Soil amoebas as biological markers to estimate the quality of swimming pool waters.

The legal biological survey of swimming pool waters is based on both the level of bacteriological contamination and the amount of material of fecal origin. The great number of soil amoebas and the occasional epidemiological risk involved led us to consider using these organisms as possible biological markers to estimate the quality of pool water and the extent of disinfection. During a 1-year survey of 54 public swimming pools, 765 superficial pool and tap water samples were collected. One portion (50 ml) drawn from 1-liter samples was filtered and cultured for amoebas. In specimens considered contaminated we detected at least 20 amoebas per liter, whereas uncontaminated samples contained fewer than 20 amoebas per liter. By keeping the threshold value voluntarily low, we were able to compare tap water with pool water and to monitor the quality of various disinfection procedures (i.e., chlorine, bromine, and Cu-Ag). The data suggest that the filters were not always protective against a high concentration of amoebas. Furthermore, these disinfection procedures were not equally efficient according to estimates based on biological criteria. In addition, the quality of swimming pool water also depends on the quality of its source tap water. Thus, the numeration of soil amoebas can be used as an additional biological marker to estimate the quality of swimming pool water.     

Detection and risk assessment of adenoviruses in swimming pool water

AIMS: The role of swimming pool water as a source of human adenovirus (HAd) infection has previously been demonstrated. In this study, the risk of infection of HAds detected in a survey of swimming pool water from two indoor and one outdoor swimming pools over a period of 1 year was assessed. METHODS AND RESULTS: The HAds were concentrated from 1 l grab samples of swimming pool water using a silicon dioxide-based method. The extracted HAd DNA was amplified by means of a nested PCR method. Adenoviruses were detected in four of 26 samples (15.4%) from the indoor swimming pool A, eight of 38 samples (21.1%) from the indoor swimming pool B and three of 28 samples (10.7%) from the outdoor swimming pool C. Application of these results in an exponential risk assessment model indicated a daily risk of infection of 2.61 x 10(-3) for swimming pool A, 3.69 x 10(-3) for swimming pool B and 1.92 x 10(-3) for swimming pool C assuming a daily consumption of 30 ml of swimming pool water. CONCLUSIONS: No acceptable (tolerable) risk of infection has yet been recommended for swimming pool water. However, the quality of swimming pool water is generally expected to be similar to that of drinking water. One infection per 10 000 consumers per year has been recommended for drinking water. The risk of HAd infections calculated for the swimming pool water under investigation exceeded this acceptable risk. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that swimming pool water which conforms to generally accepted specifications for treatment, disinfection and indicator organisms constituted a risk of HAd infection, has implications for the swimming pool industry. The formulation of acceptable (tolerable) risks of infection for swimming pool water may be essential. Specifications will, therefore, have to be formulated to ensure that swimming pool water conforms to the acceptable risk of infection.     

Factors Influencing the Effectiveness of Swimming Pool Bactericides

Techniques for culturing, harvesting, and testing bacteria to evaluate bactericidal chemicals for swimming pools are described. Concentrations of 25, 50, and 100 mg of the chlorine stabilizer cyanuric acid per liter increased the time required for a 99% kill of Streptococcus faecalis by 0.5 mg of chlorine per liter at pH 7.4 and 20 C from less than 0.25 min without cyanuric acid to 4, 6, and 12 min, respectively. The effect of concentrations of ammonia nitrogen in the range found in swimming pools on the rate of kill of 0.5 mg of chlorine per liter and of chlorine plus cyanuric acid was tested. At concentrations of ammonia nitrogen greater than 0.05 mg per liter, faster rates of kill of S. faecalis were obtained with 100 mg of cyanuric acid per liter plus 0.5 mg of chlorine per liter than with 0.5 mg of chlorine per liter alone. When water samples from four swimming pools with low ammonia levels were used as test media, 0.5 mg of added chlorine per liter killed 99.9% of the added S. faecalis in less than 2 min, but water from a pool with a large number of children required 60 to 180 min of treatment.     

Detection of Microsporidia,cryptosporidia and Giardia in swimming pools: a one-year prospective study

In order to estimate the rate of microsporidia, cryptosporidia and giardia contamination of swimming pools, sequential samples of water were collected during a one-year period in six different swimming pools in Paris, France. Fourty-eight samples were submitted to filtrations. Eluates were examined for microsporidia using polymerase chain reaction (PCR) and for cryptosporidia and giardia using immunofluorescence staining. One of 48 specimens was positive for microsporidia. Using DNA sequence analysis, unknown microsporidia species were identified, which were close to an insect microsporidia Endoreticulatus schubergi. One sample was positive for cryptosporidia and none were positive for giardia. This study shows a low level of swimming pool water contamination by microsporidia, cryptosporidia or giardia, demonstrating the efficacy of cleaning filtration and disinfection procedures used in French swimming pools.     

Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains.

Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.     

Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains

Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.     

