The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.     

Elasticity and physico-chemical properties during drinking water biofilm formation

Atomic force microscope techniques and multi-staining fluorescence microscopy were employed to study the steps in drinking water biofilm formation. During the formation of a conditioning layer, surface hydrophobic forces increased and the range of characteristic hydrophobic forces diversified with time, becoming progressively complex in macromolecular composition, which in return triggered irreversible cellular adhesion. AFM visualization of 1 to 8 week drinking water biofilms showed a spatially discontinuous and heterogeneous distribution comprising an extensive network of filamentous fungi in which biofilm aggregates were embedded. The elastic modulus of 40-day-old biofilms ranged from 200 to 9000 kPa, and the biofilm deposits with a height >0.5 μm had an elastic modulus <600 kPa, suggesting that the drinking water biofilms were composed of a soft top layer and a basal layer with significantly higher elastic modulus values falling in the range of fungal elasticity.     

Identification of active bacterial communities in a model drinking water biofilm system using 16S rRNA-based clone libraries

Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rRNA gene clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, an annular reactor was used to generate model drinking water biofilms grown on polycarbonate slides. High-quality RNA was extracted from 2-month-old biofilms and used to generate 16S rRNA-based clones. Sequencing analyses of 16S rRNA-based clones suggested that the active bacterial fraction consisted of a few dominant bacterial groups related to Nevskia ramosa and to uncultured bacteria. Several of these bacterial groups were closely related to clones characterized in a DNA-based clone library also generated in this study. Altogether, these results suggest that some of the predominant drinking water bacteria identified using DNA-based techniques are indeed active.     

Effect of wall shear rate on biofilm deposition and grazing in drinking water flow chambers

The effect of four-wall shear rates (34.9, 74.8, 142.5, and 194.5 s(-1)) on bacterial deposition on glass slides in drinking water flow chambers was studied. Biofilm image acquisition was performed over a 50-day period. Bacterial accumulation and surface coverage curves were obtained. Microscopic observations allowed us to obtain information about the dynamics and spatial distribution of the biofilm. During the first stage of biofilm formation (210-518 h), bacterial accumulation was a function of the wall shear rate: the higher the wall shear rate, the faster the bacterial deposition (1.1 and 1.9 x 10(4) bacterial cells . cm(-2) for wall shear rates of 34.9 and 142.5 s(-1), respectively). A new similarity relationship characteristic of a non-dimensional time and function of the wall shear rate was proposed to describe initial bacterial deposition. After 50 days of exposure to drinking water, surface coverage was more or less identical under the entire wall shear rates (7.44 +/- 0.9%), suggesting that biofilm bacterial density cannot be controlled using hydrodynamics. However, the spatial distribution of the biofilm was clearly different. Under low wall shear rate, aggregates were composed of bacterial cells able to "vibrate" independently on the surface, whereas, under a high wall shear rate, aggregates were more cohesive. Therefore, susceptibility to the hydraulic discontinuities occurring in drinking water system may not be similar. In all the flow chambers, significant decreases in bacterial biomass (up to 77%) were associated with the presence of amoebae. This grazing preferentially targeted small, isolated cells.     

Phylogenetic diversity of drinking water bacteria in a distribution system simulator

AIMS: To characterize the composition of microbial populations in a distribution system simulator (DSS) by direct sequence analysis of 16S rDNA clone libraries. METHODS AND RESULTS: Bacterial populations were examined in chlorinated distribution water and chloraminated DSS feed and discharge water. Bacterial strains isolated from DSS discharge water on R2A medium were identified using 16S rDNA sequence analysis. The majority of the bacteria identified were alpha-proteobacteria, ranging from approx. 34% in the DSS discharge water to 94% of the DSS isolates. Species richness estimators Chao1 and ACE (abundance-based coverage estimators) indicated that the chlorinated distribution water sample was representative of the total population diversity, while the chloraminated DSS feed water sample was dominated by Hyphomicrobium sp. sequences. The DSS discharge water contained the greatest diversity of alpha-, beta-, gamma-proteobacteria, with 36% of the sequences being operational taxonomic units (OTUs, sequences with >97.0% homology). CONCLUSIONS: This work demonstrated the dominance of alpha-proteobacteria in distribution system water under two different disinfectant residuals. The shift from chlorine to monochloramine residual may have played a role in bacterial population dynamics. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of bacteria present in treated drinking water is needed in order to better determine the risk of regrowth of potentially pathogenic organisms within distribution systems.     

