Domestic hot-water boilers harbour active thermophilic bacterial communities distinctly different from those in the cold-water supply.

Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.

Comparison of rapid methods for detection of Giardia spp. and Cryptosporidium spp. (oo)cysts using transportable instrumentation in a field deployment.

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.

Rapid detection and enumeration of Giardia lamblia cysts in water samples by immunomagnetic separation and flow cytometric analysis.

Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated.

Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry.

Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation-independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml by filtration (pore size 0.45 microm), (2) then cells are resuspended with sterile filtered buffer and double-stained with FITC- and Alexa-conjugated Legionella-specific antibodies, (3) subsequently, the cells are immunomagnetically caught, and (4) finally, fluorescently labeled Legionella cells were flow cytometrically detected and quantified. The efficiency of each step was tested separately. The whole method allows detection of L. pneumophila in 180 min with a detection limit of around 500 cells/l and a recovery of Legionella cells of 52.1 % out of spiked tap water. Fluorescence microscopy and flow cytometric cell-counting correlated well.

Cryptosporidium spp. in drinking water. Samples from rural sites in Switzerland.

In most rural areas and small communities in Switzerland the drinking water is supplied to the consumers after a minimum or even no treatment at all. However, it is just in these areas where drinking water from sources of agricultural activities can be contaminated by liquid manure and faeces of pasturing animals. The Swiss drinking water regulations are limited to the monitoring of E. coli, Enterococcus spp. and total plate counts only. Hence, resistant pathogens, as for example Cryptosporidium spp., remain unnoticed. During a drinking water survey, which lasted from June 2003 to December 2004, water samples were collected from 3 selected rural sites in Switzerland. The drinking water was investigated for Cryptosporidium spp., E. coli, Enterococcus spp., Clostridium perfringens and other parameters. In all samples oocysts of Cryptosporidium spp. were detected at elevated concentrations of up to 0.18 oocysts/l. Between 28% and 75% of the oocysts were found to be vital by the excystation method. Sampled oocysts collected from the three sites were subjected to genotyping and in one case the isolate was found to belong to the genotype of C. parvum. No evidence for increased incidents of diarrhoea in the past years was noted by local authorities.

In glucose-limited continuous culture the minimum substrate concentration for growth, Smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii, an environmentally abundant bacterium.

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters µ(max) and K(s) were determined for growth with glucose. For both strains, µ(max) was determined in batch culture after different precultivation conditions. In the case of C. heintzii, µ(max) was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h(-1) C. heintzii grew immediately with a µ(max) of 0.17 ± 0.03 h(-1). After five transfers in batch culture, µ(max) had increased only slightly to 0.18 ± 0.03 h(-1). A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D = 0.075 h(-1) into glucose-excess batch medium E. coli grew only after an acceleration phase of ~3.5 h with a µ(max) of 0.52 h(-1). After 120 generations and several transfers into fresh medium, µ(max) had increased to 0.80 ± 0.03 h(-1). For long-term adapted chemostat-cultivated cells, a K(s) for glucose of 15 µg l(-1) for C. heintzii, and of 35 µg l(-1) for E. coli, respectively, was determined in (14)C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h(-1). Using the determined pure culture parameter values for K(s) and µ(max), it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s(min)). The values estimated for s(min) were dependent on growth rate; at D = 0.05 h(-1), it was 12.6 and 0 µg l(-1) for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h(-1), s(min) for E. coli had to be raised to 34.9 µg l(-1) whereas s(min) for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K(s) value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s(min).

Screening Swiss water bodies for potentially pathogenic free-living amoebae.

Free-ling amoebae (FLA) including Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia pedata, can cause opportunistic infections leading to severe brain pathologies. Human infections with pathogenic FLA have been increasingly documented in many countries. In Switzerland, thus far, the occurrence and distribution of potentially pathogenic FLA has not been investigated. Swiss water biotopes, including swimming pools, lakes, rivers and ponds, have now been screened for the presence of FLA, and assessment of their pathogenicity potential for a mammalian host has been undertaken. Thus, a total of 17 isolates were recovered by in vitro cultivation from these different aquatic sources. Characterization by sequence analysis of Acanthamoeba spp.-specific and 'FLA-specific PCR products amplified from 18s rDNA based on morphological traits, thermotolerance, and cytotoxicity towards murine fibroblasts yielded the following findings: Echinamoeba cf. exundans (3 isolates), Hartmannella spp. (3), Vannella spp. (4), Protacanthamoebica cf. bohemica (1), Acanthamoeba cf. castellanii (1) and Naegleria spp. (5). B. mandrillaris and N. fowleri did not range amongst these isolates. None of the isolates exhibited pronounced cytotoxicity and all failed to grow at 42 degrees C; therefore, they do not present any potential for CNS pathogenicity for humans.

