Absence of DNA sequence homology with genes of the Escherichia coli hemB locus in Shiga-toxin producing E. coli (STEC) O157 strains

By molecular cloning of chromosomal DNA of a human faecal Escherichia coli O6:non-motile strain, we identified a 1350-bp DNA segment which is commonly present in laboratory and wild-type E. coli strains but had no homology to DNA of Shiga-toxin producing E. coli O157, O145 and enteropathogens E. coli O55 strains. The nucleotide sequence of the 1350-bp segment cloned on plasmid pEO67 was determined (GenBank accession number AF087670) and a 97.2% sequence homology was found to a region of the E. coli hemB locus with an unknown gene function. The introduction of pEO67 into an STEC O157:H- strain had a stimulating effect on the growth of the recipient strain which was most expressed when bacteria were grown in iron depleted M9 medium with hemin added as the exogenous iron source. This growth effect was not observed with E. coli K-12 carrying pEO67. We suggest that the cloned gene is involved in iron uptake of E. coli and that the alteration in this part of the hemB locus is clonally inherited in genetically closely related STEC O157 and O55 strains.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

Estimation of viable Escherichia coli O157 in surface waters using enrichment in conjunction with immunological detection

The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples. In principle, E. coli O157 populations (C(initial E. coli O157)) can be derived from enrichment data according to the equation: C(initial E. coli O157) = C(initial coliforms) x C(final E. coli O157)/C(final coliforms)), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining C(final E. coli O157) in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of C(initial coliforms). Estimates of C(final coliforms) were determined from turbidity data. Initial E. coli O157 populations (C(initial E. coli O157)) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of < 1 cell per 100 ml. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k= 0.47 +/- 0.13 h(-1), n= 72). There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique. Although variability in growth rates was expected to yield some low values, the fact that most E. coli O157 concentrations were < 1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of C(initial coliforms) and k(coliforms), MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 10(3) to 10(8) cells ml(-1) enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E. coli O157 cell per 100 ml surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli.     

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.     

Growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7 on beef extract medium as influenced by package atmosphere and storage temperature.

The effect of atmospheric composition and storage temperature on growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7, inoculated onto brain heart infusion agar containing 0.3% beef extract (BEM), was determined. BEM plates were packaged in barrier bags in air, 100% CO2, 100% N2, 20% CO2: 80% N2, and vacuum and were stored at 4, 10, and 37 degrees C for up to 20 days. Package atmosphere and inoculum status (i.e., uninjured or heat-injured) influenced (P < 0.01) growth and survival of E. coli O157:H7 stored at all test temperatures. Growth of heat-injured E. coli O157:H7 was slower (P < 0.01) than uninjured E. coli O157:H7 stored at 37 degrees C. At 37 degrees C, uninjured E. coli O157:H7 reached stationary phase growth earlier than heat-injured populations. Uninjured E. coli O157:H7 grew during 10 days of storage at 10 degrees C, while heat-injured populations declined during 20 days of storage at 10 degrees C. Uninjured E. coli O157:H7 stored at 10 degrees C reached stationary phase growth within approximately 10 days in all packaging atmospheres except CO2. Populations of uninjured and heat-injured E. coli O157:H7 declined throughout storage for 20 days at 4 degrees C. Survival of uninjured populations stored at 4 degrees C, as well as heat-injured populations stored at 4 and 10 degrees C, was enhanced in CO2 atmosphere. Survival of heat-injured E. coli O157:H7 at 4 and 10 degrees C was not different (P > 0.05). Uninjured and heat-injured E. coli O157:H7 are able to survive at low temperatures in the modified atmospheres used in this study.     

Chemical characterization of the lipopolysaccharides from enteropathogenic Escherichia coli O142 and O158.

