Corroding copper and steel exposed to intermittently flowing tap water promote biofilm formation and growth of Legionella pneumophila.

The information about the impact of copper pipes on the growth of Legionella pneumophila in premise plumbing is controversial. For this reason, pipe segments of copper, stainless steel (SS), mild steel (MS), polyethylene, chlorinated polyvinylchloride (CPVC) and glass (controls) were exposed to intermittently flowing (20 min stagnation time) nonchlorinated tap water of 37 °C or 16 °C (ambient temperature) during six months to study the impact of metals on biofilm formation and growth of L. pneumophila. Biofilm concentrations (BfC, measured as ATP) on copper were 3 (at 37 °C) to 6 (at 16 °C) times higher than on SS. The maximum colony counts of L. pneumophila on the materials tested at 37 °C showed a quadratic relationship with the associated BfCs, with highest values on copper and MS. The average Cu concentration on the glass control of copper (glass-copper) was more than two log units lower than the Fe concentration on glass-MS, suggesting that copper released less corrosion by-products than MS. The release of corrosion by-products with attached biomass from MS most likely enhanced biofilm formation on glass-MS. Cloning and 16S RNA gene sequence analysis of the predominating biofilm bacteria revealed that an uncultured Xanthobacteraceae bacterium and Reyranella accounted for 75% of the bacterial community on copper at 37 °C. The nitrite-oxidizing Nitrospira moscoviensis, which can also utilize hydrogen (H2) and formate, accounted for >50% of the bacterial abundance in the biofilms on MS and glass-MS at 37 °C. The predominating presence of the strictly anaerobic non-fermentative Fe(III)-reducing Geobacter and the Fe(II)-oxidizing Gallionella on MS exposed to tap water of 16 °C indicated anoxic niches and the availability of H2, low molecular weight carboxylic acids (LMWCAs) and Fe(II) at the MS surface. LMWCAs likely also promoted bacterial growth on copper, but the release mechanisms from natural organic matter at the surface of corroding metals are unclear. The effects of water stagnation time and flow dynamics on biofilm formation on copper requires further investigation.

Primary Colonizing Betaproteobacteriales Play a Key Role in the Growth of Legionella pneumophila in Biofilms on Surfaces Exposed to Drinking Water Treated by Slow Sand Filtration.

Slow sand filtration with extensive pretreatment reduces the microbial growth potential of drinking water to a minimum level at four surface water supplies in The Netherlands. The potential of these slow sand filtrates (SSFs) to promote microbial growth in warm tap water installations was assessed by measuring biofilm formation and growth of Legionella bacteria on glass and chlorinated polyvinylchloride (CPVC) surfaces exposed to SSFs at 37 ± 2°C in a model system for up to six months. The steady-state biofilm concentration ranged from 230 to 3,980 pg ATP cm-2 on glass and 1.4 (±0.3)-times-higher levels on CPVC. These concentrations correlated significantly with the assimilable organic carbon (AOC) concentrations of the warm water (8 to 24 µg acetate-C equivalents [ac-C eq] liter-1), which were raised about 2 times by mixing cold and heated (70°C) SSFs. All biofilms supported growth of Legionella pneumophila with maximum concentrations ranging from 6 × 102 to 1.5 × 105 CFU cm-2 Biofilms after ≤50 days of exposure were predominated by Betaproteobacteriales, mainly Piscinibacter, Caldimonas, Methyloversatilis, and an uncultured Rhodocyclaceae bacterium. These rapidly growing primary colonizers most likely served as prey for the host amoebae of L. pneumophilaAlphaproteobacteria, mostly Xanthobacteraceae, e.g., Bradyrhizobium, Pseudorhodoplanes, and other amoeba-resistant bacteria, accounted for 37.5% of the clones retrieved. A conceptual model based on a quadratic relationship between the L. pneumophila colony count and the biofilm concentration under steady-state conditions is used to explain the variations in the Legionella CFU pg-1 ATP ratios in the biofilms.IMPORTANCE Proliferation of L. pneumophila in premise plumbing poses a public health threat. Extended water treatment using physicochemical and biofiltration processes, including slow sand filtration, at four surface water supplies in The Netherlands reduces the microbial growth potential of the treated water to a minimum level, and the distributed drinking water complies with high quality standards. However, heating of the water in warm tap water installations increases the concentration of easily assimilable organic compounds, thereby promoting biofilm formation and growth of L. pneumophila Prevention of biofilm formation in plumbing systems by maintenance of a disinfectant residual during distribution and/or further natural organic matter (NOM) removal is not feasible in the supplies studied. Temperature management in combination with optimized hydraulics and material selection are therefore essential to prevent growth of L. pneumophila in premise plumbing systems. Still, reducing the concentration of biodegradable compounds in drinking water by appropriate water treatment is important for limiting the Legionella growth potential.

