Complete Whole Genome Sequences of Escherichia coli Surrogate Strains and Comparison of Sequence Methods with Application to the Food Industry.

In 2013, the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) began transitioning to whole genome sequencing (WGS) for foodborne disease outbreak- and recall-associated isolate identification of select bacterial species. While WGS offers greater precision, certain hurdles must be overcome before widespread application within the food industry is plausible. Challenges include diversity of sequencing platform outputs and lack of standardized bioinformatics workflows for data analyses. We sequenced DNA from USDA-FSIS approved, non-pathogenic E. coli surrogates and a derivative group of rifampicin-resistant mutants (rifR) via both Oxford Nanopore MinION and Illumina MiSeq platforms to generate and annotate complete genomes. Genome sequences from each clone were assembled separately so long-read, short-read, and combined sequence assemblies could be directly compared. The combined sequence data approach provides more accurate completed genomes. The genomes from these isolates were verified to lack functional key E. coli elements commonly associated with pathogenesis. Genetic alterations known to confer rifR were also identified. As the food industry adopts WGS within its food safety programs, these data provide completed genomes for commonly used surrogate strains, with a direct comparison of sequence platforms and assembly strategies relevant to research/testing workflows applicable for both processors and regulators.

Effect of Product Dimensions and Surface Browning Method on Salmonella Contamination in Frozen, Surface-Browned, Breaded Chicken Products Treated with Antimicrobials.

Not-ready-to-eat breaded chicken products formulated with antimicrobial ingredients were tested for the effect of sample dimensions, surface browning method and final internal sample temperature on inoculated Salmonella populations. Fresh chicken breast meat portions (5 × 5 × 5 cm), inoculated with Salmonella (7-strain mixture; 5 log CFU/g), were mixed with (5% v/w total moisture enhancement) (i) distilled water (control), (ii) caprylic acid (CAA; 0.0625%) and carvacrol (CAR; 0.075%), (iii) CAA (0.25%) and ε-polylysine (POL; 0.5%), (iv) CAR (0.15%) and POL (0.5%), or (v) CAA (0.0625%), CAR (0.075%) and POL (0.5%). Sodium chloride (1.2%) and sodium tripolyphosphate (0.3%) were added to all treatments. The mixtures were then ground and formed into 9 × 5 × 3 cm (150 g) or 9 × 2.5 × 2 cm (50 g) portions. The products were breaded, browned in (i) an oven (208 °C, 15 min) or (ii) deep fryer (190 °C, 15 s), packaged, and stored at -20 °C (8 d). Overall, maximum internal temperatures of 62.4 ± 4.0 °C (9 × 2.5 × 2 cm) and 46.0 ± 3.0 °C (9 × 5 × 3 cm) were reached in oven-browned samples, and 35.0 ± 1.1 °C (9 × 2.5 × 2 cm) and 31.7 ± 2.6 °C (9 × 5 × 3 cm) in fryer-browned samples. Irrespective of formulation treatment, total (after frozen storage) reductions of Salmonella were greater (P < 0.05) for 9 × 2.5 × 2 cm oven-browned samples (3.8 to at least 4.6 log CFU/g) than for 9 × 5 × 3 cm oven-browned samples (0.7 to 2.5 log CFU/g). Product dimensions did not (P ≥ 0.05) affect Salmonella reductions (0.6 to 2.8 log CFU/g) in fryer-browned samples. All antimicrobial treatments reduced Salmonella to undetectable levels (<0.3 log CFU/g) in oven-browned 9 × 2.5 × 2 cm samples. Overall, the data may be useful for the selection of antimicrobials, product dimensions, and surface browning methods for reducing Salmonella contamination.

Effects of cooking methods and chemical tenderizers on survival of Escherichia coli O157:H7 in ground beef patties.

This study evaluated chemical tenderizers and cooking methods to inactivate Escherichia coli O157:H7 in ground beef patties (model system for non-intact beef). Ground beef was inoculated with E. coli O157:H7 and mixed with (i) nothing (control), (ii) calcium chloride (CC) and flavoring agents (FA), (iii) CC, FA, and acetic acid (AA), (iv) sodium chloride (SC), sodium tripolyphosphate (ST), and potassium lactate (PL), and (v) the combination of SC, ST, PL, and AA. Patties were stored in aerobic or vacuum bags at -20, 4, and 12°C. Samples were grilled, broiled, or pan-fried to 60 or 65°C. Total bacterial and E. coli O157:H7 populations remained unchanged during storage. Broiling was more effective in reducing E. coli O157:H7 than grilling and pan-frying, and acidified tenderizers reduced E. coli O157:H7 more than non-acidified tenderizers in broiling. Higher reductions were observed at 65°C than 60°C in broiled and grilled samples. These results indicate that acidified tenderizers and broiling may be useful in non-intact beef safety.

