PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when ≥10³ CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥10⁴ CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥10² CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥10⁴ CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.     

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. 10¹ to 10⁵ 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. 10⁸ 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. 10² to 10⁵ 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.     

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 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ 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 × 10⁴ 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.     

A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10⁴ CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.     

Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r² = 0.95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r² = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

The Pepsin Hydrolysate of Bovine Lactoferrin Causes a Collapse of the Membrane Potential in Escherichia coli O157:H7

In the present study, the ability of bovine lactoferrin hydrolysate (LfH) to disrupt the cytoplasmic membrane of Escherichia coli O157:H7 was investigated. Lactoferrin and LfH antimicrobial activities were compared against E. coli O157:H7 and E. coli O157:H7 spheroplasts. The effect of LfH on the cytoplasmic membrane of E. coli O157:H7 cells was determined by evaluating potassium efflux (K⁺), dissipation of ATP and membrane potential (ΔΨ). LfH produced a rapid efflux of potassium ions, a decrease in intracellular levels of ATP coupled with a substantial increase in extracellular ATP levels and a complete dissipation of the ΔΨ. The results suggest that LfH causes a collapse of the membrane integrity by pore formation in the inner membrane, leading to the death of the cell. Moreover, the mechanism of action of LfH on E. coli O157:H7 appears to involve an interference with the inner membrane integrity based on experiments using E. coli O157:H7 spheroplasts.     

Interaction between the Microbial Community and Invading Escherichia coli O157:H7 in Soils from Vegetable Fields

The survival of Escherichia coli O157:H7 in soils can contaminate vegetables, fruits, drinking water, etc. However, data on the impact of E. coli O157:H7 on soil microbial communities are limited. In this study, we monitored the changes in the indigenous microbial community by using the phospholipid fatty acid (PLFA) method to investigate the interaction of the soil microbial community with E. coli O157:H7 in soils. Simple correlation analysis showed that the survival of E. coli O157:H7 in the test soils was negatively correlated with the ratio of Gram-negative (G⁻) to Gram-positive (G⁺) bacterial PLFAs (G⁻/G⁺ ratio). In particular, levels of 14 PLFAs were negatively correlated with the survival time of E. coli O157:H7. The contents of actinomycetous and fungal PLFAs in the test soils declined significantly (P, <0.05) after 25 days of incubation with E. coli O157:H7. The G⁻/G⁺ ratio declined slightly, while the ratio of bacterial to fungal PLFAs (B/F ratio) and the ratio of normal saturated PLFAs to monounsaturated PLFAs (S/M ratio) increased, after E. coli O157:H7 inoculation. Principal component analysis results further indicated that invasion by E. coli O157:H7 had some effects on the soil microbial community. Our data revealed that the toxicity of E. coli O157:H7 presents not only in its pathogenicity but also in its effect on soil microecology. Hence, close attention should be paid to the survival of E. coli O157:H7 and its potential for contaminating soils.     

Distribution of Escherichia coli O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g⁻¹ between samples from the same fecal pat. The density in most positive samples was <100 CFU g⁻¹, the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.     

Effects of carbonylcyanide-m-chlorophenylhydrazone (CCCP) and acetate on Escherichia coli O157:H7 and K-12: uncoupling versus anion accumulation

Non-growing cells of Escherichia coli O157:H7 and K-12 that were incubated anaerobically in sodium phosphate buffer at pH 6.5 consumed glucose at a rate of approximately 8 μmol·(mg protein)⁻¹·h⁻¹ and had intracellular pH values of 7.3 and 7.5, respectively. The uncoupler, carbonylcyanide-m-chlorophenylhydrazone (CCCP), caused a marked decrease in intracellular pH, ATP and potassium of both strains. Low concentrations of CCCP stimulated glucose consumption rate, but higher concentrations were inhibitory. Acetate also caused a decrease in intracellular pH, but it never caused a large decrease in glucose consumption rate. Acetate decreased the intracellular ATP of E. coli K-12, but it had no effect on the ATP of O157:H7. Acetate had no effect on the intracellular potassium of E. coli O157:H7, and acetate-treated K-12 cells had even more potassium than untreated controls. Based on these results, acetate and CCCP appear to have different effects on E. coli. The comparison of E. coli O157:H7 and K-12 indicated that intracellular pH, acetate accumulation and intracellular potassium were related. E. coli K-12 maintained a higher intracellular pH than O157:H7, accumulated more acetate and had a greater intracellular potassium.     

