Low salt exposure results in inactivation of Toxoplasma gondii bradyzoites during formulation of dry cured ready-to-eat pork sausage.

The production of safe and healthy food products represents one of the main objectives of the food industry. The presence of microorganisms in meat and products containing meat can result in a range of human health problems, as well as economic losses to producers of these products. However, contaminated meat products continue to initiate serious and large-scale outbreaks of disease in consumers. In addition to outbreaks of diseases caused by bacteria and viruses, parasitic organisms, such as Toxoplasma gondii, are responsible for foodborne infections worldwide, and in the case of T. gondii, is considered the 2nd leading cause of death from foodborne illness in the U.S. Transmission of Toxoplasma gondii has historically been linked to the consumption of raw or undercooked meat products, including pork. Specific concerns with respect to pork products are ready-to-eat (RTE) pork meals. These are pork or products containing pork that are prepared by curing or drying, and are not intended to be cooked before being consumed. Previous studies have demonstrated that T. gondii is inactivated during dry cured sausage preparation, apparently in the batter during fermentation. In this study, we have analyzed timing of inactivation of T. gondii in freshly prepared pepperoni batter to confirm our previous findings, to determine how quickly inactivation occurs during fermentation, and to confirm what parameters of the sausage preparation are involved in inactivation of the parasite. Results from the current and previous study indicate that rapid inactivation of T. gondii bradyzoites occurs in low salt batter for dry cured sausage within 4 h of initiation of fermentation.

Rapid inactivation of Toxoplasma gondii bradyzoites during formulation of dry cured ready-to-eat pork sausage.

Curing processes for pork meat in the U.S. currently require individual validation of methods to demonstrate inactivation of Trichinella spiralis, a nematode parasite historically associated with pork. However, for protozoan parasites, no such strictures exist. It has been assumed, with little evidence, that curing processes required to inactivate Trichinella also inactivate Toxoplasma gondii. Currently no model of meat chemistry exists that can be correlated with inactivation of T. gondii. Given the possibility of the presence of T. gondii in pork meat, and the frequent use of pork for ready-to-eat (RTE) products not intended to be cooked, curing methods which inactivate T. gondii early in the curing process would be of great value to producers. In this study, we tested the effect of five variables - salt/brine concentration, water activity (aw), pH, temperature, and time on inactivation of T. gondii bradyzoites in tissue cysts using low and high endpoints for common curing treatments during preparation of dry cured pork sausage. Survival of T. gondii bradyzoites at each stage of preparation was assessed using a mouse bioassay. Results indicated that encysted T. gondii bradyzoites do not survive the early stages of the dry curing process within the endpoint parameters tested here, even at levels of NaCl that are lower than typically used for dry curing (1.3%). Exposure of T. gondii encysted bradyzoites to curing components in the formulated batter resulted in rapid inactivation of bradyzoites. These data suggest that the use of dry curing components may be effective for controlling T. gondii potentially transmitted through RTE meats, rendering them safe from risk with respect to T. gondii transmission to human consumers.

Curing conditions to inactivate Trichinella spiralis muscle larvae in ready-to-eat pork sausage.

Curing processes are one method by which pork products, which are considered ready to eat (RTE) and have not been otherwise tested or treated, can be rendered safe from risk for exposure to Trichinella muscle larvae (ML). Curing processes in the U.S. currently require individual validation of methods to demonstrate inactivation of Trichinella. This is a major undertaking for each process; currently no model of meat chemistry exists that can be correlated with inactivation of Trichinella. Given the potential for new RTE products (e.g., lower salt), the availability of a wider range of tested methods for inactivation of Trichinella in pork would be of substantial value to the industry. In this study, five variables were tested - salt/brine concentration, water activity (aw), pH, temperature, and time, using low and high endpoints for common curing treatments for dry cured pork sausage. The data demonstrated that NaCl concentrations above 1.3%, in combination with fermentation to pH 5.2 or below, resulted in inactivation of > 96% of Trichinella ML in stuffed sausages within 24-28 h. All ML were inactivated by 7-10 days post-stuffing. These curing processes reliably predict inactivation of Trichinella spiralis, and can be used within the defined upper and lower endpoint parameters to reduce or eliminate the need for individual product validation.

Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco.