Efficacy of chemical interventions against Escherichia coli O157:H7 and multidrug-resistant and antibiotic-susceptible Salmonella on inoculated beef trimmings.

Studies were conducted to compare the decontamination efficacy of six chemical treatments against Escherichia coli O157:H7 and multidrug-resistant and antibiotic-susceptible Salmonella inoculated on beef trimmings. The inocula, comprising four-strain mixtures of rifampin-resistant E. coli O157:H7 and antibiotic-susceptible or multidrug-resistant (MDR and/or MDR-AmpC) Salmonella Newport and Salmonella Typhimurium, were inoculated (3 log CFU/cm(2)) separately onto samples (10 by 5 by 1 cm) derived from beef chuck rolls. Samples were left untreated (control), were immersed for 30 s in acidified sodium chlorite (0.1%, pH 2.5), peroxyacetic acid (0.02%, pH 3.8), sodium metasilicate (4%, pH 12.6), Bromitize Plus (0.0225% active bromine, pH 6.6), or AFTEC 3000 (pH 1.2), or were immersed for 5 s in SYNTRx 3300 (pH 1.0). Levels of surviving Salmonella on treated trimmings were not influenced by serotype or antibiotic resistance phenotype and were generally similar (P ≥ 0.05) or lower (P < 0.05) than levels of surviving E. coli O157:H7 regardless of antimicrobial treatment. Overall, depending on chemical treatment (reductions within each chemical treatment were similar among all tested inocula), initial counts of E. coli O157:H7 (2.7 to 3.1 log CFU/cm(2)) were reduced (P < 0.05) by 0.2 to 1.4 log CFU/cm(2). Similarly, initial counts of the tested Salmonella inocula (2.8 to 3.3 log CFU/cm(2)) were reduced (P < 0.05) by 0.4 to 1.4 (Salmonella Newport, antibiotic susceptible), 0.3 to 1.4 (Salmonella Newport, MDR-AmpC), 0.2 to 1.5 (Salmonella Typhimurium, antibiotic susceptible), 0.4 to 1.3 (Salmonella Typhimurium, MDR), and 0.4 to 1.5 (Salmonella Typhimurium, MDR-AmpC) log CFU/cm(2), depending on antimicrobial treatment. Reductions obtained with sodium metasilicate were 1.3 to 1.5 log CFU/cm(2), regardless of inoculum, and reductions obtained with the five remaining antimicrobial treatments were 0.2 to 0.7 log CFU/cm(2) (depending on treatment). Findings of this study should be useful to regulatory authorities and the meat industry as they consider Salmonella contamination on beef trimmings.

Sensitivity of Shiga toxin-producing Escherichia coli, multidrug-resistant Salmonella, and antibiotic-susceptible Salmonella to lactic acid on inoculated beef trimmings.

Studies were performed to determine whether lactic acid treatments used to reduce Escherichia coli O157:H7 on beef trimmings are also effective in controlling non-O157 Shiga toxin-producing E. coli (nSTEC), and multidrug-resistant and antibiotic-susceptible Salmonella. Beef trimming pieces (10 by 5 by 1 cm) were inoculated (3 log CFU/cm(2)) separately with four-strain mixtures of rifampin-resistant E. coli O157:H7, O26, O45, O103, O111, O121, and O145. Similarly, in a second study, trimmings were separately inoculated with rifampin-resistant E. coli O157:H7, and antibiotic-susceptible or multidrug-resistant (MDR and/or MDR-AmpC) Salmonella Newport and Salmonella Typhimurium. Inoculated trimmings were left untreated (control) or were immersed for 30 s in 5% lactic acid solutions (25 or 55°C). No differences (P ≥ 0.05) were obtained among surviving counts of E. coli O157:H7 and those of the tested nSTEC serogroups on lactic acid-treated (25 or 55°C) samples. Counts (3.1 to 3.3 log CFU/cm(2)) of E. coli O157:H7 and nSTEC were reduced (P < 0.05) by 0.5 to 0.9 (25°C lactic acid) and 1.0 to 1.4 (55°C lactic acid) log CFU/cm(2). Surviving counts of Salmonella on treated trimmings were not influenced by serotype or antibiotic resistance phenotype and were similar (P ≥ 0.05) or lower (P < 0.05) than surviving counts of E. coli O157:H7. Counts (3.0 to 3.3 log CFU/cm(2)) were reduced (P < 0.05) by 0.5 to 0.8 (E. coli O157:H7) and 1.3 to 1.5 (Salmonella) log CFU/cm(2) after treatment of samples with 25°C lactic acid. Corresponding reductions following treatment with lactic acid at 55°C were 1.2 to 1.5 (E. coli O157:H7) and 1.6 to 1.9 (Salmonella) log CFU/cm(2). Overall, the results indicated that lactic acid treatments used against E. coli O157:H7 on beef trimmings should be similarly or more effective against the six nSTEC serogroups and against multidrug-resistant and antibiotic-susceptible Salmonella Newport and Salmonella Typhimurium.

