Gastrointestinal health effects associated with the consumption of drinking water produced by point-of-use domestic reverse-osmosis filtration units

During a prospective epidemiological study of gastrointestinal health effects associated with the consumption of drinking water produced by reverse-osmosis domestic units, a correlation was demonstrated between the bacterial counts on R2A medium incubated at 35 degrees C and the reported gastrointestinal symptoms in families who used these units. A univariate correlation was found with bacterial counts on R2A medium at 20 degrees C but was confounded by the bacterial counts at 35 degrees C. Other variables, such as family size and amount of water consumed, were not independently explanatory of the rate of illness. These observations raise concerns for the possibility of increased disease associated with certain point-of-use treatment devices for domestic use when high levels of bacterial growth occur.     

Bacterial colonization of domestic reverse-osmosis water filtration units

We have analyzed the bacterial content of water from the reservoirs of 300 reverse-osmosis units installed in households. The heterotrophic plate counts on R2A medium (20 and 35 degrees C) ranged from 0 to 10(7) colony forming units per millilitre (cfu/mL). Most reservoirs contained water with bacterial counts between 10(4) and 10(5) cfu/mL. The bacteria identified were Pseudomonas (not aeruginosa), Alcaligenes or Moraxella, Acinetobacter, Flavobacterium, and Chromobacterium. This report emphasizes the importance of bacterial colonization by heterotrophic bacteria in water reservoirs from domestic reverse-osmosis units.     

A new medium for the enumeration and subculture of bacteria from potable water

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new medium for the enumeration and subculture of bacteria from potable water.

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Microbiological and virological analysis of water from two water filtration plants and their distribution systems

The microbial flora of the water produced by two water filtration plants and their drinking water distribution system were evaluated: the Pont-Viau (PV) and the Repentigny (RE) water filtration plants. Untreated water entering the plants contained 3.6 (PV) and 16.8 most probable number of infectious units (mpniu)/L (RE) enteric viruses and total coliform bacteria counts were 300,000 (PV) and 500,000 cfu/L (RE). Treated water leaving the plant was essentially free of all the bacterial indicators measured (total, stressed, and fecal coliforms; Aeromonas hydrophila; Pseudomonas aeruginosa; Clostridium perfringens; enterococci) as well as of human enteric viruses. Heterotrophic plate counts at 20 and 35 degrees C were low in the freshly treated water leaving the plants, but bacterial regrowth was observed in both distribution systems at all sampling sites. Average counts for the heterotrophic plate count (20 degrees C) were between 10(6) and 10(7) cfu/L and counts were clearly increased with the distance from the plant. The most numerous bacterial genera encountered were Bacillus, Flavobacterium, and Pseudomonas (nonaeruginosa).     

Detection of fecal coliforms in water by using [14C]mannitol

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Detection of fecal coliforms in water by using [14C]mannitol.

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Comparison of microbial numbers in soils by using various culture media and temperatures

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30 degrees C. Higher numbers of total bacterial and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30 degrees C and in sorghum soil at 25 degrees C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30 degrees C were significantly higher than at 25 degrees C. Microbial quantification was best when using TSA medium for total and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi.     

Nonphotosynthetic pigmented bacteria in a potable water treatment and distribution system.

The occurrence of pigmented bacteria in potable water, from raw source water through treatment to distribution water, including dead-end locations, was compared at sample sites in a large municipal water system. Media used to enumerate heterotrophic bacteria and differentiate pigmented colonies were standard method plate count (SPC), m-SPC, and R2A agars, incubated up to 7 days at 35 degrees C. The predominant pigmented bacteria at most sample locations were yellow and orange, with a small incidence of pink organisms at the flowing distribution site. Seasonal variations were seen, with the yellow and orange organisms shifting in dominance. SPC agar was the least productive medium for both heterotroph counts and pigmented bacteria differentiation. At the flowing distribution site, percentages of pigmented bacteria on SPC medium ranged from 2.3 to 9.67 times less than on m-SPC and from 2.3 to 9.86 times less than on R2A. At the same site, seasonal trends in the percentage of pigmented bacteria were the same for m-SPC and R2A media, and the highest and lowest percentages occurred in the fall and winter, respectively. At site 6, there appeared to be an inverse relationship between the yellow and orange pigmented groups, but upon analysis, this did not hold and all correlations between yellow and orange pigmented bacteria were positive. The study results indicate that pigmented bacteria could readily be detected by using plate counting media developed for heterotroph enumeration in potable waters with incubation periods of 7 days. Pigmented bacteria can be used as an additional marker for monitoring changes in water quality. High numbers of heterotrophs, including pigmented forms, were found at dead-end locations, usually in the absence of a free chlorine residual and when the water temperature was greater than 16 degrees C. The association of some pigmented bacteria with nosocomial and other infections raises concern that the organisms may have originated from the potable water supply. High levels of pigmented bacteria could pose an increased health risk to immunologically compromised individuals. Therefore, the bacterial quality of the distribution water should be controlled to prevent the development of high concentrations of heterotrophic plate count bacteria, including the pigmented forms.     

Nonphotosynthetic pigmented bacteria in a potable water treatment and distribution system

The occurrence of pigmented bacteria in potable water, from raw source water through treatment to distribution water, including dead-end locations, was compared at sample sites in a large municipal water system. Media used to enumerate heterotrophic bacteria and differentiate pigmented colonies were standard method plate count (SPC), m-SPC, and R2A agars, incubated up to 7 days at 35 degrees C. The predominant pigmented bacteria at most sample locations were yellow and orange, with a small incidence of pink organisms at the flowing distribution site. Seasonal variations were seen, with the yellow and orange organisms shifting in dominance. SPC agar was the least productive medium for both heterotroph counts and pigmented bacteria differentiation. At the flowing distribution site, percentages of pigmented bacteria on SPC medium ranged from 2.3 to 9.67 times less than on m-SPC and from 2.3 to 9.86 times less than on R2A. At the same site, seasonal trends in the percentage of pigmented bacteria were the same for m-SPC and R2A media, and the highest and lowest percentages occurred in the fall and winter, respectively. At site 6, there appeared to be an inverse relationship between the yellow and orange pigmented groups, but upon analysis, this did not hold and all correlations between yellow and orange pigmented bacteria were positive. The study results indicate that pigmented bacteria could readily be detected by using plate counting media developed for heterotroph enumeration in potable waters with incubation periods of 7 days. Pigmented bacteria can be used as an additional marker for monitoring changes in water quality. High numbers of heterotrophs, including pigmented forms, were found at dead-end locations, usually in the absence of a free chlorine residual and when the water temperature was greater than 16 degrees C. The association of some pigmented bacteria with nosocomial and other infections raises concern that the organisms may have originated from the potable water supply. High levels of pigmented bacteria could pose an increased health risk to immunologically compromised individuals. Therefore, the bacterial quality of the distribution water should be controlled to prevent the development of high concentrations of heterotrophic plate count bacteria, including the pigmented forms.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9-12 months. The plate counts in R2A medium incubated at 22 degrees and 37 degrees C were low initially, increasing to 10(4)-10(5) cfu/ml within a few days of bottling. The number of bacteria recovered at 22 degrees C from PVC bottles was fairly constant during the storage period, but the population isolated at 37 degrees C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis. Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Discrepancies in Bacterial Recovery from Dental Unit Water Samples on R2A Medium and a Commercial Sampling Device

Monitoring the number of bacterial colony-forming units is an important step in assuring compliance with the recommendation that water from dental units contain <200 CFU mL^–1. Media that have been used for this purpose include R2A, a standard plate counting medium for water samples, and the Millipore HPC Sampler device, designed to facilitate sampling in dental offices. Discrepancies between the two media have been observed. This study tested the hypothesis that differences in counts on the two media were due to the failure of some bacteria to grow on the HPC sampler or to grow at less efficiency than on R2A. Of four different bacterial colony phenotypes tested in three independent experimental trials, one phenotype did not grow on the HPC device, and another grew inconsistently and at lower efficiency. These results confirmed the hypothesis. From these findings, users of the HPC sampler should be aware that microbial undercounts may occur.     

Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4 degrees to 25 degrees C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 micron Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4 degrees C greater than 15 degrees C greater than 25 degrees C greater than 37 degrees C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R1drd-19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15 degrees C or of loss of plasmid function during starvation.     

Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

Changes in enterococcal population during storage of reconstituted infant food

A six-fold increase in the enterococcal population was observed in reconstituted infant food samples after storage for 2 h at 37 degrees C. The increase in enterococcal counts at 40 degrees C and 45 degrees C was approximately five-fold during the same period. However, the corresponding total viable counts increased by twelve fold at these temperatures after 2 h. After 12 h, the enterococcal and total viable counts increased to 39 x 10(4) and 36 X 10(7) colony forming units per ml, at 37 degrees C respectively. A similar pattern in enterococcal and total bacterial count was observed at 40 degrees and 45 degrees C. TNase was detected in reconstituted infant food samples held at 37 degrees, 40 degrees and 45 degrees C, after 12 h, while pH values declined to 5.0, 5.1 and 5.2, respectively at the above temperatures. From TNase positive samples, an isolate S. faecium IF-100 capable to produce TNase was recovered. Storage of reconstituted infant food samples in the refrigerator (5 degrees C) resulted in a gradual increase in enterococcal population which reached 39 X 10(3) c.f.u. per ml after 12 days. However, TNase was not detected in any of these samples.