Jun 12, 1989 - Growth of Enterobacter cloacae on various media was compared after disinfection. ... Media were obtained from Difco Laboratories (Detroit,.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1989, p. 3226-3228
Vol. 55, No. 12
0099-2240/89/123226-03$02.00/0 Copyright C 1989, American Society for Microbiology
NOTES Enumeration of Enterobacter cloacae after Chloramine Exposure S. K. WATTERS,1 B. H. PYLE,1 M. W. LECHEVALLIER,2 AND G. A. McFETERS1* Department of Microbiology, Montana State University, Bozeman, Montana 59717,1 and Belleville Laboratory, American Water Works Service Co., Inc., Belleville, Illinois 622202 Received 12 June 1989/Accepted 5 September 1989
Growth of Enterobacter cloacae on various media was compared after disinfection. This was done to examine the effects of monochloramine and chlorine on the enumeration of coliforms. The media used were TLY (nonselective; 5.5% tryptic soy broth, 0.3% yeast extract, 1.0% lactose, and 1.5% Bacto-Agar), m-T7 (selective; developed to recover injured coliforms), m-Endo (selective; contains sodium sulfite), TLYS (TLY with sodium sulfite), and m-T7S (m-T7 with sodium sulfite). Sodium sulfite in any medium improved the recovery of chloramine-treated E. cloacae. However, sodium sulfite in TLYS and m-T7S did not significantly improve the detection of chlorine-treated E. cloacae, and m-Endo was the least effective medium for recovering chlorinated bacteria. Differences in recovery of chlorine- and chloramine-treated E. cloacae are consistent with mechanistic differences between the disinfectants.
suspended in phosphate buffer (1.86 mM KH2PO4, 2.4 mM Na2HPO4, 0.04 mM MgCl2; pH 7.0), and treated with chlorine (0.05 ppm [0.05 ,ug/ml], 0°C) or monochloramine (1.0 ppm, 20°C). Species and concentrations of chlorine remained stable throughout the experiments. Initial cell densities were adjusted to approximately 108 CFU/ml before chloramination and approximately 105 CFU/ml before chlorination. Chlorine experiments were performed with low cell densities so that no chlorine demand was created. Samples were removed at timed intervals, dechlorinated with sodium thiosulfate (final concentration, 0.01%), and spread on agar plates in triplicate. Enumeration media included m-T7, mEndo, TLY (5.5% tryptic soy broth, 0.3% yeast extract, 1.0% lactose, 1.5% Bacto-Agar), TLYS (TLY plus 0.05% sodium sulfite), and m-T7S (m-T7 plus 0.1% sodium sulfite). Plates were incubated at 35°C and counted after 48 h. Three replicates of each experiment were performed. Survival was calculated as log (nlno), where n was the number of CFU after a given time interval and no was the number of CFU at time 0. The statistical significance of differences between the media, determined by the t test by using MSU STAT (developed by Richard Lund, Montana State University), was calculated with data from the 15-min interval for chloramine experiments and the 60-s interval for chlorine experiments. Sodium sulfite in any medium improved the recovery of chloramine-treated E. cloacae (Fig. 1). Higher counts were observed on m-T7S, TLYS, and m-Endo (a selective medium containing sodium sulfite) than on TLY and m-T7. The greatest recovery of chloramine-exposed bacteria was seen on m-T7S, followed by TLYS, m-Endo, TLY, and m-T7, respectively. Longer exposure of cells to monochloramine enhanced the differences between the media. Differences in the recovery of injured E. cloacae are shown in Table 1. TLYS and m-T7S were both significantly more effective for the recovery of chloramine-treated E. cloacae than TLY or m-T7 was. TLY and m-Endo were significantly more effec-
Chloramination of drinking water is becoming a popular alternative to chlorination. Free chlorine frequently generates hazardous levels of trihalomethanes in water containing organic material, in contrast to the low levels of these potential carcinogens associated with chloramination (17). With the increased use of chloramines, it is important to determine if media currently being used to enumerate indicator bacteria in chlorinated water are appropriate for use with chloraminated water. Enumeration of coliforms in chlorinated drinking water is complicated by the observation that many of the surviving bacteria are injured (14). Injured indicator bacteria frequently lose the ability to grow on selective media commonly used to detect coliforms in water (1, 3, 5, 11-13). This results in an underestimation of the actual number of coliforms present, so a new medium, m-T7, was developed to detect injured coliforms (9). Little information is available on the ability of chloramines to cause bacterial injury. Jacangelo and co-workers (6-8) explored the mechanisms of chloramine inactivation and suggested that reducing agents may reverse