

title: The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease
abstract: Legionella pneumophila is a bacterial parasite of many species of freshwater protozoa and occasionally an intracellular pathogen of humans. While protozoa are known to play a key role in the persistence of L. pneumophila in the environment, there has been limited research addressing the potential role of L. pneumophila-infected protozoa in the pathogenesis of human infection. In this report, the potential role of an L. pneumophila-infected amoeba as an infectious particle in replicative L. pneumophila lung infection was investigated in vivo with the amoeba Hartmannella vermiformis, a natural reservoir of L. pneumophila in the environment. L. pneumophila-infected H. vermiformis organisms were prepared by coculture of the amoebae and virulent L. pneumophila cells in vitro. A/J mice, which are susceptible to replicative L. pneumophila lung infection, were subsequently inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms (10(6) amoebae containing 10(5) bacteria), and intrapulmonary growth of the bacteria was assessed. A/J mice inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms developed replicative L. pneumophila lung infections. Furthermore, L. pneumophila-infected H. vermiformis organisms were more pathogenic than an equivalent number of bacteria or a coinoculum of L. pneumophila cells and uninfected amoebae. These results demonstrate that L. pneumophila-infected amoebae are infectious particles in replicative L. pneumophila infections in vivo and support the hypothesis that inhaled protozoa may serve as cofactors in the pathogenesis of pulmonary disease induced by inhaled respiratory pathogens.

title: Interaction of Mycobacterium avium with environmental amoebae enhances virulence
abstract: Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium.

title: Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae
abstract: Bacteria and protozoa coexist in a wide range of biofilm communities of natural, technical and medical importance. Generally, this interaction is characterized by the extensive grazing activity of protozoa on bacterial prey populations. We hypothesized that the close spatial coexistence in biofilms should allow opportunistic pathogenic bacteria to utilize their eukaryote-targeting arsenal to attack and exploit protozoan host cells. Studying cocultures of the environmental pathogen Pseudomonas aeruginosa and the amoeba Acanthamoeba castellanii, we found that P. aeruginosa rapidly colonized and killed biofilm-associated amoebae by a quorum-sensing independent mechanism. Analysis of the amoeba-induced transcriptome indicated the involvement of the P. aeruginosa type III secretion system (T3SS) in this interaction. A comparison of mutants with specific defects in the T3SS demonstrated the use of the secretion apparatus and the effectors ExoU, ExoS and ExoT in the killing process, of which ExoU had the greatest impact. T3SS-mediated virulence towards A. castellanii was found to be controlled by the global regulators RpoN and RpoS and through modulation of cAMP and alginate biosynthesis. Our findings suggest that conserved virulence pathways and specifically the T3SS play a central role in bacteria–protozoa interactions in biofilms and may be instrumental for the environmental persistence and evolution of opportunistic bacterial pathogens.

title: Survival of Mycobacterium avium in a model distribution system.
abstract: A pilot study was designed to examine the impact of nutrient levels, pipe materials, and disinfection on the survival of M. avium in model drinking water distribution system biofilms. Studies showed that the survival of the organism was dependant upon a complex interaction between pipe surface, nutrient levels, and disinfectants. The findings showed that when no disinfection was applied, M. avium could be recovered from biofilms at nutrient levels of 50microg/L assimilable organic carbon. M. avium concentrations were lower on copper pipe surfaces following disinfection with free chlorine as compared to monochloramine. However, due to the interference of corrosion products, chloramination of iron pipe surfaces controlled M. avium levels better than free chlorine. These data demonstrate the significance of pipe materials on the survival of M. avium complex in biofilms. Elimination of competitive heterotrophic bacteria on copper pipe surfaces by the application of disinfection resulted in a population of nearly 100% M. avium. Heat treatment of M. avium biofilms was affected by the pipe composition and organic content of the water. Effluent temperatures >53 degrees C were required to control the occurrence of M. avium in the pipeline system. Although additional studies are required using improved detection methods, the results of this investigation suggest that reducing the biodegradable organic material in drinking water, control of corrosion, maintenance of an effective disinfectant residual, and management of hot water temperatures can help limit the occurrence of M. avium complex in drinking water biofilms.

