Internal quality control samples for water bacteriology

A method is described for the preparation and application of a bacteriological quality control system for use with tests for plate counts and the enumeration of indicator organisms in water. Statistical limits are obtained and control charts prepared which are analagous to those used in analytical chemistry and clinical biochemistry laboratories. This approach has been applied successfully in this laboratory but needs to be tested more widely. Such numerically-based systems have seldom been attempted in microbiology, especially where multiple organisms have been included for the simultaneous control of several tests.     

Validation of the H<Subscript>2</Subscript>S method to detect bacteria of fecal origin by cultured and molecular methods

Using biochemical and molecular methods, this research determined whether or not the H₂S test did correctly identify sewage-contaminated waters by being the first to use culturing and molecular methods to identify the types and numbers of fecal indicator organisms, pathogens, and other microbes present in sewage samples with positive H₂S test results. For the culture-based method, samples were analyzed for the presence of fecal bacteria by spread plating the sewage sample onto differential and selective media for Aeromonas spp., Escherichia coli, sulfite-reducing clostridia, H₂S-producing bacteria, and Salmonella/Shigella spp. The isolates were then: (1) tested to determine whether they were H₂S-producing organisms and (2) identified to the genus and species level using biochemical methods. The molecular method used to characterize the microbial populations of select samples was terminal restriction fragment length polymorphisms. These experiments on sewage provided evidence that positive H₂S tests consistently contained fecal bacteria and pathogens. There were strong relationships of agreement between the organisms identified by both methods tested. This study is an important advance in microbial water quality detection since it is focused on the evaluation of a novel, low-cost, water microbiology test that has the potential to provide millions of people worldwide access to water quality detection technology. Of prime consideration in evaluating water quality tests is the determination of the test’s accuracy and specificity, and this article is a fundamental step in providing that information.     

The utilisation of arginine by oral streptococci grown glucose-limited in a chemostat

Abstract Legionella pneumophila occurring in drinking water was subjected to environmental stress through holding tests at ambient and elevated temperatures and by chemical disinfection. The bacterium in its native environment was more resistant to adverse conditions, as compared with laboratory-grown organisms. Of the several chemical disinfectants acceptable to drinking water treatment and tested for Legionella inactivation, ozonation was the most efficient method. The C · t ' products indicated that free chlorine was superior to mono- and dichloramines.     

A vacuum benthos sampler suitable for diverse habitats

The vacuum benthos sampler consists of a vacuum chamber equipped with a collecting net connected to the intake of a 12 volt pump, a battery, and a standpipe. Contents of the standpipe are vacuumed while substrate is removed and washed with the exhaust hose. The vacuum chamber is designed for rapid changing of nets during replicate sampling. This sampler is equally efficient in flowing and standing water. It was more effective than a modified Hess sampler for collecting a large variety of benthos from flowing (ca 0.25–0.75 m/s) riffles. Required operation time is variable, but 93% of invertebrates caught in 10 min were captured in the first 5 min during our tests, and there was a 94% mean recovery of released organisms during 10 min of subsequent operation. Advantages over previous suction samplers include interception of organisms before they pass through a pump, return of outlet water to the standpipe, capability of sampling in shallow (20–30 mm) water, and that it can be carried and operated by one person.     

The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk

Toxic cyanobacteria pose a significant hazard to human health and the environment. The recent characterisation of cyanotoxin synthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and their toxins. Conventional polymerase chain reaction (PCR) tests targeting cyanotoxin biosynthesis genes provide a rapid and sensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of these simple PCR tests into quantitative methods has additionally enabled the monitoring of dynamic bloom populations and the identification of particularly problematic species. More recently, DNA microarray technology has been applied to cyanobacterial diagnostics offering a high-throughput option for detecting and differentiating toxic genotypes in complex samples. Together, these molecular methods are proving increasingly important for monitoring water quality.     

