Quantification of host-specific <Emphasis Type="Italic">Bacteroides–Prevotella</Emphasis> 16S rRNA genetic markers for assessment of fecal pollution in freshwater

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides–Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9–11 log₁₀ copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides–Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r ² = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50–800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.     

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

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.     

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.     

Usefulness of the hydrogen sulfide test for assessment of water quality in Bangladesh

Aim: To evaluate the usefulness of the hydrogen sulfide (H₂S) test for assessing water quality in Bangladesh. Methods and Results: We tested 382 water samples from a variety of sources using locally produced H₂S test kits and laboratory‐based membrane filtration for the detection of Escherichia coli. Compared with membrane filtration, H₂S tests, when incubated for 24 h, had both a sensitivity and positive predictive value (PPV) of <40% when analysis was restricted to water samples with E. coli levels below 100 colony forming units (CFU) per 100 ml. In contrast, for E. coli levels from 1000 to 9999 CFU per 100 ml, sensitivity was 94% and PPV 88%; specificity was 97% and negative predictive value was 99%. Conclusions: The hydrogen sulfide test, when incubated at 24 h, is a promising alternative for assessing water quality where E. coli levels may be high. An improved understanding of the incremental impact of contamination level on health is needed to better determine its usefulness. Significance and Impact of the Study: The hydrogen sulfide test is inexpensive, easy to use and portable. Its use may allow rapid assessment of water quality in situations where cost or logistics prevent use of other testing methods, such as in remote areas or during floods and other natural disasters.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Evaluation of the Autoanalysis Colilert test for detection and enumeration of total coliforms

The Autoanalysis Colilert (AC) test was compared with the membrane filter (MF), 10-tube multiple-tube fermentation (MTF) technique, and the presence-absence test as described in Standard Methods for the Examination of Water and Wastewater for the detection and enumeration of total coliforms in water. The methods were evaluated with 31 samples from seven different sources. Each sample was analyzed by each of the techniques, using replicate 100-ml sample volumes. A total of 582 confirmed tubes were positive by the MTF test, and 533 tubes were positive by the AC test. Statistical analysis of the most-probable-number comparability data showed a statistically significant difference in the number of positive tubes, with the MTF test resulting in more positive tubes. There were no statistically significant differences in precision between the two methods. All the methods were comparable in detection of total coliforms. Levels of heterotrophic bacteria generally encountered in drinking water did not interfere with detection or enumeration of coliforms by the AC test.     

Evaluation of the Autoanalysis Colilert test for detection and enumeration of total coliforms.

The Autoanalysis Colilert (AC) test was compared with the membrane filter (MF), 10-tube multiple-tube fermentation (MTF) technique, and the presence-absence test as described in Standard Methods for the Examination of Water and Wastewater for the detection and enumeration of total coliforms in water. The methods were evaluated with 31 samples from seven different sources. Each sample was analyzed by each of the techniques, using replicate 100-ml sample volumes. A total of 582 confirmed tubes were positive by the MTF test, and 533 tubes were positive by the AC test. Statistical analysis of the most-probable-number comparability data showed a statistically significant difference in the number of positive tubes, with the MTF test resulting in more positive tubes. There were no statistically significant differences in precision between the two methods. All the methods were comparable in detection of total coliforms. Levels of heterotrophic bacteria generally encountered in drinking water did not interfere with detection or enumeration of coliforms by the AC test.     

Microalgal removal of CO<Subscript>2</Subscript> from flue gases: Changes in medium pH and flue gas composition do not appear to affect the photochemical yield of microalgal cultures

Our research objectives are to determine under what conditions microalgal-based CO₂ capture from flue gases is economically attractive. Specifically, our objective here was to select microalgae that are temperature, pH and flue gas tolerant. Microalgae were grown under five different temperatures, three different pH and five different flue gas mixtures besides 100% CO₂ (gas concentrations that the cells were exposed to ranged 5.7–100% CO₂, 0–3504 ppm SO₂, 0–328 ppm NO, and 0–126 ppm NO₂). Our results indicate that the microalgal strains tested exhibit a substantial ability to withstand a wide range of temperature (54 strains tested), pH (20 strains tested) and flue gas composition (24 strains tested) likely to be encountered in cultures used for carbon sequestration from smoke stack gases. Our results indicate that microalgal photosynthesis is a limited but viable strategy for CO₂ capture from flue gases produced by stationary combustion sources.     

