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Vol. 5, No. 3
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Dispatches
On April 21, 2003, the following correction was made to this article:
In the Table, in the sixth column and in the footnote
to the table, the units for the inactivation rate were changed to min-1.
Chlorine Inactivation of Escherichia coli
O157:H7
Eugene W. Rice, Robert M. Clark, and Clifford H. Johnson
U.S. Environmental Protection Agency, Cincinnati, Ohio, USA
| 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.
| Table. Chlorine inactivation of Escherichia
coli O157:H7 and wild-type E. coli a |
|
|
Log10CFU/ml |
|
|
|
|
|
|
|
|
After exposure time of |
|
|
| Isolate |
Initial
inoculum |
30 sec |
60 sec |
120 sec |
Inactivation
rate (min-1) |
r2 |
|
| E. coli O157:H7 |
|
|
|
|
|
|
| N009-6-1 |
5.63 |
2.60 |
1.88 |
0.82 |
-2.96 |
0.82 |
| N6001-8-10 |
5.78 |
2.52 |
1.44 |
0.72 |
-3.06 |
0.68 |
| N6021-5-1 |
5.78 |
2.54 |
1.52 |
0.66 |
-3.06 |
0.54 |
| N60049-26-1 |
5.68 |
2.35 |
1.40 |
0.54 |
-3.00 |
0.86 |
| N6059-7-2 |
5.72 |
2.42 |
1.74 |
0.86 |
-3.02 |
0.72 |
| N6104-5-9 |
5.62 |
2.40 |
1.69 |
0.72 |
-2.96 |
0.89 |
| N6114-7-2 |
5.63 |
2.52 |
1.66 |
0.89 |
-2.96 |
0.82 |
| Mean |
5.69 |
2.48 |
1.62 |
0.74 |
-2.93 |
0.82 |
| E. coli (wild type) |
|
|
|
|
|
|
| A |
5.53 |
2.66 |
1.80 |
1.52 |
-2.51 |
0.61 |
| B |
5.79 |
2.60 |
1.48 |
0.81 |
-2.68 |
0.60 |
| C |
5.68 |
2.48 |
0.92 |
0.84 |
-2.61 |
0.61 |
| D |
5.52 |
2.34 |
0.95 |
0.39 |
-2.50 |
0.61 |
| Mean |
5.63 |
2.52 |
1.28 |
0.89 |
-2.93 |
0.71 |
|
| aIn chlorine demand-free chlorinated
(CDF) buffer, 5ºC, pH 7.0, 1.1 mg/L free chlorine, 1.2 mg/L total
chlorine. Duplicate chlorine inactivation experiments were conducted
in CDF buffer at pH 7.0. All experiments were conducted at 5ºC in
a recirculating, refrigerated water bath. The chlorinated buffer was
prepared by the addition of reagent-grade sodium hypochlorite (Fisher
Scientific, Fair Lawn, NJ). Reaction vessels were continuously mixed
(250 rpm) by using an overhead stirring apparatus equipped with sterile
stainless steel paddles. Chlorine concentrations were determined by
the N,N-dimethyl-p-phenylenediamine colorimetric method (9).
Samples were removed from the reaction vessels at the desired exposure
times, and the chlorine was immediately neutralized by the addition
of 0.5 ml of 10% (wt/vol) sodium thiosulphate. Vessels containing
CDF buffer without chlorine served as controls for determining unexposed
concentrations of the bacteria. Initial levels and the number of survivors
after chlorine exposure were determined by the membrane filtration
procedure using mT7 agar incubated for 22 to 24 hours at 35ºC. This
medium was chosen because of its ability to recover oxidant-stressed
organisms (9). Levels of bacteria were determined
by duplicate filtrations of appropriate dilutions for each exposure
time.The log10-transformed data were used to determine
the levels of inactivation for each isolate. The means for the inactivation
data for the E. coli O157:H7 isolates and for the wild-type
E. coli isolates at each exposure time were used to compare
the inactivation rates between the pathogenic and the wild-type organisms.
The following first order model was used to describe the inactivation
rate: y = y1010-at,
, where t = time in seconds, y = CFU/ml at any time t,
y10 = CFU/ml at time zero, and a = the inactivation
rate in min-1. The log transformation of this equation
was used to calculate the inactivation rate. A regression analysis
using least squares was conducted for experiments with each individual
isolate and for the mean values for each of the two types of isolates
(serotype O157 and wild-type) to determine the inactivation rates
("a" values). |
|
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.
Acknowledgment
We thank Dr. Robert V. Tauxe for his encouragement and
guidance regarding this project.
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.
Address for correspondence: Eugene W. Rice, U.S.
Environmental Protection Agency, 26 West M.L. King Dr., Cincinnati, OH 45268, USA; fax:
513-569-7328; e-mail: rice.gene@epa.gov.
References
- Olsen J, Miller G, Breuer T, Kennedy M, Higgins C, McGee G, et al. A waterborne outbreak
of E. coli O157:H7 infections: evidence for acquired immunity. In: Program and
Abstracts of the 36th Annual Meeting of Infectious Diseases Society of America, Denver,
Colorado; 1998 Nov 12-15; [abstract 782]. Alexandria (VA): Infectious Disease Society of
America; 1998. p. 62.
- Blake P. Escherichia coli O157:H7 outbreak among visitors to a water park. In:
Program and Abstracts of the 36th Annual Meeting Infectious Diseases Society of America,
Denver, Colorado; 1998 Nov 12-15; [abstract 537]. Alexandria (VA): Infectious Diseases
Society of America; 1998. p. 178.
- Swerdlow DL, Woodruff BA, Brady RC, Griffin PM, Tippen S, Donnell HD, et al. A
waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody
diarrhea and death. Ann Int Med 1992;117:812-19.
- Dev VJ, Main M, Gould I. Waterborne
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- Ackman D, Marks S, Mack P, Caldwell M, Root T, Birkhead G. Swimming-associated
hemorrhagic colitis due to Escherichia coli O157:H7 infection: evidence of
prolonged contamination of a fresh water lake. Epidemiol Infect 1997;119:1-8.
- Brewster DH, Brown MI, Robertson D, Houghton GL, Bimson J, Sharp JCM.
An outbreak of Escherichia coli O157 associated with a children's paddling pool.
Epidemiol Infect 1994;112:441-7.
- Keene WE, McAnulty JM, Hoesly FC, Williams LP Jr, Hedberg K, Oxman GL,
et al.
A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7
and Shigella sonnei. N Engl J Med 1994;331:579-84.
- Rice EW, Johnson CH, Reasoner DJ.
Detection of Escherichia coli O157:H7 in water from coliform enrichment cultures.
Lett Appl Microbiol 1996;23:179-82.
- American Public Health Association. Standard methods for the examination of water and
wastewater. 19th ed. Washington: The Association; 1995.
- Water Quality Disinfection Committee. Survey of water utility disinfection practices. J
Am Water Works Assoc 1992;84:121-8.
- Rice EW, Johnson CH, Wild DK, Reasoner DJ. Survival of Escherichia coli O157:H7
in drinking water associated with a waterborne disease outbreak of hemorrhagic colitis.
Lett Appl Microbiol 1992;15:38-40.
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