Occurrence of moulds in drinking water

AIMS: In order to determine the occurrence of filamentous fungi in public drinking water systems in Norway, water from 14 water supply networks from all over the country was sampled and analysed. Networks with both ground and surface water sources were included in this study. METHODS AND RESULTS: During a one-year period, 273 water samples were collected. Frequencies of fungi in samples from raw water, treated water and from home and hospital installations were determined on the basis of incubation of 100 ml membrane-filtered samples on dichloran 18% glycerol agar media. Filamentous fungi were recovered from 62% of all samples. In ground water 42.3% of the samples were positive for mould growth, while surface waters yielded 69.7% positive samples. CONCLUSIONS: The risk to recover moulds from surface water is three times higher compared with ground water. It is more likely to detect moulds in cold waters and showers than in hot waters. SIGNIFICANCE AND IMPACT OF THE STUDY: By analysing the water reaching the consumers, the results reported in present study indicate that filamentous fungi in drinking water is not negligible, and that moulds should be considered as part of the microbiological analysis parameters in drinking water.     

Identification and phylogeny of eukaryotic 18S rDNA phylotypes detected in chlorinated finished drinking water samples from three Parisian surface water treatment plants

Aims:  We performed a preliminary assessment of the eukaryotic 18S rDNA diversity present in finished drinking water samples from three different surface water treatment plants supplying water to the city of Paris (France).
Methods and Results:  A molecular analysis was performed on a sample from each site based on sequencing of PCR amplified and cloned 18S ribosomal RNA genes. Overall, the 18S rDNA sequences combined from all samples could be affiliated to the Amoebozoa (20·8% of the phylotypes), Ciliophora (25%), Metazoa (33·3%), Fungi (8·3%), Cercozoa (4·2%) and unclassified eukaryotes (8·3%) groups.
Conclusions:  The 18S rDNA sequences affiliated to the Amoebozoa, Ciliophora and Metazoa lineages were found to be the most abundant phylotypes observed in the drinking water samples. Phylotypes found to be present in two, or all three, samples (41·7% of the total) may represent groups with members adapted to drinking water treatment plant (DWTP) ecosystem conditions.
Significance and Impact of the Study:  This study shows that finished drinking water can contain 18S rDNA sequences representing a variety of eukaryotic taxa. Further research is needed to better characterize the eukaryotic biodiversity of DWTPs and the effects of the finished drinking water diversity on the downstream water distribution network.     

Risk assessment of adenoviruses detected in treated drinking water and recreational water

AIMS: Human adenoviruses (HAds) have previously been detected in sewage and polluted river and dam water, as well as treated drinking water. The 51 serotypes of HAds cause a wide range of infections with clinical manifestations associated with the gastrointestinal, respiratory and urinary tracts, and the eyes. Water may play a meaningful role in the transmission of many of these HAd serotypes, specifically the enteric HAds which are transmitted via the faecal-oral route. The presence of these viruses in water used for drinking and recreational purposes is considered to constitute a potential health risk. In this study, the risk of infection by the group of HAds previously detected over a period of 1 year in selected drinking water supplies, as well as river and dam water used for recreational purposes, was assessed. METHODS AND RESULTS: Adenoviruses were previously detected in nine of 204 (4.41%) samples of two drinking water supplies (A and B) treated and disinfected according to international specifications, in four of 51 (7.8%) samples of river water and nine of 51 (17.7%) samples of dam water. Application of these previously published results in an exponential risk assessment model indicated an annual risk of infection of 1.01 x 10(-1) and 1.7 x 10(-1) for drinking water supplies A and B, respectively, assuming a daily consumption of 2 l day(-1). The daily risk of infection constituted by HAds in the river water was calculated as 1.71 x 10(-4), and in the dam water as 3.12 x 10(-5), assuming a consumption of 30 ml of water per day. CONCLUSIONS: The risk of infection exceeded the tolerable risk of one infection per 10 000 consumers per year proposed for drinking water. However, the results for river and dam water used for recreational purposes were within the tolerable risk of one infection per 1000 bathers per day proposed for environmental waters used for recreational purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that the risk of HAd infection calculated for the drinking water supplies and the recreational water may overestimate the actual risk of infection, as conservative values were assumed for some of the variables. For a more accurate assessment of the potential risk of infection research should at least include a thorough investigation of the water consumption of individuals in South Africa, and the efficiency of recovery of the glass wool adsorption-elution method.     