Lipopolysaccharides (LPS) from two enteropathogenic strains of E. coli O142 and O158 were isolated by hot phenol-water extraction procedure. Polyacrylamide gel electrophoretic pattern of the LPS showed the typical ladder like pattern of smooth type of LPS. The LPS of E. coli O158 was found to contain L-rhamnose, D-glucose and N-acetyl-D-galactosamine as major constituents together with D-galactose, N-acetyl-D-glucosamine, L-glycero-D-manno-heptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) whereas LPS from E. coli O142 contained L-rhamnose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as major constituents together with D-glucose, D-galactose, N-acetyl-D-glucosamine, L-glycero-D-mannoheptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO). LPS was degraded by mild acid hydrolysis to yield a degraded polysaccharide fraction and an insoluble lipid-A fraction. The main fatty acids of the lipid-A fraction of the LPS were C12:O, C14:O, and 3-OH C14:O for O158 strain whereas E. coli O142 lipid-A consisted of C12:O, C14:O, 3-OH C14:O, and C16:O. The degraded polysaccharide fraction on gel permeation chromatography gave a high moleculer weight O-chain fraction and a core oligosaccharide and a fraction containing degraded sugars. The chemical composition of LPS and its fragmented products are reported in this communication.     

Use of copper cast alloys to control Escherichia coli O157 cross-contamination during food processing

The most notable method of infection from Escherichia coli O157 (E. coli O157) is through contaminated food products, usually ground beef. The objective of this study was to evaluate seven cast copper alloys (61 to 95% Cu) for their ability to reduce the viability of E. coli O157, mixed with or without ground beef juice, and to compare these results to those for stainless steel. E. coli O157 (NCTC 12900) (2 x 10(7) CFU) mixed with extracted beef juice (25%) was inoculated onto coupons of each copper cast alloy or stainless steel and incubated at either 22 degrees C or 4 degrees C for up to 6 h. E. coli O157 viability was determined by plate counts in addition to staining in situ with the respiratory indicator fluorochrome 5-cyano-2,3-ditolyl tetrazolium. Without beef extract, three alloys completely killed the inoculum during the 6-h exposure at 22 degrees C. At 4 degrees C, only the high-copper alloys (>85%) significantly reduced the numbers of O157. With beef juice, only one alloy (95% Cu) completely killed the inoculum at 22 degrees C. For stainless steel, no significant reduction in cell numbers occurred. At 4 degrees C, only alloys C83300 (93% Cu) and C87300 (95% Cu) significantly reduced the numbers of E. coli O157, with 1.5- and 5-log kills, respectively. Reducing the inoculum to 10(3) CFU resulted in a complete kill for all seven cast copper alloys in 20 min or less at 22 degrees C. These results clearly demonstrate the antimicrobial properties of cast copper alloys with regard to E. coli O157, and consequently these alloys have the potential to aid in food safety.     

Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation

AIMS: To compare a range of enrichment broths and enrichment temperatures for the isolation of Escherichia coli O157 by immunomagnetic separation (IMS) from sandy, loam and clay soils. METHODS AND RESULTS: Soils were spiked with cocktails of four atoxigenic strains of E. coli O157 and four strains of commensal E. coli. The organisms were stressed by subjecting soils to cycles of freeze/thawing, followed by drying at 20 degrees C for up to 4 days. Nine enrichment broths were trialled based on buffered peptone water, tryptone soya broths and EC broths supplemented with a range of selective additions. Enrichments were incubated for 6 h and assessed by target recovery after IMS on cefixime tellurite sorbitol MacConkey agar (CTSMAC) incubated at 37 degrees C for 24 h. A comparison of enrichment temperatures (37 and 42 degrees C) was also performed. Buffered peptone water (with or without vancomycin) and tryptone soya broth (with or without novobiocin) gave significant increases in recovery of E. coli O157 compared to others tested. In addition, broths incubated at 42 degrees C were superior to those at 37 degrees C for the recovery of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that sub-lethally damaged E. coli O157 surviving in soil can be sensitive to antimicrobial additions. The choice and concentration of these additions is vitally important to optimize target recovery. Some IMS protocols, established for the isolation of E. coli O157, may be unsuitable for the examination of soil samples.     

Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.     

Acquisition of the rfb-gnd cluster in evolution of Escherichia coli O55 and O157

The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb and gnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (allele D) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd allele D. Though gnd alleles B and C in E. coli O157 that are distantly related to E. coli O157:H7 are more similar to allele A than to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3' to the O55 gnd allele in diverse E. coli lineages has sequences homologous to tnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157 rfb regions. We conclude that rfb and gnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.     