Comparative assessment of different biocides in swimming pool water

For disinfection of swimming pool water chlorine of chlorine-based products are normally used. In practice, these products have proven their worth regarding killing of pathogenic micro-organisms. Detailed values of their biocidal activity in swimming pool water were not found in literature. In the given study the efficacy of sodium hypochlorite (NaOCl) versus five micro-organisms was investigated.It is known that chlorination of swimming pool water may lead to formation of specific unwanted products like haloform. Nowadays, the concentration of those by-products in swimming pool water is limited and specific measures exist to minimize their formation. Nevertheless, there is increasing interest in alternative methods without by-product formation like e.g. hydrogen peroxide (H₂O₂) treatment.In the given study the antimicrobial activity of sodium hypochlorite was compared with that of different hydrogen peroxide-based products. The test procedure used was specifically designed to simulate practical conditions in a swimming pool but at the same time to lead to adequate reproducibility. Five test organisms were selected being relevant for the swimming pool area: Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, Staphylococcus aureus and Candida albicans.The swimming pool water for the test was artificially prepared. Water hardness, temperature and pH value were adjusted to a defined level. Regarding simulation of organic load it was found that a mixture of urea, creatinine and several amino acids was most appropriate.Addition of the test organisms was done in three portions: one big in the beginning and two smaller after 10 and 20 min to simulate recontamination by bathers. Total test period was 30 min. The number of surviving cells was determined after 30 s as well as after 10, 20 and 30 min.Sodium hypochlorite was tested at a concentration of 1 ppm active chlorine. Compared to that three products based on hydrogen peroxide were investigated: pure hydrogen peroxide, hydrogen peroxide + silver nitrate and a trade product based on hydrogen peroxide.Sodium hypochlorite resulted in total kill of the inoculated organisms after 10, 20 and 30 min corresponding to a log 4 reduction. In contrast to that the biocidal effect achieved by the hydrogen peroxide-based products was significantly lower than one log cycle notwithstanding a very high concentration of up to 150 ppm.The test results confirm the very good killing activity of sodium hypochlorite versus micro-organisms relevant for the swimming pool area. Products based on hydrogen peroxide, with or without silver ions, are from a microbiological point of view no real alternative to chlorine disinfection in swimming pools.     

Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.     

Influence of Stabilizer Concentration on Effectiveness of Chlorine as an Algicide

Cyanuric acid, used as chlorine stabilizer in swimming pool waters, has a relatively minor effect on the algicidal efficiency of free chlorine. The toxicity of free chlorine to three swimming pool algae was reduced slightly by 25 mg of cyanuric acid per liter if inhibiting, but less than algicidal, concentrations of chlorine were employed. Higher stabilizer concentrations (50, 100, and 200 mg/liter) generally resulted in no further reduction in the algicidal efficiency of free chlorine beyond that observed at 25 mg/liter.     

Water hygiene of two swimming pools: microbial indicators

The microbiological quality of the water of two swimming pools was evaluated to determine which organisms might be the best indicators of pollution. Membrane filtration gave low numbers of the common indicators of faecal pollution but high counts of the indigenous skin bacteria. As illnesses normally contracted in a swimming pool are mostly those of the skin rather than intestinal, it is suggested that the best indicators of the hygienic condition of water in a swimming pool are staphylococci because of their resistance to disinfection, high numbers in the environment, and ease of recovery.     

Water hygiene of two swimming pools: microbial indicators

The microbiological quality of the water of two swimming pools was evaluated to determine which organisms might be the best indicators of pollution. Membrane filtration gave low numbers of the common indicators of faecal pollution but high counts of the indigenous skin bacteria. As illnesses normally contracted in a swimming pool are mostly those of the skin rather than intestinal, it is suggested that the best indicators of the hygienic condition of water in a swimming pool are staphylococci because of their resistance to disinfection, high numbers in the environment, and ease of recovery.     

Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine.

The efficacy of copper and silver ions, in combination with low levels of free chlorine (FC), was evaluated for the disinfection of hepatitis A virus (HAV), human rotavirus (HRV), human adenovirus, and poliovirus (PV) in water. HAV and HRV showed little inactivation in all conditions. PV showed more than a 4 log10 titer reduction in the presence of copper and silver combined with 0.5 mg of FC per liter or in the presence of 1 mg of FC per liter alone. Human adenovirus persisted longer than PV with the same treatments, although it persisted significantly less than HRV or HAV. The addition of 700 micrograms of copper and 70 micrograms of silver per liter did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of FC per liter, although on some occasions it produced a level of inactivation similar to that induced by a higher dose of FC alone. Virus aggregates were observed in the presence of copper and silver ions, although not in the presence of FC alone. Our data indicate that the use of copper and silver ions in water systems may not provide a reliable alternative to high levels of FC for the disinfection of viral pathogens. Gene probe-based procedures were not adequate to monitor the presence of infectious HAV after disinfection. PV does not appear to be an adequate model viral strain to be used in disinfection studies. Bacteroides fragilis bacteriophages were consistently more resistant to disinfection than PV, suggesting that they would be more suitable indicators, although they survived significantly less than HAV or HRV.     

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.     

Determinants of the microbiological characteristics of South Australian swimming pools.

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Determinants of the microbiological characteristics of South Australian swimming pools

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Selenate reduction by bacteria from a selenium-rich environment.

Samples collected from Kesterson Reservoir were screened for bacterial presence and selenate reduction capability. Selenate concentrations of 100 mg/liter were not toxic to indigenous bacteria. Of the 44 samples collected, 20 possessed microbial populations capable of reducing selenate. Reduction was observed in 4% of the water samples, 92% of the sediment samples, and 100% of the soil samples. Microbial reduction of 100 mg of selenate per liter was complete within 1 week of incubation. Up to 75 mg of selenate per liter was reduced beyond selenite to an insoluble red precipitate. Data collected indicate that indigenous bacteria have a significant role in the biogeochemical cycling of selenium.     

Selenate reduction by bacteria from a selenium-rich environment

Samples collected from Kesterson Reservoir were screened for bacterial presence and selenate reduction capability. Selenate concentrations of 100 mg/liter were not toxic to indigenous bacteria. Of the 44 samples collected, 20 possessed microbial populations capable of reducing selenate. Reduction was observed in 4% of the water samples, 92% of the sediment samples, and 100% of the soil samples. Microbial reduction of 100 mg of selenate per liter was complete within 1 week of incubation. Up to 75 mg of selenate per liter was reduced beyond selenite to an insoluble red precipitate. Data collected indicate that indigenous bacteria have a significant role in the biogeochemical cycling of selenium.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species.

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.