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.

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.

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.

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.

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.

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.

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.

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes.

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.

Characterization of bacteriocins from Enterococcus faecium with activity against Listeria monocytogenes.

Laboratory cultures and environmental isolates of bacteria were screened for antagonism towards Listeria monocytogenes using an agar spot test. Seven of the 163 strains that were tested, one Streptococcus bovis, one Enterococcus casseliflavus, two E. avium and three E. faecium, consistently displayed antilisterial activity. Cell-free, pH-neutralized supernatants prepared from the three E. faecium strains (JBL1061, JBL1083 and JBL1351) exhibited strong antilisterial activity against L. monocytogenes, and were subjected to more detailed analyses. The antagonistic factors produced by these three strains were sensitive to chloroform and several proteolytic enzymes, resistant to heat (121 degrees C, 20 min), and stable over a wide pH range (3.0-10.0). Moreover, they were listericidal without causing cell lysis. These data suggest that a bacteriocin(s) is involved in the inhibition of L. monocytogenes by E. faecium JBL1061, JBL1083 and JBL1351.

Antilisterial activity of pediocin AcH in model food systems in the presence of an emulsifier or encapsulated within liposomes.

The listericidal activity of pediocin AcH was evaluated in slurries (5, 10, or 25% in dH2O) of nonfat dry milk, butterfat, beef muscle tissue, or beef tallow. Slurries were inoculated with Listeria monocytogenes (2-strain mixture; 2.5 x 10(6) cfu/ml) and then with pediocin AcH (30,000 arbitrary units (AU) per ml of slurry). Although pediocin activity was reduced in slurries, sufficient pediocin remained to decrease the listeriae population. For all slurries tested, the greatest decrease in counts of Listeria (1.2-1.8 log10 cfu decrease) and decrease in pediocin activity (12-54% recovery of original activity) occurred within 1.5 min of addition of pediocin to slurries. Thereafter, counts of Listeria did not change appreciably, but pediocin activity continued to decrease in most treatments for up to 60 min. In general, greater activity was recovered from: (i) slurries of lower (5%) compared to higher (25%) concentrations of food; and (ii) dairy- compared to meat-based slurries. Next, pediocin AcH was encapsulated within phosphatidyl-choline-based liposomes before addition to slurries (10%), or was used unencapsulated in slurries (10%) containing the emulsifier Tween 80. Greater pediocin activity (29-62% increase; average over all concentrations) was recovered from slurries containing encapsulated compared to free pediocin AcH. Likewise, greater pediocin activity was recovered from slurries containing an emulsifier (4-90% increase; average over all concentrations) compared to otherwise similar slurries without Tween 80. The additional recovery of pediocin activity afforded by liposomes or Tween 80 underscores the potential for direct application of biopreservatives to provide another hurdle for L. monocytogenes in foods.

The effects of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytogenes in turkey slurries.

The antilisterial effects of sodium diacetate (0.1, 0.3 and 0.5%) alone or in combination with sodium nitrite (30 ppm), sodium lactate (2.5%) or pediocin (5000 arbitrary units/ml) were evaluated in slurries (25% meat in sterile deionized H2O) prepared from vacuum-packaged, ready-to-eat turkey breast meat and challenged with Listeria monocytogenes. In the absence of food additives, counts of L. monocytogenes increased from 4.5 log10 cfu/ml to ca. 8 log10 cfu/ml within 1 day at 25 degrees C and within 14 days at 4 degrees C. Similarly, the pathogen grew to ca. 8 log10 cfu/ml within 1 d at 25 degrees C and within 28 days at 4 degrees C in slurries containing nitrite or lactate. In the presence of pediocin, after an initial decrease of 0.9 log10 cfu/ml, numbers of the pathogen reached ca. 8 log10 cfu/ml within 5 days at 25 degrees C and within 28 days at 4 degrees C. However, 0.3 and 0.5% diacetate in turkey slurries were listericidal at 4 and 25 degree C, respectively. In the presence of nitrite with diacetate, there was no appreciable difference in growth of L. monocytogenes compared with diacetate alone. Antilisterial activity was potentiated in treatments containing lactate with 0.3% diacetate at 25 degrees C and lactate with 0.1% diacetate at 4 degrees C, compared to similar treatments containing diacetate or lactate alone. A listericidal effect (ca. 7 log10 cfu/ml difference compared to slurries without additives) was observed in treatments containing pediocin with 0.5% diacetate at 25 degrees C and pediocin with 0.3% diacetate at 4 degrees C. The pH of slurries containing 0.3 or 0.5% diacetate was 5.5 and 5.2, respectively, whereas nitrite (pH 6.2), lactate (pH 6.3) or pediocin (pH 6.2) in slurries had a negligible effect on pH compared to the control (pH 6.2). The increased antilisterial activity in slurries with diacetate in combination with other additives was due to synergistic effects and not just pH. Thus, sodium diacetate alone can be used to delay growth of L. monocytogenes in turkey, and an additional level of safety can be achieved using diacetate in combination with sodium lactate or pediocin.

