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.     

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.     

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.     

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.     

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.     

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.     

Nematodes from river drift and surface drinking water supplies in southern Ontario

Sixty-six genera of nematodes representing 37 families were collected over a 13-month period from untreated and treated water from three water treatment plants in southern Ontario. Two plants receive water from the Grand River which drains agricultural, residential and industrial regions, the third from a small stream in an agricultural district. Specimens were isolated by filtration from weekly samples of untreated and treated water, most were identified to genus but a few to species.Estimated mean density in the organic drift of the river ranged from 0.58 individuals per litre in winter, when it was under ice, to 10.57 per litre during the spring thaw. Most of the 66 genera found passed through the treatment plants and over 50% of these were still motile.Eleven genera were abundant at some time of the year, six known only from soil-dwelling forms and five from aquatic or soil/aquatic species. There was no indication that sewage purification works were a major source of any genus. There were a few plant parasites.Aquatic nematodes occurred during most of the year although they were more abundant during the warmer months, high densities of soil nematodes were a feature of the spring thaw and rains and during heavy autumn rains. More than 50% of the specimens were juveniles and some common genera lacked males.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Bio-stabilization of drinking water in supply systems as observed in the tropics

Changes in the bacterial quality of drinking water were examined in the supply system of tropical Dar es Salaam, Tanzania. The water treatment fulfilled WHO standard guidelines. However, the water quality allowed for significant regrowth of bacteria, demonstrating that the WHO guidelines are insufficient to check whether or not the drinking water has an acceptable regrowth potential. An experimental pipe system was examined on site. A growth model was established, based on a zero order bacterial detachment kinetics for the biofilms colonizing the inner surface of the pipes, with a release rate factor R, and a first order growth kinetics in the water flowing in the pipes with a growth rate factor . The rate factors were estimated to be R=1.9×10⁸ m^-2 h^-1 and =2.5 h^-1. It was concluded that growth in the biofilm played a dominant role in the bio-stabilization processes, while the initial microbial quality of the water works water and the growth in the water phase were of minor importance. The bio-stabilization process is understood herein as the reduction in the concentration of nutrients and assimilable organic carbon in the water, and thus as the reduction of the growth potential of the water. Bacterial growth and recontamination were examined during storage at the user site. The results showed that heterotrophic bacteria grew, while faecal coliforms decayed during storage. It was concluded that the users handled the water hygienically.The authors are with the Technical University of Denmark, IMT, CDC-Build, 208 DK-2800 Lyngby, Denmark     

Rats retain chromium in tissues following chronic ingestion of drinking water containing hexavalent chromium

Humans have sometimes been exposed to as much as 10 ppm Cr(VI) in drinking water from contaminated wells. The risks to these individuals are not well understood because the digestive tract reduces some of the Cr(VI) to the less bioavailable Cr(III) prior to absorption, and the disposition of the remaining Cr(VI) has not been well studied. We determined tissue Cr concentrations in rats after chronic ingestion of Cr(VI) in drinking water at concentrations relevant to human exposure levels. Adult male and female Fischer 344 rats consumed ad libitum 0, 0.5, 3, or 10 ppm Cr(VI) as K₂CrO₄ in drinking water for 44 wk. Rats then were given deionized water 4–6 d prior to sample collection. Females given 3 or 10 ppm Cr(VI) consumed more Cr(VI) per unit of body weight than did males. Bone Cr concentrations were significantly elevated in rats that drink 10 ppm Cr(VI). Renal Cr concentrations were significantly elevated in male rats that drink 3 or 10 ppm Cr(VI) and in female rats dosed with 10 ppm Cr(VI). Female rats had elevated liver Cr concentrations after drinking 3 or 10 ppm Cr(VI). Testicular Cr concentrations were slightly elevated in rats that drank 10 ppm Cr(VI). Brain, ovarian, and whole-blood Cr concentrations were below detection limits in all exposure groups. Although tissue Cr accumulation may have resulted from absorption of Cr(III), it is poorly absorbed. Therefore, the increased tissue retention may also have resulted, in part, from increased absorption of Cr(VI) and its subsequent uptake from the systemic circulation.     

Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

AIMS: To evaluate the prevalence of Cryptosporidium and Giardia in surface water supplies from the province of Alava, northern Spain, and to investigate possible associations among the presence of these pathogenic protozoa with microbiological, physicochemical and atmospheric parameters. METHODS AND RESULTS: A total of 284 samples of drinking and recreational water supplies were analysed. Cryptosporidium oocysts were found in 63.5% of river samples, 33.3% of reservoirs samples, 15.4% and 22.6% of raw water samples from conventional and small water treatment facilities (respectively), 30.8% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. Giardia cysts were found in 92.3% of river samples, 55.5% of reservoirs samples, 26.9% and 45.2% of raw water samples from conventional and small water treatment facilities (respectively), 19.2% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. The presence of Cryptosporidium and Giardia had significant Pearson's correlation coefficients (P < 0.01) with the turbidity levels of the samples, and a number of significant associations were also found with the count levels for total coliforms and Escherichia coli. The samples were positive for Cryptosporidium significantly (P < 0.05) more frequently during the autumn season than during the spring and winter seasons. No significant differences were found in the seasonal pattern of Giardia. A moderate association (r = 0.52) was found between rainfall and the presence of Cryptosporidium oocysts. CONCLUSIONS: Cryptosporidium and Giardia are consistently found at elevated concentrations in surface waters for human consumption from the province of Alava, northern Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Water treatments based on rapid filtration process and/or chlorination only are often unsatisfactory to provide safe drinking water, a situation that represents an important public health problem for the affected population because of the risk of waterborne outbreaks.