Growth of Vibrio cholerae O1 Ogawa Eltor in freshwater.

Growth of Vibrio cholerae O1 Ogawa Eltor was studied with a growth assay in which autoclaved and filtered (0.22 microm) freshwater was inoculated at low cell density (5 x 10(3) cells ml(-1)) and proliferation was followed with flow cytometry. Against the common view, V. cholerae was able to grow extensively in different kinds of freshwater. The bacterium multiplied in river water, lake water and effluent of a wastewater treatment plant up to a cell density of 1.55 x 10(6) cells ml(-1). In these samples, apparent assimilable organic carbon (AOC(app)) concentrations ranged from 52 up to 800 microg l(-1) and the results demonstrate a positive trend between the AOC(app) concentration and final cell concentration, suggesting that AOC was a key parameter governing growth of V. cholerae. No growth was observed in waters (tap and bottled drinking water) containing less than approximately 60 microg AOC(app) l(-1). When pure cultures of V. cholerae were grown on identical lake water at different temperatures (20, 25 and 30 degrees C) the maximum specific growth rates (micromax) achieved were 0.22 h(-1), 0.32 h(-1) and 0.45 h(-1), respectively. In addition, growth was characterized in lake water samples amended with different concentrations of NaCl. The highest micromax of V. cholerae was recorded at moderate salinity levels (5 g NaCl l(-1), micromax=0.84 h(-1)), whereas at 30 g NaCl l(-1) (micromax=0.30 h(-1)) or 0 g NaCl l(-1) (micromax)=0.40 h(-1)) specific growth rates were significantly reduced. In the water tested here, micro(max) of V. cholerae was always around 50 % of that exhibited by a freshwater community of indigenous bacteria enriched from the water sampling site. Direct batch competition experiments between V. cholerae and the lake water bacterial community were performed at different temperatures in which V. cholerae was enumerated in the total community using fluorescent-surface antibodies. In all cases V. cholerae was able to grow and constituted around 10 % of the final total cell concentration of the community. No significant effect of temperature was observed on the outcome of the competition. Mathematical modelling of the competition at the different temperatures based on the calculated micromax values confirmed these experimental observations. The results demonstrate that V. cholerae is not only able to survive, but also able to grow in freshwater samples. In these experiments the bacterium was able to use a large fraction (12-62 %) of the AOC(app) available to the bacterial AOC-test community, indicating that V. cholerae has the ability to gain access to the substrates present in freshwater even in competition with an autochthonous bacterial lake water consortium.

Effect of integration of a GFP reporter gene on fitness of Ralstonia eutropha during growth with 2,4-dichlorophenoxyacetic acid.

Green fluorescent proteins (GFPs) are frequently used as marker and reporter systems to assess the fate and activity of microbial strains with the ability to degrade xenobiotic compounds. To evaluate the potential of this tool for tracking herbicide-degrading microorganisms in the environment a promoterless gfp was linked to the tfd C promoter, which is activated during degradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), and integrated into the chromosome of the 2,4-D-degrading strain Ralstonia eutropha JMP 134. The effects of the inserted gfp gene on the kinetics of 2,4-D degradation by R. eutropha in batch and chemostat culture were compared to those of the wild-type strain. In batch culture with 2,4-D as the only carbon and energy source the maximum specific growth rate of the gfp-marked strain did not differ significantly from the wild type. However, compared to the wild type, the 2,4-D steady-state concentration in 2,4-D-limited chemostat cultures of the gfp-marked strain was higher at all dilution rates tested. The reduced competitiveness of the gfp-marked strain at low substrate concentrations was confirmed in a competition experiment for 2,4-D in continuous culture at a dilution rate of 0.075 h-1. Reproducibly, the gfp-marked strain was displaced by the wild-type strain. The study clearly demonstrates that fitness of constructs cannot be assessed by measuring micro max with selected substrates in batch cultures only but that a thorough kinetic analysis is needed, which also considers slow, carbon-limited growth conditions as they occur in the environment.