Evaluation of dietary influences on Escherichia coli O157:H7 shedding by sheep.

The effect of diet, an abrupt diet change, and fasting on the shedding of Escherichia coli O157:H7 was investigated with experimentally inoculated sheep as a ruminant model. Sheep were fed a grass hay diet (G), which was low in protein and digestible energy and high in fiber, or a mixture of corn and pelleted alfalfa (C), which was high in protein and digestible energy and low in fiber. After a single oral inoculation of E. coli O157:H7, all the animals shed fecal E. coli O157:H7. However, sheep that were fed G shed the bacterium almost twice as long as, and in larger numbers than, did sheep that were fed C. The number of culture-positive animals increased after the diet was abruptly changed from C to G and decreased with the opposite change (G to C). A 24-h fast did not influence E. coli O157:H7 shedding. Horizontal transmission of infection between animals occurred. Recent shedding of E. coli O157:H7 did not affect recolonization with E. coli O157:H7. The findings presented in this study indicate that preharvest control of diet may reduce the risk of E. coli O157:H7-positive animals entering the food chain.     

Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers

The survival of Escherichia coli O157:H7 in feces from steers fed corn (CO) or barley (BA) was evaluated at -10, +4 and +22 degrees C. Fecal pats were inoculated with a four-strain mixture of nalidixic-acid resistant E. coli O157:H7 at two levels: 10(3) CFU g(-1) (low, L) and 105 CFU g(-1) (high, H). At -10 degrees C, duration of survival of E. coli O157:H7 was reduced (p < 0.05) in CO-L (35 days) compared to BA-L (49 days), likely due to the effects of fecal volatile fatty acids in combination with a fecal pH of <6.5. At 4 degrees C, E. coli O157:H7 was detected in BA-H, CO-H, CO-L and BA-L for 77, 77, 56 and 63 days, respectively, with no difference (p > 0.05) observed in the duration of survival or rate of decline of E. coli O157:H7 among treatments. Survival of E. coli O157:H7 was twice as likely (p < 0.05) at 22 degrees C than at 4 degrees C and -10 degrees C. While pH and dry matter content increased, and volatile fatty acid concentrations decreased over 84 days at all three temperatures, these changes were most pronounced at 22 degrees C. Survival of E. coli O157:H7 for extended periods of time in feces from both corn- and barley-fed animals was demonstrated, thus fecal material may serve as a vector for the transmission of the organism. The greater survival of E. coli O157:H7 at 22 degrees C suggests that temperature may play a role in the seasonality of transmission and prevalence of this bacterium in feedlot cattle. The reported greater prevalence of E. coli O157:H7 in cattle fed barley as compared to those fed corn does not appear to be related to elevated risk of transmission arising from differential survival of the bacterium in feces.     

Survival and growth of Escherichia coli O157:H7 on salad vegetables.

The influence of modified-atmosphere packaging, storage temperature, and time on survival and growth of Escherichia coli O157:H7 inoculated onto shredded lettuce, sliced cucumber, and shredded carrot was determined. Growth of psychotrophic and mesophilic microorganisms and changes in pH and sensory qualities of vegetables, as judged by subjective evaluation, were also monitored. Packaging under an atmosphere containing 3% oxygen and 97% nitrogen had no apparent effect on populations of E. coli O157:H7, psychotrophs, or mesophiles. Populations of viable E. coli O157:H7 declined on vegetables stored at 5 degrees C and increased on vegetables stored at 12 and 21 degrees C for up to 14 days. The most rapid increases in populations of E. coli O157:H7 occurred on lettuce and cucumbers stored at 21 degrees C. These results suggest that an unknown factor(s) associated with carrots may inhibit the growth of E. coli O157:H7. The reduction in pH of vegetables was correlated with initial increases in populations of E. coli O157:H7 and naturally occurring microfloras. Eventual decreases in E. coli O157:H7 in some samples, e.g., those stored at 21 degrees C, are attributed to the toxic effect of accumulated acids. Changes in visual appearance of vegetables were not influenced substantially by growth of E. coli O157:H7. The ability of E. coli O157:H7 to growth on raw salad vegetables subjected to processing and storage conditions simulating those routinely used in commercial practice has been demonstrated.     

