Using Dice Games to Teach Hazards, Risk, and Outcomes in HACCP Classes

This article describes the incorporation of a dice game (piggy) to teach food safety hazards and risk in an engaging way in HACCP classes. Each player accumulates points by rolling two dice, but loses points in a turn when rolling a 7, or all accumulated points when rolling two consecutive doubles. This game helps explain the difference between a concrete event, a hazard (number 7) and risk of that hazard occurring (the probability of rolling number 7). Two consecutive doubles inflict a more severe loss and can help explain the variability in outcomes of food safety hazards

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

<p>Abstract</p> <p>Background</p> <p>Host cell invasion by the foodborne pathogen <it>Campylobacter jejuni </it>is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/-and Src/Yes/Fyn^-/-deficient mice, and wild-type control cells, to investigate <it>C. jejuni</it>-induced mechanisms leading to Cdc42 activation and bacterial uptake.</p> <p>Results</p> <p>Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and <it>C. jejuni </it>invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/-knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using <it>C. jejuni </it>mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.</p> <p>Conclusion</p> <p>Collectively, our findings led us propose that <it>C. jejuni </it>infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.</p

A simplified and cost-effective enrichment protocol for the isolation of Campylobacter spp. from retail broiler meat without microaerobic incubation

<p>Abstract</p> <p>Background</p> <p>To simplify the methodology for the isolation of <it>Campylobacter </it>spp. from retail broiler meat, we evaluated 108 samples (breasts and thighs) using an unpaired sample design. The enrichment broths were incubated under aerobic conditions (subsamples A) and for comparison under microaerobic conditions (subsamples M) as recommended by current reference protocols. Sensors were used to measure the dissolved oxygen (DO) in the broth and the percentage of oxygen (O₂) in the head space of the bags used for enrichment. <it>Campylobacter </it>isolates were identified with multiplex PCR assays and typed using pulsed-field gel electrophoresis (PFGE). Ribosomal intergenic spacer analyses (RISA) and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial communities of subsamples M and A after 48 h enrichment.</p> <p>Results</p> <p>The number of <it>Campylobacter </it>positive subsamples were similar for A and M when all samples were combined (<it>P </it>= 0.81) and when samples were analyzed by product (breast: <it>P </it>= 0.75; thigh: <it>P </it>= 1.00). Oxygen sensors showed that DO values in the broth were around 6 ppm and O₂values in the head space were 14-16% throughout incubation. PFGE demonstrated high genomic similarity of isolates in the majority of the samples in which isolates were obtained from subsamples A and M. RISA and DGGE results showed a large variability in the bacterial populations that could be attributed to sample-to-sample variations and not enrichment conditions (aerobic or microaerobic). These data also suggested that current sampling protocols are not optimized to determine the true number of <it>Campylobacter </it>positive samples in retail boiler meat.</p> <p>Conclusions</p> <p>Decreased DO in enrichment broths is naturally achieved. This simplified, cost-effective enrichment protocol with aerobic incubation could be incorporated into reference methods for the isolation of <it>Campylobacter </it>spp. from retail broiler meat.</p

Major Host Factors Involved in Epithelial Cell Invasion of Campylobacter jejuni: Role of Fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in Activating Rho GTPase Rac1

Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin(−/−), integrin beta1(−/−), and focal adhesion kinase (FAK)(−/−) deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin(−/−), integrin beta1(−/−), and FAK(−/−) knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells

Unusual Manifestation of Live Staphylococcus saprophyticus, Corynebacterium urinapleomorphum, and Helicobacter pylori in the Gallbladder with Cholecystitis

Culture-independent studies have identified DNA of bacterial pathogens in the gallbladder under pathological conditions, yet reports on the isolation of corresponding live bacteria are rare. Thus, it is unclear which pathogens, or pathogen communities, can colonize the gallbladder and cause disease. Using light microscopy, scanning electron microscopy, culture techniques, phylogenetic analysis, urease assays and Western blotting, we investigated the presence of live bacterial communities in the gallbladder of a cholecystitis patient after cholecystectomy. 16S rRNA gene sequencing of isolated bacterial colonies revealed the presence of pathogens most closely resembling Corynebacterium urinapleomorphum nov. sp., Staphylococcus saprophyticus and Helicobacter pylori. The latter colonies were confirmed as H. pylori by immunohistochemistry and biochemical methods. H. pylori cultured from the gallbladder exhibited both the same DNA fingerprinting and Western cagA gene sequence with ABC-type EPIYA (Glu-Pro-Ile-Tyr-Ala) phosphorylation motifs as isolates recovered from the gastric mucus of the same patient, suggesting that gastric H. pylori can also colonize other organs in the human body. Taken together, here we report, for the first time, the identification and characterization of a community consisting of live S. saprophyticus; C. urinapleomorphum, and H. pylori in the gallbladder of a patient with acute cholecystitis. Their potential infection routes and roles in pathogenesis are discussed