The effect of high-hydrostatic-pressure processing (HPP) on the survival of a 5-strain rifampicin-resistant cocktail of Listeria monocytogenes in Queso Fresco (QF) was evaluated as a postpackaging intervention. Queso Fresco was made using pasteurized, homogenized milk, and was starter-free and not pressed. In phase 1, QF slices (12.7 × 7.6 × 1 cm), weighing from 52 to 66 g, were surface inoculated with L. monocytogenes (ca. 5.0 log10 cfu/g) and individually double vacuum packaged. The slices were then warmed to either 20 or 40°C and HPP treated at 200, 400, and 600 MPa for hold times of 5, 10, 15, or 20 min. Treatment at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log10 cfu/g at all hold times and temperatures. High-hydrostatic-pressure processing at 40°C, 400 MPa, and hold time ≥ 15 min was effective but resulted in wheying-off and textural changes. In phase 2, L. monocytogenes was inoculated either on the slices (ca. 5.0 log10 cfu/g; ON) or in the curds (ca. 7.0 log10 cfu/g; IN) before the cheese block was formed and sliced. The slices were treated at 20°C and 600 MPa at hold times of 3, 10, and 20 min, and then stored at 4 and 10°C for 60 d. For both treatments, L. monocytogenes became less resistant to pressure as hold time increased, with greater percentages of injured cells at 3 and 10 min than at 20 min, at which the lethality of the process increased. For the IN treatment, with hold times of 3 and 10 min, growth of L. monocytogenes increased the first week of storage, but was delayed for 1 wk, with a hold time of 20 min. Longer lag times in growth of L. monocytogenes during storage at 4°C were observed for the ON treatment at hold times of 10 and 20 min, indicating that the IN treatment may have provided a more protective environment with less injury to the cells than the ON treatment. Similarly, HPP treatment for 10 min followed by storage at 4°C was the best method for suppressing the growth of the endogenous microflora with bacterial counts remaining below the level of detection for 2 out of the 3 QF samples for up to 84 d. Lag times in growth were not observed during storage of QF at 10°C. Although HPP reduced L. monocytogenes immediately after processing, a second preservation technique is necessary to control growth of L. monocytogenes during cold storage. However, the results also showed that HPP would be effective for slowing the growth of microorganisms that can shorten the shelf life of QF.

Thermal inactivation of a single strain each of serotype O26:H11, O45:H2, O103:H2, O104:H4, O111:H⁻, O121:H19, O145:NM, and O157:H7 cells of Shiga toxin-producing Escherichia coli in wafers of ground beef.

For each of two trials, freshly ground beef of variable fat content (higher: 70:30 %lean:%fat; lower: 93:7 %lean:%fat) was separately inoculated with ca. 7.0 log CFU/g of a single strain of Escherichia coli serotypes O26:H11, O45:H2, O103:H2, O104:H4, O111:H⁻, O121:H19, O145:NM, and O157:H7. Next, ca. 3-g samples of inoculated beef were transferred into sterile filter bags and then flattened (ca. 1.0 mm thick) and vacuum sealed. For each temperature and sampling time, three bags of the inoculated wafers of beef were submerged in a thermostatically controlled water bath and heated to an internal temperature of 54.4°C (130°F) for up to 90 min, to 60°C (140°F) for up to 4 min, or to 65.6°C (150°F) for up to 0.26 min. In lower fat wafers, D-values ranged from 13.5 to 23.6 min, 0.6 to 1.2 min, and 0.05 to 0.08 min at 54.4, 60.0, and 65.6°C, respectively. Heating higher fat wafers to 54.4, 60.0, and 65.6°C generated D-values of 18.7 to 32.6, 0.7 to 1.1, and 0.05 to 0.2 min, respectively. In addition, we observed reductions of ca. 0.7 to 6.7 log CFU/g at 54.4°C after 90 min, ca. 1.1 to 6.1 log CFU/g at 60.0°C after 4 min, and 0.8 to 5.8 log CFU/g at 65.6°C after 0.26 min. Thus, cooking times and temperatures effective for inactivating a serotype O157:H7 strain of E. coli in ground beef were equally effective against the seven non-O157:H7 Shiga toxin-producing strains investigated herein.

Protection of probiotic bacteria in a synbiotic matrix following aerobic storage at 4 °C.

