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.

Probability of detecting and quantifying faecal contaminations of drinking water by periodically sampling for E. coli: a simulation model study.

Drinking water supply companies monitor the presence of Escherichia coli in drinking water to verify the effectiveness of measures that prevent faecal contamination of drinking water. Data are lacking, however, on the sensitivity of the monitoring programmes, as designed under the EU Drinking Water Directive. In this study, the sensitivity of such a monitoring programme was evaluated by hydraulic model simulations of contamination events and calculations of the detection probability of the actual sampling programme of 2002. In the hydraulic model simulations of 16-h periods of 1l h(-1) ingress of untreated domestic sewage, the spread of the contamination through the network and the E. coli concentration dynamics were calculated. The results show that when large parts of the sewage reach reservoirs, e.g. when they originate from the treatment plant or a trunk main, mean detection probabilities are 55-65%. When the contamination does not reach any of the reservoirs, however, the detection probability varies from 0% (when no sampling site is reached) to 13% (when multiple sites are reached). Mean detection probabilities of nine simulated ingress incidents in mains are 5.5% with an SD of 6.5%. In reality, these detection probabilities are probably lower as the study assumed no inactivation or clustering of E. coli, 100% recovery efficiency of the E. coli detection methods and immediate mixing of contaminations in mains and reservoirs. The described method provides a starting point for automated evaluations and optimisations of sampling programmes.

Sampling and quantifying invertebrates from drinking water distribution mains.

Water utilities in the Netherlands aim at controlling the multiplication of (micro-) organisms by distributing biologically stable water through biologically stable materials. Disinfectant residuals are absent or very low. To be able to assess invertebrate abundance, methods for sampling and quantifying these animals from distribution mains were optimised and evaluated. The presented method for collecting invertebrates consists of unidirectionally flushing a mains section with a flow rate of 1 ms(-1) and filtering the flushed water in two separate flows with 500 microm and 100 microm mesh plankton gauze filters. Removal efficiency from mains was evaluated in nine experiments by collecting the invertebrates removed from the mains section by intensive cleaning immediately subsequent to sampling. Of 12 taxa distinguished, all except case-building Chironomidae larvae (2%) and Oligochaeta (30%) were removed well (51-75%). Retention of invertebrates in 100 microm filters was evaluated by filtering 39 filtrates using 30 microm filters. Except for flexible and small invertebrates such as Turbellaria (13%), Nematoda (11%) and Copepoda larvae (24%), most taxa were well retained in the 100 microm filters (53-100%). During sample processing, the method for taking sub-samples with a 10 ml pipette from the suspension of samples with high sediment concentrations was found to perform well in 75% of the samples. During a 2-year national survey in the Netherlands and consecutive investigations, the method appeared to be very suitable to assess the abundance of most invertebrate taxa in drinking water distribution systems and to be practicable for relatively inexperienced sampling and lab technicians. Although the numbers of small, less abundant or sessile taxa were not accurately assessed using the method, these taxa probably should not be the primary focus of monitoring by water utilities, as consumer complaints are not likely to be caused by these invertebrates. The accuracy of quantifying small invertebrates was further improved, however, by filtering the 100microm filtrate with a 30microm mesh plankton gauze filter.