Prevalence, characterization and sources of Listeria monocytogenes in blue crab (Callinectus sapidus) meat and blue crab processing plants.

Seven blue crab processing plants were sampled to determine the prevalence and sources of Listeria spp. and Listeria monocytogenes for two years (2006-2007). A total of 488 raw crabs, 624 cooked crab meat (crab meat) and 624 environmental samples were tested by standard methods. Presumptive Listeria spp. were isolated from 19.5% of raw crabs, 10.8% of crab meat, and 69.5% of environmental samples. L. monocytogenes was isolated from 4.5% of raw crabs, 0.2% of crab meat, and 2.1% of environmental samples. Ninety-seven percent of the isolates were resistant to at least one of the ten antibiotics tested. Eight different serotypes were found among 76 L. monocytogenes isolates tested with the most common being 4b, 1/2b and 1/2a. Automated EcoRI ribotyping differentiated 11 ribotypes among the 106 L. monocytogenes isolates. Based on ribotyping analysis, the distribution of the ribotypes in each processing plant had a unique contamination pattern. A total of 92 ApaI and 88 AscI pulsotypes among the 106 L. monocytogenes isolates were found and distinct pulsotypes were observed in raw crab, crab meat and environmental samples. Ribotypes and serotypes recovered from crab processing plants included subtypes that have been associated with listeriosis cases in other food outbreaks. Our findings suggest that molecular methods may provide critical information about sources of L. monocytogenes in crab processing plants and will augment efforts to improve food safety control strategies such as targeting specific sources of contamination and use of aggressive detergents prior to sanitizing.

Fate of Shiga toxin-producing O157:H7 and non-O157:H7 Escherichia coli cells within blade-tenderized beef steaks after cooking on a commercial open-flame gas grill.

We compared the fate of cells of both Shiga toxin-producing Escherichia coli O157:H7 (ECOH) and Shiga toxin-producing non-O157:H7 E. coli (STEC) in blade-tenderized steaks after tenderization and cooking on a gas grill. In phase I, beef subprimal cuts were inoculated on the lean side with about 5.5 log CFU/g of a five-strain mixture of ECOH or STEC and then passed once through a mechanical blade tenderizer with the lean side facing up. In each of two trials, 10 core samples were removed from each of two tenderized subprimals and cut into six consecutive segments starting from the inoculated side. Ten total cores also were obtained from two nontenderized (control) subprimals, but only segment 1 (the topmost segment) was sampled. The levels of ECOH and STEC recovered from segment 1 were about 6.0 and 5.3 log CFU/g, respectively, for the control subprimals and about 5.7 and 5.0 log CFU/g, respectively, for the tenderized subprimals. However, both ECOH and STEC behaved similarly in terms of translocation, and cells of both pathogen cocktails were recovered from all six segments of the cores obtained from tenderized subprimals, albeit at lower levels in segments 2 to 6 than those found in segment 1. In phase II, steaks (2.54 and 3.81 cm thick) cut from tenderized subprimals were subsequently cooked (three steaks per treatment) on a commercial open-flame gas grill to internal temperatures of 48.9, 54.4, 60.0, 65.6, and 71.1°C. Regardless of temperature or thickness, we observed 2.0- to 4.1-log and 1.5- to 4.5-log reductions in ECOH and STEC levels, respectively. Both ECOH and STEC behaved similarly in response to heat, in that cooking eliminated significant numbers of both pathogen types; however, some survivors were recovered due, presumably, to uneven heating of the blade-tenderized steaks.

Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil.