title: Epidemiology of infection by nontuberculous mycobacteria. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in acid, brown-water swamps of the southeastern United States and their association with environmental variables.
abstract: Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (MAIS) organisms were isolated and identified from waters, soils, aerosols, and droplets ejected from water collected from four geographically separate aquatic environments (Okefenokee Swamp, GA; Dismal Swamp, VA; Claytor Lake, VA; and Cranberry Glades, WV) during several seasons. Recovery of MAIS was significantly higher from waters, soils, and aerosols collected from the two acid, brown-water swamps located in the southeastern coastal plain. High MAIS numbers correlated with warmer temperature, low pH, low dissolved oxygen, high soluble zinc, high humic acid, and high fulvic acid. This research, in relation to previous findings for the geographic distribution and physiologic ecology of MAIS, supports the conclusion that waters, soils, and aerosols of the acid, brown-water swamps of the southeastern United States coastal plain represent major environmental sources likely connected with the higher incidence of human infection in this region.

title: Humic and fulvic acids stimulate the growth of Mycobacterium avium.
abstract: Mycobacterium avium, an environmental, opportunistic pathogenic mycobacterium, has been isolated frequently and in high numbers from waters in Finland and from acid, brown water swamps of the southeastern coastal USA. M. avium has also been recovered in high numbers from Finnish drinking water and frequently isolated from Finnish AIDS patients. Boreal forests and brown water swamps are similar in that they are rich in humic and fulvic acids and of low pH and dissolved oxygen. Growth of representative isolates of M. avium in natural water was stimulated markedly by the addition of humic and fulvic acids. Further, the M. avium isolates grew at pH levels as low at 4.0 and at oxygen levels equal to 4% of atmospheric levels. The high numbers of M. avium in boreal waters and brown water swamps are likely due to their ability to proliferate in those humic- and fulvic-rich, acidic, micro-aerobic environments.

title: Pseudomonas aeruginosa: Growth in Distilled Water from Hospitals
abstract: Pseudomonas aeruginosa can grow relatively fast in distilled water obtained in hospitals and achieve high cell contaminations which remain stable for long periods of time. Cells grown in distilled water react quite differently to chemical and physical stresses than cells grown in standard laboratory culture media.

title: Role of stagnation and obstruction of water flow in isolation of Legionella pneumophila from hospital plumbing
abstract: The stagnation of water in two of four hospital hot-water storage tanks found to contain Legionella pneumophila was reduced by keeping the two tanks continually on-line for 1 year. L. pneumophila colony counts in these two tanks fell quickly to low levels, whereas the organisms persisted in the two tanks that were not in use. L. pneumophila continued to be isolated from 50 to 100% of the hospital showerheads which were sampled during this period. We also examined aerators and other hospital faucet fixtures which obstruct water flow. L. pneumophila was isolated from 22 of 30 faucet aerators and 2 of 16 vacuum breakers but not from 26 nonobstructed faucets or 6 backflow preventers. Over a 7-month period, after nine faucet aerators were sterilized, 10 of 60 surveillance cultures revealed L. pneumophila, despite the inability to isolate the organism from the potable-water tanks in use. These data suggest that prevention of stagnation in hot-water tanks may be effective in reducing L. pneumophila concentrations in potable-water systems serving high-risk populations. We have also shown that faucet aerators, by providing a surface for L. pneumophila to colonize, can become secondary reservoirs for the organism in hospital plumbing.