Comparison of decontamination efficacy of antimicrobial treatments for beef trimmings against Escherichia coli O157:H7 and 6 non-O157 Shiga toxin-producing E. coli serogroups.

UNLABELLED: The decontamination efficacy of 6 chemical treatments for beef trimmings were evaluated against Escherichia coli O157:H7 and 6 non-O157 Shiga toxin-producing E. coli (nSTEC) serogroups. Rifampicin-resistant 4-strain mixtures of E. coli O157:H7 and nSTEC serogroups O26, O45, O103, O111, O121, and O145 were separately inoculated (3 to 4 log CFU/cm(2)) onto trimmings (10 × 5 × 1 cm; approximately 100 g) fabricated from beef chuck rolls, and were immersed for 30 s in solutions of acidified sodium chlorite (0.1%, pH 2.5), peroxyacetic acid (0.02%, pH 3.8), sodium metasilicate (4%, pH 12.5), Bromitize(®) Plus (0.0225% active bromine, pH 6.6), or AFTEC 3000 (pH 1.2), or for 5 s in SYNTRx 3300 (pH 1.0). Each antimicrobial was tested independently together with an untreated control. Results showed that all tested decontamination treatments were similarly effective against the 6 nSTEC serogroups as they were against E. coli O157:H7. Irrespective of pathogen inoculum, treatment of beef trimmings with acidified sodium chlorite, peroxyacetic acid, or sodium metasilicate effectively (P < 0.05) reduced initial pathogen counts (3.4 to 3.9 log CFU/cm(2)) by 0.7 to 1.0, 0.6 to 1.0, and 1.3 to 1.5 log CFU/cm(2), respectively. Reductions of pathogen counts (3.1 to 3.2 log CFU/cm(2)) by Bromitize Plus, AFTEC 3000, and SYNTRx 3300 were 0.1 to 0.4 log CFU/cm(2), depending on treatment. Findings of this study should be useful to regulatory authorities and the meat industry as they consider nSTEC contamination in beef trimmings. PRACTICAL APPLICATIONS: Findings of this study should be useful to: (i) meat processors as they design and conduct studies to validate the efficacy of antimicrobial treatments to control pathogen contamination on fresh beef products; and (ii) regulatory agencies as they consider approaches for better control of the studied pathogens.

Activity of caprylic acid, carvacrol, ε-polylysine and their combinations against Salmonella in not-ready-to-eat surface-browned, frozen, breaded chicken products.

