Impact of growth conditions on resistance of Klebsiella pneumoniae to chloramines.

The resistance of Klebsiella pneumoniae to inorganic monochloramine (1.5 mg/liter; 3:1 Cl2:N ratio, pH 8.0) was examined in relation to growth phase, temperature of growth, and growth under decreased nutrient conditions. Growth phase did not impact resistance to chloramines. Mid-exponential and stationary-phase cells, grown in a yeast extract-based medium, had CT99 values and standard deviations of 4.8 +/- 0.1 and 4.6 +/- 0.2 mg.min/liter, respectively. Growth temperature did not alter chloramine resistance at short contact times. CT99 values of cells grown at 15 and 23 degrees C were 4.5 +/- 0.2 and 4.6 +/- 0.2 mg.min/liter, respectively. However, at longer contact times, CT99.99 values of cells grown at 15 and 23 degrees C were 14 and 8 mg.min/liter, respectively, suggesting a small resistant subpopulation for cells grown at the lower temperature. Growth under decreased nutrient conditions resulted in a concomitant increase in resistance to chloramines. When K. pneumoniae was grown in undiluted Ristroph medium and Ristroph medium diluted by 1:100 and 1:1,000, the CT99 values were 4.6 +/- 0.2, 9.6 +/- 0.4, and 24 +/- 7.0 mg.min/liter, respectively. These results indicate that nutrient availability has a greater impact than growth phase or growth temperature in promoting the resistance of K. pneumoniae to inorganic monochloramine.     

Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.

This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell.     

Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.

This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell.     

Inactivation of biofilm bacteria

The current project was developed to examine inactivation of biofilm bacteria and to characterize the interaction of biocides with pipe surfaces. Unattached bacteria were quite susceptible to the variety of disinfectants tested. Viable bacterial counts were reduced 99% by exposure to 0.08 mg of hypochlorous acid (pH 7.0) per liter (1 to 2 degrees C) for 1 min. For monochloramine, 94 mg/liter was required to kill 99% of the bacteria within 1 min. These results were consistent with those found by other investigators. Biofilm bacteria grown on the surfaces of granular activated carbon particles, metal coupons, or glass microscope slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than were unattached cells. In contrast, resistance of biofilm bacteria to monochloramine disinfection ranged from 2- to 100-fold more than that of unattached cells. The results suggested that, relative to inactivation of unattached bacteria, monochloramine was better able to penetrate and kill biofilm bacteria than free chlorine. For free chlorine, the data indicated that transport of the disinfectant into the biofilm was a major rate-limiting factor. Because of this phenomenon, increasing the level of free chlorine did not increase disinfection efficiency. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. These studies showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria. The research provides important insights into strategies for control of biofilm bacteria.     

Inactivation of biofilm bacteria.

The current project was developed to examine inactivation of biofilm bacteria and to characterize the interaction of biocides with pipe surfaces. Unattached bacteria were quite susceptible to the variety of disinfectants tested. Viable bacterial counts were reduced 99% by exposure to 0.08 mg of hypochlorous acid (pH 7.0) per liter (1 to 2 degrees C) for 1 min. For monochloramine, 94 mg/liter was required to kill 99% of the bacteria within 1 min. These results were consistent with those found by other investigators. Biofilm bacteria grown on the surfaces of granular activated carbon particles, metal coupons, or glass microscope slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than were unattached cells. In contrast, resistance of biofilm bacteria to monochloramine disinfection ranged from 2- to 100-fold more than that of unattached cells. The results suggested that, relative to inactivation of unattached bacteria, monochloramine was better able to penetrate and kill biofilm bacteria than free chlorine. For free chlorine, the data indicated that transport of the disinfectant into the biofilm was a major rate-limiting factor. Because of this phenomenon, increasing the level of free chlorine did not increase disinfection efficiency. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. These studies showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria. The research provides important insights into strategies for control of biofilm bacteria.     

Factors promoting survival of bacteria in chlorinated water supplies

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a second mechanism).     

Factors promoting survival of bacteria in chlorinated water supplies.

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a second mechanism).     

Cloning and sequencing of the gene encoding an aldehyde dehydrogenase that is induced by growing Alteromonas sp. Strain KE10 in a low concentration of organic nutrients

The protein composition of Alteromonas sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter(-1)) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD(+)-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in Escherichia coli, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.     

Ammonia-oxidizing bacteria in a chloraminated distribution system: seasonal occurrence, distribution and disinfection resistance.

