High-throughput and quantitative procedure for determining sources of Escherichia coli in waterways by using host-specific DNA marker genes

Escherichia coli is currently used as an indicator of fecal pollution and to assess water quality. While several genotypic techniques have been used to determine potential sources of fecal bacteria impacting waterways and beaches, they do not allow for the rapid analysis of a large number of samples in a relatively short period of time. Here we report that gene probes identified by Hamilton and colleagues (M. J. Hamilton, T. Yan, and M. J. Sadowsky, Appl. Environ. Microbiol. 72:4012-4019, 2006) were useful for the development of a high-throughput and quantitative macroarray hybridization system to determine numbers of E. coli bacteria originating from geese/ducks. The procedure we developed, using a QBot robot for picking and arraying of colonies, allowed us to simultaneously analyze up to 20,736 E. coli colonies from water samples, with minimal time and human input. Statistically significant results were obtained by analyzing 700 E. coli colonies per water sample, allowing for the analysis of approximately 30 sites per macroarray. Macroarray hybridization studies done on E. coli collected from water samples obtained from two urban Minnesota lakes and one rural South Carolina lake indicated that geese/ducks contributed up to 51% of the fecal bacteria in the urban lake water samples, and the level was below the detection limit in the rural lake water sample. This technique, coupled with the use of other host source-specific gene probes, holds great promise as a new quantitative microbial source tracking tool to rapidly determine the origins of E. coli in waterways and on beaches.     

The effect of wind speed and direction on the distribution of sewage-associated bacteria

A study of the relationship between wind and the distribution of sewage-associated bacteria was undertaken at a location where the sewage was discharged into the sea adjacent to the mouth of a river. The numbers of presumptive Escherichia coli were determined in 149 sea-water samples taken from three locations at distances of 1.9, 2.4 and 4.3 km from the outfall. On each sampling occasion, data on the wind speed and direction in the 3 h prior to collection of the samples were also collected. Analysis of these data demonstrated a significant role for wind speed and direction. With respect to wind direction, the numbers of presumptive E. coli present in a sample were significantly higher when the sample site lay downwind of the outfall. Wind speed was shown to have an influence on the numbers of presumptive E. coli only when the sample site was downwind of the outfall. In an analysis of 61 samples, an inverse correlation (r2 = 0.73) between salinity and log presumptive E. coli numbers was demonstrated. These data demonstrate that wind speed and direction at the time of sampling significantly influence the numbers of presumptive E. coli detected in any sea-water sample. It is argued that failure to pay sufficient attention to these parameters in the design of monitoring programmes may result in the generation of data that could provide a seriously distorted picture of the microbiological status of a water body.     

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.     

Isolation of Aeromonas hydrophila from a metropolitan water supply: seasonal correlation with clinical isolates

The occurrence of Aeromonas spp. in the metropolitan water supply of Perth, Western Australia, Australia, was monitored at several sampling points during a period of 1 year. Water within the distribution system conformed to international standards for drinking water but contained Aeromonas spp. in numbers comparable to those in raw surface water, although this water was free of Escherichia coli. Coliforms and E. coli were found in raw surface waters, and Aeromonas spp. were found in raw water from surface and underground sources. Chemical treatment, followed by chlorination at service reservoirs, resulted in water free of E. coli and a decrease in the number of Aeromonas spp. Aeromonas spp. were found in the greatest numbers in summer. Multiple regression analysis showed that growth of Aeromonas spp. in chlorinated water was related to water temperature, residual chlorine, and interaction between these variables. The incidence of Aeromonas-associated gastroenteritis, determined from isolates referred to us for enterotoxin testing, paralleled the pattern of isolation of Aeromonas spp. in water within the distribution systems. We suggest that the presence of Aeromonas spp. in drinking water needs public health appraisal and that further work should be undertaken to permit reevaluation of standards for the quality of drinking water.     

Isolation of Aeromonas hydrophila from a metropolitan water supply: seasonal correlation with clinical isolates.

The occurrence of Aeromonas spp. in the metropolitan water supply of Perth, Western Australia, Australia, was monitored at several sampling points during a period of 1 year. Water within the distribution system conformed to international standards for drinking water but contained Aeromonas spp. in numbers comparable to those in raw surface water, although this water was free of Escherichia coli. Coliforms and E. coli were found in raw surface waters, and Aeromonas spp. were found in raw water from surface and underground sources. Chemical treatment, followed by chlorination at service reservoirs, resulted in water free of E. coli and a decrease in the number of Aeromonas spp. Aeromonas spp. were found in the greatest numbers in summer. Multiple regression analysis showed that growth of Aeromonas spp. in chlorinated water was related to water temperature, residual chlorine, and interaction between these variables. The incidence of Aeromonas-associated gastroenteritis, determined from isolates referred to us for enterotoxin testing, paralleled the pattern of isolation of Aeromonas spp. in water within the distribution systems. We suggest that the presence of Aeromonas spp. in drinking water needs public health appraisal and that further work should be undertaken to permit reevaluation of standards for the quality of drinking water.     

Quantitative detection of Escherichia coli O157 in surface waters by using immunomagnetic electrochemiluminescence.

