Evaluation of quantitative real time PCR for the measurement of Helicobacter pylori at low concentrations in drinking water.

A rapid DNA extraction and quantitative, real time polymerase chain reaction (QRTPCR) analysis method targeting the ureA gene of Helicobacter pylori was evaluated for the measurement of these organisms on membrane filters at levels that might be expected to be found in drinking water samples. No interference was seen from high levels of background organisms and related, non-target species were detected at approximately 4-5 log(10) lower levels of sensitivity than H. pylori by this assay. A standard curve was generated for the method from analyses of filters containing known numbers of added H. pylori cells. Cell numbers on these filters were determined by staining with a species-specific fluorescent antibody and solid phase cytometry analyses. The mean detection sensitivity of the method was 10 H. pylori cells per filter with a 95% confidence sensitivity of 40 cells and a 95% confidence precision interval of +/-0.57 log(10) based on duplicate analyses of the samples. One liter drinking water samples from several locations in the US were inoculated with the same H. pylori cell suspensions used to generate the standard curve and gave measurements that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method. This method may be useful for the rapid screening of drinking water for H. pylori.

Confirmational Identification of Escherichia coli, a Comparison of Genotypic and Phenotypic Assays for Glutamate Decarboxylase and beta-d-Glucuronidase.

[This corrects the article on p. 3350 in vol. 62, PMID: 8795225.].

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.

Genotoxic activity detected in soils from a hazardous waste site by the Ames test and an SOS colorimetric test.

Ten soil samples from a hazardous waste site were compared for their genotoxic activity by the Ames test (Salmonella reverse mutation assay) and a modified SOS colorimetric test. Polynuclear aromatic hydrocarbons known to produce frameshift mutations were found in high levels in the soils. Salmonella typhimurium TA98, sensitive to frameshift mutations, was selected as the Ames tester strain. Escherichia coli K12 PQ37 (sulA::lacZ) was the SOS tester strain. Organic extracts were prepared from the soil samples by Soxhlet extraction. One set of the soil samples was extracted with methylene chloride and a second set with cyclohexane. Two criteria from reproducible dose-related increases in response to the soil were used to compare the positive responses: 1) the concentrations required for doubling responses and 2) a minimum concentration required to produce statistically significant increases from background controls. Analysis of variance indicated that with S9 mix, Ames and SOS results were similar for the same soils and solvent extractions. However, without S9 mix, the SOS test was significantly more sensitive than the Ames test to the genotoxins extracted from the soils. Both the Ames and SOS tests detected lower concentrations of genotoxins in methylene chloride than in cyclohexane extracts. The simplicity of the method, reduction in expenses, and results within 1 working day all contribute to the advantages of the SOS test.

Comparison of the Salmonella (Ames) test, umu tests, and the SOS Chromotests for detecting genotoxins.

The limits of detection of 10 genotoxins representing 7 chemical classes with varying structures and modes of action were compared using the Ames test (Salmonella plate-incorporation test) with 2 tester strains, 2 standard colorimetric methods (the umu test and SOS Chromotest), and modifications of the umu and SOS Chromotests developed during the course of this study. The purpose of the study was to determine the sensitivity and reproducibility of each of the six methods. The sensitivities of the methods were compared using two criteria: the concentrations required for doubling responses, and the minimum concentrations required to produce statistically significant increases from background controls. The Ames test with strains TA98 and TA100 was ranked as the most sensitive method more often than the others, but the results indicated that the umu tests were statistically equivalent to the Ames test. The original SOS Chromotest kit method was highly sensitive in detecting the direct acting genotoxins, but neither SOS test was as sensitive as the other methods in detecting indirect acting genotoxins. The umu microtiter plate test is the least expensive of the assays and would be the most suitable for screening large numbers of environmental samples.

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water.

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.

Holding effects on coliform enumeration in drinking water samples.

Standard procedures for analyzing drinking water stress the need to adhere to the time and temperature conditions recommended for holding samples collected for microbiological testing. The National Drinking Water Laboratory Certification Program requires compliance with these holding limits, but some investigators have reported difficulties in meeting them. Research was conducted by standard analytical methods to determine if changes occurred when samples were held at 5 and 22 degrees C and analyzed at 0, 24, 30, and 48 h. Samples were analyzed for coliforms by the membrane filter and fermentation-tube procedures and for heterotrophs by the pour plate method. A total of 17 treated water samples were collected from a large municipal distribution system from August to December 1981, and 12 samples were collected from January to May 1983. The samples were dosed with coliforms previously isolated from the water system, Enterobacter cloacae in 1981 and Citrobacter freundii in 1983. The coliform counts declined linearly over time, and the rates of decline were significant at both 5 and 22 degrees C. Within 24 h at 22 degrees C, levels of E. cloacae and C. freundii decreased by 47 and 26%, respectively, and at 5 degrees C, E. cloacae numbers declined by 23%. Results from these representative laboratory-grown coliforms reinforced those previously obtained with naturally occurring coliforms under the same experimental conditions. Significantly, some samples with initially unacceptable counts (greater than 4/100 ml) met the safe drinking water limits after storage at 24 h at 5 and 22 degrees C and would have been classified as satisfactory.(ABSTRACT TRUNCATED AT 250 WORDS)