Helicobacter pylori DNA in drinking water in Japan

Helicobacter pylori has been detected in drinking water in Peru and Sweden, suggesting the possibility of water-borne transmission. To date there have been few reports of H. pylori being detected in water; one was of the ureA gene of H. pylori in wells and springs in rural Japan. We examined water sampled in or near urban areas of Japan for H. pylori DNA by three assays using the polymerase chain reaction (PCR). Near Tokyo, samples were obtained: 10 of tap water, 6 of well water, 10 of river water, and 10 of sea water. Samples were filtered with membranes with 0.05- or 0.22-microm pores, which bacterial cells are caught by. Bacterial nucleic acids were extracted and purified and the PCR was done to amplify adhesin specific for H. pylori and the ureA gene, if present. Real-time PCR that measured the yield in terms of fluorescence was done with primers for 16S rRNA. None of the samples of tap, river, or sea water contained adhesin, ureA or 16S rRNA. None of the 6 samples of well water contained adhesin or ureA, but 2 of the 6 samples contained 16S rRNA. Some of the users of the well had had H. pylori infection in the past. H. pylori DNA was detected in well water and the users had been infected, so water-borne transmission via well water may occur even in towns in Japan.     

典型亚热带热分层水库秋季细菌群落垂直分布

水库在我国东南沿海地区是重要的饮用水水源地,对地区经济发展和社会稳定起到重要作用。选择亚热带地区典型的热分层水库——福建莆田东圳水库,于2011年秋季稳定分层期,以水体温度的垂直变化特征为依据进行分层采样。应用PCRDGGE和克隆测序的方法研究浮游细菌群落的垂直分布特征,利用多元统计分析揭示细菌群落与热分层水体理化指标之间的关系。结果显示:溶解氧、电导率、叶绿素a、总氮、氨氮及硝氮在上下层水体中的分布有显著差异,下层缺氧区细菌的ShannonWiener指数和DGGE条带数明显高于上层好氧区,表明东圳水库热分层水体中存在明显的物理、化学及生物分层现象。测序结果表明β-变形菌可能是东圳水库中占优势的细菌类群,统计结果提示溶解氧是显著影响细菌群落组成的环境因子。热分层水体的物理化学分层与水体细菌群落结构密切相关,提示水库生态学研究应对水体热分层给予重视。     

Prevention of bacterial diarrhea by pasteurization of drinking water in Kenya

Diarrheal disease is one of the major causes of morbidity and mortality in developing countries. Drinking water is a primary transmission route of infectious diarrheagenic bacteria in a rural area of Kenya (Microbiol. Immunol. 41: 773-778, 1997). We tried to prevent diarrhea at villages with approximately 1,500 households in Kenya by pasteurizing drinking water. A durable simple thermoindicator which changes color at 70 C was used as an indicator of pasteurization. The number of households in which drinking water was coliform bacteria-free increased from 10.7% to 43.1% after adoption of a pasteurization practice. Consequently, the incidence of severe diarrhea among people drinking pasteurized water was significantly lower than in people taking raw water (odds ratio=0.55, P=0.0016). The reduction ratio of the incidence after pasteurization was nearly equivalent with that after the adoption of a boiling method. Employment of women leaders as fieldworkers and demonstration of bacterial colony disappearance on agar plates by pasteurization also affected reduction of the diarrheal incidence.     