Swine can carry Salmonella strains that may be transmitted to humans by pork products. This investigation determined the distribution and types of Salmonella in 12 swine finishing herds and a slaughter facility in Santa Catarina, Brazil. A total of 1258 samples, consisting of environmental, feed, carcass, lymph node, and fecal material were collected and submitted to bacteriological isolation of Salmonella. From 487 positive samples, 1255 isolates were recovered and confirmed to be Salmonella. The distribution of positive samples was as follows: finishing pen floors 26% (16/61); feed 29% (42/143); feces 44% (52/119); pooled feces 59% (35/59); slaughter holding pens 90% (36/40); lymph nodes 46% (220/478); pre-chilled carcass surfaces 24% (24/98); and post-chilled carcass surfaces 24% (62/260). The most prevalent serovars were Typhimurium, Panama, Senftenberg, Derby, and Mbandaka. By pulsed-field gel electrophoresis, 1071 isolates were subtyped using XbaI, and duplicate isolates were removed. From the remaining 747 isolates, 163 macrorestriction profiles (pulsotypes) were identified. Six pulsotypes were considered very frequent, occurring in 33 isolates or more. The multiple correspondence analyses showed correlations between pulsotypes from shedding pigs (feces), herd environment (pen floors), and subiliac and prescapular lymph nodes and between lairage and carcass surface samples before and after chilling. All sources of Salmonella investigated contributed to the carrier state; however, pre-slaughter contamination at lairage was the variable most strongly associated with carcass contamination. A total of 59 different antimicrobial resistance profiles were observed in 572 Salmonella isolates. From these isolates, 17% (97/572) were susceptible to all 15 antibiotics tested, 83% (475/572) were resistant to at least one, and 43% (246/572) were resistant to four or more antibiotics (multi-resistant). The AmpGenKanTet profile was the most prevalent in carcass isolates and was associated with farm origin.

Inactivation of Shiga toxin-producing O157:H7 and non-O157:H7 Shiga toxin-producing Escherichia coli in brine-injected, gas-grilled steaks.

We quantified translocation of Escherichia coli O157:H7 (ECOH) and non-O157:H7 verocytotoxigenic E. coli (STEC) into beef subprimals after brine injection and subsequently monitored their viability after cooking steaks cut therefrom. Beef subprimals were inoculated on the lean side with ca. 6.0 log CFU/g of a five-strain cocktail of rifampin-resistant ECOH or kanamycin-resistant STEC, and then passed once through an automatic brine-injector tenderizer, with the lean side facing upward. Brine solutions (9.9% ± 0.3% over fresh weight) consisted of 3.3% (wt/vol) of sodium tripolyphosphate and 3.3% (wt/vol) of sodium chloride, prepared both with (Lac(+), pH = 6.76) and without (Lac(-), pH = 8.02) a 25% (vol/vol) solution of a 60% potassium lactate-sodium diacetate syrup. For all samples injected with Lac(-) or Lac(+) brine, levels of ECOH or STEC recovered from the topmost 1 cm (i.e., segment 1) of a core sample obtained from tenderized subprimals ranged from ca. 4.7 to 6.3 log CFU/g; however, it was possible to recover ECOH or STEC from all six segments of all cores tested. Next, brine-injected steaks from tenderized subprimals were cooked on a commercial open-flame gas grill to internal endpoint temperatures of either 37.8 °C (100 °F), 48.8 °C (120 °F), 60 °C (140 °F), or 71.1 °C (160 °F). Regardless of brine formulation or temperature, cooking achieved reductions (expressed as log CFU per gram) of 0.3 to 4.1 of ECOH and 0.5 to 3.6 of STEC. However, fortuitous survivors were recovered even at 71.1 °C (160 °F) for ECOH and for STEC. Thus, ECOH and STEC behaved similarly, relative to translocation and thermal destruction: Tenderization via brine injection transferred both pathogens throughout subprimals and cooking highly contaminated, brine-injected steaks on a commercial gas grill at 71.1 °C (160 °F) did not kill all cells due, primarily, to nonuniform heating (i.e., cold spots) within the meat.

Removal of Salmonella Enteritidis from commercial unpasteurized liquid egg white using pilot scale cross flow tangential microfiltration.

