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Applied and Environmental Microbiology, September 2003, p. 5463-5471, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5463-5471.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine

Margaret M. Williams{dagger} and Ellen B. Braun-Howland*

Wadsworth Center, New York State Department of Health, Albany, New York 12201

Received 18 September 2002/ Accepted 9 June 2003

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 (NH2Cl) 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 NH2Cl. 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 ß 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.

* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201. Phone: (518) 473-7925. Fax: (518) 402-5683. E-mail: bhowland{at}wadsworth.org.

{dagger} Present address: U.S. Environmental Protection Agency, NRMRL/WSWRD/MCCB, Cincinnati, OH 45268.

Applied and Environmental Microbiology, September 2003, p. 5463-5471, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5463-5471.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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