Enhancing the antimicrobial effects of bovine lactoferrin against Escherichia coli O157:H7 by cation chelation, NaCl and temperature

AIM: To evaluate the effect of NaCl, growth medium and temperature on the antimicrobial activity of bovine lactoferrin (LF) against Escherichia coli O157:H7 in the presence of different chelating agents. METHODS AND RESULTS: LF (32 mg ml(-1)) was tested against E. coli O157:H7 strain 3081 in Luria broth (LB) and All Purpose Tween (APT) broth with metal ion chelators sodium bicarbonate (SB), sodium lactate (SL), sodium hexametaphosphate (SHMP), ethylene diamine tetraacetic acid (EDTA) or quercetin at 0.5 and 2.5% NaCl at 10 and 37 degrees C. LF and the chelators were tested against four other E. coli O157:H7 strains in LB at 2.5% NaCl and 10 degrees C. LF alone was bacteriostatic against strains 3081 and LCDC 7283 but other strains grew. Antimicrobial effectiveness of LF was reduced in APT broth but enhanced by SB at 2.5% NaCl and 10 degrees C where 4.0 log(10) CFU ml(-1) inoculated cells were killed. EDTA enhanced antimicrobial action of the LF-SB combination. SL alone was effective against E. coli O157:H7 but a reduction in its activity at 2.5% NaCl and 10 degrees C was reversed by LF. The combinations LF-SHMP and LF-quercetin were more effective at 37 degrees C and NaCl effects varied. CONCLUSIONS: LF plus SB or SL were bactericidal toward the same 3/5 E. coli O157:H7 strains and inhibited growth of the others at 2.5% NaCl and 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of LF with either SL or SB shows potential for reducing viability of E. coli O157:H7 in food systems containing NaCl at reduced, but growth permissive temperature.     

Applicability of a model for non-pathogenic Escherichia coli for predicting the growth of pathogenic Escherichia coli

A model was developed for the temperature dependence of growth rate of a non-pathogenic Escherichia coli strain. The suitability of that model for predicting the growth rate of pathogenic E. coli strains was assessed. Growth rates of pathogenic strains were found to be adequately described by the model. Model predictions were also found to describe sufficiently well-published growth rate data for non-pathogenic E. coli on mutton carcase surfaces and E. coli O157:H7 in ground roasted beef, milk, and on cantaloupes and water melons. In addition, E. coli O157:H7 was found to grow in the region of 44–45·5 °C.     

Involvement of enterobactin in norepinephrine-mediated iron supply from transferrin to enterohaemorrhagic Escherichia coli

Exposure of bacteria to members of the stress-associated family of catecholamine hormones, principally norepinephrine, has been demonstrated to increase both growth and production of virulence-related factors. Mutation of genes for enterobactin synthesis and uptake revealed an absolute requirement for enterobactin in norepinephrine-stimulated growth of Escherichia coli O157:H7. The autoinducer produced by norepinephrine-stimulated E. coli could not substitute for enterobactin. We also demonstrate that norepinephrine promotes iron shuttling between transferrin molecules, thereby enabling the bacterial siderophore enterobactin to more readily acquire iron for growth. These results suggest one of the possible mechanisms by which the hormonal output of stress may affect enterohaemorrhagic E. coli pathogenicity.     

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Methods for the isolation of water-borne Escherichia coli O157

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.     

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef

AIM: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. METHODS AND RESULTS: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41.5 degrees C in tryptone soya broth supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l(-1))] and supplemented with cefixime (50 microg ml(-1)) and potassium tellurite (2.5 mg l(-1)). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 microg ml(-1)), cefsulodin (5 mg l(-1)) and vancomycin (8 mg l(-1)). Minced beef samples were inoculated with a number of strains of E. coli O26 (n=9) and O111 (n=8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. CONCLUSIONS: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef.