Jeffrey E. Call

Minimum and Maximum Temperatures for Growth and Verotoxin Production by Hemorrhagic Strains of Escherichia coli

The influence of temperature on growth and verotoxin production by Escherichia coli strains was studied in brain heart infusion (BHI) broth both in shake cultures at various temperatures and in a temperature-gradient incubator. All strains of E. coli surveyed grew from at least 10 to 45°C, with some strains growing at 8° C. Verotoxin production (determined using the Vero cell-assay system) was a function of both temperature and time, with the highest titers produced at temperatures supporting the fastest growth (based on days to visible turbidity) and highest viable cell counts. However, for strains producing verotoxin, toxin production was detected at any temperature supporting growth. Three strains (of 16 tested) increased 1000-fold in viable count in 4 to 6 days at 10°C. The data presented here indicate that most E. coli strains surveyed can easily grow at ca. 10°C and thus suggest the potential for growth in temperature-abused refrigerated foods.

Evaluation of fermentation, drying, and/or high pressure processing on viability of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in raw pork and Genoa salami.

We evaluated the effectiveness of fermentation, drying, and high pressure processing (HPP) to inactivate Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in Genoa salami produced with trichinae-infected pork. In addition, we evaluated the effectiveness of using HPP to inactivate T. spiralis larvae in pig masseter tissue. In part A, Genoa salami batter (about 2.3 log larvae/g) prepared with trichinae-infected pork was separately spiked with a five-strain cocktail of each microbial pathogen (about 7.0 log CFU/g) and subsequently fermented at 20 degrees C and about 90 to 95% RH for 6h and then at 27 degrees C and about 90 to 95% RH for 26 h before being dried at 20 degrees C and about 65 to 75% RH for 40 h and then at 17 degrees C and about 65 to 75% RH to/for: A) 25 d (65 mm casing), B) a target a(w) of 0.92 (65 mm casing), C) 35 d (105 mm casing), or D) a target a(w) of 0.94 (105 mm casing). Inactivation of L. monocytogenes, E. coli O157:H7, and Salmonella spp. after fermentation and drying ranged from about 1.1 to 1.3, about 1.1 to 2.2, and about 4.2 to 4.8 log CFU/g, respectively. After drying, three replicate salami samples in each of two trials for each treatment were subjected to HPP. Pressurization at 600 MPa or at 483 MPa for 1 to 12 min reduced pathogen numbers by an additional 1.6 to >or=5.0 (L. monocytogenes), 4.7 to >or=5.8 (E. coli O157:H7), and 1.9 to 2.4 (Salmonella)log CFU/g. After storage for 28 d at 4 degrees C, L. monocytogenes levels decreased by up to an additional 3.0 log CFU/g, whereas an additional decrease of up to about 1.1 and 1.7 log CFU/g was observed for E. coli O157:H7 and Salmonella, respectively. In contrast, in each of three trials, T. spiralis was inactivated (about 2.3 log larvae/g) in Genoa salami by all treatments of fermentation and drying as confirmed by both microscopy and mouse bioassays. In part B, in each of two trials, a 10-g portion (2 replicates per treatment) of infected pig masseter muscle (about 3.4 log larvae/g) were pressurized at 483 and 600 MPa for 0.5 to 5 min. T. spiralis was inactivated in pig masseter by all treatments of HPP as confirmed by both microscopy and mouse bioassays. Thus, fermentation and drying and/or HPP of contaminated Genoa salami or pork are effective for inactivating L. monocytogenes, E. coli O157:H7, Salmonella spp., and/or T. spiralis larvae. These data validate that HPP can be used as an alternate to curing for trichinae control and as a post-process intervention to meet performance standards and/or compliance guidelines for the three microbial pathogens evaluated herein.

Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine.

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx1, and stx2 genes and isolates harboring the eaeA, stx1, and hly933 genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.

Viability of a Five-Strain Mixture of Listeria monocytogenes in Vacuum-Sealed Packages of Frankfurters, Commercially Prepared with and without 2.0 or 3.0% Added Potassium Lactate, during Extended Storage at 4 and 10° C†‡

