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 Aeromonas hydrophila in a drinking-water distribution system: a field and pilot study

A 16-month study was conducted on the presence of Aeromonas hydrophila in drinking water in Indiana, U.S.A. Enumeration was conducted in source water, in various sites within a water treatment plant, and in the distribution system in both bulk water and biofilm, as well as in a simulated (annular reactors) drinking-water distribution system. Presumptive Aeromonas spp. counts on source waters regularly approached 10(3)-10(4) CFU/100 mL, during summer months and granular activated carbon - filtered water counts ranged from <1 to 490 CFU/100 mL. In source water, presumptive Aeromonas levels were related to water temperature. Aeromonas hydrophila was never detected in the treatment plant effluent or distributed bulk water, showing disinfectant efficiency on suspended bacteria; however, isolates of A. hydrophila were identified in 7.7% of the biofilm samples, indicating a potential for regrowth and contamination of drinking-water distribution systems.     

Effects of different nutrients on bacterial growth in a pilot distribution system

The growth of bacterial communities in drinking water distribution systems can lead to the development of problems incompatible with water quality requirements. This study was carried out in order to determine which factors promote bacterial growth in distribution networks. A pilot distribution system was used to perform these experiments. After addition of three different inorganic elements to the network (N, P and S) the results obtained show that they did not contribute to the growth or bacteria either in the circulating water or on the surface of the pipes of the distribution system. However, when organic carbon was added an increase in the number of circulating bacteria was observed though the number of bacteria in the biofilms of the network was constant. These results indicate that in the drinking water of the Barcelona distribution system the factor that controls the growth of bacteria is organic carbon. Moreover, bacteria from the biofilm growing on the surface of the pipes may cause the problems of bacterial growth.     

A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity

Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.     

Comparison between the evaluation of bacterial regrowth capability in a turbidimeter and biodegradable dissolved organic carbon bioreactor measurements in water

In recent years, two different approaches to the study of biodegradable organic matter in distribution systems have been followed. The assimilable organic carbon (AOC) indicates the portion of the dissolved organic matter used by bacteria and converted to biomass, which is directly measured as total bacteria, active bacteria or colony-forming units and indirectly as ATP or increase in turbidity. In contrast, the biodegradable dissolved organic carbon (BDOC) is the portion of the dissolved organic carbon that can be mineralized by heterotrophic micro-organisms, and it is measured as the difference between the inflow and the outflow of a bioreactor. In this study, at different steps in a water treatment plant, the bacterial regrowth capability was determined by the AOC method that measures the maximum growth rate by using a computerized Monitek turbidimeter. The BDOC was determined using a plug flow bioreactor. Measurements of colony-forming units and total organic carbon (TOC) evolution in a turbidimeter and of colony-forming units at the inflow/outflow of the bioreactor were also performed, calculating at all sampling points the coefficient yield ( Y = cfu/ΔTOC) in both systems. The correlations between the results from the bioreactor and turbidimeter have been calculated ; a high correlation level was observed between BDOC values and all the other parameters, except for Y calculated from bacterial suspension measured in the turbidimeter.     

The occurrence of Aeromonas species in drinking water supplies of an area of the Dolomite Mountains, Italy

A study was made of the occurrence of Aeromonas spp. in drinking water supplies in a mountain area in northeast Italy (the Dolomites). On account of its location, the water in question is exposed to a low level of pollution and systematic chemical disinfection is not necessary. Out of 7395 water samples analysed over a 3 year period, 1623 (21·95%) were found to be positive for Aeromonas , with levels ranging from 1 to 240 cfu 100 ml⁻¹ ; 72·4% of the strains were identified as Aer. hydrophila , 14·7% as Aer. caviae and 12·9% as Aer. sobria. The percentage of recovery from surface water (approximately 40%) was found to be higher than that of ground water (springs : 24·9% ; wells : 28·6%). Aeromonas spp. were isolated from 21·7% of samples from the distribution network and showed no significant variations compared with water from reservoirs. There was no evidence, therefore, of after-growth in the distribution system. No correlation was found between the concentrations of Aeromonas spp. and faecal indicator organisms. As the distribution of Aeromonas spp. was unrelated to anthropic pollution, it is believed that the search for these micro-organisms should be adopted as a further indicator of drinking water quality, especially in waters such as those in the present investigation not undergoing systematic purification treatment.     

Antimicrobial properties of moderately halotolerant bacteria from cenotes of the Yucatan peninsula

AIMS: Isolation and antimicrobial evaluation of aquatic bacterial strains from two cenotes. METHODS AND RESULTS: A total of 258 bacterial strains were isolated from the water and sediment of two cenotes in the Yucatan peninsula, all of which were screened against six pathogenic micro-organisms. Antimicrobial activity was detected in 46 of the isolated strains against at least one of the target strains tested. Antimicrobially active isolates were identified as: Aeromonas, Bacillus, Burkholderia, Photobacterium, Pseudomonas, Serratia, Shewanella, Stenotrophomonas genera, and 13 remained unidentified. All antimicrobially active strains were able to grow in salt medium at a concentration of 75 g l(-1), thus classifying as moderately halotolerant bacteria. Most of the antimicrobially active strains exhibited a broad action spectrum, where 61% was because of uncharacterized antimicrobial substances, 25% because of bacteriocins and 13% because of siderophores. Ten strains were able to biosynthesize biosurfactant metabolites. CONCLUSIONS: Native bacteria from the Yucatan peninsula showed an interesting antimicrobial activity, diverse mode of action and moderate halotolerance to salt. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on bacterial isolates from cenotes of the Yucatan peninsula and their antimicrobial characterization, with great potential for future biotechnological applications.     

Bacterial nutrients in drinking water.

Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15 degrees C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 micrograms of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to less than 50 micrograms/liter.     

Bacterial nutrients in drinking water

Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15 degrees C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 micrograms of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to less than 50 micrograms/liter.     

Development and Application of a Bioluminescence-Based Test for Assimilable Organic Carbon in Reclaimed Waters

Assimilable organic carbon (AOC) is an important parameter governing the growth of heterotrophic bacteria in drinking water. Despite the recognition that variations in treatment practices (e.g., disinfection, coagulation, selection of filter media, and watershed protection) can have dramatic impacts on AOC levels in drinking water, few water utilities routinely measure AOC levels because of the difficulty of the method. To simplify the method, the Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX test bacteria were mutagenized by using luxCDABE operon fusion and inducible transposons to produce bioluminescent strains. The growth of these strains can easily be monitored with a programmable luminometer to determine the maximum cell yield via luminescence readings, and these values can be fitted to the classical Monod growth curve to determine bacterial growth kinetics and the maximum growth rate. Standard curves using acetate carbon (at concentrations ranging from 0 to 1,000 μg/liter) resulted in coefficients of determination (r²) between luminescence units and acetate carbon levels of 0.95 for P-17 and 0.89 for NOX. The bioluminescence test was used to monitor reclaimed water, in which average AOC levels range between 150 and 1,400 μg/liter acetate carbon equivalents. Comparison of the conventional AOC assay and the bioluminescent assay produced an r² of 0.92.Biodegradable organic matter is used by heterotrophic bacteria for carbon and energy. Easily biodegradable carbon can lead to high levels of bacterial growth (2, 7, 8). The assimilable organic carbon (AOC) assay offers a standardized measurement of the heterotrophic bacterial growth potential of treated water and was originally developed by van der Kooij (13, 14). van der Kooij''s method utilized two bacterial strains (Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX) chosen for their nutritional requirements. AOC test results are considered to be more an indicator of the biological growth potential of the water and less a direct measurement of biodegradable carbon because the limiting nutrient may not be carbon (10). AOC is an important parameter governing the growth of bacteria in drinking water. AOC levels as low as 10 μg/liter can result in excessive growth of heterotrophic plate count bacteria in the absence of a chlorine residual. Even in the presence of a chlorine residual, AOC levels of >100 μg/liter have been associated with problems related to coliform and mycobacterium regrowth and possible regulatory noncompliance. High organic carbon levels, warm temperatures, and low levels of disinfectant residuals lead to water distribution system problems, including iron pipe corrosion, the growth of opportunistic pathogens (e.g., Mycobacterium avium, Aeromonas hydrophila, and Legionella pneumophila) in distribution system pipe biofilms, and bacterial regrowth in distributed water (9, 11, 13, 15). In distributed water, bacterial regrowth is perhaps the most significant mechanism for water quality deterioration between the treatment plant and the end user.AOC is the fraction of natural organic matter that is most readily used by bacteria for multiplication and is of greatest interest to drinking water utilities. Despite the recognition that variations in treatment practices (e.g., disinfection, coagulation, selection of filter media, and watershed protection) can have dramatic impacts on AOC levels in drinking water, few water utilities routinely measure AOC levels because of the complexity and difficulty of the method. Previous work attempted to simplify the method by measuring ATP instead of determining plate counts (10), but problems with commercial ATP measurement reagents discouraged utility laboratory adoption of this technique (3). The plate count and ATP methods are complex, time-consuming, and cumbersome, requiring a week or more of turnaround time before results are available. The methods are also expensive because of the technical labor involved in assaying ATP levels from filter-concentrated cells or in spread plating samples and determining plate CFU counts. These issues hindered routine determination of AOC levels and, therefore, strategies to optimize treatment for AOC removal. To simplify the method, P-17 and NOX test bacteria were mutagenized with luxCDABE operon fusion and inducible transposons to produce bioluminescent strains (4), but the engineered strains were shown to be marginally effective in low-sensitivity analog luminometers.The present work describes a rapid AOC test that uses the bioluminescent strains in conjunction with a sensitive, photon-counting luminometer. Combining the instrumentation and the bioluminescent derivatives has resulted in an easy-to-use AOC test that has been successfully applied to various water matrices. Standard curves were developed to determine the responses of the bioluminescent strains to various acetate carbon concentrations. Luminescence levels were converted to acetate carbon equivalents (based on standard curves) by using the Monod model, and maximum growth yield values were evaluated and compared. In addition, a yearlong study was conducted to measure the biological stability within reclaimed-water distribution systems from four geographically diverse reclaimed-water facilities that employed a variety of physical, chemical, and biological treatment combinations to treat wastewater effluents. In this study, average AOC levels were 10 times higher than those typically found in drinking water distribution systems and ranged from 150 to 1,400 μg/liter.     

Evaluation of the environmental impact of microbial aerosols generated by wastewater treatment plants utilizing different aeration systems

Using three sampler devices (SAS, Andersen Six-Stages and All Glass Impinger), the environmental impact of bacterial and fungal aerosols generated by municipal wastewater treatment plants operating with different methods of sludge oxygenation were evaluated. The highest microbial concentrations were recovered above the tanks (2247 cfu m-3) and in downwind positions (1425 cfu m-3), where a linear correlation (P < 0.05) was found between the quantity of sewage treated and the entities of microbial aerosol dispersion. Moreover, an exponential increase (P < 0.05) in the bacteria recovered from the air occurred at increasing times of treatment. However, after long-term plant operation, high bacterial and fungal concentrations were found in almost all of the sites around the plant. Coliforms, enterococci, Escherichia coli and staphylococci were almost always recovered in downwind positions. Considerable fractions (20-40%) of sampled bacteria were able to penetrate the final stages of the Andersen apparatus and thus, are likely to be able to penetrate the lungs. The plant operating with a fine bubble diffused air system instead was found to generate rather low concentrations of bacteria and fungi; moreover, staphylococci and indicator micro-organisms were almost absent. Finally, salmonellae, Shigella, Pseudomonas aeruginosa and Aeromonas spp. were not detected in either of the plants. The results indicate a remarkable dispersion of airborne bacteria and fungi from tanks in which oxygen is supplied via a mechanical agitation of sludge, and suggest the need to convert them to diffused aeration systems which pose a lesser hazard for human health.     

Screening of bacteria with antiviral activity from fresh water salmonid hatcheries

Bacteria isolated from two salmonid hatcheries were screened for antiviral activity against infectious hematopoietic necrosis virus (IHNV) to ascertain the presence of bacteria with anti-IHNV activity in the aquatic environment. Out of 710 bacterial isolates from the water and sediment samples, 190 strains showed anti-IHNV activities of more than 50% plaque reduction. These antiviral activities were detected predominantly in Pseudomonas, Aeromonas/Vibrio, and coryneforms. In one hatchery, the bacteria with antiviral activities were more prevalent in sediment samples than in water samples. Seventy-seven percent of the isolates with higher antiviral activities (greater than 90% plaque reduction) belonged to Pseudomonas.     

The effect of terrestrial effluents on the incidence of Aeromonas spp. in coastal waters

The numbers of Aeromonas species were monitored for one year at 18 stations on the Barcelona coast. Their concentrations exceed those of faecal coliforms and faecal streptococci in all samples, both on the shoreline and at 500 m from the coast. The mean values of the three bacterial counts reflected the proximity of the terrestrial effluents. There was a positive correlation between aeromonads and faecal indicators on the shoreline but not at 500 m offshore. This reflected their common origin and different survival rates in seawater. When sterile sea water was inoculated with A. hydrophila ATCC 7966, the numbers decreased initially by three orders of magnitude before they began to multiply. The final count was related to the concentration of organic matter in the water.     

The effect of terrestrial effluents on the incidence of Aeromonas spp. in coastal waters

The numbers of Aeromonas species were monitored for one year at 18 stations on the Barcelona coast. Their concentrations exceed those of faecal coliforms and faecal streptococci in all samples, both on the shoreline and at 500 m from the coast. The mean values of the three bacterial counts reflected the proximity of the terrestrial effluents. There was a positive correlation between aeromonads and faecal indicators on the shoreline but not at 500 m offshore. This reflected their common origin and different survival rates in seawater. When sterile sea water was inoculated with A. hydrophila ATCC 7966, the numbers decreased initially by three orders of magnitude before they began to multiply. The final count was related to the concentration of organic matter in the water.     

Distribution of the human faecal bacterium Bacteroides fragilis, its bacteriophages and their relationship to current sewage pollution indicators in bathing water

Although several bacteria are currently used as possible indicators of human pathogens in sewage-polluted sea water, they are often viewed as inadequate and especially inadequate as indicators of viral pathogens. This study investigates the distribution of Bacteroides fragilis and closely related Bacteroides spp. and their associated bacteriophages in sea water frequently used for recreational purposes. These organisms may provide a potentially more appropriate indicator. Bacteroides fragilis is one of about 10 species which are loosely placed together in the 'B. fragilis' group. Samples down-current from a sewage outfall were examined for the presence of B. fragilis group organisms and associated bacteriophages. Numbers were correlated with current bacterial and possible viral indicators at these sites. These B. fragilis group isolates were used as hosts to successfully isolate bacteriophages. The host range of these bacteriophages was investigated. It is hoped to expand this study by using these B. fragilis group hosts and their bacteriophages to identify a more suitable, European-wide, indicator of bacterial pathogens which can also be used to detect bacteriophages which are suitable as viral indicators.     

Absence of association between total heterotrophic and total coliform bacteria from a public water supply.