In vitro potential genotoxic effects of surface drinking water treated with chlorine and alternative disinfectants

A battery of in vitro short-term tests revealing different genetic end-points was set up in order to study surface-water genotoxicity after disinfection with different biocides: sodium hypochlorite (NaClO), chlorine dioxide (ClO(2)) and peracetic acid (PAA). The surface water both before and after disinfection was concentrated by adsorption on C(18) silica cartridges and the concentrates containing non-volatile organics were divided into different portions for chemical analyses and biological assays. The following in vitro tests were conducted on the water concentrates dissolved in DMSO: the Salmonella mutagenicity assay with S. typhimurium strains TA98 and TA100; the SOS Chromotest with Escherichia coli, the Microtox and Mutatox assays with Vibrio fischeri; and gene conversion, point mutation and mitochondrial DNA mutability assays with D7 diploid Saccharomices cerevisiae strain. The results show that the SOS Chromotest and the yeast assays are highly sensitive in detecting genotoxicity. The surface-water extracts were very often toxic to most of the test organisms considered, partially masking their potential mutagenic activity. Therefore, the assays with E. coli and with S. cerevisiae are more likely to show a mutagenic effect because these organisms are generally less sensitive to most toxic compounds. Among the tested disinfectants, NaClO and ClO(2) increased water genotoxicity, whereas PAA was able to slightly reduce raw water activity. However, because the organic compounds in the lake water varied with the season of the year, the disinfection processes, at times, both increased and decreased the raw water activity.     

Proficiency testing of water microbiology laboratories in The Netherlands

In a 3-year period, four series of simulated water samples containing selected test strains were distributed to more than 50 laboratories in The Netherlands for bacteriological testing. Participating laboratories examined the samples by enrichment or membrane filtration methods, or both, for total coliform organisms, thermotolerant coliform organisms, faecal streptococci and standard plate counts (37 degrees and 22 degrees C) according to Dutch standard methods. The results were quantitatively satisfactory: the distribution of positive and negative results with subsamples conformed to stochastic variation; the standard deviation of membrane or plate counts was usually in the range which may be expected from a Poisson distribution, and there was good correspondence between average counts in participating laboratories and those expected from controls in the organizing laboratory. Problems of a qualitative nature were frequently encountered, however. Among them were a false positive response with a strain of Enterobacter cloacae in the thermotolerant coliform test; a false positive result with Clostridium perfringens in enrichment tests for total or thermotolerant coliform organisms and false positive results with Micrococcus varians in the faecal streptococcus test by membrane filtration. It is concluded that quality assessment should be a consistent activity in water microbiology laboratories. For this purpose, stable and well characterized reference materials are needed.     

Progress of research on the toxicology of antibiotic pollution in aquatic organisms

Antibiotics are widely used to improve human and animal health and treat infections. Antibiotics are often used in livestock farms and fisheries to prevent diseases and promote growth. Recently, there has been increasing interest in the presence of antibiotics in aquatic environments. Low levels of antibiotic components are frequently detected in surface water, seawater, groundwater, and even drinking water. Antibiotics are consistently and continually discharged into the natural environment as parent molecules or metabolites, which are usually soluble and bioactive, and this results in a pseudo and persistent pollution. The effects of environmental antibiotic toxicity on non-target organisms, especially aquatic organisms, have become an increasing concern. Although antibiotics have been detected worldwide, their ecological and developmental effects have been poorly investigated, particularly in non-target organisms. This review describes the toxicity and underlying mechanism of antibiotic contamination in aquatic organisms, including the effects on vertebrate development. A considerable number of antibiotic effects on aquatic organisms have been investigated using acute toxicity assays, but only very little is known about the long-term effects. Aquatic photosynthetic autotrophs, such as Pseudokirchneriella subcapitata, Anabaena flos-aquae, and Lemna minor, were previously used for antibiotic toxicity tests because of low cost, simple operation, and high sensitivity. Certain antibiotics show a different degree of potency in algal toxicity tests on the basis of different test algae. Antibiotics inhibit protein synthesis, chloroplast development, and photosynthesis, ultimately leading to growth inhibition; some organisms exhibit growth stimulation at certain antibiotic concentrations. Daphnia magna and other aquatic invertebrates have also been used for checking the toxicity priority of antibiotics. When investigating the acute effect of antibiotics (e.g., growth inhibition), concentrations in standard laboratory organisms are usually about two or three orders of magnitude higher than the maximal concentrations in the aquatic environment, resulting in the underestimation of antibiotic hazards. Vertebrate organisms show a promising potential for chronic toxicity and potentially subtle effects of antibiotics, particularly on biochemical processes and molecular targets. The adverse developmental effects of macrolides, tetracyclines, sulfonamides, quinolones, and other antibiotic groups have been evaluated in aquatic vertebrates such as Danio rerio and Xenopus tropicalis. In acute toxicity tests, low levels of antibiotics have systematic teratogenic effects on fish. The effects of antibiotics on oxidative stress enzymes and cytochrome P450 have been investigated. Cytotoxicity, neurotoxicity, and genotoxicity have been observed for certain antibiotic amounts. However, there are no firm conclusions regarding the chronic toxicity of antibiotics at environmentally relevant levels because of the lack of long-term exposure studies. Herein, future perspectives and challenges of antibiotic toxicology were discussed. Researchers should pay more attention to the following points: chronic toxicity and potentially subtle effects of environmentally relevant antibiotics on vertebrates; effects of toxicity on biochemical processes and mode of action; combined toxicity of antibiotics and other antibiotics, metabolites, and heavy metals; and environmental factors such as temperature and pH.     