title: Microaerobic growth and anaerobic survival of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum.
abstract: Representative strains of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) grew at equal rates in laboratory medium at 21% (air) and 12% oxygen. Growth in 6% oxygen proceeded at a 1.4-1.8-fold lower rate. Colony formation was the same at 21% (air) and 6% oxygen. The MAIS strains survived rapid shifts from aerobic to anaerobic conditions as measured by two experimental approaches (Falkinham (1996) ). MAIS cells grown aerobically to log phase in broth were diluted, spread on agar medium, and incubated anaerobically for up to 20 days at 37°C. Although no colonies formed anaerobically, upon transfer to aerobic conditions, greater than 25% of the colony forming units (CFU) survived after 20 days of anaerobic incubation (Prince et al. (1989) ). MAIS cells grown in broth aerobically to log phase were sealed and vigorous agitation led to oxygen depletion (Wayne model). After 12 days anaerobic incubation, M. avium and M. scrofulaceum survival were high (>50%), while M. intracellulare survival was lower (22%). M. avium cells shifted to anaerobiosis in broth had increased levels of glycine dehydrogenase and isocitrate lyase. Growth of MAIS strains at low oxygen levels and their survival following a rapid shift to anaerobiosis is consistent with their presence in environments with fluctuating oxygen levels.

title: The assimilatory and dissimilatory nitrate reductases of Pseudomonas aeruginosa are encoded by different genes.
abstract: The phenotypes of certain mutant strains of Pseudomonas aeruginosa were reported to be pleiotropic for nitrate reduction; these strains were selected for their inability to dissimilate nitrate and were found also to have lost the ability to assimilate nitrate. We now report that the isolation procedure selected two mutations, one in genes encoding the synthesis of dissimilatory nitrate reductase (narA, narB or narE) and another in one of the genes (nas) encoding the synthesis of assimilatory nitrate reductase. Thus in P. aeruginosa dissimilatory and assimilatory nitrate reductases are genetically distinct. However, a loss of both enzymes is necessary to prevent slow dissimilatory growth on nitrate. Assimilatory nitrate reductase requires molybdenum to function, as does dissimilatory nitrate reductase. Lesions in narD affect incorporation of molybdenum into both enzymes, and hence exert a pleiotropic effect.

title: Factors Influencing Numbers of Mycobacterium avium, Mycobacterium intracellulare, and Other Mycobacteria in Drinking Water Distribution Systems
abstract: Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter−1. The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r2 = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm2). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.

title: Colonization of hospital water systems by legionellae, mycobacteria and other heterotrophic bacteria potentially hazardous to risk group patients
abstract: Occurrences of legionellae and nontuberculous mycobacteria were followed in water systems of a tertiary care hospital where nosocomial infections due to the two genera had been verified. The aim was to examine whether their occurrence in the circulating hot water can be controlled by addition of a heat‐shock unit in the circulation system, and by intensified cleaning of the tap and shower heads. One hot water system examined had an inbuilt heat‐shock system causing a temporary increase of temperature to 80 °C, the other was an ordinary system (60 °C). The heat‐shock unit decreased legionella colony counts in the circulating hot water (mean 35 cfu/l) compared to the ordinary system (mean 3.6×103 cfu/l). Mycobacteria constantly present in the incoming cold water (mean 260 cfu/l) were never isolated from the circulating hot water. Water sampled at peripheral sites such as taps and showers contained higher concentrations of legionellae, mycobacteria, and mesophilic and Gram‐negative heterotrophs than the circulating waters. The shower water samples contained the highest bacterial loads. The results indicate the need to develop more efficient prevention methods than the ones presently used. Prevention of mycobacteria should also be extended to incoming cold water.

title: Plumbing of hospital premises is a reservoir for opportunistically pathogenic microorganisms: a review
abstract: Several bacterial species that are natural inhabitants of potable water distribution system biofilms are opportunistic pathogens important to sensitive patients in healthcare facilities. Waterborne healthcare-associated infections (HAI) may occur during the many uses of potable water in the healthcare environment. Prevention of infection is made more challenging by lack of data on infection rate and gaps in understanding of the ecology, virulence, and infectious dose of these opportunistic pathogens. Some healthcare facilities have been successful in reducing infections by following current water safety guidelines. This review describes several infections, and remediation steps that have been implemented to reduce waterborne HAIs.