A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 10(8) cells of a non-O157 strain of E. coli ml(-1). Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 10(2) to 10(5) E. coli O157 cells ml of concentrated water(-1). To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000 E. coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E. coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25 E. coli O157 cells ml of raw water(-1), 25 cells 100 ml of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli

This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.     

An adapted ImmunoMagnetic cell separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces

Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-microm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-microm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-microm diameter Dynal bead.     

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.     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献   

Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks

Fluorescence in situ hybridization (FISH) was used for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of beta-d-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of E. coli in one of five pipes, whereas all pipes were positive with the FISH methods. E. coli was detected in 56% of the coupons using PNA FISH, but no E. coli was detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples that were negative according to culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media. E. coli contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration). We show here that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.     

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter(-1). The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r(2) = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm(2)). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.     

Evidence of septic system failure determined by a bacterial biochemical fingerprinting method

AIMS: To provide evidence of septic system failure by comparing two faecal indicator bacteria, enterococci and Escherichia coli, from defective septic tanks and adjacent creeks. METHODS AND RESULTS: A biochemical fingerprinting method was used to type and compare enterococci and E. coli strains from 39 septic tanks with creek water samples. Phenotypic diversity of enterococci (0.5 +/- 0.3) and E. coli (0.5 +/- 0.3) in septic tanks were significantly lower than those found in water samples (0.8 +/- 0.1, P < 0.0001 for enterococci and 0.9 +/- 0.1, P < 0.0001 for E. coli). Among 1072 enterococci isolates tested from septic tanks, 203 biochemical phenotypes (BPTs) were found of which 98 BPTs from 33 septic tanks were identical to several water samples. Similarly, among 621 E. coli isolates tested from septic tanks, 159 BPTs were found of which 53 BPTs from 26 septic tanks were also identical to water samples. The number of the latter bacteria was significantly (P = 0.01) higher in water samples collected from downstream compared with that of upstream in the study area. A high similarity between the populations of both indicator bacteria was also found between defective septic tanks and downstream water samples further indicating the contamination of both creeks by defective septic systems. CONCLUSIONS: Biochemical fingerprinting of faecal indicator bacteria is a useful and rapid method to provide direct evidence for septic system failure. Combination of both faecal indicator bacteria (enterococci and E. coli) provides a better judgement of the performance of a septic system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to provide direct evidence of septic system failure by identifying the presence of specific bacterial types in septic tanks and surface waters. Based on our findings, we suggest that the performance evaluation of a septic system should be accompanied by direct analysis of faecal indicator bacteria.     

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Aims:  The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water.
Methods and Results:  Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis ( r  = 0·62 and 0·77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci.
Conclusions:  The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria.
Significance and Impact of the Study:  The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications.     

A comparison of methods used to enumerate Escherichia coli in conventionally treated sewage sludge

AIMS: This study examined the suitability of three analytical methods for isolating and enumerating Escherichia coli from conventionally treated sewage sludge. METHODS AND RESULTS: Crude sewage, mesophilic anaerobic digested (MAD) sludge, and final product sludge samples were taken from six sewage treatment works for analysis. Two of the three methods tested were membrane filtration techniques, utilizing chromogenic E. coli/coliform (CEC) media and membrane-lactose glucuronide agar (MLGA); the third method was a most probable number (MPN) technique utilizing Colilert in Quantitray 2000 (Idexx). The methods were evaluated for variation, consistency, false-positive and false-negative results, as well as method correlation. The methods gave good and consistent recovery of E. coli for a range of conventionally treated sewage matrices. All of the methods had a false-positive rate of <3%, although MLGA had a high false-negative rate (35.5%) compared with Colilert (3.81%) and the CEC method (6.75%). This resulted in slightly lower presumptive counts but comparable numbers of confirmed counts. CONCLUSIONS: The three detection methods tested, chromogenic, MLGA and Colilert gave comparable recoveries, and did not vary by greater than one order of magnitude (1 log). SIGNIFICANCE AND IMPACT OF THE STUDY: Forthcoming revisions to the Use of Sludge in Agriculture Regulations (1989) will categorize sewage sludge as untreated, conventionally treated or enhanced treated in accordance to microbiological standards. The standard will be based upon numbers of E. coli removed through the sludge treatment process and the numbers remaining in the final product. It is recommended that the Colilert 2000 (Idexx, Westbrook, Maine) and CEC methods would be equally suitable to assess the reduction of indigenous E. coli in conventionally treated sludges, and that MLGA be used with follow-up confirmatory testing.     

Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables

AIMS: Raw fruits and vegetables have been increasingly associated with human infections caused by Shiga toxin-producing Escherichia coli. This study evaluates the isolation and detection of E. coli O26, O111 and O157 from vegetable samples using immunomagnetic particles. METHODS AND RESULTS: Standard cultivation and immunomagnetic separation (IMS) procedures were compared. It was found that immunomagnetic particles could efficiently concentrate E. coli cells, detecting significantly more bacteria than with standard cultivation procedures. CONCLUSION: Bacteria were detected in 93-100% of the inoculated samples using the IMS procedure, but only 36-93% samples tested by standard cultivation procedures were found to be positive. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that E. coli O26, O111 and O157 immunomagnetic particles can be a very useful and efficient tool for the detection of E. coli strains in raw vegetables, and could probably be used with samples of animal origin.