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Previous Article | Next Article 
Applied and Environmental Microbiology, May 2007, p. 3437-3439, Vol. 73, No. 10
0099-2240/07/$08.00+0 doi:10.1128/AEM.00051-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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Centers for Disease Control and Prevention, Atlanta, Georgia,1 U.S. Environmental Protection Agency, Cincinnati, Ohio2
Received 9 January 2007/ Accepted 18 March 2007
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ABSTRACT |
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INTRODUCTION |
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In the present study, seven bacterial biothreat agents (10 isolates) were exposed to preformed monochloramine, and Ct values were calculated for 2-log10 and 3-log10 inactivation. These studies were conducted at three temperatures representative of a range found within water distribution systems, 5°C, 15°C, and 25°C (pH 8 for all temperatures).
Organisms included in this study were Bacillus anthracis Sterne 34F2, B. anthracis Ames, Burkholderia pseudomallei KC872, Burkholderia mallei M-9, Brucella suis MO562, Brucella melitensis ATCC 23456, Francisella tularensis NY98, F. tularensis LVS, Yersinia pestis A1122, and Y. pestis Harbin. B. anthracis was cultured on soil extract-peptone-beef extract agar (3) for 7 days to encourage spore formation. The cells and spores were then transferred to centrifuge tubes and treated with 50% ethanol for 1 h and then washed five times with sterile ultrapure water before they were stored in 50% ethanol at 5°C. F. tularensis isolates were cultured on Cysteine heart agar (3), and all other isolates were cultured on Trypticase soy agar with 5% sheep blood (Becton Dickinson Microbiology Systems, Sparks, MD) for 24 h before testing. B. anthracis spores were adjusted to 107 CFU/ml, and other bacterial suspensions were adjusted to 108 CFU/ml in 0.05 M KH2PO4, pH 8.0. All glassware and stir bars were treated to become chlorine demand-free before sterilization. Monochloramine solutions were prepared by the addition of NH4Cl and reagent-grade NaOCl in a 2.2:1 (N:Cl) molar ratio (equivalent to approximately 2:1 Cl2:N weight ratio) to 500 ml 0.05 M KH2PO4 adjusted to pH 8. One milliliter of bacterial suspension was added to a test flask containing 99 ml of the monochloramine solutions that had been equilibrated to the appropriate temperature and monochloramine concentrations (1 or 2 mg/liter). All tests were performed in triplicate. Aliquots of 3 ml were removed from the test flasks at given times and placed immediately into tubes containing 10 µl of 5% sodium thiosulfate to neutralize the disinfectant. Serial dilutions and spread plating were performed, plates were incubated (25°C for Y. pestis, 35°C for all other organisms), and CFU were counted and checked for up to 7 days after treatment. Testing methods are further described elsewhere (5). Free available chlorine (FAC) and monochloramine, as measured by total chlorine, were monitored using the N,N-diethyl-p-phenylenediamine (DPD) colorimetric analysis method (1). FAC and monochloramine were checked before the inoculation of each solution and after the final test period for all test runs, except for the B. anthracis tests, which were checked at every sampling time. Decay curves were generated using the mean log10 of the CFU counts at each sample time. The time each organism was inactivated by 99.0% or 99.9% was determined by linear regression of the appropriate segment of the decay curve. Disinfectant concentrations at the times of interest were estimated by linear regression. The Ct values for each inactivation level and test temperature were then determined by multiplying the inactivation time by the estimated monochloramine concentration.
The Ct values for inactivation of the organism tested at 2 and 3 orders of magnitude are given in Table 1. The B. anthracis spores were less susceptible to monochloramine disinfection than all other bacteria tested, with Ct values 10- to 1,000-fold greater.