Effectiveness of a cross flow microfiltration (MF) process for removal of a cocktail of Salmonella enterica serovar Enteritidis species from commercial unpasteurized liquid egg white (LEW) from a local egg breaking plant, while maintaining its functional properties was evaluated. To facilitate MF, LEW was wedge screened, homogenized and then diluted (1:2 w/w) with distilled water containing 0.5% sodium chloride. Diluted unpasteurized LEW was inoculated with five strains of S. Enteritidis (ATCC 4931, ATCC BAA-708, ATCC 49215, ATCC 49218, and ATCC BAA-1045) to a level of approximately 10(7)CFU/mL of LEW and microfiltered using a ceramic membrane. Process parameters influencing egg white functional properties and pathogen removal efficiency were evaluated. Average permeates flux increased by almost 126% when pH of LEW was adjusted from pH 8 to pH 7 at 25 degrees C. Microbial removal efficiency was at least, on average, 6.8Log(10)CFU/mL (limit of detection < or =0.5Log(10)CFU/mL). Functional property analysis indicated that the MF process did not alter the foaming power of LEW.

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.

Thermal inactivation of Escherichia coli O157:H7 in blade-tenderized beef steaks cooked on a commercial open-flame gas grill.

Beef subprimals were inoculated on the lean side with ca. 4.0 log CFU/g of a cocktail of rifampin-resistant (Rif(r)) Escherichia coli O157:H7 strains and then passed once through a mechanical blade tenderizer with the lean side facing upward. Inoculated subprimals that were not tenderized served as controls. Two core samples were removed from each of three tenderized subprimals and cut into six consecutive segments starting from the inoculated side. A total of six cores were also obtained from control subprimals, but only segment 1 (topmost) was sampled. Levels of E. coli O157:H7 recovered from segment 1 were 3.81 log CFU/g for the control subprimals and 3.36 log CFU/g for tenderized subprimals. The percentage of cells recovered in segment 2 was ca. 25-fold lower than levels recovered from segment 1, but E. coli O157:H7 was recovered from all six segments of the cores obtained from tenderized subprimals. In phase II, lean-side-inoculated (ca. 4.0 log CFU/g), single-pass tenderized subprimals were cut into steaks of various thicknesses (1.91 cm [0.75 in.], 2.54 cm [1.0 in.], and 3.18 cm [1.25 in.]) that were subsequently cooked on a commercial open-flame gas grill to internal temperatures of 48.8 degrees C (120 degrees F), 54.4 degrees C (130 degrees F), and 60 degrees C (140 degrees F). In general, regardless of temperature or thickness, we observed about a 2.6- to 4.2-log CFU/g reduction in pathogen levels following cooking. These data validate that cooking on a commercial gas grill is effective at eliminating relatively low levels of the pathogen that may be distributed throughout a blade-tenderized steak.

Fate of surface-inoculated Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium on kippered beef during extended storage at refrigeration and abusive temperatures.

The behavior of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium on kippered beef was evaluated. Individual pieces of the product were separately inoculated on the top and bottom surfaces with each three- to six-strain pathogen cocktail at ca. 6.0 log CFU per piece and stored at 4, 10, 21, or 30 degrees C for up to 28 days in each of two trials. When kippered beef was inoculated with E. coli O157:H7, Salmonella Typhimurium, or L. monocytogenes and stored at 4, 10, 21, or 30 degrees C for up to 28 days, pathogen numbers decreased ca. 0.4 to 0.9, 1.0 to 1.8, 3.0 to > or = 5.25, and > or = 5.0 to 5.25 log CFU per piece, respectively. Average D-values for E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes stored at 4 to 30 degrees C for 28 days were ca. 41 to 4.6, 40.8 to 5.3, and 29.5 to 4.3 days, respectively. As expected, the higher the storage temperature, the greater the level and rate of inactivation for all three pathogens. These data establish that kippered beef does not provide an environment conducive to proliferation of these pathogens.

Behavior of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium in teewurst, a raw spreadable sausage.