Viability of Escherichia coli O157:H7 in pepperoni during the manufacture of sticks and the subsequent storage of slices at 21, 4 and -20 degrees C under air, vacuum and CO2.

A raw, pepperoni batter (75% pork:25% beef with a fat content of about 32%) was inoculated with a pediococcal starter culture (about 10(8) cfu/g) and a five-strain cocktail of Escherichia coli O157:H7 (> or = 2 x 10(7) cfu/g), mixed with non-meat ingredients, and then hand-stuffed into 55 mm fibrous casings to form sticks. The numbers of the pathogen were determined before stuffing, after fermentation, after drying/slicing, and after periods of storage. For storage, slices were packaged under air, vacuum or CO2 and stored at -20, 4 and 21 degrees C. Sticks were fermented at 36 degrees C and 85% relative humidity (RH) to < or = pH 4.8 and then dried at 13 degrees C and 65% RH to a moisture/protein ratio (M/Pr) of < or = 1.6:1. Fermentation and drying resulted in the numbers of the pathogen decreasing by about 2 log10 units. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers. The greatest reductions in numbers were observed during storage at 21 degrees C, when numbers decreased to about 2 and 3.8 log10 cfu/g within 14 days in product stored under air and vacuum, respectively, and a 5 log10 reduction was observed for both atmospheres within 28 days. Regardless of the storage atmosphere, numbers did not decrease below 3.6 or 3.7 log10 cfu/g after 90 days of storage at -20 or 4 degrees C, respectively. These data confirm that fermentation and drying are sufficient to eliminate only about 2 log10 cfu/g of E. coli O157:H7 from fermented sausage, and that additional strategies, such as storage for at least 2 weeks at ambient temperature in air, are required to achieve a 5 to 6 log10 reduction in the numbers of the pathogen in sliced pepperoni.

Genomic fingerprinting of 80 strains from the WHO multicenter international typing study of listeria monocytogenes via pulsed-field gel electrophoresis (PFGE).

An international multicenter typing study of Listeria monocytogenes was initiated by the World Health Organization (Food Safety Unit, Geneva) in order to evaluate the usefulness of various phenotypic and genotypic typing methods for L. monocytogenes, to select and standardize the most appropriate methods to define common nomenclature of varieties and to select specific reference strains. Pulsed-field gel electrophoresis was used in four laboratories for molecular characterization of a set of 80 'coded' strains distributed to all participating laboratories. The endonucleases ApaI and SmaI, used in all four laboratories, yielded between 21 and 28 restriction endonuclease digestion profiles (REDP). AscI was used, in addition, in laboratory A and displayed 21 REDP. The combination of ApaI, SmaI or AscI REDP established 25 to 33 genomic groups. depending on the laboratory and the number of viable strains. Agreement of typing data among the four laboratories ranged from 79 to 90%. Forty-nine (69%) of the 71 strains viable in all four laboratories were placed into exactly the same genomic groups in all four laboratories. The epidemiological relevance of the strains became known after decoding and it was shown that most of the epidemiologically related strains were correctly identified by the four groups of investigators. i.e., most related strains were placed into the same genomic groups by all four laboratories. Similar results were obtained when 11 duplicate cultures were analyzed-on average 84% of the duplicates were identified. Comparison of REDP obtained by laboratory A with REDP from previously analyzed set of 176 L. monocytogenes strains allowed the prediction of the serovar-groups of the 80 strains. These predictions of serovar-groups were later confirmed by serotyping results obtained by laboratories involved in the WHO multicenter typing study of L. monocytogenes. In general this study reconfirmed that PFGE is a very accurate and reproducible method for fine structure comparison and molecular typing of L. monocytogenes.

