Salmonella Enteritidis in meat, poultry, and pasteurized egg products regulated by the U.S. Food Safety and Inspection Service, 1998 through 2003.

The U.S. Food Safety and Inspection Service (FSIS) tests for Salmonella in meat, poultry, and egg products through three regulatory testing programs: the Pathogen Reduction-Hazard Analysis and Critical Control Point (PR-HACCP) program, the ready-to-eat program for meat and poultry products, and the pasteurized egg products program. From 1998 through 2003, 293,938 samples collected for these testing programs were analyzed for the presence of Salmonella enterica serotypes. Of these samples, 12,699 (4.3%) were positive for Salmonella, and 167 (1.3%) of the positive samples (0.06% of all samples) contained Salmonella Enteritidis. The highest incidence of Salmonella Enteritidis was observed in ground chicken PR-HACCP samples (8 of 1,722 samples, 0.46%), and the lowest was found in steer-heifer PR-HACCP samples (0 of 12,835 samples). Salmonella Enteritidis isolates were characterized by phage type, pulsed-field gel electrophoretic pattern, and antimicrobial susceptibility. Phage typing of 94 Salmonella Enteritidis isolates identified PT13 (39 isolates) and PT8 (36 isolates) as the most common types. One isolate from a ready-to-eat ham product was characterized as PT4. Electrophoretic analysis of 148 Salmonella Enteritidis isolates indicated genetic diversity among the isolates, with 28 unique XbaI electrophoretic patterns identified. Of these 148 isolates, 136 (92%) were susceptible to each of 16 antimicrobials tested. Two isolates were resistant to ampicillin alone, and 10 isolates were resistant to two or more antimicrobials. Isolation of Salmonella Enteritidis from FSIS-regulated products emphasizes the need for continued consumer education on proper food handling and cooking practices and continued work to decrease the prevalence of Salmonella in meat, poultry, and pasteurized egg products.

Testing for Salmonella in raw meat and poultry products collected at federally inspected establishments in the United States, 1998 through 2000.

The Food Safety and Inspection Service (FSIS) issued Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems; Final Rule (the PR/HACCP rule) on 25 July 1996. To verify that industry PR/HACCP systems are effective in controlling the contamination of raw meat and poultry products with human disease-causing bacteria, this rule sets product-specific Salmonella performance standards that must be met by slaughter establishments and establishments producing raw ground products. These performance standards are based on the prevalence of Salmonella as determined from the FSIS's nationwide microbial baseline studies and are expressed in terms of the maximum number of Salmonella-positive samples that are allowed in a given sample set. From 26 January 1998 through 31 December 2000, federal inspectors collected 98,204 samples and 1,502 completed sample sets for Salmonella analysis from large, small, and very small establishments that produced at least one of seven raw meat and poultry products: broilers, market hogs, cows and bulls, steers and heifers, ground beef, ground chicken, and ground turkey. Salmonella prevalence in most of the product categories was lower after the implementation of PR/HACCP than in pre-PR/HACCP baseline studies and surveys conducted by the FSIS. The results of 3 years of testing at establishments of all sizes combined show that >80% of the sample sets met the following Salmonella prevalence performance standards: 20.0% for broilers, 8.7% for market hogs, 2.7% for cows and bulls, 1.0% for steers and heifers, 7.5% for ground beef, 44.6% for ground chicken, and 49.9% for ground turkey. The decreased Salmonella prevalences may partly reflect industry improvements, such as improved process control, incorporation of antimicrobial interventions, and increased microbial-process control monitoring, in conjunction with PR/HACCP implementation.

Reduction of Aqueous Chlorine by Organic Material.

