Comparison of Commercial Test Kits for Detection of Salmonella and E. coli O157:H7 in Alfalfa Spent Sprout Irrigation Water.

BACKGROUND: Sprout growers in the United States are required to test spent sprout irrigation water (SSIW) or in-process sprouts for Escherichia coli O157:H7 and Salmonella species. Pathogen screening kits are commercially available; however, few have been validated for analysis of sprouts or SSIW. OBJECTIVE: This study evaluated AOAC-certified test kits (lateral flow devices [LFDs], enzyme immunoassays [EIAs], and molecular assays) in comparison with culture methods described in the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for detection of Salmonella and E. coli O157:H7 in alfalfa SSIW. METHOD: Twenty-five milliliter aliquots of alfalfa SSIW, either uninoculated or inoculated with Salmonella or E. coli O157:H7 at a low (∼0.5-0.7 CFU/25 mL) or high level (∼10-20 CFU/25 mL), were subjected to the enrichment and assay protocols recommended by each test. Pathogen presence was confirmed following FDA BAM procedures and, if applicable, test kit manufacturer protocols. RESULTS: Twelve of the 13 Salmonella test kits evaluated (except VIDAS UP) performed well and detected Salmonella in 100% of SSIW samples contaminated at 0.61 CFU/mL. Performance varied among E. coli O157:H7 test kits, with four (Reveal, MicroSEQ, GDS, MDA) of 12 kits designed for next-day detection, and four (Reveal, VIP Gold, MicroSEQ, GDS) of seven kits designed for same-day detection capable of detecting the pathogen in 100% samples contaminated at 0.90 CFU/mL. CONCLUSIONS: Enrichment conditions play a key role in determining the performance of test kits and the success of confirmation. HIGHLIGHTS: This study is the first to compare a wide range of commercial test kits for detection of Salmonella and E. coli O157:H7 in SSIW.

Evaluation of Inactivating Norovirus, Hepatitis A, and Listeria monocytogenes on Raspberries by Sanitizer Spray.

HIGHLIGHTS: Chlorine and PAA spray reduced MNV and L. monocytogenes from raspberries by <1.0 log. Residual PAA on raspberries further reduced MNV and Listeria during postspray frozen storage. PAA decayed more slowly than active chlorine on raspberry surfaces. The data suggest that PAA could aid in risk reduction of pathogens on raspberries.

Impact of thermal processing on ELISA detection of peanut allergens.

This study examined the effect of heat treatment on the solubility of peanut proteins and compared the performances of two commercial ELISA kits (Veratox Quantitative Peanut Allergen Test and BioKits Peanut Assay Kit) for quantitation of peanut residues as affected by different heat treatments (moist and dry heat) and detection targets (mixture of proteins vs specific protein). Both laboratory-prepared and commercial peanut flour preparations were used for the evaluation. The two ELISA kits tended to underestimate the levels of protein in samples that were subjected to elevated heat, respectively, by more than 60- or 400-fold lower for the autoclaved samples and by as much as 70- or 2000-fold lower for the dark-roast commercial flour samples. The BioKits test, which employs antibodies specific to a heat labile protein (Ara h 1), in general exhibited a greater degree of underestimation. These results suggest that commercial ELISA kits may not be able to accurately determine the amount of proteins present in thermally processed foods due to changes in the solubility and immunoreactivity of the target proteins. Users need to be aware of such limitations before applying ELISA kits for evaluation of food allergen control programs.

Evaluation of PCR detection of Salmonella in alfalfa sprouts and spent irrigation water collected during sprouting of naturally contaminated seed.

This study evaluated the efficacy of a PCR-based system (DuPont Qualicon BAX) for detection of Salmonella in sprouts and spent irrigation water collected during sprouting of seeds naturally contaminated with Salmonella. Alfalfa seeds were grown in Mason jars at 20 and 30°C for 3 days. Levels of Salmonella present in the water and sprouts were determined by most-probable-number (MPN) analysis. Background microflora levels were also determined. Samples of spent irrigation water and sprouts were enriched overnight individually in tetrathionate broth and in buffered peptone water with novobiocin at 42°C and then run in the BAX system. Samples were also enriched according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA BAM) method for Salmonella as a comparison. Salmonella levels were lower at 20°C compared with 30°C for some trials, and background microflora levels ranged from 10(7) to 10(8) CFU/g or ml at 20°C and 10(8) to 10(9) CFU/g or ml at 30°C. In trials with a Salmonella level >1.1 MPN/g or ml, both the BAX and FDA BAM methods were able to detect Salmonella in all samples. In trials with lower levels (0.21 MPN/g or ml or lower) of Salmonella, BAX was able to detect more positive samples than FDA BAM. For one trial with <0.003 MPN/g or ml of Salmonella, the presence of the pathogen was not indicated by either the BAX or the FDA BAM method. The results suggest that PCR detected low levels of Salmonella in sprouts or spent irrigation water collected from sprouting of naturally contaminated seeds.