Effects of materials of construction on tastes and odours in drinking water

Products and materials used incontact with drinking water have the potentialto cause tainting of drinking water. Whilsthealth effects are not necessarily associatedwith such effects, for consumers, flavour orodour are the main parameters they use toassess the wholesomeness of their drinkingwater. There are a number of possible causes oftainting from products and materials and theseare discussed. In many countries products usedin contact with drinking water are screened toavoid odour or flavour problems. However,despite these safeguards tainting problems canand do still occur. Some relate tomisapplication of products in distributionsystems, poor domestic plumbing practices orthe use of domestic appliances after the pointof supply.     

Polaromonas and Hydrogenophaga species are the predominant bacteria cultured from granular activated carbon filters in water treatment

Aim:  Identification of the predominating cultivable bacteria in granular activated carbon (GAC) filters used in a variety of water treatment plants for selecting representative strains to study the role of bacteria in the removal of dissolved organic matter.
Methods and Results:  Bacterial isolates were collected from 21 GAC filters in nine water treatment plants treating either ground water or surface water with or without oxidative pretreatment. Enrichment of samples in dilute liquid medium improved culturability of the bacteria by approximately log unit, to 9% up to 70% of the total cell counts. Genomic fingerprinting and 16S rDNA sequence analysis revealed that most (68%) of the isolates belonged to the Betaproteobacteria and 25% were identified as Alphaproteobacteria . The number of different genera within the Betaproteobacteria was higher in the GAC filters treating ozonated water than in the filters treating nonozonated water. Polaromonas was observed in nearly all of the GAC filters (86%), and the genera Hydrogenophaga , Sphingomonas and Afipia were observed in 43%, 33% and 29% of the filter beds, respectively. AFLP analysis revealed that the predominating genus Polaromonas included a total of 23 different genotypes.
Conclusions:  This study is the first to demonstrate that Polaromonas , which has mainly been observed in ultraoligotrophic freshwater environments, is a common component of the microbial community in GAC filters used in water treatment.
Significance and Impact of the Study:  The predominance of ultraoligotrophic bacteria in the GAC filters indicates that very low concentrations of substrates are available for microbial growth. Polaromonas species are suited for further studies on the nutritional versatility and growth kinetics enabling the modelling of biodegradation processes in GAC filters.     

Influence of biofilms on iron and manganese deposition in drinking water distribution systems

Although health risk due to discoloured water is minimal, such water continues to be the source of one of the major complaints received by most water utilities in Australia. Elevated levels of iron (Fe) and/or manganese (Mn) in bulk water are associated with discoloured water incidents. The accumulation of these two elements in distribution systems is believed to be one of the main causes for such elevated levels. An investigation into the contribution of pipe wall biofilms towards Fe and Mn deposition, and discoloured water events is reported in this study. Eight laboratory-scale reactors were operated to test four different conditions in duplicate. Four reactors were exposed to low Fe (0.05?mg?l^?1) and Mn (0.02?mg?l^?1) concentrations and the remaining four were exposed to a higher (0.3 and 0.4?mg?l^?1 for Fe and Mn, respectively) concentration. Two of the four reactors which received low and high Fe and Mn concentrations were chlorinated (3.0?mg?l^?1 of chlorine). The biological activity (measured in terms of ATP) on the glass rings in these reactors was very low (～1.5 ng cm^?2 ring). Higher concentrations of Fe and Mn in bulk water and active biofilms resulted in increased deposition of Fe and Mn on the glass rings. Moreover, with an increase in biological activity, an increase in Fe and Mn deposition was observed. The observations in the laboratory-scale experiments were in line with the results of field observations that were carried out using biofilm monitors. The field data additionally demonstrated the effect of seasons, where increased biofilm activities observed on pipe wall biofilms during late summer and early autumn were found to be associated with increased deposition of Fe and Mn. In contrast, during the cooler months, biofilm activities were a magnitude lower and the deposited metal concentrations were also significantly less (ie a drop of 68% for Fe and 86% for Mn). Based on the laboratory-scale investigations, detachment of pipe wall biofilms due to cell death or flow dynamics could release the entrapped Fe and Mn into the bulk water, which could lead to a discoloured water event. Hence, managing biofilm growth on drinking water pipelines should be considered by water utilities to minimize accumulation of Fe and Mn in distribution networks.