Nitrification in chloraminated drinking water can have a number of adverse effects on water quality, including a loss of total chlorine and ammonia-N and an increase in the concentration of heterotrophic plate count bacteria and nitrite. To understand how nitrification develops, a study was conducted to examine the factors that influence the occurrence of ammonia-oxidizing bacteria (AOB) in a chloraminated distribution system. Samples were collected over an 18-month period from a raw-water source, a conventional treatment plant effluent, and two covered, finished-water reservoirs that previously experienced nitrification episodes. Sediment and biofilm samples were collected from the interior wall surfaces of two finished-water pipelines and one of the covered reservoirs. The AOB were enumerated by a most-probable-number technique, and isolates were isolated and identified. The resistance of naturally occurring AOB to chloramines and free chlorine was also examined. The results of the monitoring program indicated that the levels of AOB, identified as members of the genus Nitrosomonas, were seasonally dependent in both source and finished waters, with the highest levels observed in the warm summer months. The concentrations of AOB in the two reservoirs, both of which have floating covers made of synthetic rubber (Hypalon; E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.), had most probable numbers that ranged from less than 0.2 to greater than 300/ml and correlated significantly with temperature and levels of heterotrophic plate count bacteria. No AOB were detected in the chloraminated reservoirs when the water temperature was below 16 to 18 degrees C. The study indicated that nitrifiers occur throughout the chloraminated distribution system. Higher concentrations of AOB were found in the reservoir and pipe sediment materials than in the pipe biofilm samples. The AOB were approximately 13 times more resistant to monochloramine than to free chlorine. After 33 min of exposure to 1.0 mg of monochloramine per liter (pH 8.2, 23 degrees C), 99% of an AOB culture was inactivated. The amounts of this disinfectant that are currently used (1.5 mg/liter at a 3:1 ratio of chlorine to ammonia-N) may be inadequate to control the growth of these organisms in the distribution system.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine.

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Nonuniform spatial patterns of respiratory activity within biofilms during disinfection.

Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.     

Effect of the method of preparing monochloramine upon inactivation of MS2 coliphage, Escherichia coli, and Klebsiella pneumoniae.

Monochloramine prepared in situ by first adding chlorine to a suspension of microorganisms, followed by subsequent addition of ammonia, inactivated the MS2 coliphage more rapidly than did exposure of phage to monochloramine prepared either by adding chlorine to ammonia or by adding chlorine and ammonia simultaneously. The rapid viral inactivation was apparently due to the exposure of MS2 to free chlorine before the addition of ammonia. The average 99% CT value of MS2 when exposed to free chlorine was 1.3 and 1.1 at 5 and 15 degrees C, respectively. The average 99% CT values of MS2 briefly exposed to the combined action of free chlorine followed by the addition of ammonia to form monochloramine in situ were 19.3 and 1.5 at 5 and 15 degrees C, respectively. No 99% CT values were calculated for the inactivation of MS2 with preformed monochloramine because less than 1 log (90%) of inactivation occurred during a 4-h contact time. Inactivation of MS2 by monochloramine was more rapid at 15 than at 5 degrees C and when the chlorine to nitrogen weight ratio was 5:1 compared with 3:1. Monochloramine was a more efficient inactivating agent for the coliforms Escherichia coli and Klebsiella pneumoniae than it was for the MS2 coliphage.     

Thermal inactivation of Enterococcus faecium: effect of growth temperature and physiological state of microbial cells

AIMS: To provide data on the effects on culture temperature and physiological state of cells on heat resistance of Enterococcus faecium, which may be useful in establishing pasteurization procedures. METHODS AND RESULTS: The heat resistance of this Ent. faecium (ATCC 49624 strain) grown at different temperatures was monitored at various stages of growth. In all cases, the bacterial cells in the logarithmic phase of growth were more heat sensitive. For cells which had entered in the stationary phase, D70 values of 0.53 min at 5 degrees C, 0.74 min at 10 degrees C, 0.83 min at 20 degrees C, 0.79 min at 30 degrees C, 0.63 min at 37 degrees C, 0.48 min at 40 degrees C and 0.41 min at 45 degrees C were found. By extending the incubation times cells were more heat resistant as stationary phase progressed, although a different pattern was observed for cells grown at different temperatures. At the lower temperatures heat resistance increased progressively, reaching D70 values of 1.73 min for cells incubated at 5 degrees C for 50 days and 1.04 min for those grown at 10 degrees C for 16 days. At other temperatures assayed heat resistance became stable for late stationary phase cells, reaching D70 values of 1.05, 1.08 and 1.01 min for cultures incubated at 20, 30 and 37 degrees C. Heat resistance of cells obtained at higher temperatures, 40 and 45 degrees C, was significantly lower, with D70 values of 0.76 and 0.67 min, respectively. Neither the growth temperature nor the growth phase modified the z-values significantly. CONCLUSIONS: D70 values obtained for Ent. faecium (ATCC 49624) varies from 0.33 to 1.73 min as a function of culture temperature and physiological state of cells. However, z values calculated were not significantly influenced by these factors. A mean value of 4.50 +/- 0.39 degrees C was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall results strongly suggest that, to establish heat processing conditions of pasteurized foods ensuring elimination of Ent. faecium, it is advisable to take into account the complex interaction of growth temperature and growth phase of cells acting on bacterial thermal resistance.     

