Stephen D. Weagant

Survival of Escherichia coli O157:H7 in Mayonnaise and Mayonnaise-Based Sauces at Room and Refrigerated Temperatures

Three Escherichia coli Ol57:H7 (EHEC) strains were inoculated separately into portions of commercially prepared mayonnaise held at 25 or 7°C, then examined periodically for survival of detectable EHEC. Four mayonnaise-based sauces including: a) mayonnaise-mustard sauce, b) blue cheese dressing, c) thousand island dressing and d) seafood sauce, were each inoculated with one EHEC strain. Samples of these sauces were held at 5°C, and assayed periodically for survival of detectable EHEC. Both direct plate count and selective enrichment recovery were employed as assay procedures. Escherichia coli O157:H7 strains, when inoculated and mixed into mayonnaise and stored at 25°C, became undetectable after 72 h storage when assayed by direct plating or by selective enrichment. The same strains inoculated into mayonnaise and stored at 7°C were detectable up to 35 days when assayed by direct plating or by selective enrichment. Escherichia coli O157:H7 inoculated into mayonnaise-based sauces and held at 5°C were detectable past 35 days in three of the four sauces. Loss of EHEC culturability occurred within 3 days in mayonnaise-mustard sauce.

The Incidence of Listeria Species in Frozen Seafood Products

Samples of frozen seafood products from several countries were tested for the presence of Listeria monocytogenes and other Listeria species using the U.S. Food and Drug Administration (FDA) Listeria isolation method. Of 57 samples tested, 35 contained Listeria species and 15 of 57 samples contained L. monocytogenes . Samples found positive included raw shrimp, cooked and peeled shrimp, cooked crabmeat, raw lobster tails, langostinos, scallops, squid and surimi-based imitation seafoods. Positive samples were obtained from nine different countries around the world.

Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H− Serotype

ABSTRACT A multiplex real-time PCR method to simultaneously detect the stx1 and stx2 genes of Shiga toxin-producing Escherichia coli and a unique conserved single-nucleotide polymorphism in the E. coli O157:H7/H−uidA gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were ≥1.89, and as few as 6 CFU/reaction could be detected.

An improved rapid technique for isolation of Escherichia coli 0157:H7 from foods

A newly revised enrichment and agar-plating system was tested for selectivity and sensitivity in recovery of unstressed and cold-stressed Escherichia coli O157:H7 from foods. Various foods inoculated with known levels of enterohemorrhagic E. coli O157:H7 (EHEC) were tested by enrichment for 6 h at 37°C in modified tryptic soy broth (mTSB) base supplemented with vancomycin, cefsulodin and cefixime, referred to as EHEC enrichment broth (EEB). Subsequently, portions were spread-plated on sorbitol-MacConkey agar supplemented with tellurite and cefixime (TCSMAC). Further selective enrichment was also examined using immunomagnetic separation (IMS) from the EEB prior to spread-plating on TCSMAC agar. These methods were compared to a procedure of enrichment in mTSB (supplemented with novobiocin) at 37°C for 24 h followed by spread-plating of decimal dilutions on hemorrhagic colitis 4-methylumbelliferyl-B-D-glucuronide (HC-MUG) agar. The new enrichment isolation technique was found to be sensitive at a level of one EHEC organism per 10 g of food in four food types. This represents an approximate l00-fold to 1,000-fold enhancement in sensitivity over the comparative method for foods with high levels of competitive microflora. These enrichment-isolation protocols also were compared in analysis of naturally contaminated raw or undercooked ground beef samples implicated in foodborne illness. EEB-TCSMAC with and without IMS were combined with rapid biochemical tests, and with O157 latex agglutination and confirmation of toxin genes by polymerase chain reaction (PCR) to provide a completed test within 30 h of initiating testing. The new system was successful in 15 of 17 samples, where only 6 of 17 were found positive by the comparative technique.

Genetic Analysis for Virulence Factors in Escherichia coli O104:H21 That Was Implicated in an Outbreak of Hemorrhagic Colitis

ABSTRACT Isolates of enterohemorrhagic Escherichia coli (EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (stx2) and to express Stx2. They did not have the eaeA gene for γ-intimin, which is typically found in O157:H7, or the α- or β-intimin derivatives, which are common in other EHEC and enteropathogenic E. coli serotypes. Results of the multiplex PCR also indicated that the ehxA gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set ofehxA-specific primers, which indicated the presence ofehxA. To resolve this discrepancy, the ehxAregion in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the ehxAgene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3′ end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the ehxA primers used in the multiplex PCR. Comparison of the ehxA sequences of O104:H21 strains with those of other Stx-producing E. colistrains showed that they more closely resembled those of O8:H19 strains, which have cluster II ehxA genes, than those of O157:H7 strains, which have cluster I ehxA sequences. By modifying the upstream ehxA primer, the multiplex PCR was able to detect ehxA genes in both O157:H7 and O104:H21 strains.

Comparison of Template Preparation Methods from Foods for Amplification of Escherichia coli O157 Shiga-Like Toxins Type I and II DNA by Multiplex Polymerase Chain Reaction

Escherichia coli O157:H7 has been responsible for several recent food-borne outbreaks in the United States. To protect the public health, it is essential that rapid and sensitive methods be developed for detection of this pathogen in foods. Methods were compared for preparation of template DNA for the polymerase chain reaction (PCR) from enrichments of food homogenates seeded with E. coli O157:H7. Samples were enriched for 6 h at 37°C in modified tryptic soy broth supplemented with vancomycin, cefsulodin, and cefixime. Aliquots of the enrichments (10 ml or 1 ml) were analyzed by either washing twice with physiological saline or incubating with antibodies to O157 coupled to immunomagnetic beads (Dynal®) followed by resuspending and boiling the samples. A portion of the preparation was used in a multiplex PCR to amplify a 274-bp fragment from the sltI gene and a 364-bp fragment from the sltII gene. PCR amplification of 1-ml portions of enrichment broth was successful at inoculation levels of about 10 cells per g of food. Increasing the test sample volume to 10 ml and/or using an immunomagnetic separation step improved the PCR detection sensitivity to about 1 cell per g; the entire analysis can be completed within 12 h.

