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.     

Novel method for studying the public health significance of macroinvertebrates occurring in potable water

An experimental procedure was developed to study the association of selected bacteria and macroinvertebrates and their response to free available chlorine disinfection. The organisms selected for study were Escherichia coli ( LacZ545 ), Enterobacter cloacae (ATCC 23355), and the amphipod Hyalella azteca . E. coli was shown to bind tightly (1.6 X 10(4) CFU per amphipod ) to this macroinvertebrate and to resist repeated attempts to wash it off. E. cloacae was shown to bind much less tightly (1.4 X 10(3) CFU per amphipod ) to H. azteca and was less resistant to removal by washing. The extent of association is a function of macroinvertebrate size (surface area), but the procedure produces repeatable results usable for controlled experimentation. This method, together with the dual bacterial identification criteria (morphology and antibiotic resistance), was used to study the response of unassociated and associated E. coli and E. cloacae to disinfection with free available chlorine at 1.0 mg/liter. Unassociated E. coli populations decreased to less than 1% of their zero time controls within 1 min of contact time, whereas more than 2% of the associated E. coli populations remained viable after 60 min of contact at 1.0 mg of free available chlorine per liter. Unassociated E. cloacae populations decreased to less than 1% of their zero time controls within 1 min of contact time, whereas ca. 15% of the associated E. cloacae populations remained viable after 60 min of contact at 1.0 mg of free available chlorine per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel method for studying the public health significance of macroinvertebrates occurring in potable water.

An experimental procedure was developed to study the association of selected bacteria and macroinvertebrates and their response to free available chlorine disinfection. The organisms selected for study were Escherichia coli ( LacZ545 ), Enterobacter cloacae (ATCC 23355), and the amphipod Hyalella azteca . E. coli was shown to bind tightly (1.6 X 10(4) CFU per amphipod ) to this macroinvertebrate and to resist repeated attempts to wash it off. E. cloacae was shown to bind much less tightly (1.4 X 10(3) CFU per amphipod ) to H. azteca and was less resistant to removal by washing. The extent of association is a function of macroinvertebrate size (surface area), but the procedure produces repeatable results usable for controlled experimentation. This method, together with the dual bacterial identification criteria (morphology and antibiotic resistance), was used to study the response of unassociated and associated E. coli and E. cloacae to disinfection with free available chlorine at 1.0 mg/liter. Unassociated E. coli populations decreased to less than 1% of their zero time controls within 1 min of contact time, whereas more than 2% of the associated E. coli populations remained viable after 60 min of contact at 1.0 mg of free available chlorine per liter. Unassociated E. cloacae populations decreased to less than 1% of their zero time controls within 1 min of contact time, whereas ca. 15% of the associated E. cloacae populations remained viable after 60 min of contact at 1.0 mg of free available chlorine per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of Legionella pneumophila to chlorine in tap water.

A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.     

Susceptibility of Legionella pneumophila to chlorine in tap water

A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.     

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.     

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.     

Note: susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0.1, 0.2 and 0.5 mg l-1), pH (6, 7 and 8) and temperature (4, 21 and 32 degrees C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH6 than at pH8 for both strains. Under the standard conditions of temperature 21 degrees C, pH7 and chlorine concentration 0.2 mg l-1, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Assessment of the bacteriological activity associated with granular activated carbon treatment of drinking water.

Bacteriological analyses were performed on the effluent from a conventional water treatment pilot plant in which granular activated carbon (GAC) had been used as the final process to assess the impact of GAC on the microbial quality of the water produced. Samples were collected twice weekly for 160 days from the effluents of six GAC columns, each of which used one of four different empty-bed contact times (7.5, 15, 30, and 60 min). The samples were analyzed for heterotrophic plate counts and total coliforms. Effluent samples were also exposed to chloramines and free chlorine for 60 min (pH 8.2, 23 degrees C). Bacterial identifications were performed on the disinfected and nondisinfected effluents. Additional studies were conducted to assess the bacteriological activity associated with released GAC particles. The results indicated that heterotrophic plate counts in the effluents from all columns increased to 10(5) CFU/ml within 5 days and subsequently stabilized at 10(4) CFU/ml. The heterotrophic plate counts did not differ at different empty-bed contact times. Coliforms (identified as Enterobacter spp.) were recovered from the nondisinfected effluent on only two occasions. The disinfection results indicated that 1.5 mg of chloramines per liter inactivated approximately 50% more bacteria than did 1.0 mg of free chlorine per liter after 1 h of contact time. Chloramines and chlorine selected for the development of different bacterial species--Pseudomonas spp. and Flavobacterium spp., respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of the bacteriological activity associated with granular activated carbon treatment of drinking water