Investigation on the mechanism of H<Subscript>2</Subscript>S removal by biological activated carbon in a horizontal biotrickling filter

The use of supporting media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and a shelter that protects the microorganisms from inhibitory compounds. In our previous studies, activated carbon (AC) alone used as a support medium for H₂S biological removal was proved prompt and efficient in a bench-scale biofilter and biotrickling filter. In this study, the mechanisms of H₂S elimination using microbial immobilized activated carbon, i.e., biological activated carbon (BAC), are investigated. A series of BAC as supporting medium were taken from the inlet to outlet of a bench-scale horizontal biotrickling filter to examine the different effects of physical/chemical adsorption and microbial degradation on the overall removal of H₂S. The surface properties of BAC together with virgin and exhausted carbon (after H₂S breakthrough test, non-microbial immobilization) were characterized using the sorption of nitrogen (Braunner–Emmett–Teller test), scanning electron microscopy (SEM), surface pH, thermal, carbon–hydrogen–nitrogen–sulfur (CHNS) elemental and Fourier transform infrared (FTIR) analyses. Tests of porosity and surface area provide detailed information about the pore structure of BAC along the bed facilitating the understanding of potential pore blockages due to biofilm coating. A correlation between the available surface area and pore volume with the extent of microbial immobilization and H₂S uptake is evidenced. SEM photographs show the direct carbon structure and biofilm coated on carbon surface. FTIR spectra, differential thermogravimetric curves and CHNS results indicate less diversity of H₂S oxidation products on BAC than those previously observed on exhausted carbon from H₂S adsorption only. The predominant oxidation product on BAC is sulfuric acid, and biofilm is believed to enhance the oxidation of H₂S on carbon surface. The combination of biodegradation and physical adsorption of using BAC in removal of H₂S could lead to a long-term (i.e., years) good performance of biotrickling filters and biofilters based on BAC compared to carbon adsorption only.     

Dissimilatory Reduction of Fe(III) by Thermophilic Bacteria and Archaea in Deep Subsurface Petroleum Reservoirs of Western Siberia

Twenty-five samples of stratal fluids obtained from a high-temperature (60–84°C) deep subsurface (1700–2500 m) petroleum reservoir of Western Siberia were investigated for the presence of dissimilatory Fe(III)-reducing microorganisms. Of the samples, 44% and 76% were positive for Fe(III) reduction with peptone and H₂ respectively as electron donors. In most of these samples, the numbers of culturable thermophilic H₂-utilizing iron reducers were in the order of 10–100 cells/ml. Nine strains of thermophilic anaerobic bacteria and archaea isolated from petroleum reservoirs were tested for their ability to reduce Fe(III). Eight strains belonging to the genera Thermoanaerobacter, Thermotoga, and Thermococcus were found capable of dissimilatory Fe(III) reduction, with peptone or H₂ as electron donor and amorphous Fe(III) oxide as electron acceptor. These results demonstrated that Fe(III) reduction may be a common feature shared by a wide range of anaerobic thermophiles and hyperthermophiles in deep subsurface petroleum reservoirs. Received: 1 March 1999 / Accepted: 5 April 1999     

Faecal contamination of drinking water sources of Dhaka city during the 2004 flood in Bangladesh and use of disinfectants for water treatment

AIMS: To describe the extent of faecal pollution and point of use water treatment strategy during and after the 2004 flood in Dhaka. METHODS: A total of 300 water samples were collected from 20 different drinking water sources in Kamalapur, Dhaka city from August 2004 to January 2005. The level of faecal contamination was estimated using measurements of faecal indicator bacteria (total coliforms, faecal coliforms and faecal streptococci) and isolation of Vibrio cholerae was carried out following standard procedures. Total dissolved solids, dissolved oxygen, hardness, chloride and pH were also monitored. The efficacy of four disinfectants including Halotab, Zeoline-200, alum potash and bleaching powder were tested as point of use water treatment agents. The unacceptable level of contamination of total coliforms (TC), faecal coliforms (FC) and faecal streptococci (FS) ranged from 23.8% to 95.2%, 28.6% to 95.2% and 33.3% to 90.0%, respectively. The isolation rates of V. cholerae O1 and O139 were both 0.33%, and non-O1/non-O139 was 7.0%. CONCLUSION: Water collected during and after floods was contaminated with TC, FC, FS and V. cholerae. Although alum potash, bleaching powder, Halotab and Zeoline-200 were all effective general disinfectants, Halotab and Zeoline-200 were superior to bleaching powder and alum potash against FC. SIGNIFICANCE AND IMPACT OF THE STUDY: During and after floods, point of use water treatment could reduce waterborne diseases among flood-affected people.     

Bacteriophages of enteric bacteria in drinking water, comparison of their distribution in two countries

The presence of bacteriophages infecting enteric bacteria was tested in more than 1500 drinking water samples in Israel and Spain. Bacteriophages tested were somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. The three groups of bacteriophage were isolated in 100 ml water samples by the presence/absence test with similar frequencies, which ranged from 4·4% for somatic coliphages to 6·1% for bacteriophages infecting Bact. fragilis. In contrast, the frequency of isolation of bacteriophages was significantly higher than the frequency of isolation of faecal coliforms, which averaged only 1·9%. No significant differences were observed between the frequencies of isolation between the samples tested in Spain and those tested in Israel. The percentage of groundwater samples containing faecal coliforms and somatic coliphages was reduced significantly by chlorination, despite known deficiencies. However, there was no effect on the occurrence of F-specific bacteriophages and Bact. fragilis bacteriophages.     