title: Widespread molecular detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States.
abstract: In the United States, 6,868 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009-2010. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and recovered from a variety of natural freshwater environments. Human exposure to L. pneumophila Sg1 may occur from aerosolization and subsequent inhalation of household and facility water. In this study, two primer/probe sets (one able to detect L. pneumophila and the other L. pneumophila Sg1) were determined to be highly sensitive and selective for their respective targets. Over 272 water samples, collected in 2009 and 2010 from 68 public and private water taps across the United States, were analyzed using the two qPCR assays to evaluate the incidence of L. pneumophila Sg1. Nearly half of the taps showed the presence of L. pneumophila Sg1 in one sampling event, and 16% of taps were positive in more than one sampling event. This study is the first United States survey to document the occurrence and colonization of L. pneumophila Sg1 in cold water delivered from point of use taps.

title: Opportunistic pathogens enriched in showerhead biofilms
abstract: The environments we humans encounter daily are sources of exposure to diverse microbial communities, some of potential concern to human health. In this study, we used culture-independent technology to investigate the microbial composition of biofilms inside showerheads as ecological assemblages in the human indoor environment. Showers are an important interface for human interaction with microbes through inhalation of aerosols, and showerhead waters have been implicated in disease. Although opportunistic pathogens commonly are cultured from shower facilities, there is little knowledge of either their prevalence or the nature of other microorganisms that may be delivered during shower usage. To determine the composition of showerhead biofilms and waters, we analyzed rRNA gene sequences from 45 showerhead sites around the United States. We find that variable and complex, but specific, microbial assemblages occur inside showerheads. Particularly striking was the finding that sequences representative of non-tuberculous mycobacteria (NTM) and other opportunistic human pathogens are enriched to high levels in many showerhead biofilms, >100-fold above background water contents. We conclude that showerheads may present a significant potential exposure to aerosolized microbes, including documented opportunistic pathogens. The health risk associated with showerhead microbiota needs investigation in persons with compromised immune or pulmonary systems.

title: Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods
abstract: Methods using polymerase chain reaction (PCR) and gene probes to detect viable Legionella pneumophila were investigated with cells exposed to biocide or elevated temperature. Exposure to hypochlorite caused viable nonculturable cells to form. Culturable and viable nonculturable cells showed positive PCR amplification, whereas nonviable cells did not. Viable cells were also specifically detected with mip mRNA as the target, reverse transcription (to form cDNA), and PCR amplification. After exposure to elevated temperature, only viable culturable cells were detected, which corresponded with positive PCR amplification.

title: The viable but nonculturable state in bacteria.
abstract: It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

title: Recent findings on the viable but nonculturable state in pathogenic bacteria.
abstract: Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this 'viable but nonculturable' (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.

title: Integration of Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on domestic plumbing materials.
abstract: Drinking water biofilms were grown on coupons of plumbing materials, including ethylene-propylene-diene-monomer (EPDM) rubber, silane cross-linked polyethylene (PE-X b), electron-ray cross-linked PE (PE-X c) and copper under constant flow-through of cold tap water. After 14 days, the biofilms were spiked with Pseudomonas aeruginosa, Legionella pneumophila and Enterobacter nimipressuralis (10(6) cells/mL each). The test bacteria were environmental isolates from contamination events in drinking water systems. After static incubation for 24 h, water flow was resumed and continued for 4 weeks. Total cell count and heterotrophic plate count (HPC) of biofilms were monitored, and P. aeruginosa, L. pneumophila and E. nimipressuralis were quantified, using standard culture-based methods or culture-independent fluorescence in situ hybridization (FISH). After 14 days total cell counts and HPC values were highest on EPDM followed by the plastic materials and copper. P. aeruginosa and L. pneumophila became incorporated into drinking water biofilms and were capable to persist in biofilms on EPDM and PE-X materials for several weeks, while copper biofilms were colonized only by L. pneumophila in low culturable numbers. E. nimipressuralis was not detected in any of the biofilms. Application of the FISH method often yielded orders of magnitude higher levels of P. aeruginosa and L. pneumophila than culture methods. These observations indicate that drinking water biofilms grown under cold water conditions on domestic plumbing materials, especially EPDM and PE-X in the present study, can be a reservoir for P. aeruginosa and L. pneumophila that persist in these habitats mostly in a viable but non-culturable state.