The viability of Listeria monocytogenes was monitored on frankfurters containing added potassium lactate that were obtained directly from a commercial manufacturer. Eight links (ca. 56 g each) were transferred aseptically from the original vacuum-sealed bulk packages into nylon-polyethylene bags. Each bag then received a 4-ml portion of a five-strain mixture of the pathogen. Frankfurters containing 2.0 or 3.0% potassium lactate were evaluated using 20 CFU per package, and frankfurters containing 3.0% potassium lactate were evaluated using 500 CFU per package. The packages were vacuum-sealed and stored at 4 or 10 degrees C for up to 90 or 60 days, respectively. During storage at 4 degrees C, pathogen numbers remained at about 1.6 log10 CFU per package over 90 days in packages containing frankfurters with 2.0% potassium lactate that were inoculated with about 20 CFU. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 20 CFU and stored at 4 degrees C, pathogen numbers remained at about 1.4 log10 CFU per package over 90 days. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 500 CFU and stored at 4 degrees C, pathogen numbers remained at about 2.4 log10 CFU per package over 90 days. However, in the absence of any added potassium lactate, pathogen numbers increased to 4.6 and 5.0 log10 CFU per package after 90 days of storage at 4 degrees C for starting levels of 20 and 500 CFU per package, respectively. During storage at 10 degrees C, pathogen numbers remained at about 1.4 log10 CFU per package over 60 days in packages containing frankfurters with 2.0% potassium lactate that were inoculated with about 20 CFU. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 20 CFU and stored at 10 degrees C, pathogen numbers remained at about 1.1 log10 CFU per package over 60 days of storage. In the absence of any added potassium lactate, pathogen numbers increased to 6.5 log10 CFU per package after 28 days and then declined to 5.0 log10 CFU per package after 60 days of storage at 10 degrees C. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 500 CFU per package, pathogen numbers remained at about 2.4 log10 CFU per package over 60 days of storage at 10 degrees C, whereas in the absence of any added potassium lactate, pathogen numbers increased to about 6.6 log10 CFU per package within 40 days and then declined to about 5.5 log10 CFU per package after 60 days of storage. The viability of L. monocytogenes in frankfurter packages stored at 4 and 10 degrees C was influenced by the pH and the presence or levels of lactate but not by the presence or levels of indigenous lactic acid bacteria or by the proximate composition of the product. These data establish that the addition of 2.0% (P < 0.0004) or 3.0% (P < 0.0001) potassium lactate as an ingredient in frankfurters can appreciably enhance safety by inhibiting or delaying the growth of L. monocytogenes during storage at refrigeration and abuse temperatures.

Retail Survey of Brazilian Milk and Minas Frescal Cheese and a Contaminated Dairy Plant To Establish Prevalence, Relatedness, and Sources of Listeria monocytogenes Isolates

ABSTRACT A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.

Recovery rate of Listeria monocytogenes from commercially prepared frankfurters during extended refrigerated storage.

To assess the prevalence of Listeria monocytogenes in vacuum-sealed packages of frankfurters, about 33,000 packages (1 lb each) were obtained by a third-party contractor from 12 volunteer commercial manufacturers over a 2-year period. The 12 producers, each of which contributed about 2,700 packages of frankfurters from one production run, comprised 9 large and 3 small plants located in eight U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) districts in 10 states. Five days after manufacture, 500 packages were sampled at the USDA/Agricultural Research Service (ARS) Eastern Regional Research Center (ERRC) in Wyndmoor, Pa., by the USDA/ARS package rinse method. At regular intervals during subsequent storage at 4 and 10 degrees C, an additional 200 packages were tested for the pathogen at each sampling point. From a statistical perspective, L. monocytogenes was not recovered from any of the products of nine of the producers, whereas the pathogen was recovered at rates of 1.5% (plant 367), 2.2% (plant 439), and 16% (plant 133) from the products of the remaining three plants. In total, 532 of 32,800 (1.6%) packages of frankfurters tested positive for the pathogen. The recovery rates did not change appreciably over time, there was no appreciable difference in L. monocytogenes recovery rates with respect to frankfurter storage temperature (4 or 10 degrees C), and the seasonality of manufacture had no influence on recovery rate. Molecular subtyping of multiple L. monocytogenes-positive isolates from each plant revealed that profile A (serotype 1/2a) was displayed by about 90% of the 1,105 isolates tested. However, in some cases it was also possible to recover more than one profile from a given plant. This study provides estimates of the prevalence, types, and viability of L. monocytogenes associated with commercially prepared frankfurters during extended refrigerated storage.

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.

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®) 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.

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.

Prevalence of Campylobacter within a Swine Slaughter and Processing Facility

In this work, the occurrence of Campylobacter in a swine slaughter and processing facility was studied. Thirty composite carcass samples, representing 360 swine carcasses, were taken immediately after exsanguination, immediately after polishing, after the final wash, and after overnight chilling at 2 degrees C. Thirty matching composite rectal samples were also taken immediately after exsanguination, and 60 nonmatching individual colon samples were collected from the same lot of swine during evisceration. Also, 72 environmental samples were collected from equipment used in the slaughter operation (42 samples) and the processing operation (30 samples). Campylobacter was isolated by direct plating on Campy-Line agar (CLA) or Campy-Cefex agar (CCA), as well as by Bolton broth enrichment and subsequent inoculation onto CLA or CCA. For all four recovery methods combined, Campylobacter was detected on 33% (10 of 30) of the composite carcasses immediately after exsanguination, 0% (0 of 30) after polishing, 7% (2 of 30) immediately before chilling, and 0% (0 of 30) after overnight chilling. The pathogen was recovered from 100% (30 of 30) of the composite rectal samples and 80% (48 of 60) of the individual colon samples. Campylobacter was detected in 4.8% (2 of 42) and 3.3% (1 of 30) of the slaughter and processing equipment samples, respectively. The recovery rate achieved with direct plating on CLA was significantly higher (P < 0.05) than those achieved with the other three recovery methods. For the 202 isolates recovered from all of the various samples tested, Campylobacter coli was the predominant species (75%) and was followed by Campylobacter spp. (24%) and Campylobacter jejuni (1%). These results indicate that although Campylobacter is highly prevalent in the intestinal tracts of swine arriving at the slaughter facility, this microorganism does not progress through the slaughtering operation and is not detectable on carcasses after overnight chilling.