The fate of Listeria monocytogenes, Salmonella Typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on teewurst, a traditional raw and spreadable sausage of Germanic origin. Multi-strain cocktails of each pathogen (ca. 5.0 log CFU/g) were used to separately inoculate teewurst that was subsequently stored at 1.5, 4, 10, and 21 degrees C. When inoculated into commercially-prepared batter just prior to stuffing, in general, the higher the storage temperature, the greater the lethality. Depending on the storage temperature, pathogen levels in the batter decreased by 2.3 to 3.4, ca. 3.8, and 2.2 to 3.6 log CFU/g for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, during storage for 30 days. When inoculated onto both the top and bottom faces of sliced commercially-prepared finished product, the results for all four temperatures showed a decrease of 0.9 to 1.4, 1.4 to 1.8, and 2.2 to 3.0 log CFU/g for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, over the course of 21 days. With the possible exceptions for salt and carbohydrate levels, chemical analyses of teewurst purchased from five commercial manufacturers revealed only subtle differences in proximate composition for this product type. Our data establish that teewurst does not provide a favourable environment for the survival of E. coli O157:H7, S. Typhimurium, or L. monocytogenes inoculated either into or onto the product.

Validation of a commercial process for inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on the surface of whole muscle beef jerky.

We validated the lethality of three time and temperature regimens for commercial processing of whole muscle beef jerky. A total of ca. 8.9 log CFU per strip of multiple-strain cocktails of Escherichia coli O157:H7, Salmonella Typhimurium, or Listeria monocytogenes were separately applied onto the surface of beef strips that were treated as follows: (i) inoculated but not marinated or (ii) inoculated and then marinated. A total of three beef strips for each treatment in each of three trials were separately inoculated with a cocktail of one of the three pathogens and placed on the top, middle, and bottom racks of a loading truck. The strips on the rack were loaded into a commercial smokehouse and cooked and dried for 1.5, 2.5, or 3.5 h at a target temperature of 180 degrees F (82.2 degrees C) with constant (natural hickory) smoking, but without the addition of humidity. Regardless of how the strips were treated or where the strips were placed on the loading rack, drying for 1.5, 2.5, or 3.5 h to a target temperature of 180 degrees F (average of 177.2 +/- 5.6 degrees F [80.7 +/- 3.1 degrees C]), with constant smoke at an initial average relative humidity of 63.1% to a final average relative humidity of 20.9% resulted in a decrease of > or = 7.3 log CFU per strip (> or = 6.9 log CFU/g) for each of the three pathogen cocktails. Of note, marinated strips that were cooked and dried for 2.5 and 3.5 h or nonmarinated strips cooked or dried for 3.5 h also satisfied the U.S. Food Safety and Inspection Service standard of identity (moisture-to-protein ratio < or = 0.75:1) and/or shelf-stability (water activity < or = 0.8) requirements for jerky.

Retail survey of Brazilian milk and Minas frescal cheese and a contaminated dairy plant to establish prevalence, relatedness, and sources of Listeria monocytogenes isolates.

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.

Animal and environmental impact on the presence and distribution of Salmonella and Escherichia coli in hydroponic tomato greenhouses.

From 2003 to 2004, we studied the impact of environmental influences on the microbiological quality of a hydroponic tomato farm. The presence of Salmonella was investigated on 906 samples of tomatoes and 714 environmental samples. The farm comprised 14 greenhouses and a technologically advanced packinghouse, and operated under a sanitary agricultural practices plan. The objective of the present study was to determine the operating sources of contamination. During the course of the study, two independent natural events affected the farm. In 2003, water runoff entered some of the greenhouses. A year later, wild animals (opossums, mice, and sparrows) gained entry into several of the greenhouses. Salmonella and Escherichia coli were found in samples of tomatoes, water puddles, soil, shoes, and the feces of local wild and farm animals. Salmonella Montevideo, Salmonella Newport, and strains of the F serogroup were isolated from tomatoes. Almost all of the Salmonella Newport strains were isolated from samples collected during or immediately after the flood. Analysis by pulsed-field gel electrophoresis revealed that some Salmonella Montevideo isolates from tomatoes, opossums, and mice displayed identical XbaI or AvrII patterns, suggesting that these wild animals represented one source of contamination. F serogroup strains were found mostly on samples of goat feces and personnel shoes when standard working practices were in place. Shoes were found to be an important vehicle for dissemination of Salmonella into the greenhouses. The level of protection provided by hydroponic greenhouses does not exclude the eventuality that enteric pathogenic bacteria can gain access through various avenues.

