Oleksandr A. Byelashov

Control of Listeria monocytogenes on vacuum-packaged frankfurters sprayed with lactic acid alone or in combination with sodium lauryl sulfate.

U.S. regulations require that processors employ lethal or inhibitory antimicrobial alternatives in production of ready-to-eat meat and poultry products that support growth of Listeria monocytogenes and may be exposed to the processing environment after a lethality treatment. In this study, lactic acid (LA; 5%, vol/vol) and sodium lauryl sulfate (SLS; 0.5%, wt/vol) were evaluated individually or as a mixture (LASLS) for control of L. monocytogenes on frankfurters. Frankfurters were inoculated with a 10-strain mixture of L. monocytogenes, sprayed for 10 s (20 bar, 23 +/- 2 degrees C) with antimicrobials or distilled water (DW) before (LASLS or DW) or after (LA, SLS, LASLS, or DW) inoculation (4.8 +/- 0.1 log CFU/cm2), vacuum packaged, and stored at 4 degrees C for 90 days. Samples were analyzed for numbers of the pathogen (on PALCAM agar) and for total microbial counts (on tryptic soy agar with yeast extract) during storage. Spraying with DW, LA, or SLS after inoculation reduced numbers of L. monocytogenes by 1.3 +/- 0.2, 1.8 +/- 0.5, and 2.0 +/- 0.4 log CFU/cm2, respectively. The LASLS mixture applied before or after inoculation reduced pathogen populations by 1.8 +/- 0.4 and 2.8 +/- 0.2 log CFU/cm2, respectively. No further reduction by any treatment was observed during storage. The bacterial growth curves (fitted by the model of Baranyi and Roberts) indicated that the lag-phase duration of the bacterium on control samples (13.85 to 15.18 days) was extended by spraying with all solutions containing LA. For example, LA suppressed growth of L. monocytogenes for 39.14 to 41.01 days. Pathogen growth rates also were lower on frankfurters sprayed after inoculation with LA or LASLS compared to those sprayed with DW. Therefore, spraying frankfurters with a mixture of LA and SLS may be a useful antilisterial alternative treatment for ready-to-eat meat and poultry products.

Reduction of Listeria monocytogenes on frankfurters treated with lactic acid solutions of various temperatures.

United States regulations require ready-to-eat meat and poultry processors to control Listeria monocytogenes using interventions which may include antimicrobials that reduce post-processing contamination by at least 1 log-cycle; if the treatment achieves > or = 2 log reductions, the plant is subject to less frequent microbial testing. Lactic acid (LA) may be useful as a post-lethality intervention and its antimicrobial properties may increase with temperature of application. The aim of this study was to evaluate the effect of LA solution concentration and temperature on L. monocytogenes counts of inoculated frankfurters and to identify parameters (concentration, temperature, and time) that achieve 1 and 2 log-unit immediate reductions. Frankfurters were surface-inoculated with a 10-strain mixture of L. monocytogenes (4.4 +/- 0.1 log CFU/cm(2)) and then immersed in distilled water or LA solutions (0-3%) of 4, 25, 40, or 55 degrees C for 0-120 s. A regression equation for L. monocytogenes reduction included significant (P < 0.05) effects by the terms of concentration, time, temperature, and the interaction of concentration and temperature; other tested parameters (other interactions, quadratic and cubic terms), within the experimental range examined, did not affect (P > or = 0.05) the extent of reduction. Results indicated that the effectiveness of LA against L. monocytogenes, in addition to concentration, increased with solution temperature (in the range of 0.6-2.8 log CFU/cm(2)). The developed equation may allow processors to vary conditions of treatment with LA to achieve a 1 or 2 log-unit reduction of the pathogen and comply with United States regulations.

Fate of Listeria monocytogenes during freezing, thawing and home storage of frankfurters.

Little information is available regarding the fate of Listeria monocytogenes during freezing, thawing and home storage of frankfurters even though recent surveys show that consumers regularly store unopened packages in home freezers. This study examined the effects of antimicrobials, refrigerated storage, freezing, thawing method, and post-thawing storage (7 degrees C) on L. monocytogenes on frankfurters. Inoculated (2.1 log CFU/cm(2)) frankfurters formulated without (control) or with antimicrobials (1.5% potassium lactate plus 0.1% sodium diacetate) were vacuum-packaged, stored at 4 degrees C for 6 or 30 d and then frozen (-15 degrees C) for 10, 30, or 50 d. Packages were thawed under refrigeration (7 degrees C, 24 h), on a countertop (23 +/- 2 degrees C, 8 h), or in a microwave oven (2450 MHz, 1100 watts, 220 s followed by 120 s holding), and then stored aerobically (7 degrees C) for 14 d. Bacterial populations were enumerated on PALCAM agar and tryptic soy agar plus 0.6% yeast extract. Antimicrobials completely inhibited (p < 0.05) growth of L. monocytogenes at 4 degrees C for 30 d under vacuum-packaged conditions, and during post-thawing aerobic storage at 7 degrees C for 14 d. Different intervals between inoculation and freezing (6 or 30 d) resulted in different pathogen levels on control frankfurters (2.1 or 3.9 log CFU/cm(2), respectively), while freezing reduced counts by <1.0 log CFU/cm(2). Thawing treatments had little effect on L. monocytogenes populations (<0.5 log CFU/cm(2)), and post-thawing fate of L. monocytogenes was not influenced by freezing or by thawing method. Pathogen counts on control samples increased by 1.5 log CFU/cm(2) at d-7 of aerobic storage, and reached 5.6 log CFU/cm(2) at d-14. As indicated by these results, consumers should freeze frankfurters immediately after purchase, and discard frankfurters formulated without antimicrobials within 3 d of thawing and/or opening.