Assessment of the microbial growth potential of slow sand filtrate with the biomass production potential test in comparison with the assimilable organic carbon method.

Slow sand filtration is the final treatment step at four surface-water supplies in the Netherlands. The microbial growth potential (MGP) of the finished water was measured with the assimilable organic carbon (AOC) method using pure cultures and the biomass production potential (BPP) test. In the BPP test, water samples were incubated untreated at 25 °C and the active-biomass concentration was measured by adenosine tri-phosphate (ATP) analysis. Addition of a river-water inoculum improved the test performance and characteristic growth and maintenance profiles of the water were obtained. The maximum ATP concentration attained within seven days and the cumulative biomass production after 14 days of incubation (BPC14, d ng ATP L-1) showed highly significant and strong linear relationships with the AOC in the slow sand filtrates. The lowest AOC and BPC14 levels were observed in the supplies applying dune filtration without ozonation in post treatment, with AOC/TOC = 1.7 ± 0.3 µg acetate-C equivalents mg-1 C and BPC14/TOC = 16.3 ± 2.2 d ng ATP mg-1 C, corresponding with 1.2 ± 0.19 ng ATP mg-1 C. These characteristics may represent the lowest specific MGP of natural organic matter achievable by biofiltration at temperatures ≤20 °C. The AOC and BPC14 concentrations in the slow sand filtrate of the supply treating lake water by ozonation with granular-activated-carbon filtration and slow sand filtration as post treatment increased with decreasing temperature. The BPP test revealed that this slow sand filtrate sampled at 2 °C contained growth-promoting compounds that were not detected with the AOC test. These observations demonstrate the utility of the BPP test for assessing the MGP of drinking water and show the performance limits of biofiltration for MGP reduction.

Incubation of premise plumbing water samples on Buffered Charcoal Yeast Extract agar at elevated temperature and pH selects for Legionella pneumophila.

Worldwide, over 90% of the notified cases of Legionnaires' disease are caused by Legionella pneumophila. However, the standard culture medium for the detection of Legionella in environmental water samples, Buffered Charcoal Yeast Extract (BCYE) agar of pH 6.9 ± 0.4 with or without antimicrobial agents incubated at 36 ± 1 °C, supports the growth of a large diversity of Legionella species. BCYE agar of elevated pH or/and incubation at elevated temperature gave strongly reduced recoveries of most of 26 L. non-pneumophila spp. tested, but not of L. pneumophila. BCYE agar of pH 7.3 ± 0.1, incubated at 40 ± 0.5 °C (BCYE pH 7.3/40 °C) was tested for selective enumeration of L. pneumophila. Of the L. non-pneumophila spp. tested, only L. adelaidensis and L. londiniensis multiplied under these conditions. The colony counts on BCYE pH 7.3/40 °C of a L. pneumophila serogroup 1 strain cultured in tap water did not differ significantly from those on BCYE pH 6.9/36 °C when directly plated and after membrane filtration and showed repeatability's of 13-14%. By using membrane filtration L. pneumophila was detected in 58 (54%) of 107 Legionella-positive water samples from premise plumbing systems under one or both of these culture conditions. The L. pneumophila colony counts (log-transformed) on BCYE pH 7.3/40 °C were strongly related (r2 = 0.87) to those on BCYE pH 6.9/36 °C, but differed significantly (p < 0.05) by a mean of - 0.12 ± 0.30 logs. L. non-pneumophila spp. were detected only on BCYE pH 6.9/36 °C in 49 (46%) of the samples. Hence, BCYE pH 7.3/40 °C can facilitate the enumeration of L. pneumophila and their isolation from premise plumbing systems with culturable L. non-pneumophila spp., some of which, e.g. L. anisa, can be present in high numbers.