Hot water postprocess pasteurization of cook-in-bag turkey breast treated with and without potassium lactate and sodium diacetate and acidified sodium chlorite for control of Listeria monocytogenes.

Surface pasteurization and food-grade chemicals were evaluated for the ability to control listeriae postprocess on cook-in-bag turkey breasts (CIBTB). Individual CIBTB were obtained directly from a commercial manufacturer and surface inoculated (20 ml) with a five-strain cocktail (ca. 7.0 log) of Listeria innocua. In each of two trials, the product was showered or submerged for up to 9 min with water heated to 190, 197, or 205 degrees F (ca. 87.8, 91.7, or 96.1 degrees C) in a commercial pasteurization tunnel. Surviving listeriae were recovered from CIBTB by rinsing and were then enumerated on modified Oxford agar plates following incubation at 37 degrees C for 48 h. As expected, higher water temperatures and longer residence times resulted in a greater reduction of L. innocua. A ca. 2.0-log reduction was achieved within 3 min at 205 and 197 degrees F and within 7 min at 190 degrees E In related experiments, the following treatments were evaluated for control of Listeria monocytogenes on CIBTB: (i) a potassium lactate-sodium diacetate solution (1.54% potassium lactate and 0.11% sodium diacetate) added to the formulation in the mixer and 150 ppm of acidified sodium chlorite applied to the surface with a pipette, or (ii) a potassium lactate-sodium diacetate solution only, or (iii) no potassium lactate-sodium diacetate solution and no acidified sodium chlorite. Each CIBTB was inoculated (20 ml) with ca. 5 log CFU of a five-strain mixture of L. monocytogenes and then vacuum sealed. In each of two trials, half of the CIBTB were exposed to 203 degrees F water for 3 min in a pasteurization tunnel, and the other half of the CIBTB were not; then, all CIBTB were stored at 4 degrees C for up to 60 days, and L. monocytogenes was enumerated by direct plating onto modified Oxford agar. Heating resulted in an initial reduction of ca. 2 log CFU of L. monocytogenes per CIBTB. For heated CIBTB, L. monocytogenes increased by ca. 2 log CFU per CIBTB in 28 (treatment 1), 28 (treatment 2), and 14 (treatment 3) days. Thereafter, pathogen levels reached ca. 7 log CFU per CIBTB in 45, 45, and 21 days for treatments 1, 2, and 3, respectively. In contrast, for nonheated CIBTB, L. monocytogenes levels increased from ca. 5 log CFU per CIBTB to ca. 7 log CFU per CIBTB in 28, 21, and 14 days for treatments 1, 2, and 3, respectively. Lastly, in each of three trials, we tested the effect of hot water (203 degrees F for 3 min) postprocess pasteurization of inoculated CIBTB on the lethality of L. monocytogenes and validated that it resulted in a 1.8-log reduction in pathogen levels. Collectively, these data establish that hot water postprocess pasteurization alone is effective in reducing L. monocytogenes on the surface of CIBTB. However, as used in this study, the potassium lactate-sodium diacetate solution and acidified sodium chlorite were only somewhat effective at controlling the subsequent outgrowth of this pathogen during refrigerated storage.