Effect of heat treatment on the quantitative detection of egg protein residues by commercial enzyme-linked immunosorbent assay test kits.

This study examined the changes in the solubility of egg proteins as affected by different heat treatments and compared the performances of three commercial test kits for the quantitation of protein residues in heat-treated samples. National Institute of Standards and Technology (NIST) whole egg standard reference material #8415 and Henningsen spray-dried whole egg powder were subjected to heating in the presence of water at 60 and 100 degrees C, autoclaving for 5 or 10 min, or dry heating at 60-400 degrees C for 10 min. The amount of protein in the heated samples was assayed using the bicinchoninic acid total protein assay as well as egg-specific commercial enzyme-linked immunosorbent assay (ELISA) kits. Elevated heat resulted in a lower level of proteins extracted. Neogen's Veratox kit, which is reactive to multiple proteins in egg, greatly underestimated the amount of residual proteins in the boiled or autoclaved samples. Tepnel BioSystems' Biokits assay, which employs antibodies specific to a heat-stable marker protein (ovomucoid), registered a higher level of protein in these samples. Both test kits substantially underestimated the amount of residual proteins in samples dry-heated at temperatures >176 degrees C. The Morinaga test, using an improved extraction buffer, registered the highest level of protein in the heat-treated NIST samples but not the Henningsen samples. The underestimation by the commercial test kits was attributed to changes in the immunoreactivity of residual proteins after heat treatments and not the differences in the amount of protein extracted. These results suggest that thermal processing may affect the quantitative analysis of allergens and needs to be taken into account in the validation of commercial ELISA test kits.

Sodium lactate, sodium diacetate and pediocin: Effects and interactions on the thermal inactivation of Listeria monocytogenes on bologna.

The effects and interactions of temperature (56.3-60 degrees C), sodium lactate (SL; 0-4.8%), sodium diacetate (SD; 0-0.25%) and pediocin (0-10,000 AU) on Listeria monocytogenes on bologna were studied and a predictive inactivation model was developed. Bologna was manufactured with different SL/SD concentrations in the formulation, dipped in pediocin solution and treated at different temperatures using combinations of parameters determined by central composite design. D-values were calculated and analyzed using second order response regression. Predicted D-values were also calculated. The observed D-values for L. monocytogenes on bologna ranged from 2.10 to 35.59 min. Temperature alone decreased predicted D-values from 99.02 min at 56.3 degrees C to 44.71 min at 60.0 degrees C. Adding SL decreased D-values (85.43-22.71 min) further; however, heat and SD combined was the most effective for reducing L. monocytogenes on bologna. An SD level of 0.25% at 58.2 degrees C had the overall lowest predicted D-value (15.95 min). Combination treatments increased or decreased D-values, depending on the temperature. Pediocin (2500 and 5000 AU) and heat decreased D-values, but exhibited a protective effect at higher concentrations (>or=7500 AU). The results showed that interactions between additives in formulations can vary at different temperatures/concentrations, thereby affecting thermal inactivation of foodborne pathogens in meat products. Hence, food processors should modify food formulations carefully, and verify with adequate testing so that product safety is not compromised.

Minimum leak size determination, under laboratory and commercial conditions, for bacterial entry into polymeric trays used for shelf-stable food packaging.

