Volume 5, Number 3—June 1999
Chlorine Inactivation of Escherichia coli O157:H7
We analyzed isolates of Escherichia coli O157:H7 (which has recently caused waterborne outbreaks) and wild-type E. coli to determine their sensitivity to chlorination. Both pathogenic and nonpathogenic strains were significantly reduced within 1 minute of exposure to free chlorine. Results indicate that chlorine levels typically maintained in water systems are sufficient to inactivate these organisms.
Escherichia coli O157:H7 is becoming increasingly recognized as a waterborne pathogen. Two recent outbreaks during summer 1998, one involving a drinking water supply in Wyoming (1) and another involving recreational water exposure at a water park in Georgia (2), have underscored the role of water in transmission. Contaminated drinking water (3,4) and recreational water have been associated with outbreaks of hemorrhagic colitis caused by E. coli O157:H7 (5-7). Chlorination of water is one of the primary public health measures used to ensure that both potable water and water used in recreational settings are free of microbial pathogens. Our study was undertaken to determine the chlorine resistance of E. coli O157:H7 and compare this resistance with that of wild-type E. coli.
Seven strains of E. coli O157:H7, isolated from cattle from geographically distinct areas (Florida, Idaho, Illinois, Missouri, Texas, Washington, and Wisconsin), were obtained from the U.S. Department of Agriculture (D. Miller, Ames, IA). The isolates exhibited the characteristic phenotypic traits: sorbitol-negative, ß-glucuronidase–negative, lactose-positive, indole-positive, and positive for glutamate decarboxylase (8). All enterohemorrhagic isolates were active toxin producers, as determined by in vitro enzyme immunoassay (Meridian Diagnostics, Inc., Cincinnati, OH). These cattle isolates were chosen as representative strains that might contaminate water supplies after surface run-off from pastures and fields. Four wild-type E. coli isolates from cattle manure from a local dairy farm (Ohio) were characterized by biochemical test kits (bioMerieux Vitek, Hazelwood, MO). All bacterial cultures used in the disinfection experiments were grown for 18 to 20 hours at 35ºC in brain heart infusion broth, concentrated by centrifugation, and washed three times in phosphate buffer (9) before testing.
The results of the disinfection experiments, including the rates of inactivation, are shown in the Table. Initial levels for all isolates were 5.52 to 5.79 log10 CFU/ml. The mean chlorine levels at each exposure time were 1.1 mg/L free chlorine and 1.2 mg/L total chlorine. For both the pathogenic and the wild-type strains, exposure to these levels of chlorine for 1 minute reduced the viable populations by approximately four orders of magnitude. The inactivation rates and corresponding correlation coefficient (r2 ) values are listed in the Table. Little difference was observed in the rates of inactivation for the pathogenic and wild-type organisms.
These results indicate that the E. coli O157:H7 isolates used in this study were sensitive to chlorination and were similar in resistance to that of wild-type E. coli isolates. The biocidal activity of chlorine decreases with decreasing temperature (not done in this study). The 5ºC temperature we used represents a worst-case condition for both ground water or winter surface-water temperature. A survey of disinfection practices in the United States found that water utilities maintain a median chlorine residual of 1.1 mg/L and a median exposure time of 45 minutes before the point of first use in the distribution system (10). At this level of chlorination, E. coli O157:H7 is unlikely to survive conventional water treatment practices in the United States. E. coli O157:H7 survives at a similar rate to that of wild-type E. coli in nondisinfected drinking water (11). Survival patterns and sensitivity to chlorination previously observed for the strains used in this study suggest that wild-type E. coli could serve as an adequate indicator organism for fecal contamination of water. Using wild-type E. coli to indicate E. coli O157:H7 would be useful because most analytical procedures for detecting E. coli in drinking water (e.g., assays for lactose fermentation at 44ºC to 45ºC or production of the enzyme ß-glucuronidase) cannot detect pathogenic E. coli O157:H7 strains (8).
Although chlorination appears to adequately control this pathogen, not all municipal water supplies use chlorine disinfection. In addition, chlorine residual can dissipate under adverse conditions, and exposure to sunlight or organic chlorine-demand substances can greatly diminish chlorine levels. Protection of organisms associated with particulate matter, such as fecal material, can also readily decrease the biocidal activity of chlorine. These considerations are particularly important in determining the efficacy of chlorination in a recreational water setting. The results of this study indicate that the isolates studied were sensitive to chlorination. Evaluation of other isolates under differing environmental conditions would be worthy of further consideration.
Dr. Rice is a microbiologist in the Microbial Contaminants Control Branch, Water Supply and Water Resources Division, National Risk Management Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. His research focuses on detection and inactivation of waterborne pathogens and microbial indicator organisms.
We thank Dr. Robert V. Tauxe for his encouragement and guidance regarding this project.
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