title: Effect of GAC pre-treatment and disinfectant on microbial community structure and opportunistic pathogen occurrence.
abstract: Opportunistic pathogens in potable water systems are an emerging health concern; however, the factors influencing their proliferation are poorly understood. Here we investigated the effects of prior granular activated carbon (GAC) biofiltration [GAC-filtered water, unfiltered water, and a blend (30% GAC filtered and 70% unfiltered water)] and disinfectant type (chlorine, chloramine) on opportunistic pathogen occurrence using five annular reactors (ARs) to simulate water distribution systems, particularly premise plumbing. GAC pre-treatment effectively reduced total organic carbon (TOC), resulting in three levels of influent TOC investigated. Quantitative polymerase chain reaction (q-PCR) provided molecular evidence of natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., Hartmannella vermiformis and Mycobacterium avium on AR coupons. Cultivable mycobacteria and amoeba, including pathogenic species, were also found in bulk water and biofilm samples. While q-PCR tends to overestimate live cells, it provided a quantitative comparison of target organisms colonizing the AR biofilms in terms of gene copy numbers. In most cases, total bacteria and opportunistic pathogens were higher in the three undisinfected ARs, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively inhibited M. avium and H. vermiformis colonization. Pyrosequencing of 16S rRNA genes in coupon biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, this study provides insights into the potential of different disinfectants and GAC biofilters at the treatment plant and in buildings to control downstream opportunistic pathogens and broader drinking water microbial communities.

title: The importance of the viable but non-culturable state in human bacterial pathogens
abstract: Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.

title: Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii
abstract: Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.

title: Multidrug-resistant Acinetobacter Infection Mortality Rate and Length of Hospitalization
abstract: Acinetobacter infections have increased and gained attention because of the organism’s prolonged environmental survival and propensity to develop antimicrobial drug resistance. The effect of multidrug-resistant (MDR) Acinetobacter infection on clinical outcomes has not been reported. A retrospective, matched cohort investigation was performed at 2 Baltimore hospitals to examine outcomes of patients with MDR Acinetobacter infection compared with patients with susceptible Acinetobacter infections and patients without Acinetobacter infections. Multivariable analysis controlling for severity of illness and underlying disease identified an independent association between patients with MDR Acinetobacter infection (n = 96) and increased hospital and intensive care unit length of stay compared with 91 patients with susceptible Acinetobacter infection (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.2–5.2 and OR 2.1, 95% CI 1.0–4.3] respectively) and 89 uninfected patients (OR 2.5, 95% CI 1.2–5.4 and OR 4.2, 95% CI 1.5–11.6] respectively). Increased hospitalization associated with MDR Acinetobacter infection emphasizes the need for infection control strategies to prevent cross-transmission in healthcare settings.

title: Detection of Acinetobacter spp. in rural drinking water supplies
abstract: A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.

