Characterization of dysgonic, heterotrophic bacteria from drinking water

Only a small percentage of the heterotrophic bacteria encountered in water distribution systems are identifiable, because of these organisms fail to grow on the conventional media used for biochemical characterization. Organisms that would not subculture from the same standard plate count agar used for initial isolation were successfully subcultured on a low-nutrient medium, R3A. These cultures were then inoculated to a modified O/F base medium containing specific substrates. This, combined with a lower incubation temperature (30 degrees C), increased the enzymatic activity of many of the organisms. These reactions established a groundwork for tentative taxonomy.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Inhibition of growth ofLegionella species by heterotrophic plate count bacteria isolated from chlorinated drinking water

The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Characterization of psychrotolerant heterotrophic bacteria from Finnish Lapland

A total of 331 aerobic heterotrophic bacterial strains were isolated from various ecosystems of Finnish Lapland (68-69 degrees N) including forest soil, arctic alpine-tundra soil, stream water, lake and mire sediments, lichen and snow algae. Whole cell fatty acid and 16S rRNA gene sequence analysis and microscopy indicated that the isolates were dominated by Gram-negative bacteria, while only 20 Gram-positive strains were isolated. Based on 16S rRNA gene sequences the isolates were members of alpha-, beta-, gamma-Proteobacteria, Gram-positives with low G+C content, Actinobacteria and the Cytophaga/Flexibacter/Bacteroides group. More than one-third of the isolates could be tentatively identified as Pseudomonas spp. which were particularly abundant in the alpine-tundra soils where they represented 60% of all isolates. Other frequently isolated Gram-negative taxa were Burkholderia sp., Collimonas sp., Pedobacter sp., Janthinobacter sp., Duganella sp., Dyella sp. and Sphingomonas sp. Growth temperature ranges and hydrolytic enzyme activities of selected ca.100 strains were screened. The strains were psychrotolerant growing generally at temperatures ranging from 0 to 30 degrees C, as 82% of the isolates grew at 0 degrees C while only 7% grew at 35 degrees C. Protease and lipase activities at 5 degrees C were detected in more than half of the strains while approximately 20% of the strains possessed amylase and/or cellulase activities.     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0·45 μm membrane filters which were then placed upon appropriate media and incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37°C in 2·5% CO₂ for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia , and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0.45 microns membrane filters which were then placed upon appropriate media incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37 degrees C in 2.5% CO2 for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia, and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.

Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.). Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment. The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10' organisms/g (dry weight) of sediment during June and July. Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined. A single species, Clostridium bifermentens, comprised 47.7% of the total. Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp. (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%). Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures. Gas-liqid radiochromatography was used to determine the soluble metabolic end products from [U-14C]glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities. At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested. These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake.     

Characterization of indicator bacteria in municipal raw water, drinking water, and new main water samples

Municipal water samples were analyzed by membrane filter (MF) and presence-absence (P-A) tests for pollution indicator bacteria. In four years, 11 514 bacterial cultures were isolated from either raw water, drinking water, or new main water samples submitted to three environmental laboratories. The bacterial species occurring most often in all types of water samples were Escherichia coli (11.6-39.7%), Enterobacter aerogenes (18.1-26.3%), Aeromonas hydrophila (8.8-17.0%), Klebsiella pneumoniae (7.7-10.3%), and Citrobacter freundii (5.9-22.7%). A lactose - lauryl tryptose - tryptone broth was examined as an alternative medium to modified MacConkey broth in the presumptive portion of the P-A test. The intensity of acid and gas production in presumptive positive P-A bottles was compared with the types and frequencies of indicator bacteria shown by confirmatory tests. The results of detecting indicator bacteria following the analysis of 53 130 samples over a 2-year period were arranged by water source (well, lake, river, mixed) and water type (raw or drinking) to determine the influence of these parameters on the recovery of indicator bacteria. A further subdivision of the sample types into raw surface, raw ground, in-plant, plant discharge, reservoir, and distribution samples demonstrated the effect of water treatment practices.     

Antibiotic-resistant bacteria in drinking water.

We analyzed drinking water from seven communities for multiply antibiotic-resistant (MAR) bacteria (bacteria resistant to two or more antibiotics) and screened the MAR bacterial isolates obtained against five antibiotics by replica plating. Overall, 33.9% of 2,653 standard plate count bacteria from treated drinking waters were MAR. Two different raw water supplies for two communities carried MAR standard plate count bacteria at frequencies of 20.4 and 18.6%, whereas 36.7 and 67.8% of the standard plate count populations from sites within the respective distribution systems were MAR. Isolate identification revealed that MAR gram-positive cocci (Staphylococcus) and MAR gram-negative, nonfermentative rods (Pseudomonas, Alcaligenes, Moraxella-like group M, and Acinetobacter) were more common in drinking waters than in untreated source waters. Site-to-site variations in generic types and differences in the incidences of MAR organisms indicated that shedding of MAR bacteria living in pipelines may have contributed to the MAR populations in tap water. We conclude that the treatment of raw water and its subsequent distribution select for standard plate count bacteria exhibiting the MAR phenotype.     