UNLABELLED: Caprylic acid (CAA), carvacrol (CAR), ε-polylysine (POL), and their combinations were evaluated for reduction of Salmonella contamination in not-ready-to-eat surface-browned, frozen, breaded chicken products. Fresh chicken breast meat pieces (5 × 5 × 5 cm) were inoculated with Salmonella (7-strain mixture; 4-5 log CFU/g) and mixed with distilled water (control) or with CAA, CAR, and POL as single or combination treatments of 2 or 3 ingredients. Sodium chloride (1.2%) and sodium tripolyphosphate (0.3%) were added to all formulations, followed by grinding of the mixtures and forming into 9 × 5 × 3 cm portions. Sample surfaces were brushed with egg whites, coated with breadcrumbs, surface-browned in an oven (208 °C, 15 min), packaged, and stored at -20 °C (7 d). Total reductions of inoculated Salmonella in untreated (control) surface-browned, breaded products after frozen storage were 0.8 to 1.4 log CFU/g. In comparison, single treatments of CAA (0.25% to 1.0%), CAR (0.3% to 0.5%), and POL (0.125% to 1.0%) reduced counts by 2.9 to at least 4.5, 3.4 to at least 4.4, and 1.4 to 2.3 log CFU/g, respectively, depending on concentration. Pathogen counts of products treated with 2- or 3-ingredient combination treatments (0.03125% to 0.25% CAA, 0.0375% to 0.3% CAR, and/or 0.5% POL) were 0.4 to at least 3.3 log CFU/g lower (depending on treatment) than those of the untreated controls. The antimicrobial activity of 2-ingredient combinations comprised of 0.125% CAA, 0.15% CAR, or 0.5% POL was enhanced (P < 0.05) when applied as a 3-ingredient combination (that is, 0.125% CAA + 0.15% CAR + 0.5% POL). These data may be useful for the selection of antimicrobial treatments to reduce Salmonella contamination in not-ready-to-eat processed chicken products. PRACTICAL APPLICATION: Findings from the study may be useful for the selection of suitable antimicrobials, concentrations, and combinations to reduce Salmonella contamination in not-ready-to-eat surface-browned, frozen, breaded chicken products.

Antimicrobials for reduction of Salmonella contamination in uncooked, surface-browned breaded chicken products.

Surface-browned but uncooked frozen breaded chicken products have been associated with salmonellosis outbreaks due to inadequate or no cooking of the products before consumption. This study was conducted to evaluate the effect of three antimicrobials against Salmonella during manufacture of a surface-browned, uncooked frozen breaded chicken meat product. Fresh chicken breast meat portions (5 by 5 by 5 cm) were inoculated (4 to 5 log CFU/g) with Salmonella and mixed with caprylic acid (CAA; 0.5 and 1.0%), carvacrol (CAR; 0.3 and 0.5%), ε-polylysine (POL; 0.125 and 0.25%), or distilled water (control). Sodium chloride (1.2%) and sodium tripolyphosphate (0.3%) were added to all treatments, and the mixtures were ground (5% total moisture enhancement level) and formed into portions (9 by 5 by 3 cm). The products were breaded and surface browned by baking in an oven (208°C for 15 min) or deep frying in vegetable oil (190°C for 15 s), packaged in polyethylene bags, and stored at -20°C for 7 days. Total reductions of inoculated Salmonella in untreated control oven- or fryer-browned products after frozen storage were 1.2 and 0.8 log CFU/g, respectively. In comparison, treatment with CAA, CAR, or POL reduced initial pathogen counts by 3.3 to >4.5, 4.1 to >4.7, and 1.1 to 1.6 log CFU/g, respectively, regardless of the antimicrobial concentration and browning method. Treatment with 1.0% CAA (oven browned) or 0.5% CAR (oven or fryer browned) reduced Salmonella to nondetectable levels (<0.3 log CFU/g) in stored frozen products. These data may be useful for development of suitable antimicrobial treatments to reduce the risk of Salmonella contamination in surface-browned, uncooked frozen breaded chicken products.

Transfer, attachment, and formation of biofilms by Escherichia coli O157:H7 on meat-contact surface materials.

UNLABELLED: Studies examined the effects of meat-contact material types, inoculation substrate, presence of air at the liquid-solid surface interface during incubation, and incubation substrate on the attachment/transfer and subsequent biofilm formation by Escherichia coli O157:H7 on beef carcass fabrication surface materials. Materials studied as 2 × 5 cm coupons included stainless steel, acetal, polypropylene, and high-density polyethylene. A 6-strain rifampicin-resistant E. coli O157:H7 composite was used to inoculate (6 log CFU/mL, g, or cm²) tryptic soy broth (TSB), beef fat/lean tissue homogenate (FLH), conveyor belt-runoff fluids, ground beef, or beef fat. Coupons of each material were submerged (4 °C, 30 min) in the inoculated fluids or ground beef, or placed between 2 pieces of inoculated beef fat with pressure (20 kg) applied. Attachment/transfer of the pathogen was surface material and substrate dependent, although beef fat appeared to negate differences among surface materials. Beef fat was the most effective (P < 0.05) inoculation substrate, followed by ground beef, FLH, and TSB. Incubation (15 °C, 16 d) of beef fat-inoculated coupons in a beef fat homogenate (pH 4.21) allowed the pathogen to survive and grow on coupon surfaces, with maximal biofilm formation observed between 2 and 8 d of storage and when air was present at the liquid-solid interface. The results indicated that the process of fabricating beef carcasses may be conducive to the attachment of E. coli O157:H7 onto meat-contact surfaces and subsequent biofilm formation. Furthermore, it is recommended that substrates found in beef fabrication settings, rather than laboratory culture media, be used in studies designed to investigate E. coli O157:H7 biofilm development and control in these environments. PRACTICAL APPLICATION: Findings of this study provide knowledge on the effect of type of beef carcass fabrication surface material, fabrication-floor fluids and residues, and incubation conditions on attachment/transfer and subsequent biofilm formation by E. coli O157:H7. The results highlight the importance of thoroughly cleaning soiled surfaces to remove all remnants of beef fat or other organic material that may harbor or protect microbial contaminants during otherwise lethal antimicrobial interventions.