the injurious effects of chloramines. Experiments performed by Duncanson and Cabelli (4) support this theory. They have shown that the presence of sodium sulfite (a reducing agent) in media enhances the recovery of chloramine-stressed coliforms. In this study, we examined the differences in recovery of both chlorine- and chloramine-injured Enterobacter cloacae on various media. Media were obtained from Difco Laboratories (Detroit, Mich.) and chemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise stated. Solutions of chlorine and monochloramine were prepared and measured as described by LeChevallier et al. (10). E. cloacae was obtained from a collection of organisms made during a study of unexplained coliform occurrences in the facilities of the South Central Connecticut Regional Water Authority and American Water Works Service Co. This bacterium was grown on R2A agar, scraped from the surface of the agar, *
tive than m-T7. The presence of sodium sulfite in the media did not have a dramatic effect on the recovery of chlorine-treated E. cloa-
Corresponding author. 3226
VOL. 55, 1989
NOTES
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FIG. 1. Comparative enumeration of E. cloacae treated with 1.0 ppm of monochloramine (pH 7.0, 20°C). Symbols for media: 0, TLY; 0, TLYS; A, m-T7; A, m-T7S; OI, m-Endo. The error bar to the right of the graph shows the width of a 95% confidence interval for each of the mean values shown at 15 min.
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Time (sec) FIG. 2. Comparative enumeration of E. cloacae treated with 0.05 ppm of chlorine (pH 7.0, 0°C). Symbols for media: 0, TLY; 0, TLYS; A, m-T7; A, m-T7S; [1, m-Endo. The error bar to the right of the graph shows the width of a 95% confidence interval for each of the mean values shown at 60 s.
cae (Fig. 2). No significant differences between the performances of TLYS, m-T7S, TLY, and m-T7 were observed (Table 1). However, TLYS and m-T7S were significantly more effective for recovering chlorine-injured cells than m-Endo was. The rates at which free chlorine and chloramines inactivate bacteria are considerably different (2, 15). Therefore, a comparison of medium performance was done with low concentrations of chlorine for short periods and with relatively high concentrations of monochloramine for much longer periods. Statistical comparisons were performed with data from intervals during which inactivation, as detected by survival on TLY medium, was similar following either chlorine or monochloramine treatment. The increased viability observed on the media containing sodium sulfite indicates that this reducing agent promoted recovery of chloramine-injured coliforms. Mechanistic differences between the actions of chlorine and chloramine may explain why this effect is not observed after chlorination. Jacangelo and co-workers (6-8) have theorized that sulfur-containing amino acids and tryptophan are the primary targets of monochloramine and that bacterial inactivation may be the result of inhibition of protein-associated activities. They have shown that oxidation of sulfhydryl groups to disulfides is reversible. Our data also indicate that some monochloramine damage is reversible. This could be attributed to reduction of disulfide bonds but may also be due to repair of other types of oxidative injury.
Chlorine has been shown to react rapidly with a much broader range of biochemicals than chloramine does (7). The reason sodium sulfite had little effect on the recovery of chlorine-treated E. cloacae may be the number and possible irreversibility of the oxidative reactions caused by chlorine. Our results indicate that there are differences in the culturability of chlorine- and monochloramine-injured coliforms. Therefore, caution needs to be exercised when a medium for enumeration of injured coliforms is selected. Even though m-T7 is an effective medium for the recovery of chlorine-injured coliforms, it should probably be modified for use with chloramine-treated coliforms. In contrast, mEndo has been found to be of limited use for the recovery of chlorine-injured coliforms but appears to be effective for recovering chloramine-treated bacteria. This observaton is consistent with data obtained by Rice et al. (16). Sodium sulfite-containing media such as m-Endo may also be preferable for the recovery of coliforms from chlorine-treated sewage effluent or other waters containing high levels of organic compounds, in which chlorine is likely to react with ammonia to form chloramines. Although media containing sodium sulfite are effective for recovering chloramine-injured coliforms under laboratory conditions, further studies using environmental samples obtained under field conditions should be done to test this observation.