Biofilm Composition and Threshold Concentration for Growth of Legionella pneumophila on Surfaces Exposed to Flowing Warm Tap Water without Disinfectant.

Legionella pneumophila in potable water installations poses a potential health risk, but quantitative information about its replication in biofilms in relation to water quality is scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was investigated under controlled hydraulic conditions in a model system inoculated with biofilm-grown L. pneumophila The biofilm on glass (average steady-state concentration, 23 ± 9 pg ATP cm-2) exposed to treated aerobic groundwater (0.3 mg C liter-1; 1 µg assimilable organic carbon [AOC] liter-1) did not support growth of the organism, which also disappeared from the biofilm on CPVC (49 ± 9 pg ATP cm-2) after initial growth. L. pneumophila attained a level of 4.3 log CFU cm-2 in the biofilms on glass (1,055 ± 225 pg ATP cm-2) and CPVC (2,755 ± 460 pg ATP cm-2) exposed to treated anaerobic groundwater (7.9 mg C liter-1; 10 µg AOC liter-1). An elevated biofilm concentration and growth of L. pneumophila were also observed with tap water from the laboratory. The Betaproteobacteria Piscinibacter and Methyloversatilis and amoeba-resisting Alphaproteobacteria predominated in the clones and isolates retrieved from the biofilms. In the biofilms, the Legionella colony count correlated significantly with the total cell count (TCC), heterotrophic plate count, ATP concentration, and presence of Vermamoeba vermiformis This amoeba was rarely detected at biofilm concentrations of <100 pg ATP cm-2 A threshold concentration of approximately 50 pg ATP cm-2 (TCC = 1 × 106 to 2 × 106 cells cm-2) was derived for growth of L. pneumophila in biofilms.IMPORTANCELegionella pneumophila is the etiologic agent in more than 10,000 cases of Legionnaires' disease that are reported annually worldwide and in most of the drinking water-associated disease outbreaks reported in the United States. The organism proliferates in biofilms on surfaces exposed to warm water in engineered freshwater installations. An investigation with a test system supplied with different types of warm drinking water without disinfectant under controlled hydraulic conditions showed that treated aerobic groundwater (0.3 mg liter-1 of organic carbon) induced a low biofilm concentration that supported no or very limited growth of L. pneumophila Elevated biofilm concentrations and L. pneumophila colony counts were observed on surfaces exposed to two types of extensively treated groundwater, containing 1.8 and 7.9 mg C liter-1 and complying with the microbial water quality criteria during distribution. Control measures in warm tap water installations are therefore essential for preventing growth of L. pneumophila.

Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C.