Inactivation of particle-associated coliforms by chlorine and monochloramine

Sieves and nylon screens were used to separate primary sewage effluent solids into particle fractions of less than 7- or greater than 7-micron size. The efficiency of separation was determined by using a particle counter. Indigenous coliforms associated with the particle fractions were tested for their resistance to chlorine and monochloramine. Coliforms associated with the less than 7-microns fraction were inactivated more rapidly by 0.5 mg of chlorine per liter at 5 degrees C and pH 7 than coliforms associated with the greater than 7-microns fraction. Homogenization of the greater than 7-microns fraction not only resulted in an increase in the number of less than 7-microns particles, but also increased the rate of inactivation to a rate similar to that of the less than 7-microns fraction. With 1 mg of monochloramine per liter at 5 degrees C and pH 7, particle size had no appreciable effect on the rate of inactivation. At pH 8, however, the less than 7-micron fraction was inactivated more rapidly than the greater than 7-micron fraction. The time required for 99% inactivation of the particle fractions with monochloramine at pH 7 or 8 was 20- to 50-fold greater than the time required for the same amount of inactivation with chlorine at pH 7. The results indicate that coliforms associated with sewage effluent particles are inactivated more rapidly with 0.5 mg of chlorine per liter than with 1.0 mg of monochloramine per liter. However, greater than 7-micron particles can have a protective effect against the disinfecting action of chlorine.     

Inactivation of particle-associated coliforms by chlorine and monochloramine.

Sieves and nylon screens were used to separate primary sewage effluent solids into particle fractions of less than 7- or greater than 7-micron size. The efficiency of separation was determined by using a particle counter. Indigenous coliforms associated with the particle fractions were tested for their resistance to chlorine and monochloramine. Coliforms associated with the less than 7-microns fraction were inactivated more rapidly by 0.5 mg of chlorine per liter at 5 degrees C and pH 7 than coliforms associated with the greater than 7-microns fraction. Homogenization of the greater than 7-microns fraction not only resulted in an increase in the number of less than 7-microns particles, but also increased the rate of inactivation to a rate similar to that of the less than 7-microns fraction. With 1 mg of monochloramine per liter at 5 degrees C and pH 7, particle size had no appreciable effect on the rate of inactivation. At pH 8, however, the less than 7-micron fraction was inactivated more rapidly than the greater than 7-micron fraction. The time required for 99% inactivation of the particle fractions with monochloramine at pH 7 or 8 was 20- to 50-fold greater than the time required for the same amount of inactivation with chlorine at pH 7. The results indicate that coliforms associated with sewage effluent particles are inactivated more rapidly with 0.5 mg of chlorine per liter than with 1.0 mg of monochloramine per liter. However, greater than 7-micron particles can have a protective effect against the disinfecting action of chlorine.     

Growth of Bacteria in Inorganic Medium at Different Levels of Airborne Organic Substances

Invasion rates of airborne organic substances into sterile mineral medium were compared by using flasks closed with cotton stoppers, silicone stoppers, and screw caps with Teflon gaskets. The resulting increases of dissolved organic carbon were 0.5, 0.2, and 0 mg/liter per week, respectively. The compounds supported the growth of lake water bacteria and a strain of Pseudomonas fluorescens. Growth rates were correlated to the permeability of the stoppers used. The measured input of organic carbon in the sterile mineral medium is considered to be a minimum value for the actual contribution of organic compounds by the air. Multiplication rates of the bacteria suggest that the organisms prevent the escape of volatile organic substances from the medium by rapid utilization. The steady nutrient supply through the air should be considered in growth experiments with bacteria at low concentrations of nutrients.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide.

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.