Prevalence of <it>Campylobacter</it> spp. in skinless, boneless retail broiler meat from 2005 through 2011 in Alabama, USA

<p>Abstract</p> <p>Background</p> <p>The prevalence of <it>Campylobacter</it> spp. in 755 skinless, boneless retail broiler meat samples (breast, tenderloins and thighs) collected from food stores in Alabama, USA, from 2005 through 2011 was examined. <it>Campylobacter</it> spp. were isolated using enrichment and plate media. Isolates were identified with multiplex PCR assays and typed with pulsed field gel electrophoresis (PFGE). Data were analyzed by nominal variables (brand, plant, product, season, state and store) that may affect the prevalence of these bacteria.</p> <p>Results</p> <p>The average prevalence of <it>Campylobacter</it> spp. in retail broiler meat for these years was 41%, with no statistical differences in the prevalence by year (<it>P</it> > 0.05). Seasons did not affect the prevalence of <it>C. jejuni</it> but statistically affected the prevalence of <it>C. coli</it> (<it>P</it> < 0.05). The prevalence by brand, plant, product, state and store were different (<it>P</it> < 0.05). Establishments from two states had the highest prevalence (<it>P</it> < 0.05). <it>C. coli</it> and <it>C. jejuni</it> had an average prevalence of 28% and 66%, respectively. The prevalence of <it>C. coli</it> varied by brand, plant, season, state, store and year, while the prevalence of <it>C. jejuni</it> varied by brand, product, state and store. Tenderloins had a lower prevalence of <it>Campylobacter</it> spp. than breasts and thighs (<it>P</it> < 0.05). Although no statistical differences (<it>P</it> > 0.05) were observed in the prevalence of <it>C. jejuni</it> by season, the lowest prevalence of <it>C. coli</it> was recorded from October through March. A large diversity of PFGE profiles was found for <it>C. jejuni</it>, with some profiles from the same processing plants reappearing throughout the years.</p> <p>Conclusions</p> <p>The prevalence of <it>Campylobacter</it> spp. did not change during the seven years of the study; however, it did change when analyzed by brand, product and state. Seasons did not affect the prevalence of <it>C. jejuni,</it> but they did affect the prevalence of <it>C. coli</it>. Larger PFGE databases are needed to assess the temporal reoccurrence of PFGE profiles to help predict the risk associated with each profile.</p

Conjugative Transfer of Chromosomally Encoded Antibiotic Resistance from Helicobacter pylori to Campylobacter jejuni

Many strains of Helicobacter pylori are naturally competent for transformation and able to transfer chromosomal DNA among different isolates using a conjugation-like mechanism. In this study, we sought to determine whether H. pylori can transfer DNA into Campylobacter jejuni, a closely related species of the Campylobacterales group. To monitor the transfer, a chromosomally encoded streptomycin resistance cassette prearranged by a specific mutation in the rpsL gene of H. pylori was used. Mating of the bacteria on plates or in liquid broth medium produced C. jejuni progeny containing the streptomycin marker. DNA transfer was unidirectional, from H. pylori to C. jejuni, and the progeny were genetically identical to C. jejuni recipient strains. DNase I treatment reduced but did not eliminate transfer, and DNase I-treated cell supernatants did not transform, ruling out phage transduction. Recombinants also did not occur when the mating bacteria were separated by a membrane, suggesting that DNA transfer requires cell-to-cell contact. Transfer of the streptomycin marker was independent of the H. pylori comB transformation system, the cag pathogenicity island, and another type IV secretion system called tfs3. These findings indicated that a DNase I-resistant, conjugation-like mechanism may contribute to horizontal DNA transfer between different members of the Campylobacteriales group. The significance of this DNA uptake by C. jejuni in the context of acquiring antibiotic resistance is discussed