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.     

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.     

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.     

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.     

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.     

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.     

Study on inactivation of iron bacteria isolated from real drinking water distribution systems by free chlorine and chloramine

To study the inactivation characteristic of iron bacteria isolated from real drinking water distribution systems and investigate the influence of disinfectants, pH and temperature on inactivation process. Two kinds of iron bacteria were isolated from the water phase in distribution systems and identified asAcinetobacter baumannii andMicrobacterium oxydans. Bench-scale study on inactivation of the two kinds of iron bacteria were carried out, with the impact of disinfectants, pH and temperature under different levels concerned. Free chlorine and monochloramine could achieve an inactivation rate of 99.9% on bothA. Baumannii andM. Oxydans with the CT-value of 10 mg/L·min. Free chlorine was more effective than monochloramine with 1∼2 log higher inactivation rate.Microbacterium oxydans was more resistant to disinfectant thanA. Baumannii. High pH enhanced the inactivation of A.baumannii and low temperature availed the inactivation of bothA. Baumannii andMicrobacterium. For iron bacteria in the water, inactivation ratio could not reach 99% when residual chlorine was 0.05 mg/L in drinking water distribution systems according to Standards for Drinking Water Quality.     

Comparison of the efficacy of free residual chlorine and monochloramine against biofilms in model and full scale cooling towers.

The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion. Biofilms accumulate on all submerged industrial and environmental surfaces. The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm. The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower. The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers. A cooling tower and a laboratory model recirculating water system were used as biofilm reactors. Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers. Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal. Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons. Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Comparison of the Efficacy of Free Residual Chlorine and Monochloramine against Biofilms in Model and Full Scale Cooling Towers

The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion. Biofilms accumulate on all submerged industrial and environmental surfaces. The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm. The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower. The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers. A cooling tower and a laboratory model recirculating water system were used as biofilm reactors. Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers. Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal. Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons. Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

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.     

Inactivation of Adenoviruses,Enteroviruses, and Murine Norovirus in Water by Free Chlorine and Monochloramine

Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41 (HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and E11), and murine norovirus (MNV) are compared in this study. Experiments were performed with 0.2 mg of free chlorine or 1 mg of monochloramine/liter at pH 7 and 8 in buffered reagent-grade water at 5°C. CT values (disinfectant concentration × time) for 2- to 4-log₁₀ (99 to 99.99%) reductions in virus titers were calculated by using the efficiency factor Hom model. The enteroviruses required the longest times for chlorine inactivation and MNV the least time. CVB5 required the longest exposure time, with CT values of 7.4 and 10 mg·min/liter (pH 7 and 8) for 4-log₁₀ inactivation. Monochloramine disinfection was most effective for E1 (CT values ranged from 8 to 18 mg·min/liter for 2- and 3-log₁₀ reductions, respectively). E11 and HAdV2 were the least susceptible to monochloramine disinfection (CT values of 1,300 and 1,600 mg-min/liter for 3-log₁₀ reductions, respectively). Monochloramine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disinfection. These data will be useful in drinking water risk assessment studies and disinfection system planning.Disinfection is a critical step in the drinking water treatment process to inactivate infectious viruses because primary treatment is less effective for the removal of viruses. Chlorine and monochloramine are the most widely used disinfectants in the United States (2). Free chlorine is widely used as a primary disinfectant following filtration and also as a secondary disinfectant in distribution systems. Under the Long Term 2 Enhanced Surface Water Treatment Rule (38), monochloramine can also be used as a primary disinfectant, but because it requires longer contact times to achieve the same level of disinfection as free chlorine it is primarily used as a secondary disinfectant to maintain a stable disinfectant residual in the distribution system and minimize disinfection by-product formation and biofilm growth.The efficacy of chlorine disinfection for viruses has been evaluated in numerous studies over the years. Many early studies focused on the disinfection of polioviruses by chlorine (14, 17, 26, 28, 30, 39, 40, 43). Early investigators suggested a number of variables that must be controlled in the disinfection of viruses: contact time, temperature, ionic strength, pH, chlorine concentration, and virus aggregation (29, 30). These researchers concluded that comparisons and general trends of disinfection efficacy can only be discerned for viruses when the same disinfection parameters are applied.Fewer studies have investigated the disinfection efficacy of monochloramine, but monochloramine disinfection has been found to be less effective than free chlorine for viruses. In comparative studies of chlorine and monochloramine disinfection, coxsackievirus B5, adenovirus 2, and adenovirus 41 were found to be inactivated far more readily by chlorine than monochloramine (4, 5, 32). For drinking water treatment systems where monochloramine is used as a secondary disinfectant, it is important to know its efficacy for a wide range of viruses, as infectious viruses may be introduced in the distribution system where only monochloramine is present. In addition, relatively few studies have investigated the efficacy of monochloramine as systematically as free chlorine; frequently only one concentration, pH, or temperature has been investigated. Two notable exceptions were investigations that examined monochloramine disinfection of human adenovirus 2 (HAdV2) and coxsackievirus B5 (CVB5) at multiple pH levels (21, 31).In 2005, the U.S. Environmental Protection Agency (USEPA) published its second candidate contaminant list (CCL2). The CCL2 is comprised of unregulated microbial and chemical contaminants of potential public health concern that are known or anticipated to occur in drinking water systems and includes: echovirus, coxsackievirus, adenovirus, and calicivirus (36). A number of researchers have reported the disinfection efficacy of free chlorine for representatives of the CCL2 viruses (4, 5, 7, 11, 13, 18, 20, 22, 27, 33, 34, 35), but fewer studies have investigated the disinfection efficacy of monochloramine on these viruses (4, 5, 21, 31). In addition, comparison between existing studies of chlorine or monochloramine disinfection is difficult because of differences in the viruses examined, experimental parameters investigated, and analytical methods used.The present study compared the inactivation kinetics for representative CCL2 viruses with levels of free chlorine and monochloramine recommended for drinking water disinfection. Duplicate experiments with both disinfectants were carried out in pH 7 and 8 buffered chlorine-demand-free (CDF) water at 5°C, with eight viruses chosen to represent the CCL2 virus types. Coxsackieviruses B5 and B3 (CVB5 and CVB3) and echoviruses 1 and 11 (E1 and E11) were chosen based on existing data suggesting resistance to free chlorine, disease implications, and likelihood of presence in higher numbers in natural water. Three representative human adenoviruses were studied, including both serotypes of species F HAdV (40 and 41) that cause gastroenteritis and HAdV2, a representative of respiratory HAdV that may be found in water because they are present in fecal excretions (9). Murine norovirus (MNV), phylogenetically similar to human norovirus and the only norovirus that can be propagated in cell culture, was used as a surrogate for human norovirus. Kinetic inactivation curves are presented, and CT values (disinfectant concentration × time, reported in mg·min/liter) were calculated by using the efficiency factor Hom (EFH) model (16).     

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.     

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).     

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.     

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).     

Enhanced inactivation of salmonella and pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log(10) units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.