Seasonal prevalence of Shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants

The seasonal prevalence of Escherichia coli O157:H7, Salmonella, non-O157 E. coli (STEC), and stx-harboring cells was monitored at three Midwestern fed-beef processing plants. Overall, E. coli O157:H7 was recovered from 5.9% of fecal samples, 60.6% of hide samples, and 26.7% of carcasses sampled before the preevisceration wash. This pathogen also was recovered from 1.2% (15 of 1,232) of carcasses sampled at chilling (postintervention) at approximate levels of <3.0 cells per 100 cm2. In one case, the E. coli O157:H7 concentration dropped from ca. 1,100 cells per 320 cm2 at the preevisceration stage to a level that was undetectable on ca. 2,500 cm2 at the postintervention stage. The prevalence of E. coli O157:H7 in feces peaked in the summer, whereas its prevalence on hide was high from the spring through the fall. Overall, Salmonella was recovered from 4.4, 71.0, and 12.7% of fecal, hide, and preevisceration carcass samples, respectively. Salmonella was recovered from one postintervention carcass (of 1,016 sampled). Salmonella prevalence peaked in feces in the summer and was highest on hide and preevisceration carcasses in the summer and the fall. Non-O157 STEC prevalence also appeared to vary by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8% and could have influenced these results. Cells harboring stx genes were detected by PCR in 34.3, 92.0, 96.6, and 16.2% of fecal, hide, preevisceration carcass, and postintervention carcass samples, respectively. The approximate level of non-O157 STEC and stx-harboring cells on postintervention carcasses was > or = 3.0 cells per 100 cm2 for only 8 of 199 carcasses (4.0%). Overall, the prevalence of E. coli O157:H7, Salmonella, and non-O157 STEC varied by season, was higher on hides than in feces, and decreased dramatically, along with pathogen levels, during processing and during the application of antimicrobial interventions. These results demonstrate the effectiveness of the current interventions used by the industry and highlight the significance of hides as a major source of pathogens on beef carcasses.     

Effect of a reactive oxygen species-generating system for control of airborne microorganisms in a meat-processing environment

The effectiveness of reactive oxygen species (ROS)-generating AirOcare equipment on the reduction of airborne bacteria in a meat-processing environment was determined. Serratia marcescens and lactic acid bacteria (Lactococcus lactis subsp. lactis and Lactobacillus plantarum) were used to artificially contaminate the air via a six-jet Collison nebulizer. Air in the meat-processing room was sampled immediately after aerosol generation and at various predetermined times at multiple locations by using a Staplex 6 stage air sampler. Approximately a 4-log reduction of the aerial S. marcescens population was observed within 2 h of treatment (P < 0.05) compared to a 1-log reduction in control samples. The S. marcescens populations reduced further by approximately 4.5 log after 24 h of exposure to ROS treatment. Approximately 3-log CFU/m3 reductions in lactic acid bacteria were observed following 2-h ROS exposure. Further ROS exposure reduced lactic acid bacteria in the air; however, the difference in their survival after 24 h of exposure was not significantly different from that observed with the control treatment. S. marcescens bacteria were more sensitive to ROS treatment than the lactic acid bacteria. These findings reveal that ROS treatment using the AirOcare unit significantly reduces airborne S. marcescens and lactic acid bacteria in meat-processing environments within 2 h.     

Methods for recovering Escherichia coli O157:H7 from cattle fecal, hide, and carcass samples: sensitivity and improvements