Evaluation of Brining Ingredients and Antimicrobials for Effects on Thermal Destruction of Escherichia coli O157:H7 in a Meat Model System

Escherichia coli O157:H7 may become internalized during brine injection of meat. This study evaluated the effect of brining ingredients on E. coli O157:H7 in a meat model system after simulated brining, storage, and cooking. Fresh knuckles (5.3 +/- 2.4% fat) or beef shoulder (15.3 +/- 2.2% fat) were ground individually, mixed with an 8-strain composite of rifampicin-resistant E. coli O157:H7 (7 log CFU/g) and brining solutions. Treatments included no brining, distilled water, sodium chloride (NaCl, 0.5%), sodium tripolyphosphate (STP, 0.25%), sodium pyrophosphate (SPP, 0.25%), NaCl + STP, NaCl + SPP, NaCl + STP + potassium lactate (PL, 2%), NaCl + STP + sodium diacetate (SD, 0.15%), NaCl + STP + PL + SD, NaCl + STP + lactic acid (0.3%), NaCl + STP + acetic acid (0.3%), NaCl + STP + citric acid (0.3%), NaCl + STP + EDTA (20 mM) + nisin (0.0015%) or pediocin (1000 AU/g), NaCl + STP + sodium metasilicate (0.2%), NaCl + STP + cetylpyridinium chloride (CPC; 0.5%), and NaCl + STP + hops beta acids (0.00055%). Samples (30 g) were analyzed for pH, and total microbial and rifampicin-resistant E. coli O157:H7 (inoculum) populations immediately after mixing, storage (24 h at 4 degrees C), and cooking to 65 degrees C. Fat and moisture contents and water activity were measured after storage and cooking only; cooking losses also were determined. The effect of beef type on microbial counts, pH, and water activity was negligible. No reductions in microbial counts were obtained by the brining solutions immediately or after storage, except for samples treated with CPC, which reduced (P < 0.05) pathogen counts after storage by approximately 1 log cycle. Cooking reduced pathogen counts by 1.5 to 2.5 logs, while CPC-treated samples had the lowest (P < 0.05) counts compared to any other treatment. These data may be useful in developing/improving brining recipes for control of E. coli O157:H7 in moisture-enhanced beef products.

Fate of post-processing inoculated Listeria monocytogenes on vacuum-packaged pepperoni stored at 4, 12 or 25 °C

If present, Listeria monocytogenes may not be eliminated during processing of pepperoni or may be introduced during peeling, slicing, or packaging. We evaluated the fate of the pathogen on sliced inoculated pepperoni during vacuum-packaged storage, and potential differences in survival among three types of inocula, including nonacid-adapted, acid-adapted and pepperoni extract-habituated cultures. Commercial pepperoni (two replicates, three samples per treatment) was sliced and inoculated (3 to 4 log CFU/cm(2)), before vacuum-packaging and storage for up to 180 days at 4, 12 or 25 degrees C. Samples were periodically analyzed for pathogen counts (PALCAM agar) and total bacterial counts (tryptic soy agar with 0.6% yeast extract). The pH of the product was relatively stable (4.50-4.81) throughout storage. Overall, levels of the pathogen (all inocula) and total counts decreased continuously during storage at all temperatures. The pathogen died slower at 4 degrees C than at 12 and 25 degrees C, while at 12 and 25 degrees C the death rates were similar. Death rates depended on type of inoculum and generally decreased in the order: acid-adapted, extract-habituated and nonacid-adapted inoculum. At day 60, pathogen levels were below the detection limit and remained undetectable throughout the rest of the 180-day storage period, regardless of inoculum type and storage temperature. Therefore, storage of sliced vacuum-packaged pepperoni, especially at ambient temperature, prior to consumption may reduce the potential risk of listeriosis.