Evaluation of nisin-coated cellulose casings for the control of Listeria monocytogenes inoculated onto the surface of commercially prepared frankfurters.

Commercially prepared frankfurters were formulated with and without approximately 1.4% potassium lactate and 0.1% sodium diacetate and were subsequently processed in cellulose casings coated with and without nisin (approximately 50,000 IU per square inch of internal surface area) to control the outgrowth of Listeria monocytogenes during refrigerated storage. The frankfurters were inoculated with approximately 5 log CFU per package of a five-strain mixture of L. monocytogenes and then vacuum sealed before being stored at 4 degrees C for 60 to 90 days. Surviving organisms were recovered and enumerated by rinsing each package with 18 ml of sterile 0.1% peptone water and plating onto MOX selective agar. The data for each of two trials were averaged. In packages that contained frankfurters formulated with potassium lactate and sodium diacetate and prepared in nisin-coated casings, L. monocytogenes levels decreased by 1.15 log CFU per package after 90 days of storage. L. monocytogenes levels decreased by 0.95 log CFU per package in frankfurters that were prepared in casings that were not coated with nisin. In packages of frankfurters that were formulated without potassium lactate and sodium diacetate and prepared in nisin-coated casings, L. monocytogenes levels decreased by 0.88 log CFU per package after 15 days of storage but then increased appreciably thereafter over a 60-day period of refrigerated storage. There was also an appreciable increase in pathogen numbers during 60 days of storage in otherwise similar frankfurters formulated without potassium lactate and sodium diacetate prepared in casings that were not coated with nisin. These data confirm that potassium lactate and sodium diacetate display listeriostatic activity as an ingredient of commercial frankfurters. These data also establish that cellulose casings coated with nisin display only moderate antilisterial activity in vacuum-sealed packages of commercially prepared frankfurters during storage at 4 degrees C.

Effect of reheating on viability of a five-strain mixture of Listeria monocytogenes in vacuum-sealed packages of frankfurters following refrigerated or frozen storage.

The purpose of this study was to assess consumer preferences for storing and reheating frankfurters and to use this information to assess the effect of product formulation and storage times and temperatures on the viability of Listeria monocytogenes after reheating of frankfurters. Individual links were inoculated with about 8.0 log CFU per package of a five-strain mixture of the pathogen, vacuum sealed, and stored at 4 degrees C for 3 and 15 days and at -18 degrees C for 30 days. Frankfurters formulated with and without 2% added potassium lactate were heated to a surface temperature of 60, 70, 80, or 90 degrees C for up to 8 min by submersing the packages in a thermostatically controlled circulating water bath. Surviving bacteria were recovered and counted by rinsing the contents of each package with sterile peptone water and plating this solution directly onto modified Oxford selective agar plates. In general, the results revealed that about a 5-log unit reduction was achieved by reheating to a surface temperature of 70 degrees C for about 2 min or 80 or 90 degrees C for about 0.6 min regardless of storage conditions or formulation. Product formulation did not appreciably affect the viability of the pathogen after heating; there was no appreciable difference in the number of cells surviving the heat treatment in product prepared with or without potassium lactate. These findings can be used to establish reheating guidelines for consumers to ensure that frankfurters, which may become contaminated with low levels of L. monocytogenes prior to packaging and after unpackaging, are adequately reheated prior to consumption.

Validation of the USDA/ARS package rinse method for recovery of Listeria monocytogenes from naturally contaminated, commercially prepared frankfurters.