Behavior of Salmonella typhimurium DT104 during the manufacture and storage of pepperoni.

Pepperoni batter (ca. 70% pork:30% beef) was prepared and subsequently inoculated with a six-strain cocktail (ca. 4.4 x 10(7) per gram batter) of Salmonella typhimurium DT104. After fermentation at 36 degrees C and 92% relative humidity (RH) to < or = pH 4.8, counts of the pathogen decreased by about 1.3 log10 units. An additional 1.6 log10 unit decrease was observed following drying at 13 degrees C and 65% RH to a moisture protein ratio (M/Pr) of 1.6:1. After storage of pepperoni sticks for 56 days under vacuum at 4 or 21 degrees C, counts of the pathogen were about 4.6 and 6.6 log10 units lower, respectively, compared with starting levels in the batter. These data establish that fermentation and drying result in about a 3.0 log10 reduction in numbers of S typhimurium DT104 in pepperoni sticks and that storage of pepperoni sticks under vacuum at ambient temperature is more severe on the pathogen than refrigerated storage.

Inhibition of microbial growth in chub-packed ground beef by refrigeration (2 degrees C) and medium-dose (2.2 to 2.4 kGy) irradiation.

Two studies were conducted to monitor microbial growth in chub-packed ground beef. In the first study, the effect of storage temperatures (2 and 7 degrees C) on chubs was examined for up to 18 days. Storage of chubs at 2 degrees C slowed microbial growth such that total counts exceeded 7.5 log10 cfu/g within 10 days compared to within 4 days for chubs stored at 7 degrees C. Microbial counts > or = 7.5 log10 cfu/g were associated with off-odors and samples were considered spoiled. The predominant microbes were homofermentative lactococci, although a gas-producing bacterium, Hafnia alvei, was prevalent in one of three trials. In the second study, medium dose (2.2 to 2.4 kGy) X-ray irradiation extended the shelf-life of chub-packed ground beef at 2 degrees C by approximately 14 days. Microbial counts on irradiated ground beef did not exceed 7.5 log10 cfu/g during 34 days of storage, while this level was reached in non-irradiated chubs by day 13. The predominant microbes on both non-irradiated and irradiated chubs were homofermentative lactococci; H. alvei was not isolated in the second study. These data indicated that refrigerated (2 degrees C) storage significantly delayed microbial spoilage in chub-packed ground beef, and that spoilage was further delayed when refrigerated (2 degrees C) ground beef chubs were exposed to medium dose irradiation.

Viability of Escherichia coli O157:H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63 or 68 degrees C in a home-style dehydrator.

Beef jerky batter was prepared to fat contents of about 5 and 20% and inoculated with about 10(8) cfu g(-1) of a five-strain inoculum of Escherichia coli O157:H7. Pathogen numbers were determined in the raw batter and in the strips formed from it after drying at 52, 57, 63, and 68 degrees C for times that ranged from 2 to 20 h. For both the high and low fat products, pathogen numbers were reduced by about 5 log10 cfu g(-1) within 4 h drying at 68 degrees C and within 8 h drying at 63 degrees C. At 57 degrees C, a 5-log10-unit reduction was achieved within 10h drying for the 5% fat product and within 16 h drying for the 20% fat product. At 52 degrees C, a 5-log10-unit reduction was achieved within 10 h drying for the 5% fat product and within 20 h drying for the 20% fat product. In at least one of the three trials for all four drying temperatures tested, the pathogen was present following enrichment of the samples in synthetic media. The calculated D values decreased from 2.59, 2.48, 1.23, and 1.17 as the temperature increased from 52, 57, 63, and 68 degrees C and as the fat content decreased from 20 to 5%. However, there was no direct correlation between the moisture-to-protein ratio and either the doneness of the strips or the viability of the pathogen. These data indicate that the fat content and the time and temperature at which strips are dried directly impact the viability of E. coli O157:H7 in ground and formed beef jerky.

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid.

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.