Thermal inactivation of Escherichia coli O157:H7 inoculated at different depths of non-intact blade-tenderized beef steaks.

UNLABELLED: Studies evaluated thermal inactivation of Escherichia coli O157:H7 inoculated at different depths of simulated blade-tenderized non-intact steaks. Fresh beef slices (0.3 or 0.6 cm thick) were stacked on top of each other to form 2.4 or 1.2 cm thick steaks. Steaks were blade-tenderized and then inoculated with rifampicin-resistant Escherichia coli O157:H7 (8 strain mixture; 4 log CFU/cm(2)) on the surface or between slices, vacuum-packaged, and stored at 4 or -20 °C for 5 d before cooking. Steaks were cooked by pan-broiling or roasting to a geometric center temperature of 60 °C. Frozen samples were either cooked from the frozen state or after thawing to approximately 4 or 25 °C. In steaks inoculated on the external surface and cooked by pan-broiling, pathogen survivors recovered from thinner (1.2 cm) steaks were greater (P < 0.05) than those recovered from thicker (2.4 cm) steaks. Cooking steaks from a frozen state or after thawing (4 or 25 °C) did not (P ≥ 0.05) affect extent of pathogen inactivation. Survivors after pan-broiling of 2.4 cm thick steaks increased (P < 0.05) from 0.3 to 1.3 log CFU/cm(2) for surface-inoculated steaks to 2.5 to 3.2 log CFU/cm(2) for samples inoculated at the center (1.2 cm depth). In comparison, overall thermal destruction of the pathogen in steaks cooked by roasting was less, and survivor counts were generally not different (P ≥ 0.05) at each depth of inoculation. These data should be useful in development of lethality guidelines to ensure safe consumption of non-intact meat products. PRACTICAL APPLICATION: Results of this study should be useful for developing cooking guidelines, for foodservice establishments and consumers, to ensure safe consumption of non-intact meat products.

Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing.

Escherichia coli O157:H7 attached to beef-contact surfaces found in beef fabrication facilities may serve as a source of cross-contamination. This study evaluated E. coli O157:H7 attachment, survival and growth on food-contact surfaces under simulated beef processing conditions. Stainless steel and high-density polyethylene surfaces (2×5cm) were individually suspended into each of three substrates inoculated (6log CFU/ml or g) with E. coli O157:H7 (rifampicin-resistant, six-strain composite) and then incubated (168h) statically at 4 or 15°C. The three tested soiling substrates included sterile tryptic soy broth (TSB), unsterilized beef fat-lean tissue (1:1 [wt/wt]) homogenate (10% [wt/wt] with sterile distilled water) and unsterilized ground beef. Initial adherence/attachment of E. coli O157:H7 (0.9 to 2.9log CFU/cm(2)) on stainless steel and high-density polyethylene was not affected by the type of food-contact surface but was greater (p<0.05) through ground beef. Adherent and suspended E. coli O157:H7 counts increased during storage at 15°C (168h) by 2.2 to 5.4log CFU/cm(2) and 1.0 to 2.8log CFU/ml or g, respectively. At 4°C (168h), although pathogen levels decreased slightly in the substrates, numbers of adherent cells remained constant on coupons in ground beef (2.4 to 2.5log CFU/cm(2)) and increased on coupons in TSB and fat-lean tissue homogenate by 0.9 to 1.0and 1.7 to 2.0log CFU/cm(2), respectively, suggesting further cell attachment. The results of this study indicate that E. coli O157:H7 attachment to beef-contact surfaces was influenced by the type of soiling substrate and temperature. Notably, attachment occurred not only at a temperature representative of beef fabrication areas during non-production hours (15°C), but also during cold storage (4°C) temperatures, thus, rendering the design of more effective sanitation programs necessary.

Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials.

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

Inactivation of Escherichia coli O157:H7 in moisture-enhanced nonintact beef by pan-broiling or roasting with various cooking appliances set at different temperatures.

This study evaluated inactivation of Escherichia coli O157:H7 in moisture-enhanced restructured nonintact beef cooked to 65 °C using different cooking appliances set at different temperatures. Batches (2 kg) of coarse-ground beef (approximately 5% fat) were mixed with an 8-strain composite (100 mL) of rifampicin-resistant E. coli O157:H7 (6.4 ± 0.1 log CFU/g) and a solution (100 mL) of sodium chloride plus sodium tripolyphosphate to yield concentrations (wt/wt) of 0.5% and 0.25%, respectively, in the final product. Beef portions of 2.54 cm thickness (15 cm dia) were prepared and were vacuum-packaged and frozen (-20 °C, 42 h). Partially thawed (-2.5 ± 1.0 °C) portions were pan-broiled (Presto electric skillet and Sanyo grill) or roasted (Oster toaster oven and Magic Chef kitchen oven) to 65 °C. The appliances were set at, and preheated before cooking to 149 or 204 °C (electric skillet), 149 or 218 °C (grill), 149 or 232 °C (toaster oven), and 149, 204, or 260 °C (kitchen oven). Temperatures of appliances and beef samples were monitored with thermocouples, and meat samples were analyzed for surviving microbial populations. In general, the higher the appliance temperature setting, the shorter the time needed to reach 65 °C, and the higher the edge and surface temperatures of the meat samples. Temperatures of 204 to 260 °C, regardless of appliance, resulted in greater (P < 0.05) pathogen reductions (3.3 to 5.5 log CFU/g) than those obtained at 149 °C (1.5 to 2.4 log CFU/g). The highest (P < 0.05) reduction (5.5 log CFU/g) was obtained in samples cooked in the kitchen oven set at 260 °C. The results should be useful to the food service industry for selection of effective nonintact beef cooking protocols, and for use in risk assessments for nonintact meat products. Practical Application: Results of this study should be useful for developing cooking recommendations to enhance the safety of nonintact beef products, and for use in risk assessments of such products.

Thermal inactivation of acid, cold, heat, starvation, and desiccation stress-adapted Escherichia coli O157:H7 in moisture-enhanced nonintact beef.

This study was conducted to compare thermal inactivation of stress-adapted and nonadapted Escherichia coli O157:H7 in nonintact beef moisture enhanced with different brine formulations and cooked to 65°C. Coarsely ground beef was mixed with acid, cold, heat, starvation, or desiccation stress-adapted or nonadapted rifampin-resistant E. coli O157:H7 (eight-strain mixture, 5 to 6 log CFU/g) and a brine solution for a total moisture enhancement level of 10%. The brine treatments included distilled water (control), sodium chloride (0.5% NaCl) plus sodium tripolyphosphate (0.25% STP), or NaCl + STP combined with cetylpyridinium chloride (0.2% CPC), lactic acid (0.3% LA), or sodium metasilicate (0.2% SM). The treated meat was extruded into bags (15 cm diameter), semifrozen (-20°C for 4.5 h), and cut into 2.54-cm (1-in.)-thick portions. Samples were individually vacuum packaged, frozen (-20°C for 42 h), and tempered at 4°C for 2.5 h before cooking. Partially thawed (-1.8 ± 0.4°C) samples were pan broiled to an internal temperature of 65°C. Pathogen counts of partially thawed (before cooking) samples moisture enhanced with brines containing CPC, LA, or SM were 0.7 to 1.1, 0.0 to 0.4, and 0.2 to 0.4 log CFU/g, respectively, lower than those of the control. Compared with microbial count reductions obtained after pan broiling of beef inoculated with nonadapted E. coli O157:H7 cells, count reductions during cooking of meat inoculated with cold and desiccation stress-adapted, acid stress-adapted, and heat and starvation stress-adapted cells indicated sensitization, cross protection, and no effect, respectively, of these stresses on the pathogen during subsequent exposure to heat. Among all stressed cultures, CPC-treated samples (0.8 to 3.6 log CFU/g) and LA-treated samples (0.8 to 3.5 log CFU/g) had the lowest numbers of E. coli O157:H7 survivors after cooking.