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B. pseudomallei, B. mallei, B. suis, F. tularensis, and Y. pestis demonstrated a 2-log10 inactivation at Ct values of 21.9 to 52.5 at 25°C. Using similar conditions of temperature and pH, Ward et al. reported a mean Ct value of 54 mg·min/liter for Escherichia coli (17). The B. melitensis isolate was more resistant than the other gram-negative organisms, with a Ct value of 104.4 at 25°C (Table 1). If we consider that distribution systems target a residual monochloramine concentration of 2 mg/liter at the average residence time location (14) and if the Ct of Y. pestis Harbin is 25, then it would require 12.5 min to achieve a reduction of 3 log10 of this organism in a distribution system if water temperature and pH were similar to these test parameters (25°C and pH 8). Brucella melitensis, however, with a Ct of 116.6, would require 58.3 min to achieve the same 3-log10 level of inactivation.
The Ct values for all organisms increase with decreasing water temperature (Table 1) so that B. melitensis would require 112 min (almost 2 h) at 15°C and 290 min (almost 5 h) at 5°C to be reduced by 3 log10 organisms. The Brucella strains were the only alphaproteobacterial isolates of the bacteria tested. Preliminary evidence suggests that differences in membrane lipopolysaccharide core structures may exist between Brucella spp. and other gram-negative organisms, with variations within the genus reported (6). This variation within the genus is evidenced in these data in that B. suis was not as resistant to the disinfectant as B. melitensis. B. melitensis did not, however, show a marked tolerance to FAC (12), but this can likely be explained by the mechanistic differences of inactivation for the two disinfectants.
Monochloramine is thought to react with sulfur-containing amino acids and tryptophan in the bacterial cell wall (7, 8). The mechanism of FAC as a disinfectant differs in that FAC targets many aspects of the cell structure and metabolism, such as oxidation of the cell membrane, DNA damage, and respiration inhibition (11).
Bacillus spores are known to be more resistant to disinfectants than vegetative bacteria because of their complex spore coat (13), and resistance to monochloramine is no exception (Table 1). Water with the target residual of 2 mg/liter monochloramine would reduce the viability of B. anthracis Ames spores by 3 log10 in 10.0 h, 14.1 h, and 56.8 h at 25°C, 15°C, and 5°C, respectively. B. anthracis Sterne spores proved to be more resistant to inactivation, requiring 15.4 h, 32.7 h, and 126.4 h for a 3-log10 reduction of viable spores at 25°C, 15°C, and 5°C, respectively (Table 2).
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Monochloramine is a less effective disinfectant for all organisms tested when they are exposed at lower temperatures. At 5°C and 2 mg/liter monochloramine, none of the gram-negative bacteria tested can be inactivated, as defined by a 3-log10 reduction, within the 45 min mean time to the first point of use. At 5°C, 46 min to 4.8 h would be required for a 3-log10 inactivation, depending upon the organism.
B. anthracis spores cannot be reduced by 2 or 3 log10 under these median treatment conditions (45 min and 2 mg/liter monochloramine) regardless of temperature and would require hours or days of disinfectant exposure (Table 2).
Monochloramine, though a less effective disinfectant than free chlorine, is being used increasingly as a secondary disinfectant because of the tendency to form lower levels of the disinfection by-products (DBPs) closely regulated by the SWTR. Fewer taste and odor complaints from consumers also make monochloramine use attractive. Disadvantages include problems with controlling excess ammonia to avoid nitrification and the need to control pH for better efficacy. Many treatment facilities have opted to alternate between FAC and monochloramine to control nitrification problems and biofilm formation, to boost disinfection efficacy, and to reduce DBPs (18).
Water conditions such as pH (19), turbidity (9), nutrient availability (4), and the presence of biofilms (10) can alter the efficacy of chemical disinfectants; hence, the Ct values stated in this work are valid for these laboratory test conditions only. Additionally, given the variations in susceptibility observed among strains of the same species in these studies, continued testing of a variety of strains is essential to better prepare for protecting public health if potable water is ever contaminated with any of these bacterial agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 30 March 2007.
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REFERENCES |
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