The utility of the U.S. Department of Agriculture/Agricultural Research Service (USDA/ARS) package rinse method for recovering Listeria monocytogenes from the surface of contaminated foods was validated in comparison to the standard USDA/Food Safety and Inspection Service (FSIS) product composite enrichment method and two other methods using frankfurters from a lot with a known package prevalence rate of approximately 16% for this pathogen. One hundred packages from this batch of naturally contaminated, commercially prepared frankfurters were examined as follows: (i) the package exudative fluid was removed and tested using the standard USDA/FSIS product composite enrichment method; (ii) approximately 5 to 7 portions of frankfurters were removed to obtain a 25-g composite of meat that was then processed using the standard USDA/FSIS product composite enrichment method: (iii) 50 ml of 0.1% peptone water was added to each package, and the USDA/ARS package rinse method was performed on the remaining contents; and (iv) after removing the rinse fluid, the solid contents remaining in each package were directly enriched using the USDA/FSIS product composite enrichment method. These four methods identified that 7, 6, 15, and 9 of the 100 packages tested positive for the pathogen, respectively. Although no single approach yielded a positive result for every package that tested positive for L. monocytogenes by any one of the four sampling strategies, the USDA/ARS package rinse method was appreciably (P < 0.05) better than either the package exudate enrichment method or the standard USDA/FSIS product composite enrichment method at recovering the bacterium. These findings validate the sensitivity and ease of use of the USDA/ARS package rinse method using naturally contaminated frankfurters and argue strongly for its adoption for routine screening of ready-to-eat products that are prone to surface contamination with undesirable microbes such as L. monocytogenes.

Use of pulsed-field gel electrophoresis to monitor a five-strain mixture of Listeria monocytogenes in frankfurter packages.

In a previous study, the viability of a five-strain mixture of Listeria monocytogenes (including Scott A [serotype 4b, clinical isolate], 101M [serotype 4b, beef-pork sausage isolate], F6854 [serotype 1/2a, turkey frankfurter isolate], H7776 [serotype 4b, frankfurter isolate], and MFS-2 [serotype 1/2a, pork plant isolate]) was monitored during refrigerated storage of frankfurters prepared with and without 3.0% added potassium lactate. Throughout a 90-day period of storage at 4 degrees C, the initial inoculum level of 20 CFU per package remained relatively constant in packages containing frankfurters prepared with potassium lactate, but pathogen counts increased to 4.6 log10 CFU in packages containing frankfurters prepared without added potassium lactate. To determine which of the five strains persisted under these conditions, randomly selected colonies obtained after 28 and 90 days of refrigerated storage of frankfurters were analyzed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI to generate distinct banding patterns for each of the five strains. Then, with the use of PFGE as a tool for identification, the percentages of the strains on days 28 and 90 of the growth study were compared. In the absence of any added potassium lactate in the product, 43% of the 58 isolates recovered on day 28 were identified as strain Scott A, 12% were identified as strain 101M, 22% were identified as strain F6854, 10% were identified as strain H7776, and 12% were identified as strain MFS-2. However, by day 90, an appreciable number (83%) of the 60 isolates analyzed were identified as strain MFS-2. In packages containing frankfurters formulated with 3.0% potassium lactate, all five strains were present at frequencies of 5 to 36% among the 19 isolates tested on day 28; however, by day 90, strain MFS-2 made up the statistical majority (63%) of the 27 isolates tested. The results of this study indicate that strain MFS-2, a serotype 1/2a isolate recovered from a pork processing plant, was more persistent than strains Scott A, 101M, F6854, or H7776 during the extended refrigerated storage of frankfurters.

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of salmonella isolates obtained from the carcasses and feces of swine at slaughter.

Salmonella enterica isolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmed Salmonella isolates from the 84 Salmonella-positive samples obtained from the previous study. A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups. The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively. In addition, multiple isolates were obtained from 67 of the 84 Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples. Both carcass and fecal isolates of Salmonella were recovered from 13 swine, resulting in "matched" samples. Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated with Salmonella pulsotypes that were not isolated from the feces of the same animal. Conversely, from 6 of the 13 (46%) matched animals, Salmonella clonal types were isolated from the feces that were not isolated from the carcass of the same animal. These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses. In addition, these results indicate that the examination of multiple Salmonella isolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing.

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.

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, stx(1), and stx(2) genes and isolates harboring the eaeA, stx(1), and hly(933) genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.