TABLE 1. Results of statistical analysis to determine significant differences in bacterial recoveries with different media
LITERATURE CITED
Chlorine
Monochloramine Medium
Survival mean
(log[n/no]) m-T7S TLYS m-Endo TLY m-T7 a
-1.502 -2.286 -2.864 -3.375 -4.892
Nonsigni-
Survival
ficant setsa
(log[nn)
A
A, B B, C C D
mean
-2.304 -2.212 -3.004 -2.630 -2.637
Nonsigni-
fcn es ficant sets
E E F
E, F E, F Any two media with the same letter have means that are not significantly
different at a 0.01 level.
We thank Jarrod O'Leary for technical assistance and Ajaib Singh for helpful comments. This work was supported by American Water Works Association Research Foundation grant 309-87.
1. Braswell, J. R., and A. W. Hoadley. 1974. Recovery of Escherichia coli from chlorinated secondary sewage. Appl. Microbiol. 28:328-329. 2. Butterfield, C. T. 1948. Bactericidal properties of chloramines and free chlorine in water. Public Health Rep. 63:934-940. 3. Camper, A. K., and G. A. McFeters. 1979. Chlorine injury and the enumeration of waterborne coliform bacteria. Appl. Environ. Microbiol. 37:633-641. 4. Duncanson, R. A., and V. J. Cabelli. 1987. Membrane filter method for the enumeration of chlorine-damaged coliforms in drinking water, p. 57-69. 1986 Water Technology Conference proceedings, Portland, Oreg., American Water Works Association, Denver.
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NOTES
5. Hoadley, A. W., and C. M. Cheng. 1974. Recovery of indicator bacteria on selective media. J. Appl. Bacteriol. 37:45-57. 6. Jacangelo, J. G., and V. P. Olivieri. 1985. Aspects of the mode of action of monochloramine, p. 575-586. In R. L. Jolley (ed.), Water chlorination: chemistry, environmental impact and health effects, vol. 5. Lewis Publications, Inc., Chelsea, Mich. 7. Jacangelo, J. G., and V. P. Olivieri. 1987. Mechanisms of inactivation of microorganisms by combined chlorine. Research report of the American Water Works Association Research Foundation. American Public Health Association, Washington, D.C. 8. Jancangelo, J. G., V. P. Olivieri, and K. Kawata. 1987. Oxidation of sulfhydryl groups by monochloramine. Water Res. 21:1339-1344. 9. LeChevallier, M. W., S. C. Cameron, and G. A. McFeters. 1983. New medium for improved recovery of coliform bacteria from drinking water. Appl. Environ. Microbiol. 45:484-492. 10. LeChevallier, M. W., C. D. Cawthon, and R. G. Lee. 1988. Inactivation of biofilm bacteria. Appl. Environ. Microbiol. 54:2492-2499. 11. LeChevallier, M. W., and G. A. McFeters. 1985. Enumerating
12. 13. 14.
15. 16.
17.
injured coliforms in drinking water. J. Am. Water Works Assoc. 77:81-87. Lin, S. 1973. Evaluation of coliform tests for chlorinated secondary effluents. J. Water Pollut. Control Fed. 45:498-506. McFeters, G. A., and A. K. Camper. 1983. Enumeration of indicator bacteria exposed to chlorine. Adv. Appl. Microbiol. 29:177-193. McFeters, G. A., J. S. Kippin, and M. W. LeChevallier. 1986. Injured coliforms in drinking water. Appl. Environ. Microbiol. 51:1-5. National Academy of Sciences. 1980. Drinking water and health, vol. 2. National Academy Press, Washington, D.C. Rice, E. W., K. R. Fox, H. D. Nash, E. J. Read, and A. P. Smith. 1987. Comparison of media for recovery of total coliform bacteria from chemically treated water. Appl. Environ. Microbiol. 53:1571-1573. Stevens, A. A., C. J. Slocum, D. R. Seeger, and G. G. Robeck. 1978. Chlorination of organics in drinking water, p. 77-101. In R. L. Jolley (ed.), Water chlorination: environmental impact and health effects, vol. 1. Ann Arbor Science Publishers Inc., Ann Arbor, Mich.