Evaluation of techniques for enrichment and isolation of Escherichia coli O157:H7 from artificially contaminated sprouts.

Because sprouted seed products are kept wet during and after production, have high levels of nutrients, and a neutral pH, they are subject to the outgrowth of pathogens such as Escherichia coli O157:H7. For these same reasons, these products also contain high levels of heterotrophic organisms and in particular coliform bacteria. Recent outbreaks have focused attention on the need to improve methodology for isolating this pathogen from sprouts. When 40 E. coli O157:H7 strains were grown in pure culture in enterohemorrhagic E. coli enrichment broth (EEB) as prescribed in the U.S. FDA-Bacteriological Analytical Manual (FDA-BAM) and in EEB modified by varying the cefixime concentration, outgrowth for all strains in EEB was inhibited at 0.05 mg/l but for only 2 of 40 strains when the cefixime level was adjusted to 0.0125 mg/l. These two enrichment formulae were compared to modified E. coli broth (mEC), modified Tryptic Soy Broth with 20 mg/l novobiocin (mTSB + N), modified Buffered Peptone Water (mBPW), and mBPW with added 10 mg/l acriflavin, 10 mg/l cefsulodin, and 8 mg/l vancomycin (mBPW + ACV) for isolation of E. coli O157:H7 from sprouts. These comparisons were performed using low-level (0.12 to 0.42 cfu/g) artificially contaminated alfalfa and mixed salad sprouts. After enrichment, two isolation methods were compared for recovery; direct plating to Tellurite-Cefixime Sorbitol MacConkey agar (TCSMAC) and immunomagnetic separation (IMS) (Dynabeads anti-E. coli O157, Dynal, Oslo, Norway) followed by plating to TCSMAC. In addition, an immunoprecipitin detection kit, VIP (BioControl, Bellevue, WA), was evaluated for detection after enrichment. We found that five of the six enrichments were equivalent for detection or recovery while one enrichment (mTSB + N without agitation) was less productive. Incubation for 24 h was more effective in recovering E. coli O157:H7 from sprouts than 6 h for all enrichment broths. Plating after IMS was more productive than direct plating at these low levels of contamination, yielding recovery in 70 of 90 trials compared to 37 of 90 trials without IMS for six enrichments. The sensitivity of VIP for detection of E. coli O157:H7 varied depending on the enrichment broth. Because of the rapid rate of growth of E. coli O157:H7 in mBPW, the high productivity of mBPW + ACV after 24-h enrichment and its compatibility with both IMS and detection with immunoprecipitin tests, mBPW + ACV at 42 degrees C with agitation was found to be the most promising enrichment protocol for testing sprouts.

Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods

Detection of Escherichia coli O157:H7 by conventional cultural methods can be difficult in food matrices with a high background flora such as raw ground beef. Raw ground beef samples, artificially contaminated separately with five strains of E. coli O157:H7 at low (~0.2 cfu/g) and high (~2 cfu/g) levels, were enriched by two enrichment protocols; buffered peptone water (BPW) at 37 °C for 5h and 24h and modified buffered peptone water with pyruvate (mBPWp) for 5h at 37 °C followed by adding selective agents and incubating at 42 °C to 24h. Detection of added E. coli O157:H7 by real-time PCR (RTiPCR) and recovery on isolation agars was performed before and after PATHATRIX™ immunomagnetic separation (IMS). RTiPCR detection and cultural recovery of inoculated E. coli O157:H7 after 5h enrichment were poor at 0.21-0.24 cfu/g. The addition of IMS after 5h enrichment did not improve RTiPCR detection but markedly improved recovery by culturing. By extending enrichment to 24h, RTiPCR detection improved to 76% for either enrichment protocol without IMS. When 24h enrichment was followed by IMS, RTiPCR detection was also further improved. Cultural recovery after 24h enrichment was 56% and 84% without IMS and 100% and 92% after IMS for BPW and mBPWp respectively. Extended enrichment to 24h followed by IMS was found to be sensitive and reliable for detection and cultural recovery from raw ground beef using either enrichment method.

Yersiniosis Associated with Tofu Consumption: Serological, Biochemical and Pathogenicity Studies of Yersinia enterocolitica Isolates

From December 1981 to February 1982, 87 individuals (ages two months to 74 years) in the Seattle, WA, area developed the clinical symptoms of yersiniosis. Illness was related to consumption of commercial tofu (soybean curd) contaminated with Yersinia enterocolitica . The six Y. enterocolitica strains recovered from the hospitalized patients indicated that two antigenically distinct strains, 0:8 and 0:Tacoma, were involved. At the manufacturing site of the incriminated tofu, 112 Y. enterocolitica strains were recovered, of which two were serotype 0:8. The reactions of these strains were similar to those of clinical 0:8 strains in biochemical tests and in eight virulence factor tests. The LD50 for suckling mice was identical for all strains which killed mice. Although the causative organism(s) was not recovered from other samples of packaged tofu, our findings incriminated water used in the processing of tofu as the source of infection. The source of the second Y. enterocolitica strain (0:Tacoma) in this outbreak was not identified.

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.