Heterotrophic plate counts (HPC) and total coliforms (TC) are two major microbial indicators that are used to monitor the potability of water. Although the presence of heterotrophs has been hypothesized to predict the presence of TC, there have been few documented reports. Intensive sampling of raw, treated effluent and distribution water from a public water supply serving 400,000 people provided an opportunity to study the relationship between these two indicator groups of bacteria. A total of 26,158 samples were analyzed, including 12,970 from 1986 and 13,188 from 1985. There were 13,429 samples from the distribution system, 5,524 from treatment effluents, and 7,205 from raw water. The associations between HPC and TC were made on both a hits-and-misses and numerical comparison (CFU per milliliter) basis. The periodicity of the two indicators was also analyzed to determine whether the presence of one group could predict the presence of the other. Atypical bacteria were also related to the presence of these two indicator bacteria. Venn diagrams and nonparametric statistics revealed the following correlation coefficients for HPC and TC for 1985 and 1986 combined: raw water r = 0.45, treated effluent r = 0.06, and distribution system r = 0.10. Atypical bacteria showed a similar relationship with HPC. There was no predictive periodicity for HPC and TC within +/- 10 days of isolation of each other. Therefore, in a 2-year survey of a public water supply, the presence of HPC had a low correlation coefficient with TC, as determined by hits-and-misses and numerical comparisons. The enumeration of one group was found to be independent of the other.     

Absence of association between total heterotrophic and total coliform bacteria from a public water supply

Heterotrophic plate counts (HPC) and total coliforms (TC) are two major microbial indicators that are used to monitor the potability of water. Although the presence of heterotrophs has been hypothesized to predict the presence of TC, there have been few documented reports. Intensive sampling of raw, treated effluent and distribution water from a public water supply serving 400,000 people provided an opportunity to study the relationship between these two indicator groups of bacteria. A total of 26,158 samples were analyzed, including 12,970 from 1986 and 13,188 from 1985. There were 13,429 samples from the distribution system, 5,524 from treatment effluents, and 7,205 from raw water. The associations between HPC and TC were made on both a hits-and-misses and numerical comparison (CFU per milliliter) basis. The periodicity of the two indicators was also analyzed to determine whether the presence of one group could predict the presence of the other. Atypical bacteria were also related to the presence of these two indicator bacteria. Venn diagrams and nonparametric statistics revealed the following correlation coefficients for HPC and TC for 1985 and 1986 combined: raw water r = 0.45, treated effluent r = 0.06, and distribution system r = 0.10. Atypical bacteria showed a similar relationship with HPC. There was no predictive periodicity for HPC and TC within +/- 10 days of isolation of each other. Therefore, in a 2-year survey of a public water supply, the presence of HPC had a low correlation coefficient with TC, as determined by hits-and-misses and numerical comparisons. The enumeration of one group was found to be independent of the other.     

The intestinal microflora of farmed Dover sole (Solea solea) at different stages of fish development

The bacterial flora in the intestines of farmed Dover sole ( Solea solea L.) was investigated at different stages of fish development in relation to different diets fed under farming conditions. The flora of water and diets were also analyzed. The dominant generic group isolated from all water samples examined was Pseudomonas Gp IV/ Alcaligenes. The same generic group was dominant in homogenized pelagic larvae indicating a relationship between the microflora of those young fish and that of tank water. In older fish a steady increase in the percentage incidence of Vibrio / anaerogenic Aeromonas was observed in the intestines as fish grew and diets changed. The Vibrio /anaerogenic Aeromonas group was dominant in Lumbricillus rivalis used as an intermediate diet and the increased incidence of this group in fish intestines occurred after fish were fed this diet. The composite pellet used for ongrowing carried a high percentage of Gram positive micro-organisms and there was no relationship between the pellet microflora and that found in the fish intestines.     

Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.     

Universal primer PCR with DGGE for rapid detection of bacterial pathogens

A universal primer PCR (UPPCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens. The results show that this method is efficient at amplifying the conserved regions of bacterial 16S rRNA genes with universal primers and can detect causative bacterial pathogens rapidly. Six species of bacteria from fisheries (Pseudomonas fluorescens, Vibrio anguillarum, Aeromonas hydrophila, Vibrio fluvialis, Providencia rettgeri and Aeromonas sobria) were examined. Our results indicate that the approach we undertook can be adopted not only for axenic bacterial populations but also for mixed communities as well. Furthermore, we were able to achieve the rapid detection of multiple bacteria a single in sample. In addition, UPPCR-DGGE was shown to be better than previously reported UPPCR-single-stranded conformation polymorphism (SSCP)-based methods for the rapid detection of bacterial pathogens.