This study sought to determine the minimum leak size for entry of Enterobacter aerogenes under laboratory conditions, and normal flora under commercial conditions, into tryptic soy broth with yeast extract (TSBYE), homestyle chicken, and beef enchilada packaged in 355-ml polyethylene terephthalate/ethylene vinyl alcohol/polypropylene trays. Channel leaks (diameters of 50 to 200 microm) were made across the sealing area of the trays. Pinholes (diameters of 5 to 50 microm) were made by imbedding laser-drilled metal and plastic disks into the tray lids. For the laboratory simulation, all trays were submerged and agitated for 30 min at 25 degrees C in phosphate-buffered saline that contained 10(7) CFU/ml of E. aerogenes. Under commercial conditions, trays with channel leaks were processed in retorts to achieve commercial sterility. All trays were subsequently incubated at 37 degrees C for 2 weeks, and their contents plated onto eosin-methylene blue agar (for laboratory simulation) to enumerate E. aerogenes and brain heart infusion agar (for commercial conditions) to determine the presence of any bacteria. Under laboratory conditions, minimum pinhole sizes for E. aerogenes entry approximated 5 microm (TSBYE, metal disks; homestyle chicken, plastic disks), 20 microm (beef, plastic disks), and 30 microm (beef, metal disks). The minimum channel leak sizes for entry of E. aerogenes approximated 10 microm (TSBYE), 70 microm (chicken), and 200 microm (beef enchilada). Under commercial conditions, the minimum channel leak size for bacterial entry approximated 40 microm (TSBYE), 50 microm (homestyle chicken), and more than 200 microm (beef). Results showed that E. aerogenes can enter pinholes as small as 5 microm under a worst-case scenario. This information can be used to set pass and fail parameters for leak detection devices.

Control of Listeria monocytogenes with combined antimicrobials on beef franks stored at 4 degrees C.

Contamination of ready-to-eat meat products such as beef franks with Listeria monocytogenes has become a major concern for the meat processing industry and an important food safety issue. The objective of this study was to determine the effectiveness of combinations of antimicrobials as aqueous dipping solutions to control L. monocytogenes on vacuum-packaged beef franks stored at 4 degrees C for 3 weeks. Commercial beef franks were dipped for 5 min in three antimicrobial solutions: pediocin (6,000 AU), 3% sodium diacetate and 6% sodium lactate combined, and a combination of the three antimicrobials. Samples were then inoculated with 10(7) CFU/g of either four L. monocytogenes strains individually or a cocktail of the four strains, vacuum packaged, and stored at 4 degrees C for 3 weeks. Sampling was carried out at day 0 and after 2 and 3 weeks of storage. Individual strains, as well as the cocktail, exhibited different responses to the antimicrobial treatments. After 2 and 3 weeks of storage at 4 degrees C, pediocin-treated beef franks showed a less than 1-log reduction for all bacterial strains. Samples treated with the sodium diacetate-sodium lactate combination showed about a 1-log reduction after 2 weeks of storage for all strains and between a 1- and 2-log reduction after 3 weeks of storage, depending on the bacterial strain. When the three antimicrobials were combined, reductions ranged between 1 and 1.5 log units and 1.5 to 2.5 log units after 2 and 3 weeks of storage, respectively, at 4 degrees C. These results indicate that the use of combined antimicrobial solutions for dipping treatments is more effective at inhibiting L. monocytogenes than treatments using antimicrobials such as pediocin separately.

Regulation of neurotoxin complex expression in Clostridium botulinum strains 62A, Hall A-hyper, and NCTC 2916.

The kinetics of botulinum toxin gene expression have been investigated in Clostridium botulinum type A strains 62A, Hall A-hyper, and type A(B) strain NCTC 2916 during the growth cycle. The analyses were performed in TPGY and type A Toxin Production Media (TPM). The mRNA transcript levels encoding the proteins of the neurotoxin complex were determined using Northern analyses. Neurotoxin concentrations in culture supernatants and lysed cell pellets were assayed using ELISA, Western blots, and mouse bioassay. Proteolytic activation of botulinum neurotoxin during the growth cycle was evaluated by Western blots. For all three strains, mRNA transcripts for the toxin complex genes were initially detected in early log phase, reached peak levels in early stationary phase, and rapidly decreased in mid-to-late stationary phase and during lysis. Toxin expression varied depending on the strain and growth medium. Toxin production was highest in strain Hall A-hyper, followed by NCTC 2916 and 62A. For C. botulinum strain Hall A-hyper, cell lysis and toxin release into the supernatant occurred rapidly for cells grown in TPM, while cells grown in TPGY remained in stationary phase with minimal lysis and toxin release through 96 h of growth. In contrast, strains 62A and NCTC 2916 lysed more extensively than Hall A-hyper in TPGY. TPM supported higher toxin production and activation than TPGY in strains 62A and Hall A-hyper. These data support that the genes of the botulinum neurotoxin complex are temporally expressed during late-log and early stationary phase and that toxin complex formation depends on the strain and growth medium. Botulinum toxin synthesis and activation appears to be a complex process that is highly regulated by nutritional and environmental conditions. Further research is needed to elucidate the sensing mechanisms and genetic regulatory factors controlling these processes.