Preliminary observations on psychrotrophic and psychrophilic,heterotrophic bacteria from antarctic water samples

Bacteria isolated from antarctic sea and lake water samples were examined for growth at 5 °C and 25 °C. Of 250 isolates, 249 were psychrotrophic and one was psychrophilic. The effect of temperature on the growth rate of the latter (strain 755) and a typical psychrotroph (strain 203A) was described by the square root of the growth rate constant. T_MIN values calculated from this model confirmed the strains as psychrophile and psychrotroph.Identification of 93 isolates showed the majority to be Pseudomonas (82%), with Flavobacterium and Moraxella representing 13% of the isolates. The remainder were coryneforms and yeasts. Isolate 611 was studied in detail because of its unusual morphology. This strain has features in common with the genera Spirosoma, Runella and Flectobacillus, but cannot be assigned with certainty. It may represent a new taxon of curved, gram negative bacteria.     

Analysis of cytotoxicity and invasiveness of heterotrophic plate count bacteria (HPC) isolated from drinking water on blood media

Heterotrophic plate count (HPC) bacteria are naturally present in all aqueous environments. These bacteria undergo multiplication cycles in drinking water, especially in closed containers (bottled water) or in tap water when chlorine levels are dissipated, such as in dead ends in water mains or household plumbing. A study was undertaken to estimate health risk from these naturally occurring bacteria by the determination of cytotoxicity and invasiveness in a human enterocyte cell line. HPC bacteria were isolated from bottled and tap water samples by enumerating them under physical and chemical conditions analogous to human physiology. All HPC bacteria were examined at both log and lag phase of their growth cycles. Bacterial broth supernatant fluids were also tested to serve as critical negative controls. Naturally occurring HPC bacteria demonstrated low invasiveness and cytotoxicity with more than 95% of isolates showing equivalency to broth supernatant fluid. When showing either invasiveness or cytotoxicity, only a small number of cells from the culture were positive. Of those that were positive, log phase HPC bacteria were significantly more cytotoxic and invasive than those from stationary phase. Bacterial broth controls demonstrated varied, but often marked, cytotoxicity.     

Evaluation of water corrosion,scaling extent and heterotrophic plate count bacteria in asbestos and polyethylene pipes in drinking water distribution system

Corrosion and scaling is one of the most important factors influencing drinking water quality that cause health disorders and economic problems. The aim of this study was to evaluate these phenomena in two sources of surface (Makou city) and ground water (Khoy city) in water networks. Corrosion and scaling potential was surveyed by Langelier, Ryzener, aggressiveness, Larson and Puckorius Indices and with measuring water physical, chemical, and microbial parameters. Statistical paired samples t-test displayed significant difference in means value of Langelier, Ryzener, Puckorius indices between cold and warm seasons of the year in Khoy samples and significant difference in means value of Ryzener, Puckorius and aggressiveness indices between cold and warm seasons of the year in Makou samples (p-value <0.001). Heterotrophic plate count water samples investigated in two cold and hot seasons in Khoy were respectively 14 ± 16 cfu ml^?1 and 41 ± 26 cfu ml^?1 and in the town of Makou were 11 ± 7 cfu ml^?1 and 61 ± 29 cfu ml^?1, respectively. In terms of health impacts, corrosion in different mains is important, then providing proper measures for balancing water quality before entering to the network and substituting of mains to prevent economic and health problems are necessary.     

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.     

Chlorine resistance patterns of bacteria from two drinking water distribution systems.

The relative chlorine sensitivities of bacteria isolated from chlorinated and unchlorinated drinking water distribution systems were compared by two independent methods. One method measured the toxic effect of free chlorine on bacteria, whereas the other measured the effect of combined chlorine. Bacteria from the chlorinated system were more resistant to both the combined and free forms of chlorine than those from the unchlorinated system, suggesting that there may be selection for more chlorine-tolerant microorganisms in chlorinated waters. Bacteria retained on the surfaces of 2.0-microns Nuclepore membrane filters were significantly more resistant to free chlorine compared to the total microbial population recovered on 0.2-micron membrane filters, presumably because aggregated cells or bacteria attached to suspended particulate matter exhibit more resistance than unassociated microorganisms. In accordance with this hypothesis, scanning electron microscopy of suspended particulate matter from the water samples revealed the presence of attached bacteria. The most resistant microorganisms were able to survive a 2-min exposure to 10 mg of free chlorine per liter. These included gram-positive spore-forming bacilli, actinomycetes, and some micrococci. The most sensitive bacteria were readily killed by chlorine concentrations of 1.0 mg liter-1 or less, and included most gram-positive micrococci, Corynebacterium/Arthrobacter, Klebsiella, Pseudomonas/Alcaligenes, Flavobacterium/Moraxella, and Acinetobacter.     

Viable but nonculturable bacteria in drinking water.

Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.     

Percentage of gelatinolytic bacteria among heterotrophic bacteria as indicator of water quality

The relationship between the physiological group of gelatinolytic bacteria and the abundance of heterotrophic bacteria in freshwater ecosystems was described, based on analysis of 1082 different freshwater samples collected in Croatia. Percentages of gelatinolytic bacteria among the population of heterotrophic bacteria showed a significant negative correlation with the abundance of heterotrophic bacteria. The relation between the physiological group (gelatinolytic bacteria) and heterotrophic bacteria can be considered to be an indicator of the pollution degree of freshwaters. A high relative content of gelatinolytic bacteria (> 76%) always indicates the colony-forming units of heterotrophic bacteria < 1000/mL, which corresponds to the high water quality; gelatinolytic bacteria < 11% indicate polluted waters. Isolated strains of aerobically grown gelatinolytic bacteria were Gram-negative rod-shaped or Gram-positive endospore-forming rod-shaped cells.     

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.