The survival of single strains of Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus reuteri was investigated in synbiotics that included 10 mg/ml of fructo-oligosaccharides, inulin and pectic-oligosaccharides in an alginate matrix under refrigerated (4 °C) aerobic storage conditions. When the matrices were cross-linked with calcium (45 mM), 102-103 cfu/ml of L. acidophilus and L. reuteri, and 0-103 cfu/ml of B. breve and B. longum survived refrigerated aerobic storage for 28 days. Following refrigerated storage, acetic (3-9 mM), butyric (0-2 mM), propionic (5-16 mM) and lactic acids (1-48 mM) were produced during the growth of probiotics in BHI broth at 37 °C, suggesting their metabolic activity after storage was stressed. When calcium cross-linking was not used in synbiotics, the matrix remained more gel-like after inoculation when compared to the calcium cross-linked matrix. At least 107 cfu/ml of probiotic bacteria survived after 21 days of storage within these gel-like alginate matrices. Significantly higher levels of B. breve, L. acidophilus and L. reuteri were obtained from the synbiotic matrices supplemented with fructo-oligosaccharides, inulin and pectic-oligosaccharides compared to alginate alone. B. longum survival was the same (~7 logs) in all gel-like synbiotic matrices. These results show that synbiotics protected probiotic bacteria and extended their shelf-life under refrigerated aerobic conditions. Synbiotics represent a viable delivery vehicle for health-promoting bacteria.

Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk.

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 µm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-µm ceramic membrane, followed by pasteurization (72°C, 18.6s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log(10) BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 µm or 1.4 µm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-µm membrane removed 5.91±0.05 log(10) BA spores/mL of milk and the 1.4-µm membrane removed 4.50±0.35 log(10) BA spores/mL of milk. The 0.8-µm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-µm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log(10) spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log(10) BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-µm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization.

Validation of food-grade salts of organic acids as ingredients to control Listeria monocytogenes on pork scrapple during extended refrigerated storage.

This study was conducted to investigate control of Listeria monocytogenes on pork scrapple during storage at 4°C. In phase I, scrapple was formulated, with or without citrate-diacetate (0.64%), by a commercial processor to contain various solutions or blends of the following antimicrobials: (i) lactate-diacetate (3.0 or 4.0%), (ii) lactate-diacetate-propionate (2.0 or 2.5%), and (iii) levulinate (2.0 or 2.5%). Regardless of whether citrate-diacetate was included in the formulation, without the subsequent addition of the targeted antimicrobials pathogen levels increased ca. 6.4 log CFU/g within the 50-day storage period. In the absence of citrate-diacetate but when the targeted antimicrobials were included in the formulation, pathogen numbers increased by ca. 1.3 to 5.2 log CFU/g, whereas when citrate-diacetate was included with these antimicrobials, pathogen numbers increased only by ca. 0.7 to 2.3 log CFU/g. In phase II, in the absence of citrate-diacetate, when the pH of the lactate-diacetate-propionate blend (2.5%) was adjusted to pH 5.0 or 5.5 pathogen numbers remained unchanged (≤0.5 log CFU/g increase) over 50 days, whereas when citrate-diacetate was included with the lactate-diacetate-propionate blend adjusted to pH 5.0 or 5.5, pathogen numbers decreased by 0.3 to 0.8 log CFU/g. In phase III, when lower concentrations of the lactate-diacetate-propionate blend (1.5 or 1.94%) were adjusted to pH 5.5, pathogen numbers increased by ca. 6.0 and 4.7 log CFU/g, respectively, whereas when the mixture was adjusted to pH 5.0, pathogen numbers increased by ≤0.62 log CFU/g. Thus, scrapple formulated with lactate-diacetate-propionate (1.5 and 1.94% at pH 5.0) is an unfavorable environment for outgrowth of L. monocytogenes.

Control of Listeria monocytogenes on commercially-produced frankfurters prepared with and without potassium lactate and sodium diacetate and surface treated with lauric arginate using the Sprayed Lethality in Container (SLIC(R)) delivery method.