Legionella pneumophila proliferates in freshwater environments at temperatures ranging from 25 to 45°C. To investigate the preference of different sequence types (ST) for a specific temperature range, growth of L. pneumophila serogroup 1 (SG1) ST1 (environmental strains), ST47, and ST62 (disease-associated strains) was measured in buffered yeast extract broth (BYEB) and biofilms grown on plasticized polyvinyl chloride in flowing heated drinking water originating from a groundwater supply. The optimum growth temperatures in BYEB were approximately 37°C (ST1), 39°C (ST47), and 41°C (ST62), with maximum growth temperatures of 42°C (ST1) and 43°C (ST47 and ST62). In the biofilm at 38°C, the ST47 and ST62 strains multiplied equally well compared to growth of the environmental ST1 strain and an indigenous L. pneumophila non-SG1 strain, all attaining a concentration of approximately 107 CFU/cm-2 Raising the temperature to 41°C did not impact these levels within 4 weeks, but the colony counts of all strains tested declined (at a specific decline rate of 0.14 to 0.41 day-1) when the temperature was raised to 42°C. At this temperature, the concentration of Vermamoeba vermiformis in the biofilm, determined with quantitative PCR (qPCR), was about 2 log units lower than the concentration at 38°C. In columns operated at a constant temperature, ranging from 38 to 41°C, none of the tested strains multiplied in the biofilm at 41°C, in which also V. vermiformis was not detected. These observations suggest that strains of ST47 and ST62 did not multiply in the biofilm at a temperature of ≥41°C because of the absence of a thermotolerant host. IMPORTANCE: Growth of Legionella pneumophila in tap water installations is a serious public health concern. The organism includes more than 2,100 varieties (sequence types). More than 50% of the reported cases of Legionnaires' disease are caused by a few sequence types which are very rarely detected in the environment. Strains of selected virulent sequence types proliferated in biofilms on surfaces exposed to warm (38°C) tap water to the same level as environmental varieties and multiplied well as pure culture in a nutrient-rich medium at temperatures of 42 and 43°C. However, these organisms did not grow in the biofilms at temperatures of ≥41°C. Typical host amoebae also did not multiply at these temperatures. Apparently, proliferation of thermotolerant host amoebae is needed to enable multiplication of the virulent L. pneumophila strains in the environment at elevated temperatures. The detection of these amoebae in water installations therefore is a scientific challenge with practical implications.

Improved biostability assessment of drinking water with a suite of test methods at a water supply treating eutrophic lake water.

Assessment of drinking-water biostability is generally based on measuring bacterial growth in short-term batch tests. However, microbial growth in the distribution system is affected by multiple interactions between water, biofilms and sediments. Therefore a diversity of test methods was applied to characterize the biostability of drinking water distributed without disinfectant residual at a surface-water supply. This drinking water complied with the standards for the heterotrophic plate count and coliforms, but aeromonads periodically exceeded the regulatory limit (1000 CFU 100 mL(-1)). Compounds promoting growth of the biopolymer-utilizing Flavobacterium johnsoniae strain A3 accounted for c. 21% of the easily assimilable organic carbon (AOC) concentration (17 ± 2 µg C L(-1)) determined by growth of pure cultures in the water after granular activated-carbon filtration (GACF). Growth of the indigenous bacteria measured as adenosine tri-phosphate in water samples incubated at 25 °C confirmed the low AOC in the GACF but revealed the presence of compounds promoting growth after more than one week of incubation. Furthermore, the concentration of particulate organic carbon in the GACF (83 ± 42 µg C L(-1), including 65% carbohydrates) exceeded the AOC concentration. The increased biomass accumulation rate in the continuous biofouling monitor (CBM) at the distribution system reservoir demonstrated the presence of easily biodegradable by-products related to ClO2 dosage to the GACF and in the CBM at 42 km from the treatment plant an iron-associated biomass accumulation was observed. The various methods applied thus distinguished between easily assimilable compounds, biopolymers, slowly biodegradable compounds and biomass-accumulation potential, providing an improved assessment of the biostability of the water. Regrowth of aeromonads may be related to biomass-turnover processes in the distribution system, but establishment of quantitative relationships is needed for confirmation.

Polysaccharides and proteins added to flowing drinking water at microgram-per-liter levels promote the formation of biofilms predominated by bacteroidetes and proteobacteria.

