Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

Rapid identification and differentiation of agricultural faecal contamination sources using multiplex PCR

Aims:  To develop a quick, easy-to-use, robust and sensitive multiplex PCR assay to detect common sources of agricultural faecal contamination using a combination of bacterial and eukaryote-specific PCR targets.
Method and Results:  A novel multiplex PCR method was developed that utilizes primers specific for a conserved region of the eukaryote cytochrome-B gene as well as a universal 16S rRNA and the E. coli -specific uidA gene. This multiplex PCR assay was capable of identifying faecal amendments from pig, sheep, cow and goat sources in 24/30 (80%) of amended water samples.
Conclusions:  The method was capable of accurately identifying common agricultural sources.
Significance and Impact of the study:  The procedure described here is simple, rapid (<5 h) and can be used as a first step in microbial source tracking studies, particularly where agricultural faecal contamination is suspected.     

Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy

A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Development of quantitative real-time PCR assays for detection and quantification of surrogate biological warfare agents in building debris and leachate

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R(2) > 0.98) over a 7-log-unit dynamic range down to 10(1) B. atrophaeus cells or spores. Quantification of S. marcescens (R(2) > 0.98) was linear over a 6-log-unit dynamic range down to 10(2) S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.     

Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Enumeration of viable E. coli in rivers and wastewaters by fluorescent in situ hybridization

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culture-based method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water.     

Abrin-a A chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active

Abrin-a A chain (ABRaA) is a potent plant toxin, which possesses N-glycosylase activity toward eukaryotic 28S rRNA, and may have potential use in cancer therapy. To improve levels of expression in Escherichia coli, the gene encoding ABRaA was optimized by replacing rare codons with high-frequency ones, and synthesized using two-step PCR. The optimized ABRaA was cloned into the pET-His vector, and highly expressed in cytoplasm of E. coli. The yield of the purified recombinant (r) ABRaA proteins was up to 80 mg/l of induced culture. The rABRaA was one-step purified to homogeneity and its RNA-N-glycosylase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0. For the first time, ABRaA expressed as soluble form in E. coli from a PCR-synthesized gene is catalytically and functionally active.     

Selective and sensitive method for PCR amplification of Escherichia coli 16S rRNA genes in soil

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72 degrees C, which is 10 degrees C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl(2) concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting E. coli DNA in heterogeneous DNA samples, such as those extracted from soil.     

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.     

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.     

Temporal Assessment of the Impact of Exposure to Cow Feces in Two Watersheds by Multiple Host-Specific PCR Assays

Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.     

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

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.     

Microbial communities and fecal indicator bacteria associated with Cladophora mats on beach sites along Lake Michigan shores

A high biomasses of Cladophora, a filamentous green alga, is found mainly during the summer along the shores of Lake Michigan. In this study, the abundance and persistence of the fecal indicator bacterium Escherichia coli and sulfate-reducing bacteria (SRB) on Cladophora mats collected at Lake Michigan beaches were evaluated using both culture-based and molecular analyses. Additionally, 16S rRNA gene cloning and sequencing were used to examine the bacterial community composition. Overall, E. coli was detected in all 63 samples obtained from 11 sites, and the average levels at most beaches ranged from 2,700 CFU/100 g (wet weight) of Cladophora to 7,500 CFU/100 g of Cladophora. However, three beaches were found to have site average E. coli densities of 12,800, 21,130, and 27,950 CFU/100 g of Cladophora. The E. coli levels in the lake water collected at the same time from these three sites were less than the recommended U.S. Environmental Protection Agency limit, 235 CFU/100 ml. E. coli also persisted on Cladophora mats in microcosms at room temperature for more than 7 days, and in some experiments it persisted for as long as 28 days. The SRB densities on Cladophora mats were relatively high, ranging from 4.4x10(6) cells/g (6.64 log CFU/g) to 5.73x10(6) cells/g (6.76 log CFU/g) and accounting for between 20% and 27% of the total bacterial counts. Partial sequences of the 16S rRNA gene clones revealed a phylogenetically diverse community, in which the Cytophaga-Flavobacterium-Bacteroides cluster and the low-G+C-content gram-positive bacteria were the dominant organisms, accounting for 40% and 12.8%, respectively, of the total clone library. These results further reveal the potential public health and ecological significance of Cladophora mats that are commonly found along the shoreline of Lake Michigan, especially with regard to the potential to harbor microorganisms associated with fecal pollution and odor-causing bacteria.     

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples.

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.