Antibacterial activity and mechanism of action of ε-poly-l-lysine

ε-Poly-l-lysine (ε-PL)² is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) ³ levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) ⁴ indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) ⁵ regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157: H7

针对大肠杆菌O157:H7(Escherichia coli O157:H7,E.coli O157:H7)传统检测方法检测周期长的问题,建立了肉类中的E.coli O157:H7的改良环介导等温扩增(LAMP)快速检测方法。以E.coli O157:H7的O157特异性抗原rfbE基因、鞭毛H7特异性抗原fliC基因序列作为靶序列,分别设计2套增加了环引物的改良LAMP引物序列,单管同时检测,通过肉眼观察白色沉淀,判断检测结果。采用36株细菌验证了该改良LAMP引物的特异性。热裂解法提取的DNA经改良LAMP体系扩增20 min,检测E.coli O157:H7的灵敏度为1.4 CFU/mL,人工污染肉中的E.coli O157:H7检出限为1.8 CFU/g。137份实样中,检测出1份E.coli O157:H7假阳性,与行业标准SNT0973-2000符合率达到99.3%。     

Comparison of the BAX for Screening/E. coli O157:H7 Method with Conventional Methods for Detection of Extremely Low Levels of Escherichia coli O157:H7 in Ground Beef

Escherichia coli O157:H7 is an important food-borne pathogen. Often E. coli O157:H7 is difficult to detect, because it is present sporadically at very low levels together with very high levels of competitor organisms which can be difficult to distinguish phenotypically. Cultural methods are time-consuming and give variable results in the detection of E. coli O157:H7. This study examined the performance of BAX for Screening/E. coli O157:H7, a new rapid method for the detection of E. coli O157:H7, against traditional and improved cultural methods and an immunodiffusion assay. All cultural methods demonstrated inadequacy in detecting the presence of E. coli O157:H7 in inoculated samples. The limitations of these cultural methods further complicate evaluation of screening methodologies. The BAX for Screening/E. coli O157:H7 assay outperformed the other methods, with a detection rate of 96.5%, compared to 39% for the best cultural method and 71.5% for the immunodiffusion method. The BAX for Screening/E. coli O157:H7 assay proved to be a rapid, highly sensitive test for the detection of low levels of E. coli O157:H7 in ground beef.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 × 10⁵ to 1 × 10⁶ of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

Effect of Forage or Grain Diets with or without Monensin on Ruminal Persistence and Fecal Escherichia coli O157:H7 in Cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10¹⁰ CFU/animal) made resistant to nalidixic acid (Nal^r). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal^r E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal^r E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Assessments of Total and Viable Escherichia coli O157:H7 on Field and Laboratory Grown Lettuce

Leafy green produce has been associated with numerous outbreaks of foodborne illness caused by strains of Escherichia coli O157:H7. While the amounts of culturable E. coli O157:H7 rapidly decline after introduction onto lettuce in the field, it remains to be determined whether the reduction in cell numbers is due to losses in cell viability, cell injury and a subsequent inability to be detected by standard laboratory culturing methods, or a lack of adherence and hence rapid removal of the organism from the plants during application. To assess which of these options is most relevant for E. coli O157:H7 on leafy green produce, we developed and applied a propidium monoazide (PMA) real-time PCR assay to quantify viable (with PMA) and total (without PMA) E. coli O157:H7 cells on growth chamber and field-grown lettuce. E. coli O157:H7, suspended in 0.1% peptone, was inoculated onto 4-week-old lettuce plants at a level of approximately 10⁶ CFU/plant. In the growth chamber at low relative humidity (30%), culturable amounts of the nontoxigenic E. coli O157:H7 strain ATCC 700728 and the virulent strain EC4045 declined 100 to 1000-fold in 24 h. Fewer E. coli O157:H7 cells survived when applied onto plants in droplets with a pipette compared with a fine spray inoculation. Total cells for both strains were equivalent to inoculum levels for 7 days after application, and viable cell quantities determined by PMA real-time PCR were approximately 10⁴ greater than found by colony enumeration. Within 2 h after application onto plants in the field, the number of culturable E. coli ATCC 700728 was reduced by up to 1000-fold, whereas PCR-based assessments showed that total cell amounts were equivalent to inoculum levels. These findings show that shortly after inoculation onto plants, the majority of E. coli O157:H7 cells either die or are no longer culturable.     

Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 10¹ to 1.5 × 10⁵ CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

Longitudinal Study of Escherichia coli O157:H7 Dissemination on Four Dairy Farms in Wisconsin

A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination of Escherichia coli O157:H7. A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months). Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative. Farm R had two separate periods of E. coli O157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996). Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 × 10² to 8.7 × 10⁴ CFU per g. E. coli O157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X. When present in animal drinking water, E. coli O157:H7 disseminated through the cohort cattle and other cattle that used the water source. E. coli O157:H7 was found in water at <1 to 23 CFU/ml. Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm. The isolates from farm R displayed two predominant XbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively. Six additional REDP that were ≥89% similar to REDP 3 or REDP 7 were identified among the farm R isolates. Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3. Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9. These results suggest that E. coli O157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period.