Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from
potable water. The effect of
disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of
hypochlorous acid (
ClOH) for 67 min or 4 ppm of
monochloramine (NH(2)Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of
disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of
ClOH or 4 ppm of NH(2)Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of
ClOH after 5 days of growth in the absence of
disinfectant. Whole-cell in situ hybridizations using fluorescently tagged,
16S rRNA-targeted
oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of
disinfectants. Specifically, Legionella pneumophila, E. coli, and beta and delta proteobacteria were identified within biofilms both before and
after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within
disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting
waterborne disease.