National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube fermentation method

A defined substrate method was developed to simultaneously enumerate total coliforms and Escherichia coli from drinking waters without the need for confirmatory or completed tests. It is a new method based on technology that uses a hydrolyzable substrate as a specific indicator-nutrient for the target microbes. No equipment other than a 35 degrees C incubator and long-wavelength (366-nm) light is necessary. To perform the test, one only has to add water to the powdered ingredients in a tube or flask. If total coliforms are present in the water sample, the solution will change from its normal colorless state (no target microbes present) to yellow. The specific presence of E. coli will cause the same tube to fluoresce under a longwave (366-nm) UV lamp. The test, called Autoanalysis Colilert (AC), was compared with Standard Methods for the Examination of Water and Wastewater 10-tube multiple tube fermentation (MTF) in a national evaluation. Five utilities, representing six U.S. Environmental Protection Agency regions, participated. All water samples came from distribution systems. Split samples from a wide variety of water sources were analyzed for the MPN-versus-MPN comparison. A total of 1,086 tubes were positive by MTF, and 1,279 were positive by AC. There was no statistical difference between MTF and AC. Species identifications from positive tubes confirmed the sensitivity of the AC. A national evaluation of the AC test showed that it: (i) was as sensitive as Standard Methods MTF, (ii) specifically enumerated 1 total coliform per 100 ml, in a maximum of 24 h, (iii) simultaneously enumerated 1 E. coli per 100 ml in the same analysis, (iv) was not subject to false-positive or false-negative results by heterotrophic bacteria, (v) did not require confirmatory tests, (vi) grew injured coliforms, (vii) was easy to inoculate, and (viii) was very easy to interpret.     

National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube fermentation method.

A defined substrate method was developed to simultaneously enumerate total coliforms and Escherichia coli from drinking waters without the need for confirmatory or completed tests. It is a new method based on technology that uses a hydrolyzable substrate as a specific indicator-nutrient for the target microbes. No equipment other than a 35 degrees C incubator and long-wavelength (366-nm) light is necessary. To perform the test, one only has to add water to the powdered ingredients in a tube or flask. If total coliforms are present in the water sample, the solution will change from its normal colorless state (no target microbes present) to yellow. The specific presence of E. coli will cause the same tube to fluoresce under a longwave (366-nm) UV lamp. The test, called Autoanalysis Colilert (AC), was compared with Standard Methods for the Examination of Water and Wastewater 10-tube multiple tube fermentation (MTF) in a national evaluation. Five utilities, representing six U.S. Environmental Protection Agency regions, participated. All water samples came from distribution systems. Split samples from a wide variety of water sources were analyzed for the MPN-versus-MPN comparison. A total of 1,086 tubes were positive by MTF, and 1,279 were positive by AC. There was no statistical difference between MTF and AC. Species identifications from positive tubes confirmed the sensitivity of the AC. A national evaluation of the AC test showed that it: (i) was as sensitive as Standard Methods MTF, (ii) specifically enumerated 1 total coliform per 100 ml, in a maximum of 24 h, (iii) simultaneously enumerated 1 E. coli per 100 ml in the same analysis, (iv) was not subject to false-positive or false-negative results by heterotrophic bacteria, (v) did not require confirmatory tests, (vi) grew injured coliforms, (vii) was easy to inoculate, and (viii) was very easy to interpret.     

Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan

Microbial diversity and methanogenic potential in formation water samples from a dissolved-in-water type gas field were investigated by using 16S rRNA gene libraries and culture-based methods. Two formation water samples (of 46 and 53°C in temperature) were obtained from a depth of 700 to 800 m. Coenzyme F₄₂₀-autofluorescence indicated that 10³–10⁴ cells per ml of active methanogens were present, accounting for at least 10% of the total cell count. The 16S rRNA gene sequence analysis indicated that the diversity of Archaea and Bacteria of the two samples was quite limited; i.e., the archaeal libraries were dominated by the sequences related to Methanobacterium formicicum and Methanothermobacter thermautotrophicus, and the bacterial libraries were dominated by the sequences related to Hydrogenophilus and Deferribacter. Of the methanogenic substrates tested using the formation water-based medium, only H₂–CO₂ gave rise to methane formation. Those dominant archaeal and bacterial genera have the potential to use hydrogen for growth at the in situ temperatures, suggesting that the formation water of the Pliocene strata in the gas field has been provided with hydrogen, probably from underneath the strata, and thus on-going active methanogenesis has been occurring to date. An erratum to this article can be found at     

Comparison of membrane filtration and Autoanalysis Colilert presence-absence techniques for analysis of total coliforms and Escherichia coli in drinking water samples.

Over a 4-month period, 950 samples of treated drinking water were analyzed for total coliforms (TC) and Escherichia coli by both membrane filtration (MF) and Autoanalysis Colilert presence-absence (AC) techniques. The two tests agreed 97% of the time on the basis of presumptive TC results and 98.5% of the time on the basis of verified TC results. Samples which produced disagreement between the two tests were most often TC positive by MF and TC negative by AC. E. coli was recovered four times: twice by MF only, and twice by AC only but without the diagnostic fluorescence reaction. In two samples, E. coli could not be isolated from fluorescence-positive AC tests. On the basis of these results, the AC test was implemented as the routine analytical procedure for TC but not for E. coli.