Laurence Mathieu

Reversible shift in the α-, β- and γ-proteobacteria populations of drinking water biofilms during discontinuous chlorination

As disinfection strategies could support a shift of some bacterial populations, the biodiversity of drinking water biofilms depending on the disinfectant concentrations was explored. The effect of different chlorine sequences applied for several weeks (0.1-0.4-0.1 mg Cl(2)L(-1) or vice versa) was tested on the abundance of the alpha-, beta- and gamma-proteobacteria populations, used as indicators of changes in bacterial populations within drinking water biofilms. Using dynamic (industrial pilot) and batch (bench scale) conditions, our work demonstrated the ability of the 3 proteobacteria subclasses to re-organize following discontinuous chlorinations. The beta- and gamma-proteobacteria subclasses were favoured by high free residual chlorine concentrations (0.4 mg Cl(2)L(-1)) while alpha-proteobacteria population was sensitive to this oxidant level. The proteobacteria population shifts within the biofilm exposed to discontinuous chlorination were reversible. The resilience of the biofilm proteobacteria populations exposed to oxidant stress questioned the emergence of bacterial population less sensitive to chlorine.

Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH)

Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

Indigenous bacterial inocula for measuring the biodegradable dissolved organic carbon (BDOC) in waters

A collaborative study was carried out in order to compare the biodegradable dissolved organic carbon (BDOC) analysis using different indigenous mixed bacterial populations as inocula. The variables examined in the study were the laboratories, the water samples (raw, ozonated or finished water), the origin of the inoculum: suspended bacteria from river waters and attached bacteria onto sand taken from drinking water treatment plants. Variances associated with individual components of this study were isolated: the laboratories represented the highest source of variation (56.6–70.7%), while the inoculum origin contribution to the increase of variance is lower (12–26%). As a conclusion, indigenous mixed bacterial populations (suspended or attached bacteria) may be used reliably as standard inocula in BDOC determinations without introducing excessive variability.

Monitoring the effect of organic matter on biofilm growth in low nutritive waters by ATR/FT-IR spectroscopy

Bacterial biofilm growth was followed in low nutritive media by the use of attenuated total reflectance Fourier transform infrared (ATR/FT-IR) spectroscopy, an in situ non-destructive method. The aims were to determine the effect of organic matter of different origins on the development of the biofilm and to evaluate the usefulness of the method as an early warning signal for changes in organic matter in drinking water. The assays were carried out with a strain of Enterobacter cloacae attached to a zinc selenide crystal, which is compatible with vibrational spectroscopic analysis. The E. cloacae biofilm was grown in flow cells and was continuously fed with various filtered (0.2 w m pore size) water samples. The water samples (drinking water, diluted river water, and diluted algal bloom) all contained the same dissolved organic carbon (DOC) concentrations (1.5-1.7 mg lm 1) but variable quantities of biodegradable organic matter. The development of biofilms was continuously monitored for 2 weeks at 20°C. Protein amide II and polysaccharides C-O and C-O-C stretching band areas were measured around 1550 cmm 1 and 1050 cmm 1, respectively. The results indicated that ATR/FT-IR spectroscopy could discriminate between the time courses of biofilms in the three water samples tested, especially protein signals, even after relatively short periods of time (10 h). E. cloacae biofilms developed well with algal bloom and river water but not with drinking water. The absorption signals obtained within the first 10 h could lead to the development of an early warning system for changes in the nutritive status of drinking water.

Accumulation of MS2, GA, and Qβ phages on high density polyethylene (HDPE) and drinking water biofilms under flow/non-flow conditions.

Accumulation of enteric viruses on surfaces within a drinking water distribution system was investigated in a reactor using three F-specific RNA bacteriophages (MS2, GA, and Qβ) as models of human pathogenic viruses. The influence of hydrodynamic versus hydrostatic conditions and the effect of the colonization of HDPE surfaces with two-month-old biofilms were assessed for virus accumulation on surfaces. In order to work under controlled laminar conditions and to study various wall shear stresses at the same time, a new rotating disc reactor was designed. Among the wall shear rates applied in the reactor (450 to 1640 s(-1)) no significant differences were observed concerning both the total number of bacteria, which was found to be around 1.7 × 10(7) cells/cm(2) and the virus concentrations on surfaces were about 3 × 10(4), 5 × 10(5) and 3 × 10(5) eq PFU/cm(2) for MS2, GA and Qβ phages, respectively. Comparison between static versus dynamic conditions revealed that both Brownian diffusion and convective diffusion were involved in the transport of these soft colloidal particles and an increase reaching about 1 log in virus concentrations measured on surfaces appeared when hydrodynamic conditions where applied. Our results also showed the influence of the colonization by two-month-old drinking water biofilms which led to a change in the level of virus adhesion. The implication of the physico-chemical properties was also underlined since different adhesion profiles were obtained for the three bacteriophages and MS2 phage was found to be the less adherent one whatever the conditions applied.