The Meats Research Unit (MRU) methods, developed by MRU scientists of the U.S. Meat Animal Research Center, have been used to study the prevalence of Escherichia coli O157:H7 in cattle carcass, hide, and fecal samples. The sensitivity of these methods for recovery of injured E. coli O157:H7 cells from inoculated and uninoculated samples was determined, and potential improvements to these methods were evaluated. When using the conventional MRU methods, 91% of the pre-evisceration carcass samples tested positive for E. coli O157:H7 when inoculated with 5 to 10 CFU, 100% of hide samples tested positive for E. coli O157:H7 when inoculated with 30 to 50 CFU, and 96% of the fecal samples produced positive results when inoculated with 300 to 400 CFU per 10 g. The addition of a phosphate buffer to the tryptic soy broth enrichment improved recovery of E. coli O157:H7 from feces. Using the modified enrichment, 92% of the samples were identified as positive when inoculated with 10 to 30 CFU per 10 g. Substituting a commercially available wash buffer for the phosphate-buffered saline (PBS) plus Tween 20 wash buffer during immunomagnetic separation of hide samples improved recovery of the target organism at lower inoculum concentrations. When comparing uninoculated samples, substituting a PBS buffer plus a zwitterionic detergent for PBS plus Tween 20 also had a positive effect on recovery of E. coli O157:H7 from hide samples. Data presented here indicate that the MRU methods are highly effective at recovering injured E. coli O157:H7 from fecal, hide, and beef carcass samples; however, modifications can be added to increase the sensitivity.     

Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea

Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants.     

Comparison of the molecular genotypes of Escherichia coli O157:H7 from the hides of beef cattle in different regions of North America

Cattle hides become contaminated with Escherichia coli O157:H7 via pathogen transmission in the feedlot, during transport, and while in the lairage environment at the processing facility, and the bacteria can be transferred to beef carcasses during processing. Several studies have shown that E. coli O157:H7 strains possessing indistinguishable restriction digest patterns (RDPs) can be isolated from distant locations. Most of these studies, however, examined RDPs from strains isolated within a single region of the United States or Canada. The experiment described in the present study was designed to identify the molecular genotypes of E. coli O157:H7 isolates from beef cattle hides in nine major cattle-producing regions of North America. Prevalence for E. coli O157:H7 in beef cattle hide samples ranged from 9 to 85%. Pulsed-field gel electrophoresis (PFGE) analysis of XbaI-digested genomic DNA from 1,193 E. coli O157:H7 isolates resulted in 277 unique RDPs. Of the 277 unique XbaI RDPs, 54 contained isolates collected from multiple regions. After two subsequent rounds of PFGE analysis (BlnI and SpeI), there were still many isolates (n = 154) that could not be distinguished from others, even though they were collected from different regions separated by large geographical distances. On multiple occasions, strains isolated from cattle hides in Canada had RDPs that were indistinguishable after three enzyme digestions from cattle hide isolates collected in Kansas and Nebraska. This information clearly shows that strains with indistinguishable RDPs originate from multiple sources that can be separated by large distances and that this should be taken into account when the source tracking of isolates is based on PFGE.     

Development of methods for the recovery of Escherichia coil O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples

Culture methods were developed for the concurrent recovery of Escherichia coli O157:H7 and Salmonella from bovine carcass, hide, and fecal samples. Several enrichment conditions were tested for the overall growth of pure cultures; tryptic soy broth for 2 h at 25 degrees C and then for 6 h at 42 degrees C was the protocol selected for use. Immunomagnetic separation (IMS) was incorporated for sensitivity and selectivity, along with a post-IMS enrichment for the recovery of Salmonella as recommended by the manufacturer. Selective agars for plating after IMS were chosen on the basis of ease of target colony identification. Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and Rainbow agar supplemented with novobiocin and potassium tellurite were chosen for the recovery of E. coli O157:H7. Brilliant green agar with sulfadiazine and Hektoen enteric agar supplemented with novobiocin were selected for the recovery of Salmonella. The resulting methods were evaluated along with standard or previously used methods for the recovery of E. coli O157:H7 and Salmonella from bovine hide and fecal samples and carcass sponge samples. The Meats Research Unit (MRU) methods performed at least as well as the established methods, except that a secondary enrichment in tetrathionate (TT) broth prior to IMS was required for the optimal recovery of Salmonella from feces. Thus, the MRU and MRU-TT methods are effective in the recovery of both E. coli O157:H7 and Salmonella from a single bovine carcass, hide, or fecal sample.     

Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants

The effectiveness of current antimicrobial interventions used in reducing the prevalence or load of Escherichia coli O157 and indicator organisms on cattle hides and carcasses at two commercial beef processing plants was evaluated. Sponge sampling of beef cattle was performed at five locations from the initial entry of the animals to the slaughter floor to the exit of carcasses from the "hotbox" cooler. For each sample, E. coli O157 prevalence was determined and total aerobic bacteria, Enterobacteriaceae, and E. coli O157 were enumerated. E. coli O157 was found on 76% of animal hides coming into the plants, but no carcasses leaving the cooler were identified as contaminated with E. coli O157. A positive relationship was seen between the incidence of E. coli O157 in hide samples and that in preevisceration samples. Aerobic plate counts and Enterobacteriaceae counts averaged 7.8 and 6.2 log CFU/100 cm2, respectively, on hides, and 1.4 and 0.4 log CFU/100 cm2, respectively, on chilled carcasses. Aerobic plate counts and Enterobacteriaceae counts on preevisceration carcasses were significantly related to the respective levels on the corresponding hides; the carcasses of animals whose hides carried higher numbers of bacteria were more likely to carry higher numbers of bacteria. Implementation of the sampling protocol described here would allow processors to evaluate the efficacy of on-line antimicrobial interventions and allow industrywide benchmarking of hygienic practices.     

Autonomic QoS control in enterprise Grid environments using online simulation

As Grid Computing increasingly enters the commercial domain, performance and quality of service (QoS) issues are becoming a major concern. The inherent complexity, heterogeneity and dynamics of Grid computing environments pose some challenges in managing their capacity to ensure that QoS requirements are continuously met. In this paper, a comprehensive framework for autonomic QoS control in enterprise Grid environments using online simulation is proposed. This paper presents a novel methodology for designing autonomic QoS-aware resource managers that have the capability to predict the performance of the Grid components they manage and allocate resources in such a way that service level agreements are honored. Support for advanced features such as autonomic workload characterization on-the-fly, dynamic deployment of Grid servers on demand, as well as dynamic system reconfiguration after a server failure is provided. The goal is to make the Grid middleware self-configurable and adaptable to changes in the system environment and workload. The approach is subjected to an extensive experimental evaluation in the context of a real-world Grid environment and its effectiveness, practicality and performance are demonstrated.     

Potential of surface-enhanced Raman spectroscopy for the rapid identification of Escherichia coli and Listeria monocytogenes cultures on silver colloidal nanoparticles

Surface-enhanced Raman (SERS) spectra of various batches of bacteria adsorbed on silver colloidal nanoparticles were collected to explore the potential of the SERS technique for rapid and routine identification of E. coli and L. monocytogenes cultures. Relative standard deviation (RSD) of SERS spectra from silver colloidal suspensions and ratios of SERS peaks from small molecules (K(3)PO(4)) were used to evaluate the reproducibility, stability, and binding effectiveness of citrate-reduced silver colloids over batch and storage processes. The results suggested consistent reproducibility of silver colloids over batch process and also stability and consistent binding effectiveness over an eight-week storage period. A variety of mixtures of E. coli/L. monocytogenes cultures with different colloidal batches revealed that, despite large variations in relative intensities and positions of SERS active bands, characteristic and unique bands at 712 and 390 cm(-1) were consistently observed and were the strongest in E. coli and L. monocytogenes cultures, respectively. Two specific bands were used to develop simple algorithms in the evaluation of binding effectiveness of silver colloids over storage and further to identify E. coli and L. monocytogenes cultures with a 100% success. A single spectrum acquisition took 5 approximately 6 min, and a minimum of 25 microL silver colloid was directly mixed with 25 microL volume of incubated bacterial culture. The short acquisition time and small volume of incubated bacterial culture make silver colloidal nanoparticle based SERS spectroscopy ideal for potential use in the routine and rapid screening of E. coli and L. monocytogenes cultures on large scales. This is the first report of the development of simple and universal algorithms for bacterial identification from the respective exclusive SERS peaks.