title: Acanthamoeba sp. promotes the survival and growth of Acinetobacter baumanii.
abstract: Acinetobacter baumanii, which may be found in water, is an important emerging hospital-acquired pathogen. Free-living amoebae can be recovered from the same water networks, and it has been shown that these protozoa may support the growth of other bacteria. In this paper, we have studied potential relationships between A. baumanii and Acanthamoeba species. Two strains of A. baumanii isolated from hospital water were co-cultivated with the trophozoites or supernatants of two free-living amoebae strains: Acanthamoeba castellanii or Acanthamoeba culbertsoni. Firstly, the presence of the amoebae or their supernatants induced a major increase in A. baumanii growth, compared with controls. Secondly, A. baumanii affected only the viability of A. culbertsonii, with no effect on A. castellanii. Electron microscopy observations of the cultures investigating the bacterial location in the protozoa showed persistence of the bacteria within cyst wall even after 60 days of incubation. In our study, the survival and growth of A. baumanii could be favored by Acanthamoeba strains. Special attention should consequently be paid to the presence of free-living amoebae in hospital water systems, which can promote A. baumanii persistence.

title: Prevalence and distribution of Aeromonas hydrophila in the United States
abstract: The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.

title: Adhesion of Aeromonas hydrophila to water distribution system pipes after different contact times.
abstract: Scanning electron microscopy observation was used to investigate the ability of Aeromonas hydrophila to attach to various water distribution pipe surfaces, such as stainless steel, copper, and polybutylene, after different contact times at ambient and storage temperatures. Surface energy value of each surface was estimated by contact angle measurements using water, alpha-bromonaphthalene, and dimethyl sulfoxide. Our results indicated that Aeromonas cells could easily attach to all surface types after exposures as short as 1 or 4 h at both temperatures (4 and 20 degrees C). Polybutylene, a low-energy surface (41.2 mJ.m-2), followed by stainless steel (65.7 mJ.m-2), was most colonized by Aeromonas cells, whereas few cells were observed on copper, which has a surface energy of 45.8 mJ.m-2. Extracellular materials could also be observed on polybutylene surfaces, especially after 1 and 4 h of exposure at the refrigeration temperature.

title: Bactericidal effect of chlorine on motile Aeromonas spp. in drinking water supplies and influence of temperature on disinfection efficacy
abstract: The susceptibility of toxigenic Aeromonas spp. to free chlorine in drinking water supplies, and the influence of environmental temperature on the bactericidal activity of the oxidant, were evaluated. The results showed inactivation curves characterized by an initial phase of rapid reduction of viable cells followed by a slow inactivation of bacteria. The effect of the chlorine compound was markedly influenced by water temperature. At a summer water temperature (20 °C), the efficacy of the chlorine concentrations tested was found to be two to three times lower compared to that found at a winter temperature (5 °C). Resistance was moderately, but significantly, greater in Aer. hydrophilavsAer. caviae and Aer. sobria, but all Aeromonas spp. were more susceptible than Escherichia coli. Selective pressure with free chlorine did not produce Aeromonas cells with higher levels of chlorine resistance.

title: Do free-living amoebae in treated drinking water systems present an emerging health risk?
abstract: There is an expanding body of evidence that free-living amoebae (FLA) increase both the numbers and virulence of water-based, human-pathogenic, amoeba-resisting microorganisms (ARM). Legionella spp., Mycobacterium spp., and other opportunistic human pathogens are known to be both ARM and also the etiologic agents of potentially fatal human lung infections. However, comparatively little is known about the FLA that may facilitate ARM growth in drinking water. This review examines the available literature on FLA in treated drinking water systems; in total 26 studies from 18 different countries. FLA were reported to breakthrough the water treatment barrier and enter distribution systems, in addition to the expected post-treatment system ingress. Once in the distribution system there is evidence of FLA colonization and regrowth especially in reservoirs and in-premise plumbing storage tanks. At the point of use the average FLA detection rate was 45% but highly variable (n = 16, σ = 31) due to both differences in both assay methods and the type of water systems examined. This review reveals that FLA are consistently detected in treated drinking water systems around the world and present a yet unquantified emerging health risk. However, more research is urgently required before accurate risks assessments can be undertaken to assess the impacts on human health, in households and institutions, due to exposure to FLA facilitated pathogenic ARM.

title: Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network
abstract: Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.

title: Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence
abstract: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system.

title: Adherence and biofilm formation of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus to household plumbing materials
abstract: Measure adherence and biofilm formation by cells of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus on common household plumbing materials namely stainless steel, glass, zinc‐galvanized steel, copper and polyvinyl chloride (PVC).

title: Physiology and morphology of Legionella pneumophila in continuous culture at low oxygen concentration.
abstract: Two strains of Legionella pneumophila serogroup 1 monoclonal subgroup Pontiac were grown for the first time in continuous culture using a chemically defined medium. The influence of temperature on physiology and morphology was investigated by fixing the growth rate (equal to the dilution rate, D) at 0.08 h-1 and controlling the pH and dissolved oxygen concentration of the culture. Serine provided the principal source of carbon and energy but growth was limited by tyrosine. The bacterium behaved as a microaerophile in this medium, with maximal growth occurring at 0.31 (mg O2)I-1 (equivalent to a dissolved oxygen tension of 4% (v/v) air saturation at 30 degrees C). The cultures consisted of flagellated, short rods at 24 degrees C, but exhibited an increased level of pleomorphism and the loss of flagella as the temperature was increased to 37 degrees C. The presence of intracellular granules was noted, and their abundance was temperature-dependent. Polyhydroxybutyrate was present in L. pneumophila, and the proportion of the cell dry weight that it accounted for varied with temperature, being maximal at 24 degrees C. The ratio of saturated to unsaturated fatty acids in the cells decreased as the temperature was reduced towards 24 degrees C, so as to maintain membrane fluidity at low growth temperature.

title: Long-Term Effects of Hospital Water Network Disinfection on Legionella and Other Waterborne Bacteria in an Italian University Hospital
abstract: Legionella control still remains a critical issue in healthcare settings where the preferred approach to health risk assessment and management is to develop a water safety plan. We report the experience of a university hospital, where a water safety plan has been applied since 2002, and the results obtained with the application of different methods for disinfecting hot water distribution systems in order to provide guidance for the management of water risk.  The disinfection procedures included continuous chlorination with chlorine dioxide (0.4–0.6 mg/L in recirculation loops) reinforced by endpoint filtration in critical areas and a water treatment based on monochloramine (2-3 mg/L). Real-time polymerase chain reaction and a new immunoseparation and adenosine triphosphate bioluminescence analysis were applied in environmental monitoring.  After 9 years, the integrated disinfection-filtration strategy significantly reduced positive sites by 55% and the mean count by 78% (P < .05); however, the high costs and the occurrence of a chlorine-tolerant clone belonging to Legionella pneumophila ST269 prompted us to test a new disinfectant. The shift to monochloramine allowed us to eliminate planktonic Legionella and did not require additional endpoint filtration; however, nontuberculous mycobacteria were isolated more frequently as long as the monochloramine concentration was 2 mg/L; their cultivability was never regained by increasing the concentration up to 3 mg/L.  Any disinfection method needs to be adjusted/fine-tuned in individual hospitals in order to maintain satisfactory results over time, and only a locally adapted evidence-based approach allows assessment of the efficacy and disadvantages of the control measures.

title: Prolonged Outbreak of Mycobacterium chimaera Infection After Open-Chest Heart Surgery.
abstract: Invasive Mycobacterium chimaera infections were diagnosed in 2012 in 2 heart surgery patients on extracorporeal circulation. We launched an outbreak investigation to identify the source and extent of the potential outbreak and to implement preventive measures.



We collected water samples from operating theaters, intensive care units, and wards, including air samples from operating theaters. Mycobacterium chimaera strains were characterized by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Case detection was performed based on archived histopathology samples and M. chimaera isolates since 2006, and the patient population at risk was prospectively surveyed.



We identified 6 male patients aged between 49 and 64 years with prosthetic valve endocarditis or vascular graft infection due to M. chimaera, which became clinically manifest with a latency of between 1.5 and 3.6 years after surgery. Mycobacterium chimaera was isolated from cardiac tissue specimens, blood cultures, or other biopsy specimens. We were able also to culture M. chimaera from water circuits of heater-cooler units connected to the cardiopulmonary bypass, and air samples collected when the units were in use. RAPD-PCR demonstrated identical patterns among M. chimaera strains from heater-cooler unit water circuits and air samples, and strains in 2 patient clusters.



The epidemiological and microbiological features of this prolonged outbreak provided evidence for the airborne transmission of M. chimaera from contaminated heater-cooler unit water tanks to patients during open-heart surgery.

title: Detection and termination of an extended low-frequency hospital outbreak of GIM-1-producing Pseudomonas aeruginosa ST111 in Germany.
abstract: Metallo-β-lactamase German imipenemase-1 (GIM-1)-mediated carbapenem resistance is emerging in Germany but has not spread beyond a very localized region. The aim of this study was to describe the first outbreak of an extensively drug-resistant GIM-1-carrying Pseudomonas aeruginosa strain affecting 29 patients in a tertiary care hospital from 2002-2013.



The outbreak was studied retrospectively and prospectively by a combination of molecular methods (carbapenemase polymerase chain reaction [PCR]), genotyping (DiversiLab, pulsed field gel electrophoresis and multi-locus sequence typing, bioMérieux, Marcy l'Etoile, France), descriptive epidemiology, and extensive environmental investigations using swabs with liquid transport medium, blaGIM-1 PCR, directly from the medium and culture.



Of the 29 affected patients, 24 had been admitted to a surgical intensive care unit at some point, where environmental sampling revealed a high burden of blaGIM-1 in the wastewater system. The outbreak strain was found in several sinks and on a reusable hair washbasin. Initially, general infection control measures were applied; thereafter, specific measures were implemented, including the restriction of washbasin use. Continued surveillance over a period of 2 years has revealed no further case of GIM-1-carrying Pseudomonas aeruginosa.



This long-term outbreak highlights the potential of molecular methods in surveillance for multidrug-resistant pathogens and in environmental sampling and the successful containment by application of specific control measures targeting biofilms within sink drains as potential environmental reservoirs for P aeruginosa.

title: Nontuberculous Mycobacteria from Household Plumbing of Patients with Nontuberculous Mycobacteria Disease
abstract: To determine whether plumbing could be a source of nontuberculous mycobacteria (NTM) infection, during 2007–2009 I isolated NTM from samples from household water systems of NTM patients. Samples from 22/37 (59%) households and 109/394 (28%) total samples yielded NTM. Seventeen (46%) of the 37 households yielded >1 Mycobacterium spp. isolate of the same species as that found in the patient; in 7 of those households, the patient isolate and 1 plumbing isolate exhibited the same repetitive sequence-based PCR DNA fingerprint. Households with water heater temperatures <125°C (<50°C) were significantly more likely to harbor NTM compared with households with hot water temperatures >130°F (>55°C) (p = 0.0107). Although households with water from public or private water systems serving multiple households were more likely to have NTM (19/27, 70%) compared with households with a well providing water to only 1 household (5/12, 42%), that difference was not significant (p = 0.1532).

title: Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I
abstract: The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.

title: Probiotic approach to pathogen control in premise plumbing systems? A review.
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.

title: Anticipating challenges with in-building disinfection for control of opportunistic pathogens.
abstract: A new American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE) standard for control of Legionella (ASHRAE Standard 188, 2013) emphasizes use of in-building disinfection techniques to reduce the exposure of at-risk consumers to opportunistic pathogens in premise plumbing (OPPPs). This standard and other recommendations for OPPP control have implications for scaling in and corrosion of plumbing systems, which can sometimes adversely affect the efficacy of the disinfection method and physical integrity of the plumbing system, prompting this proactive critical review of challenges associated with implementation of Standard 188.