The application of bioassay techniques to water pollution problems — The United Kingdom experience

The quality of the aquatic environment has long been assessed by chemical analyses and by biological surveillance of plant and animal communities. More recently, the biological response of living organisms has been used to evaluate the environmental impact of aqueous wastes.Laboratory tests on single species have been used widely to evaluate the acute effects of potential pollutants. However, the value of such tests, often conducted on exotic species, is receiving increasing criticism. Measurements of more subtle chronic sub-lethal effects are now showing increasing promise as regulatory tools in environmental assessment and pollution control. The paper reviews the techniques being used, and those currently under development for, the water pollution control authorities in the UK. Practical examples of applications are provided and the future value of bioassays is discussed.     

Implementation of a multiple biomonitoring approach to evaluate the potential for impact from an industrial discharge

Over a 2-year period, an industrial discharger implemented a program to determine if there was a potential for in-stream impact from its discharge, and, if necessary, to eliminate that potential. Six basic study designs were used. These included: (1) ambient toxicity tests using indicator organisms; (2) in-stream waste concentration (IWC) chronic testing using indicator organisms; (3) on-site flow-through toxicity testing using indicator and resident species with receiving stream water as the diluent; (4) in situ acute toxicity studies using indicator and resident species; (5) biological surveys of the receiving stream; and (6) artificial stream studies. The outcome of the studies resulted in conclusive data on which to base the design of a diffuser to dilute the effluent 1:20. This concentration was well below the lowest acute no-observed-effect concentration (10% effluent) determined using sensitive resident test species. In this manner, impact from the effluent on the James River had been reduced so that even the most sensitive resident species were protected. As a result of the study, the facility's permit was modified so that toxicity tests were made only on effluent diluted with receiving stream water to represent dilution at 1Q10 rather than 100 percent effluent. Follow-up studies have concentrated on a series of toxicity tests which were designed to identify the toxicants in the final effluent.     

Numerical Diagnostic Key for the Identification of Enterobacteriaceae

A numerical diagnostic key for enteric organisms is described which permits the identification of typical strains and of biochemical variants with high accuracy. Unknown strains are inoculated into a basic set of five media which permit the testing of eight biochemical reactions. The positive reactions are assigned points, and the score of a strain is added up, after which the identification of the strain is obtained from a table. In many instances, the final identification is obtained with this set of biochemical tests; and, in other instances, a small number of additional tests are required to distinguish between organisms giving the same score in the basic set of biochemical tests. The key permits an accurate, rapid, and economical differentiation of the typical and the more common atypical biotypes of enteric organisms in the clinical laboratory.     

Proficiency testing of water microbiology laboratories in The Netherlands

In a 3-year period, four series of simulated water samples containing selected test strains were distributed to more than 50 laboratories in The Netherlands for bacteriological testing. Participating laboratories examined the samples by enrichment or membrane filtration methods, or both, for total coliform organisms, thermotol-erant coliform organisms, faecal streptococci and standard plate counts (37˙ and 22˙C) according to Dutch standard methods. The results were quantitatively satisfactory: the distribution of positive and negative results with subsamples conformed to stochastic variation; the standard deviation of membrane or plate counts was usually in the range which may be expected from a Poisson distribution, and there was good correspondence between average counts in participating laboratories and those expected from controls in the organizing laboratory. Problems of a qualitative nature were frequently encountered, however. Among them were a false positive response with a strain of Enterobacter cloacae in the thermotolerant coliform test; a false positive result with Clostridium perfringens in enrichment tests for total or thermotolerant coliform organisms and false positive results with Micrococcus varians in the faecal streptococcus test by membrane filtration. It is concluded that quality assessment should be a consistent activity in water microbiology laboratories. For this purpose, stable and well characterized reference materials are needed.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes.

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.     