Bacteriological analyses were performed on the effluent from a conventional water treatment pilot plant in which granular activated carbon (GAC) had been used as the final process to assess the impact of GAC on the microbial quality of the water produced. Samples were collected twice weekly for 160 days from the effluents of six GAC columns, each of which used one of four different empty-bed contact times (7.5, 15, 30, and 60 min). The samples were analyzed for heterotrophic plate counts and total coliforms. Effluent samples were also exposed to chloramines and free chlorine for 60 min (pH 8.2, 23 degrees C). Bacterial identifications were performed on the disinfected and nondisinfected effluents. Additional studies were conducted to assess the bacteriological activity associated with released GAC particles. The results indicated that heterotrophic plate counts in the effluents from all columns increased to 10(5) CFU/ml within 5 days and subsequently stabilized at 10(4) CFU/ml. The heterotrophic plate counts did not differ at different empty-bed contact times. Coliforms (identified as Enterobacter spp.) were recovered from the nondisinfected effluent on only two occasions. The disinfection results indicated that 1.5 mg of chloramines per liter inactivated approximately 50% more bacteria than did 1.0 mg of free chlorine per liter after 1 h of contact time. Chloramines and chlorine selected for the development of different bacterial species--Pseudomonas spp. and Flavobacterium spp., respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of disinfectants on Campylobacter jejuni

Because of the increasing recognition that Campylobacter jejuni is an important enteric pathogen of humans, we studied the effects of widely used disinfectants on the viability of this organism. At an inoculum size of 10(3) to 10(4) CFU/ml, 1.25 mg of hypochlorite per liter killed three strains within 1 min. At an inoculum size of 10(6) to 10(7) CFU/ml, 5 mg of hypochlorite per liter killed three strains within 15 min. Killing of similar concentrations of C. jejuni and Escherichia coli by hypochlorite was approximately the same. At the high inoculum, 0.15% phenolic compound, 10 mg of iodophor per liter, 1:50,000 quaternary ammonium compound, 70% ethyl alcohol, and 0.125% glutaraldehyde killed all three strains within 1 min. These studies demonstrate that, under the conditions we tested (pH 7.0; 24 to 26 degrees C), the recommended standard concentrations of disinfecting agents are adequate to destroy C. jejuni.     

Effects of disinfectants on Campylobacter jejuni.

Because of the increasing recognition that Campylobacter jejuni is an important enteric pathogen of humans, we studied the effects of widely used disinfectants on the viability of this organism. At an inoculum size of 10(3) to 10(4) CFU/ml, 1.25 mg of hypochlorite per liter killed three strains within 1 min. At an inoculum size of 10(6) to 10(7) CFU/ml, 5 mg of hypochlorite per liter killed three strains within 15 min. Killing of similar concentrations of C. jejuni and Escherichia coli by hypochlorite was approximately the same. At the high inoculum, 0.15% phenolic compound, 10 mg of iodophor per liter, 1:50,000 quaternary ammonium compound, 70% ethyl alcohol, and 0.125% glutaraldehyde killed all three strains within 1 min. These studies demonstrate that, under the conditions we tested (pH 7.0; 24 to 26 degrees C), the recommended standard concentrations of disinfecting agents are adequate to destroy C. jejuni.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

Physiological responses of bacteria in biofilms to disinfection.

In situ enumeration methods using fluorescent probes and a radioisotope labelling technique were applied to evaluate physiological changes of Klebsiella pneumoniae within biofilms after disinfection treatment. Chlorine (0.25 mg of free chlorine per liter [pH 7.2]) and monochloramine (1 mg/liter [pH 9.0]) were employed as disinfectants in the study. Two fluorgenic compounds, 5-cyano-2,3-ditolyl tetrazolium chloride and rhodamine 123, and tritiated uridine incorporation were chosen for assessment of physiological activities. Results obtained by these methods were compared with those from the plate count and direct viable count methods. 5-Cyano-2,3-ditolyl tetrazolium chloride is an indicator of bacterial respiratory activity, rhodamine 123 is incorporated into bacteria in response to transmembrane potential, and the incorporation of uridine represents the global RNA turnover rate. The results acquired by these methods following disinfection exposure showed a range of responses and suggested different physiological reactions in biofilms exposed to chlorine and monochloramine. The direct viable count response and respiratory activity were affected more by disinfection than were the transmembrane potential and RNA turnover rate on the basis of comparable efficiency as evaluated by plate count enumeration. Information revealed by these approaches can provide different physiological insights that may be used in evaluating the efficacy of biofilm disinfection.     

Chlorine disinfection of atypical mycobacteria isolated from a water distribution system

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C.t value (product of the disinfectant concentration and contact time) of 60 mg.min.liter(-1), frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C.t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4 degrees C to 25 degrees C) and a decrease in pH result in better inactivation.     

Bacterial nitrification in chloraminated water supplies.

Nitrifying bacteria were detected in 64% of samples collected from five chloraminated water supplies in South Australia and in 20.7% of samples that contained more than 5.0 mg of monochloramine per liter. Laboratory experiments confirmed that nitrifying bacteria are relatively resistant to the disinfectant. Increased numbers of the bacteria were associated with accelerated decays of monochloramine within distribution systems. The combination of increased concentrations of oxidized nitrogen with decreased total chlorine residuals can be used as a rapid indicator of bacterial nitrification.