Growth dynamic of Naegleria fowleri in a microbial freshwater biofilm

The presence of pathogenic free-living amoebae (FLA) such as Naegleria fowleri in freshwater environments is a potential public health risk. Although its occurrence in various water sources has been well reported, its presence and associated factors in biofilm remain unknown. In this study, the density of N. fowleri in biofilms spontaneously growing on glass slides fed by raw freshwater were followed at 32 °C and 42 °C for 45 days. The biofilms were collected with their substrata and characterized for their structure, numbered for their bacterial density, thermophilic free-living amoebae, and pathogenic N. fowleri. The cell density of N. fowleri within the biofilms was significantly affected both by the temperature and the nutrient level (bacteria/amoeba ratio). At 32 °C, the density remained constantly low (1-10 N. fowleri/cm(2)) indicating that the amoebae were in a survival state, whereas at 42 °C the density reached 30-900 N. fowleri/cm(2) indicating an active growth phase. The nutrient level, as well, strongly affected the apparent specific growth rate (μ) of N. fowleri in the range of 0.03-0.23 h(-1). At 42 °C a hyperbolic relationship was found between μ and the bacteria/amoeba ratio. A ratio of 10(6) to 10(7) bacteria/amoeba was needed to approach the apparent μ(max) value (0.23 h(-1)). Data analysis also showed that a threshold for the nutrient level of close to 10(4) bacteria/amoeba is needed to detect the growth of N. fowleri in freshwater biofilm. This study emphasizes the important role of the temperature and bacteria as prey to promote not only the growth of N. fowleri, but also its survival.

Pontiac fever among retirement home nurses associated with airborne legionella.

The aim of this study was to ascertain incidence of symptoms compatible with Pontiac fever (PF) and to assess their association with exposure to legionella bacteria among retirement home nurses who help residents take their shower. Within a non-epidemic framework, 104 nurses of 19 retirement homes were followed up during an average period of four months. Data on symptoms, number and location of showers they attended were recorded daily by each participant. Water and aerosol bacterial quality was characterised at the end of follow-up using the culturable and the in-situ hybridisation techniques. Among 11 Pontiac-like episodes, eight cases complied with the study definition of PF. Water concentrations >10(3) cfu legionella per litre were associated with an increased risk of PF, with dose-response patterns. No association was established between the aerosol legionella concentrations and PF events. A threshold value of 103 cfu legionella per litre of water might be used with a view to protection from legionella-associated occupational conditions.

Biocidal efficacy of monochloramine against planktonic and biofilm‐associated Naegleria fowleri cells

Free‐living amoebae (FLA) in aqueous systems are a problem for water network managers and health authorities because some are pathogenic, such as Naegleria fowleri, and they have also been reported to operate as reservoirs and vectors of several pathogenic bacteria. Therefore, to better control the occurrence of such amoebae, we evaluate the efficacy of monochloramine against planktonic forms (trophozoites and cysts) and also biofilm‐associated cells of N. fowleri as FLA are often associated with biofilms.

The action of chemical and mechanical stresses on single and dual species biofilm removal of drinking water bacteria

The presence of biofilms in drinking water distribution systems (DWDS) is a global public health concern as they can harbor pathogenic microorganisms. Sodium hypochlorite (NaOCl) is the most commonly used disinfectant for microbial growth control in DWDS. However, its effect on biofilm removal is still unclear. This work aims to evaluate the effects of the combination of chemical (NaOCl) and mechanical stresses on the removal of single and dual species biofilms of two bacteria isolated from DWDS and considered opportunistic, Acinectobacter calcoaceticus and Stenotrophomonas maltophilia. A rotating cylinder reactor was successfully used for the first time in drinking water biofilm studies with polyvinyl chloride as substratum. The single and dual species biofilms presented different characteristics in terms of metabolic activity, mass, density, thickness and content of proteins and polysaccharides. Their complete removal was not achieved even when a high NaOCl concentrations and an increasing series of shear stresses (from 2 to 23Pa) were applied. In general, NaOCl pre-treatment did not improve the impact of mechanical stress on biofilm removal. Dual species biofilms were colonized mostly by S. maltophilia and were more susceptible to chemical and mechanical stresses than these single species. The most efficient treatment (93% biofilm removal) was the combination of NaOCl at 175mg·l-1 with mechanical stress against dual species biofilms. Of concern was the high tolerance of S. maltophilia to chemical and mechanical stresses in both single and dual species biofilms. The overall results demonstrate the inefficacy of NaOCl on biofilm removal even when combined with high shear stresses.

Bacterial repopulation of drinking water pipe walls after chlorination.

Abstract The short-term kinetics of bacterial repopulation were evaluated after chlorination of high-density polyethylene (HDPE) colonized with drinking water biofilms and compared with bare HDPE surfaces. The effect of chlorination was partial as a residual biofilm persisted and was time-limited as repopulation occurred immediately after water resupply. The total number of bacteria reached the same levels on both the bare and chlorinated biofilm-fouled HDPE after a seven-day exposure to drinking water. Due to the presence of a residual biofilm, the hydrophobicity of chlorinated biofilm-fouled surface exhibited much lower adhesion forces (2.1 nN) compared to bare surfaces (8.9 nN). This could explain the rapid repopulation after chlorination, with a twofold faster bacterial accumulation rate on the bare HDPE surface. γ-Proteobacteria dominated the early stages of repopulation of both surfaces and a shift in the dominance occurred over the colonization time. Such observations define a timescale for cleaning frequency in industrial environments and guidelines for a rinsing procedure using drinking water.