Viability of Listeriamonocytogenes was monitored on frankfurters formulated with or without potassium lactate and sodium diacetate at a ratio of ca. 7:1 and treated with lauric arginate (LAE; 22 or 44ppm) using the Sprayed Lethality in Container (SLIC(R)) delivery method. Without antimicrobials, pathogen numbers remained relatively constant at ca. 3.3logCFU/package for ca. 30d, but then increased to ca. 8.4logCFU/package over 120d. Regardless of whether or not lactate and diacetate were included, when treated with LAE, pathogen numbers decreased from ca. 3.3logCFU/package to ca. 1.5logCFU/package within 2h, but then increased to 7.3 and 6.7logCFU/package, respectively, after 120d. When frankfurters were formulated with lactate and diacetate and treated with LAE, pathogen numbers decreased by ca. 2.0logCFU/package within 2h and remained relatively unchanged over the 120d. These data confirm that LAE provides an initial lethality towards L. monocytogenes and when used in combination with reduced levels/ratio of lactate and diacetate as an ingredient for frankfurters provides inhibition throughout shelf life.

Effect of storage and subsequent reheating on viability of Listeria monocytogenes on pork scrapple.

We evaluated the fate of Listeria monocytogenes on commercial pork scrapple, a regionally popular, ready-to-eat (RTE) meat. We also conducted an informal survey to address consumer practices for storing and reheating scrapple. Of the 129 consumers who responded to at least one of the eight questions posed in the survey, about half (46.4%; 52 of 112) considered scrapple RTE, the majority (69.7%; 76 of 109) stored it in the refrigerator, and all (100%; 112 of 112) preferred to reheat it prior to consumption. Most respondents (83.9%; 94 of 112) reheated the scrapple by pan frying for 1 to 10 min at medium to high temperature. To study pathogen behavior, slices of pork scrapple were surface inoculated with a five-strain cocktail of L. monocytogenes (ca. 2.0 log CFU/g), vacuum sealed, and stored for up to 60 days. Pathogen levels increased to 8.9, 9.5, and 9.9 log CFU/g after 44 (4 degrees C), 21 (10 degrees C), and 5 (21 degrees C) days, respectively. When slices 1.3 cm (ca. 55 g) and 1.9 cm (ca. 85 g) thick were surface inoculated with L. monocytogenes (ca. 7.0 log CFU/g) and then reheated in a skillet (191 degrees C) for 0.5 to 4 min per side or to target instantaneous internal temperatures of 48.9 to 71.1 degrees C, it was possible to achieve pathogen reductions ranging from ca. 2.2 to 6.5 log CFU/g. These data confirm that in the unlikely event of postprocessing contamination of pork scrapple by L. monocytogenes, proper reheating can appreciably reduce levels of the pathogen before consumption.

Effectiveness of cross-flow microfiltration for removal of microorganisms associated with unpasteurized liquid egg white from process plant.

Thermal preservation is used by the egg industry to ensure the microbiological safety of liquid egg white (LEW); however, it does not eliminate all microorganisms and impairs some of the delicate functional properties of LEW. In this study, a pilot-scale cross-flow microfiltration (MF) process was designed to remove the natural microflora present in commercial LEW, obtained from a local egg-breaking plant, while maintaining the nutritional and functional properties of the LEW. LEW, containing approximately 10(6 +/- 1.7) colony forming units (CFU) per milliliter of total aerobic bacteria, was microfiltered using a ceramic membrane with a nominal pore size of 1.4 microm, at a cross-flow velocity of 6 m/s. To facilitate MF, LEW was screened, homogenized, and then diluted (1 : 2, w/w) with distilled water containing 0.5% sodium chloride. Homogenized LEW was found to have a threefold lower viscosity than unhomogenized LEW. Influence of MF temperature (25 and 40 degrees C) and pH (6 and 9) on permeate flux, transmission of egg white nutrients across the membrane, and microbial removal efficiency were evaluated. The pH had a significantly greater influence on permeate flux than temperature. Permeate flux increased by almost 148% when pH of LEW was adjusted from pH 9 to pH 6 at 40 degrees C. Influence of temperature on permeate flux, at a constant pH, however, was found to be inconclusive. Microbial removal efficiency was at least 5 log(10) CFU/mL. Total protein and SDS-PAGE analysis indicated that this MF process did not alter the protein composition of the permeate, compared to that of the feed LEW, and that the foaming properties of LEW were retained in the postfiltered samples.

Validation of commercial processes for inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on the surface of whole-muscle turkey jerky.