Probabilistic models for the prediction of target growth interfaces of Listeria monocytogenes on ham and turkey breast products.

UNLABELLED: This study developed growth/no growth models for predicting growth boundaries of Listeria monocytogenes on ready-to-eat cured ham and uncured turkey breast slices as a function of lactic acid concentration (0% to 4%), dipping time (0 to 4 min), and storage temperature (4 to 10 °C). A 10-strain composite of L. monocytogenes was inoculated (2 to 3 log CFU/cm²) on slices, followed by dipping into lactic acid and storage in vacuum packages for up to 30 d. Total bacterial (tryptic soy agar plus 0.6% yeast extract) and L. monocytogenes (PALCAM agar) populations were determined on day 0 and at the endpoint of storage. The combinations of parameters that allowed increases in cell counts of L. monocytogenes of at least l log CFU/cm² were assigned the value of 1, while those limiting growth to <1 log CFU/cm² were given the value of 0. The binary data were used in logistic regression analysis for development of models to predict boundaries between growth and no growth of the pathogen at desired probabilities. Indices of model performance and validation with limited available data indicated that the models developed had acceptable goodness of fit. Thus, the described procedures using bacterial growth data from studies with food products may be appropriate in developing growth/no growth models to predict growth and to select lactic acid concentrations and dipping times for control of L. monocytogenes. PRACTICAL APPLICATION: The models developed in this study may be useful in selecting lactic acid concentrations and dipping times to control growth of Listeria monocytogenes on cured ham and uncured turkey breast during product storage, and in determining probabilities of growth under selected conditions. The modeling procedures followed may also be used for application in model development for other products, conditions, or pathogens.

Presence of antibiotic-resistant commensal bacteria in samples from agricultural, city, and national park environments evaluated by standard culture and real-time PCR methods.

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance.

Characterization and transferability of class 1 integrons in commensal bacteria isolated from farm and nonfarm environments.

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

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

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

Evaluation of brining ingredients and antimicrobials for effects on thermal destruction of Escherichia coli O157:H7 in a meat model system.

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

Inactivation of Escherichia coli O157:H7 in nonintact beefsteaks of different thicknesses cooked by pan broiling, double pan broiling, or roasting by using five types of cooking appliances.

This study compared thermal inactivation of Escherichia coli O157:H7 in nonintact beefsteaks of different thicknesses by different cooking methods and appliances. Coarsely ground beef was inoculated with rifampin-resistant E. coli O157:H7 (eight-strain composite, 6 to 7 log CFU/g) and then mixed with sodium chloride (0.45%) plus sodium tripolyphosphate (0.23%); the total water added was 10%. The meat was stuffed into bags (10-cm diameter), semifrozen (-20 degrees C, 6 h), and cut into 1.5-, 2.5-, and 4.0-cm-thick steaks. Samples were then individually vacuum packaged, frozen (-20 degrees C, 42 h), and tempered (4 degrees C, 2.5 h) before cooking. Partially thawed (-2 +/- 1 degrees C) steaks were pan broiled (Presto electric skillet and Sanyo grill), double pan broiled (George Foreman grill), or roasted (Oster toaster oven and Magic Chef standard kitchen oven) to a geometric center temperature of 65 degrees C. Extent of pathogen inactivation decreased in order of roasting (2.0 to 4.2 log CFU/g) > pan broiling (1.6 to 2.8 log CFU/g) >/= double pan broiling (1.1 to 2.3 log CFU/g). Cooking of 4.0-cm-thick steaks required a longer time (19.8 to 65.0 min; variation was due to different cooking appliances), and caused greater reductions in counts (2.3 to 4.2 log CFU/g) than it did in thinner samples (1.1 to 2.9 log CFU/g). The time to reach the target temperature increased in order of George Foreman grill (3.9 to 19.8 min) < Oster toaster oven (11.3 to 45.0 min) < Presto electric skillet (16.3 to 55.0 min) < Sanyo grill (14.3 to 65.0 min) < standard kitchen oven (20.0 to 63.0 min); variation was due to steak thickness. Results indicated that increased steak thickness allowed greater inactivation of E. coli O157:H7, as time to reach the target internal temperature increased. Roasting in a kitchen oven was most effective for pathogen inactivation.