Biopolymers are important substrates for heterotrophic bacteria in (ultra)oligotrophic freshwater environments, but information about their utilization at microgram-per-liter levels by attached freshwater bacteria is lacking. This study aimed at characterizing biopolymer utilization in drinking-water-related biofilms by exposing such biofilms to added carbohydrates or proteins at 10 µg C liter(-1) in flowing tap water for up to 3 months. Individually added amylopectin was not utilized by the biofilms, whereas laminarin, gelatin, and caseinate were. Amylopectin was utilized during steady-state biofilm growth with simultaneously added maltose but not with simultaneously added acetate. Biofilm formation rates (BFR) at 10 µg C liter(-1) per substrate were ranked as follows, from lowest to highest: blank or amylopectin (≤6 pg ATP cm(-2) day(-1)), gelatin or caseinate, laminarin, maltose, acetate alone or acetate plus amylopectin, and maltose plus amylopectin (980 pg ATP cm(-2) day(-1)). Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequence analyses revealed that the predominant maltose-utilizing bacteria also dominated subsequent amylopectin utilization, indicating catabolic repression and (extracellular) enzyme induction. The accelerated BFR with amylopectin in the presence of maltose probably resulted from efficient amylopectin binding to and hydrolysis by inductive enzymes attached to the bacterial cells. Cytophagia, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia grew during polysaccharide addition, and Alpha-, Beta-, and Gammaproteobacteria, Cytophagia, Flavobacteriia, and Sphingobacteriia grew during protein addition. The succession of bacterial populations in the biofilms coincided with the decrease in the specific growth rate during biofilm formation. Biopolymers can clearly promote biofilm formation at microgram-per-liter levels in drinking water distribution systems and, depending on their concentrations, might impair the biological stability of distributed drinking water.

Pyrosequence analysis of the hsp65 genes of nontuberculous mycobacterium communities in unchlorinated drinking water in the Netherlands.

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.

Nontuberculous mycobacteria, fungi, and opportunistic pathogens in unchlorinated drinking water in The Netherlands.

The multiplication of opportunistic pathogens in drinking water supplies might pose a threat to public health. In this study, distributed unchlorinated drinking water from eight treatment plants in the Netherlands was sampled and analyzed for fungi, nontuberculous mycobacteria (NTM), and several opportunistic pathogens by using selective quantitative PCR methods. Fungi and NTM were detected in all drinking water samples, whereas Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Aspergillus fumigatus were sporadically observed. Mycobacterium avium complex and Acanthamoeba spp. were not detected. Season had no influence on the occurrence of these organisms, except for NTM and S. maltophilia, which were present in higher numbers in the summer. Opportunistic pathogens were more often observed in premise plumbing water samples than in samples from the distribution system. The lowest number of these organisms was observed in the finished water at the plant. Thus, fungi, NTM, and some of the studied opportunistic pathogens can multiply in the distribution and premise plumbing systems. Assimilable organic carbon (AOC) and/or total organic carbon (TOC) had no clear effects on fungal and NTM numbers or on P. aeruginosa- and S. maltophilia-positive samples. However, L. pneumophila was detected more often in water with AOC concentrations above 10 µg C liter(-1) than in water with AOC levels below 5 µg C liter(-1). Finally, samples that contained L. pneumophila, P. aeruginosa, or S. maltophilia were more frequently positive for a second opportunistic pathogen, which shows that certain drinking water types and/or sampling locations promote the growth of multiple opportunistic pathogens.

Variability of invertebrate abundance in drinking water distribution systems in the Netherlands in relation to biostability and sediment volumes.

A survey of invertebrates in drinking water from treatment works, internal taps and hydrants on mains was carried out by almost all water companies in the Netherlands from September 1993 to August 1995. Aquatic sow bugs (Asellidae, 1-12 mm) and oligochaeta worms (Oligochaeta, 1-100 mm), both known to have caused rare though embarrassing consumer complaints, were found to form 98% of the mean biomass in water flushed from mains. Their numbers in the mains water ranged up to 1500 (mean 37) Asellidae m(-3) and up to 9900 (mean 135) Oligochaeta m(-3). Smaller crustaceans (0.5-2 mm) dominated the numbers in water from mains. e.g. water fleas (Cladocera and Copepoda up to 14,000 m(-3)). Common invertebrates in treated water and in tap water were Rotifera (<1 mm) and nematode worms (Nematoda, <2 mm). No Asellidae, large Oligochaeta (>5 mm) or other large invertebrates were found in 1560 samples of 200 l treated water or tap water. Large variations in invertebrate abundance were found within and between distribution systems. Of the variability of mean biomass in mains per system, 55%, 60% and 63% could statistically be explained by differences in the Biofilm Formation Rate, non-particulate organic matter and the permanganate index of the treated water of the treatment works respectively. A similar correlation was found between mean invertebrate biomass and mean sediment volumes in the distribution systems (R(2) = 52%).