Modified Biochemical Tests for Characterization of L-Phase Variants of Bacteria

Biochemical capabilities of bacterial and L-phase organisms of 15 bacterial strains were examined by using a variety of modified routine diagnostic biochemical tests. The results demonstrated that 13 of the tests examined were suitable for use with L-phase variants, that L-phase variants and revertant bacterial phase organisms retained diagnostically significant capabilities of the respective bacterial or L-phase organisms from which they were derived, and that the 13 biochemical tests could be usefully employed to relate a given L-phase variant to a given bacterial phase organism, to distinguish L-phase variants of different species, and to aid in the identification of nonreverting L-phase variants.     

THE RAPID IDENTIFICATION OF ESCHERICHIA COLI I BY THE PRODUCTION AT 44° OF BOTH INDOLE AND GAS FROM LACTOSE

SUMMARY: The modification of the 44° test proposed by Mackenzie, Taylor & Gilbert (1948) is useful for the rapid identification of Escherichia coli I in water and foods. False positive tests caused by other coli-aerogenes bacteria, or by their association with other organisms, can be considered rare. Only a few E. coli I fail to produce indole or ferment lactose at 44°, and further confirmation for routine purposes is necessary only when the results at 44° are discordant, e.g. indole positive-lactose negative or indole negative-lactose positive.     

Conventional culture for water quality assessment: is there a future?

Conventional culture for the detection, enumeration and identification of micro-organisms has been the traditional tool of the microbiologist. It is, however, time-consuming and labour-intensive and confirmed results often require several days of analysis. Culture may not grow the organisms being sought and for enumeration may only detect a small proportion of the total population. However, it does have the advantage of being simple to use and relatively inexpensive. It is also a direct means of assessing cell viability. Novel fluorogenic dyes and fluorgenic and chromogenic substrates have overcome some of these problems by providing a means of rapid and specific detection and enumeration whilst removing the need for subculture and confirmation tests. Immunological tests such as ELISA have significantly reduced analysis time by providing specific target organism detection. Molecular techniques have removed the need for culture. Improvements in sensitivity, and removal of the inhibitory nature of sample matrices, have allowed analysts to detect low levels of micro-organisms but the questions of viability and comparability with cultural techniques still remain. Are we about to see a change of culture in water quality assessment, or can cultural techniques be developed that reduce analysis time to a few hours and can rapid methods be used for detecting the presence and viability of organisms?     

Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure

Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.     

Impact of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO)

The use of saline water as secondary quality water in urban environments for sanitation is a promising alternative towards mitigating fresh water scarcity. However, this alternative will increase the salinity in the wastewater generated that may affect the biological wastewater treatment processes, such as biological phosphorus removal. In addition to the production of saline wastewater by the direct use of saline water in urban environments, saline wastewater is also generated by some industries. Intrusion of saline water into the sewers is another source of salinity entering the wastewater treatment plant. In this study, the short-term effects of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) were investigated to assess the impact of salinity on enhanced biological phosphorus removal. Hereto, PAO and GAO cultures enriched at a relatively low salinity level (0.02 % W/V) were exposed to salinity concentrations of up to 6 % (as NaCl) in anaerobic batch tests. It was demonstrated that both PAO and GAO are affected by higher salinity levels, with PAO being the more sensitive organisms to the increasing salinity. The maximum acetate uptake rate of PAO decreased by 71 % when the salinity increased from 0 to 1 %, while that of GAO decreased by 41 % for the same salinity increase. Regarding the stoichiometry of PAO, a decrease in the P-release/HAc uptake ratio accompanied with an increase in the glycogen consumption/HAc uptake ratio was observed for PAO when the salinity increased from 0 to 2 % salinity, indicating a metabolic shift from a poly-P-dependent to a glycogen-dependent metabolism. The anaerobic maintenance requirements of PAO and GAO increased as the salinity concentrations risen up to 4 % salinity.     

Use of toxicity assays for enantiomeric discrimination of pharmaceutical substances

Toxicity assays are commonly used as general indicators of environmental water pollution. In the study described here, selected toxicity tests have been used to evaluate the different toxicity levels of enantiomers of different pharmaceutical drugs that can be found as potential contaminants in water environments. Isomers of dopa, fluoxetine, and atenolol were tested with three aquatic organisms corresponding to different trophic levels: Daphnia magna (a crustacean), Pseudokirchneriella subcapitata (a microalga), and Tetrahymena thermophila (a protozoan). Different levels of toxicity were observed for each enantiomer, suggesting that significant enantioselectivity occurs in aquatic toxicity and that such enantiomeric differences must be considered when evaluating the ecological effects of these compounds.