Three strips of turkey breast meat were separately inoculated with multistrain mixtures of Escherichia coli O157:H7, Salmonella Typhimurium, or Listeria monocytogenes and placed on the top, middle, and bottom levels of a loading rack. The strips on the rack were then loaded into a smokehouse and cooked-dried for either 2.5 or 3.5 h at 73.8 degrees C (165 degrees F) or 1.5 or 2.5 h at 82.2 degrees C (180 degrees F) with constant hickory smoking and without addition of humidity. Cooking-drying marinated turkey jerky at 73.8 degrees C (165 degrees F) or 82.2 degrees C (180 degrees F) resulted in a >or= 7.1 log(10) cfu/strip reduction of all 3 pathogens. For nonmarinated jerky strips that were inoculated with E. coli O157:H7 or L. monocytogenes and cooked-dried at 82.2 degrees C (180 degrees F), a reduction of >or= 7.4 log(10) cfu/strip was observed, whereas for strips that were inoculated with Salmonella, a reduction of >or= 6.8 log(10) cfu/strip was observed. Cooking-drying nonmarinated turkey breast strips at 73.8 degrees C (165 degrees F) for 3.5 h resulted in a reduction of ca. 7.1 to 7.6 log(10) cfu/strip for all 3 pathogens, whereas for strips that were cooked-dried for 2.5 h, a reduction of ca. 5.4 to 6.2 log(10) cfu/strip was observed. Only marinated turkey jerky that was cooked-dried for 3.5 h at 73.8 degrees C (165 degrees F) satisfied the USDA-FSIS standard of identity (moisture: protein

Translocation of surface-inoculated Escherichia coli O157:H7 into beef subprimals following blade tenderization.

In phase I, beef subprimals were inoculated on the lean side with ca. 0.5 to 3.5 log CFU/g of a rifampin-resistant (rifr) cocktail of Escherichia coli O157:H7 and passed once, lean side up, through a mechanical blade tenderizer. Inoculated subprimals that were not tenderized served as controls. Ten core samples were removed from each subprimal and cut into six consecutive segments: segments 1 to 4 comprised the top 4 cm and segments 5 and 6 the deepest 4 cm. Levels of E. coli O157:H7 recovered from segment 1 of control subprimals when inoculated with ca. 0.5, 1.5, 2.5, or 3.5 log CFU/g were 0.6, 1.46, 2.5, and 3.19 log CFU/g, respectively. Following tenderization, pathogen levels recovered from segment 1 inoculated with 0.5 to 3.5 log CFU/g were 0.22, 1.06, 2.04, and 2.7 log CFU/g, respectively. Levels recovered in segment 2 were 7- to 34-fold lower than levels recovered from segment 1. Next, in phase II, the translocation of ca. 4 log CFU of the pathogen per g was assessed for lean-side-inoculated subprimals passed either once (LS) or twice (LD) through the tenderizer and for fat-side-inoculated subprimals passed either once (FS) or twice (FD) through the tenderizer. Levels in segment 1 for LS, LD, FS, and FD tenderized subprimals were 3.63, 3.52, 2.85, and 3.55 log CFU/g, respectively. The levels recovered in segment 2 were 14- to 50-fold lower than levels recovered in segment 1 for LS, LD, FS, and FD subprimals. Thus, blade tenderization transfers E. coli O157:H7 primarily into the topmost 1 cm, but also into the deeper tissues of beef subprimals.

Viability of multi-strain mixtures of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage.

The fate of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on soudjouk. Fermentation and drying alone reduced numbers of L. monocytogenes by 0.07 and 0.74 log(10)CFU/g for sausages fermented to pH 5.3 and 4.8, respectively, whereas numbers of S. typhimurium and E. coli O157:H7 were reduced by 1.52 and 3.51 log(10)CFU/g and 0.03 and 1.11 log(10)CFU/g, respectively. When sausages fermented to pH 5.3 or 4.8 were stored at 4, 10, or 21 degrees C, numbers of L. monocytogenes, S. typhimurium, and E. coli O157:H7 decreased by an additional 0.08-1.80, 0.88-3.74, and 0.68-3.17 log(10)CFU/g, respectively, within 30 days. Storage for 90 days of commercially manufactured soudjouk that was sliced and then surface inoculated with L. monocytogenes, S. typhimurium, and E. coli O157:H7 generated average D-values of ca. 10.1, 7.6, and 5.9 days at 4 degrees C; 6.4, 4.3, and 2.9 days at 10 degrees C; 1.4, 0.9, and 1.6 days at 21 degrees C; and 0.9, 1.4, and 0.25 days at 30 degrees C. Overall, fermentation to pH 4.8 and storage at 21 degrees C was the most effective treatment for reducing numbers of L. monocytogenes (2.54 log(10)CFU/g reduction), S. typhimurium (> or =5.23 log(10)CFU/g reduction), and E. coli O157:H7 (3.48 log(10)CFU/g reduction). In summary, soudjouk-style sausage does not provide a favorable environment for outgrowth/survival of these three pathogens.

Prevalence, types, and geographical distribution of Listeria monocytogenes from a survey of retail Queso Fresco and associated cheese processing plants and dairy farms in Sonora, Mexico.

In the first part of this study, samples were collected from farms, cheese processing plants (CPPs), and retail markets located in various geographical areas of Sonora, Mexico, over a 12-month period during the summer of 2004 and winter of 2005. Four (all Queso Fresco [QF] from retail markets) of 349 total samples tested positive for Listeria monocytogenes (Lm). Of these four positive samples, three were collected in the northern region and one in the southern region of Sonora. Additionally, two were collected during the winter months, and two were collected during the summer months. For the second part of the study, a total of 39 samples from a farm, a CPP, and retail markets were collected and processed according to a combination of the Norma Oficial Mexicana NOM-143-SSA1-1995.10 method (NOM) and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual method, and 27 samples from these same locations were collected and processed according to the U.S. Department of Agriculture Food Safety and Inspection Service method (USDA-FSIS). The NOM-FDA method recovered the pathogen from 6 (15%) of 39 samples (one cheese and five product contact surfaces), while the USDA-FSIS method recovered the pathogen from 5 (18.5%) of 27 samples (all product contact surfaces). In addition, the 40 isolates recovered from the 15 total samples that tested positive for Lm grouped into five distinct pulsotypes that were ca. 60% related, as determined by pulsed-field gel electrophoresis analysis. The results of this study confirmed a 3.4% prevalence of Lm in QF collected from retail markets located in Sonora and no appreciable difference in the effectiveness of either the NOM-FDA or USDA-FSIS method to recover the pathogen from cheese or environmental samples.

Effect of preinoculation growth media and fat levels on thermal inactivation of a serotype 4b strain of Listeria monocytogenes in frankfurter slurries.

Preinoculation growth conditions and fat levels were evaluated for effects on the heat resistance of Listeria monocytogenes strain MFS 102 in formulated frankfurter slurries and on frankfurter surfaces. Comparison of linear inactivation rates (D-values) for cells heated in frankfurter slurry showed that growth conditions were significant (P<0.05) factors affecting subsequent thermal resistance. The average D(60 degrees C)-values for the five preinoculation growth media tested from most resistant to least heat resistant were: tryptic soy broth with 0.6% yeast extract (TSBYE) (2.2 min) and 8.5% fat slurry (2.2 min), followed by 23% fat slurry (1.7 min) and 11% fat slurry (1.7 min), and then TSYBE with quaternary ammonium compounds added (TSBYE+Q) (1 min). The fat level in the frankfurter heating media also had a significant (P<0.05) effect on the thermal death rate of L. monocytogenes. Cells heated in 8.5% fat slurry had a significantly higher (P<0.05) D(60 degrees C)-value (2.2 min) than those heated in 11% fat (1.0 min) and 23% fat slurry (0.9 min). Growth media (TSBYE, 8.5% fat slurry, and TSBYE+Q), and fat level (15% and 20%), however, were not significant factors (P>0.05) affecting thermal inactivation rates on frankfurter surfaces. Heat inactivation rates were consistently higher on frankfurter surfaces compared to similar treatments done in frankfurter slurry. On frankfurter surfaces, a 2.3- to 5.1-log(10) reduction was achieved after 15 min depending on frankfurter surface type. The time necessary to achieve a 3-log(10) reduction using post-processing pasteurization of frankfurters in a hot water-bath at 60 degrees C almost doubled for cells grown in TSBYE and heated in 23% fat frankfurter slurry (19.6 min) versus cells grown and heated in 8.5% fat frankfurter slurry (10.8 min).

A predictive model to describe the effects of temperature, sodium lactate, and sodium diacetate on the inactivation of a serotype 4b strain of Listeria monocytogenes in a frankfurter slurry.

A modified Gompertz equation was used to model the effects of temperature (55, 60, and 65 degrees C), sodium lactate (0, 2.4, and 4.8%), and sodium diacetate (0, 0.125, and 0.25%) on inactivation of Listeria monocytogenes strain MFS 102 (serotype 4b) in frankfurter slurry. The effects of these factors were determined on the shouldering region (parameter A), maximum death rate (parameter B), and tailing region (parameter C) of microbial inactivation curves. Increased temperature or sodium diacetate concentrations increased the death rate, whereas increased sodium lactate concentrations decreased heat resistance. Complex two-way interactive effects were also observed. As both temperature and sodium lactate increased, the death rate decreased; however, as temperature and sodium diacetate increased, the death rate increased. The effect of the interaction between sodium lactate and sodium diacetate on the maximum death rate varied with temperature. Increases in both acidulants at temperatures above 56.7 degrees C decreased the death rate, whereas at temperatures below 56.7 degrees C, increases in both acidulants increased the death rate. To test for significant differences between treatments, D-values were calculated and compared. This comparison revealed that, in general, sodium lactate increased heat resistance and sodium diacetate decreased heat resistance of L. monocytogenes. This information is important for reducing and minimizing contamination during postprocessing thermal treatments.

A prfA transposon mutant of Listeria monocytogenes F2365, a serotype 4b strain, is able to survive in the gastrointestinal tract but does not cause systemic infection of the spleens and livers of intragastrically inoculated mice.

prfA is a member of the Crp/Fnr family of global regulatory genes in Listeria monocytogenes that has been shown previously to regulate several key virulence determinants both in vitro and in parenterally inoculated laboratory rodents. However, the role of prfA in the ability of L. monocytogenes to cause infection via the gastrointestinal (GI) tract has not been clearly established. In this study, we used a prfA transposon mutant of L. monocytogenes F2365, a serotype 4b strain, to assess the role of prfA in the pathogenesis of gastrointestinal listeriosis in mice. We found that the prfA mutant was able to survive in the GI tract (i.e., cecum) of mice, albeit in numbers somewhat less than those of the wild-type parent strain of L. monocytogenes. However, mice inoculated with the prfA mutant did not exhibit systemic infection of the spleen and liver, as was noted for mice inoculated with the wild-type parent strain. Survival of the prfA mutant in synthetic gastric fluid at pH 2.5 or 5 was somewhat reduced compared to that of the wild-type strain, as was its ability to invade and multiply within differentiated human intestinal epithelial cells (Caco-2 cells). Prior infection with the prfA mutant gave mice some protection against a subsequent challenge with virulent L. monocytogenes, although much less than that gained by prior gastrointestinal infection with the wild-type parent strain. These findings indicate that the global regulatory gene prfA is dispensable for colonization of the GI tract in mice but not for systemic infection.

Behavior of Listeria monocytogenes in pH-modified chicken salad during refrigerated storage.

The growth and inactivation kinetics of L. monocytogenes were evaluated at pH 4.0, 4.6, and 5.2 during storage at 5.0 degrees C, 7.2 degrees C, and 21.1 degrees C (41 degrees F, 45 degrees F, and 70 degrees F). Using commercially produced pasteurized chicken salad, the authors adjusted the pH levels with acetic acid or sodium acetate. Samples of 25 g each of the pH-modified salad were inoculated to approximately 1 x 10(6) cells per gram with a three-strain mixture of L. monocytogenes and stored for up to 119 days. Samples were enumerated for L. monocytogenes according to the Food and Drug Administration-modified most-probable-number (MPN) procedure, and log MPN was plotted against time. Inactivation was seen at all pH levels and at all temperatures. At 21.1 degrees C, a 6-log reduction was seen after 14 days at pH 4.0, after 52 days at pH 4.6, and after 38 days at pH 5.2. Inactivation at 21.1 degrees C began within hours or days at pH 4.0, and after a lag phase of 10 to 12 days at pH 4.6 and 5.2. Inactivation was slower in cold-storage temperatures. At 7.2 degrees C, a microbial reduction of 1.1 log (pH 5.2) and > 3 log (pH 4.0 and 4.6) was observed at 119 days. At 5 degrees C, a 7.5-log reduction was observed at 24 days at pH 4.0. At pH levels of 4.6 and 5.2, however, only a 4-log reduction was found at 119 days. The data generated in this study may be used to develop predictive models that could specifically address the interactions of pH and storage temperature on the viability of L. monocytogenes in prepared salads.

Viability of Listeria monocytogenes on commercially-prepared hams surface treated with acidic calcium sulfate and lauric arginate and stored at 4°C.

We demonstrated the effectiveness of delivering an antimicrobial purge/fluid into shrink-wrap bags immediately prior to introducing the product and vacuum sealing, namely the "Sprayed Lethality In Container" (SLIC™) intervention delivery method. The pathogen was Listeria monocytogenes, the antimicrobials were acidic calcium sulfate (ACS; calcium sulfate plus lactic acid; 1:1 or 1:2 in dH(2)O) and lauric arginate (LAE; Ethyl-N-dodecanoyl-l-arginate hydrochloride; 5% or 10% in dH(2)O), and the product was commercially prepared "table brown" ham (ca. 3 pounds each). Hams were surface inoculated with a five-strain cocktail of L. monocytogenes (ca. 7.0 log(10) CFU per ham), added to shrink-wrap bags that already contained ACS or LAE, vacuum-sealed, and stored at 4°C for 24h. Pathogen levels decreased by 1.2, 1.6, 2.4, and 3.1 log(10) CFU/ham and 0.7, 1.6, 2.2, and 2.6 log(10) CFU/ham in samples treated with 2, 4, 6, and 8mL of a 1:1 and 1:2 solution of ACS, respectively. In samples treated with 2, 4, 6, and 8mL of a 5% solution of LAE, pathogen levels decreased by 3.3, 6.5, 5.6, and 6.5 log(10) CFU/ham, whereas when treated with a 10% solution of LAE pathogen levels decreased ca. 6.5 log(10) CFU/ham for all application volumes tested. The efficacy of ACS and LAE were further evaluated in shelf-life studies wherein hams were surface inoculated with either ca. 3.0 or 7.0 log(10) CFU of L. monocytogenes, added to shrink-wrap bags that contained 0, 4, 6, or 8mL of either a 1:2 solution of ACS or a 5% solution of LAE, vacuum-sealed, and stored at 4°C for 60 days. For hams inoculated with 7.0 log(10) CFU, L. monocytogenes levels decreased by ca.1.2, 1.5, and 2.0 log(10) CFU/ham and 5.1, 5.4, and 5.5 log(10) CFU/ham within 24h at 4°C in samples treated with 4, 6, and 8mL of a 1:2 solution of ACS and a 5% solution of LAE, respectively, compared to control hams that were not treated with either antimicrobial. Thereafter, pathogen levels remained relatively unchanged (±1.0 log(10) CFU/ham ) after 60 days at 4°C in hams treated with 4, 6, and 8mL of a 1:2 solution of ACS and increased by ca. 2.0-5.0 log(10) CFU/ham in samples treated with 4, 6, and 8mL of a 5% solution of LAE. For hams inoculated with 3.0 log(10) CFU, L. monocytogenes levels decreased by 1.3, 1.9, and 1.8 log(10) CFU/ham within 24h at 4°C in samples treated with 4, 6, and 8mL of a 1:2 solution of ACS, respectively, compared to control hams that were not treated. Likewise, levels of the pathogen were reduced to below the limit of detection (i.e., 1.48 log(10) CFU/ham) in the presence of 4, 6, and 8mL of a 5% solution of LAE within 24h at 4°C. After 60 days at 4°C, pathogen levels remained relatively unchanged (±0.3 log(10) CFU/ham) in hams treated with 4, 6, and 8mL of a 1:2 solution of ACS. However, levels of L. monocytogenes increased by ca. 2.0 log(10) CFU/ham in samples treated with 4 and 6mL of a 5% LAE solution within 60 days but remained below the detection limit on samples treated with 8mL of this antimicrobial. These data confirmed that application via SLIC™ of both ACS and LAE, at the concentrations and volumes used in this study, appreciably reduced levels of L. monocytogenes on the surface of hams within 24h at 4°C and showed potential for controlling outgrowth of the pathogen over 60 days of refrigerated storage.