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean.

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).

Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments.

Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at µg liter(-1) levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 µg C liter(-1) in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10(-2) liter·µg(-1) of C·h(-1)) ≫ maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10(-2) liter·µg(-1) of C·h(-1)). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell(-1) level was achieved at ≤10 µg C liter(-1) than at >10 µg C liter(-1) with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at µg liter(-1) levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.

Concentration and diversity of uncultured Legionella spp. in two unchlorinated drinking water supplies with different concentrations of natural organic matter.

Two unchlorinated drinking water supplies were investigated to assess the potential of water treatment and distribution systems to support the growth of Legionella spp. The treatment plant for supply A distributed treated groundwater with a low concentration (<0.5 ppm of C) of natural organic matter (NOM), and the treatment plant for supply B distributed treated groundwater with a high NOM concentration (8 ppm of C). In both supplies, the water temperature ranged from about 10°C after treatment to 18°C during distribution. The concentrations of Legionella spp. in distributed water, analyzed with quantitative PCR (Q-PCR), averaged 2.9 (± 1.9) × 10(2) cells liter(-1) in supply A and 2.5 (± 1.6) × 10(3) cells liter(-1) in supply B. No Legionella was observed with the culture method. A total of 346 clones (96 operational taxonomical units [OTUs] with ≥97% sequence similarity) were retrieved from water and biofilms of supply A and 251 (43 OTUs) from supply B. The estimation of the average value of total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77% of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity were observed in the supply with the low NOM concentration.

Detection of protozoan hosts for Legionella pneumophila in engineered water systems by using a biofilm batch test.

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

Effect of water composition, distance and season on the adenosine triphosphate concentration in unchlorinated drinking water in the Netherlands.

The objective of our study was to determine whether water composition, distance to the treatment plant and season significantly affect the adenosine triphosphate (ATP) concentration in distributed drinking water, in order to resolve the suitability of ATP as an indicator parameter for microbial regrowth. Results demonstrated that the ATP concentration in distributed water averaged between 0.8 and 12.1 ng ATP L(-1) in the Netherlands. Treatment plants with elevated biofilm formation rates in treated water, showed significantly higher ATP concentrations in distributed drinking water and ATP content was significantly higher in the summer/autumn compared to the winter period at these plants. Furthermore, transport of drinking water in a large-sized distribution system resulted in significantly lower ATP concentrations in water from the distal than the proximal part of the distribution system. Finally, modifications in the treatment significantly affected ATP concentrations in the distributed drinking water. Overall, the results from our study demonstrate that ATP is a suitable indicator parameter to easily, rapidly and quantitatively determine the total microbial activity in distributed drinking water.

Ammonia-oxidizing bacteria and archaea in groundwater treatment and drinking water distribution systems.

The ammonia-oxidizing prokaryote (AOP) community in three groundwater treatment plants and connected distribution systems was analyzed by quantitative real-time PCR and sequence analysis targeting the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Results demonstrated that AOB and AOA numbers increased during biological filtration of ammonia-rich anoxic groundwater, and AOP were responsible for ammonium removal during treatment. In one of the treatment trains at plant C, ammonia removal correlated significantly with AOA numbers but not with AOB numbers. Thus, AOA were responsible for ammonia removal in water treatment at one of the studied plants. Furthermore, an observed negative correlation between the dissolved organic carbon (DOC) concentration in the water and AOA numbers suggests that high DOC levels might reduce growth of AOA. AOP entered the distribution system in numbers ranging from 1.5 x 10(3) to 6.5 x 10(4) AOPs ml(-1). These numbers did not change during transport in the distribution system despite the absence of a disinfectant residual. Thus, inactive AOP biomass does not seem to be degraded by heterotrophic microorganisms in the distribution system. We conclude from our results that AOA can be commonly present in distribution systems and groundwater treatment, where they can be responsible for the removal of ammonia.

Free-living protozoa in two unchlorinated drinking water supplies, identified by phylogenic analysis of 18S rRNA gene sequences.

Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20 degrees C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.

Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR.

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.