Survival of Escherichia coli O157:H7 in Meat Product Brines Containing Antimicrobials

UNLABELLED Brine solution injection of beef contaminated with Escherichia coli O157:H7 on its surface may lead to internalization of pathogen cells and/or cross-contamination of the brine, which when recirculated, may serve as a source of new product contamination. This study evaluated survival of E. coli O157:H7 in brines formulated without or with antimicrobials. The brines were formulated in sterile distilled water (simulating the composition of freshly prepared brines) or in a nonsterile 3% meat homogenate (simulating the composition of recirculating brines) at concentrations used to moisture-enhance meat to 110% of initial weight, as follows: sodium chloride (NaCl, 5.5%) + sodium tripolyphosphate (STP, 2.75%), NaCl + sodium pyrophosphate (2.75%), or NaCl + STP combined with potassium lactate (PL, 22%), sodium diacetate (SD, 1.65%), PL + SD, lactic acid (3.3%), acetic acid (3.3%), citric acid (3.3%), nisin (0.0165%) + ethylenediamine tetraacetic acid (EDTA, 200 mM), pediocin (11000 AU/mL) + EDTA, sodium metasilicate (2.2%), cetylpyridinium chloride (CPC, 5.5%), or hops beta acids (0.0055%). The brines were inoculated (3 to 4 log CFU/mL) with rifampicin-resistant E. coli O157:H7 (8-strain composite) and stored at 4 or 15 °C (24 to 48 h). Immediate (0 h) pathogen reductions (P < 0.05) of 1.8 to ≥ 2.4 log CFU/mL were observed in brines containing CPC or sodium metasilicate. Furthermore, brines formulated with lactic acid, acetic acid, citric acid, nisin + EDTA, pediocin + EDTA, CPC, sodium metasilicate, or hops beta acids had reductions (P < 0.05) in pathogen levels during storage; however, the extent of pathogen reduction (0.4 to > 2.4 log CFU/mL) depended on the antimicrobial, brine type, and storage temperature and time. These data should be useful in development or improvement of brine formulations for control of E. coli O157:H7 in moisture-enhanced meat products. PRACTICAL APPLICATION Results of this study should be useful to the meat industry for developing or modifying brine formulations to reduce the risk of E. coli O157:H7 in moisture-enhanced meat products.

Characterization and Transferability of Class 1 Integrons in Commensal Bacteria Isolated from Farm and Nonfarm Environments

This study assessed the distribution of class 1 integrons in commensal bacteria isolated from agricultural and nonfarm environments, and the transferability of class 1 integrons to pathogenic bacteria. A total of 26 class 1 integron-positive isolates were detected in fecal samples from cattle operations and a city park, water samples from a beef ranch and city lakes, and soil, feed (unused), manure, and compost samples from a dairy farm. Antimicrobial susceptibility testing of class 1 integron-positive Enterobacteriaceae isolates from city locations displayed multi-resistance to 12-13 out of the 22 antibiotics tested, whereas class 1 integron-positive Enterobacteriaceae isolates from cattle operations only displayed tetracycline resistance. Most class 1 integrons had one gene cassette belonging to the aadA family that confers resistance to streptomycin and spectinomycin. One isolate from a dog fecal sample collected from a city dog park transferred its class 1 integron to a strain of Escherichia coli O157:H7 at a frequency of 10(-7) transconjugants/donor by in vitro filter mating experiments under the stated laboratory conditions. Due to the numerous factors that may affect the transferability testing, further investigation using different methodologies may be helpful to reveal the transferability of the integrons from other isolates. The presence of class 1 integrons among diverse commensal bacteria from agricultural and nonfarm environments strengthens the possible role of environmental commensals in serving as reservoirs of antibiotic resistance genes.

Evaluation of changes in Listeria monocytogenes populations on frankfurters at different stages from manufacturing to consumption.

This study evaluated the fate of inoculated Listeria monocytogenes on frankfurters stored under conditions simulating those that may be encountered between manufacturing and consumption. Frankfurters with or without 1.5% potassium lactate and 0.1% sodium diacetate (PL/SD) were inoculated (1.8 +/- 0.1 log CFU/cm(2)) with a 10-strain composite of L. monocytogenes, vacuum-packaged, and stored under conditions simulating predistribution storage (24 h, 4 degrees C), temperature abuse during transportation (7 h, 7 degrees C followed by 7 h, 12 degrees C), and storage before purchase (60 d, 4 degrees C; SBP). At 0, 20, 40, and 60 d of SBP, samples were exposed to conditions simulating delivery from stores to homes or food establishments (3 h, 23 degrees C), and then opened or held vacuum-packaged at 4 or 7 degrees C for 14 d (SHF). Pathogen counts remained relatively constant on frankfurters with PL/SD regardless of product age and storage conditions; however, they increased on product without antimicrobials. In vacuum-packaged samples, during SHF at 4 degrees C, the pathogen grew faster (P < 0.05) on older product (20 d of SBP) compared to product that was fresh (0 d of SBP); a similar trend was observed in opened packages. At 7 degrees C, the fastest growth (0.35 +/- 0.02 log CFU/cm(2)/d) was observed on fresh product in opened packages; in vacuum-packages, growth rates on fresh and aged products were similar. By day 40 of SBP the pathogen reached high numbers and increased slowly or remained unchanged during SHF. This information may be valuable in L. monocytogenes risk assessments and in development of guidelines for storage of frankfurters between package opening and product consumption.