Aqueous chlorine, used to reduce surface bacteria populations on carcasses of slaughter animals after evisceration, during chilling, and after transport, dissipates in the presence of organic matter. This study characterized the amount of residual chlorine present when aqueous HOCI was exposed to bovine serum albumin, bovine lean muscle, porcine adipose tissue, or Trypticase soy agar (TSA) surfaces. Test chlorine solutions, made using Ca (OCl)2, contained 0, 50, 100, 200, 400, 800, 1,600, or 3,200 ppm chlorine, the latter two concentrations being used only in the case of albumin. Chlorine depletion by albumin was almost instantaneous, but was influenced by the amount of albumin present and the initial chlorine concentration. Chlorine exposed to organic surfaces was reduced most readily by lean muscle, then by TSA, and least by adipose tissue. Available chlorine was reduced by about 62% when the volume of aqueous chlorine was 22 ml/cm2 of lean muscle and by about 89% when the volume of aqueous chlorine was 0.69 ml/cm2. With increasing exposure time, the exposure to lean and fat decreased available chlorine by an average of about 10% in 4 min, 27% in 32 min, and 45% in 96 min. Thirteen pure bacterial cultures and two mixed cultures associated with meat were exposed to aqueous chlorine to characterize the effectiveness of the chlorine. All cultures except Bacillus cereus and Enterococcus faecalis were destroyed within 15 s by 3 ppm chlorine. Based on the data, the authors conclude that (a) available chlorine reduction is dependent on exposure time, chlorine concentration, and amount/source of organic material and (b) bacterial inactivation by aqueous chlorine is species specific. These data are of value for estimating chlorine dose for carcass decontamination during washing/chilling and for confirming that bacterial resistance to HOCI is species specific.

Assessment of Three Nucleic Acid Hybridization Systems for Detection of Campylobacter spp. in Poultry Products.

Three commercially available nucleic acid hybridization systems were evaluated in combination with the United States Department of Agriculture-Food Safety and Inspection Service (USDA/FSIS) cultural protocol for the detection of Campylobacter spp. from a variety of poultry products. Samples were enriched for 24 h in Hunt broth and then plated onto modified charcoal Campylobacter differential agar. Suspensions of growth from the selective agar plates were then analyzed by the probe assays. The GENE-TRAK® Campylobacter Assay (revised format) and the GEN-PROBE® ACCUPROBE™ Campylobacter Culture Confirmation Test showed sensitivities and specificities of 100% upon testing of 30 raw chicken rinses. The original format GENE-TRAK® test had a sensitivity of 93% and a specificity of 100% when these samples were tested. Ninety percent of the raw chicken rinses were found to contain Campylobacter spp. By either of the two more sensitive probes or by the USDA/FSIS cultural method. Eighty-three percent of the rinses registered Campylobacter -positive by the original format GENE-TRAK® probe. When inoculated ready-to-eat poultry samples were examined, the revised format GENE- TRAK® test and the ACCUPROBE™ assay had sensitivities of 83% and specificities of 100%. The original format GENE-TRAK® test showed a 75% sensitivity and a 100% specificity with these samples. The USDA/FSIS cultural method had a sensitivity of 79% and a specificity of 100% with the inoculated samples. The detection limit of the revised format GENE-TRAK® and the ACCUPROBE™ assays upon testing pooled cell suspensions of four Campylobacter jejuni poultry isolates was approximately 106 CPU/ml.

Isolation of Escherichia coli 0157:H7 Using 0157 Specific Antibody Coated Magnetic Beads.

Escherichia coli 0157 specific antibody, coated on magnetic beads, was used to concentrate and remove the E. coli 0157:H7 from mixed cultures and meat samples. The problem of nontarget organism carryover was addressed by adding Protamine to the culture-bead sample, washing the beads three times in saline, and changing the test tubes with each wash. These modifications reduced the nontarget colony counts obtained from uninoculated meat samples. This procedure enabled consistent recovery of E. coli 0157:H7 from inoculated meat samples. The percentage of E. coli 0157:H7 cells captured, compared to the total number of cells captured, ranged from 48 to 100%. Two strains of E. coli 0157, H7 and :non-H7, appeared to compete with one another and thus reduce or prevent isolation.

Evaluation of a Colorimetric DNA Hybridization Test for Detection of Salmonellae in Meat and Poultry Products.

A commercially available colorimetric DNA hybridization test was compared with the USDA/FSIS conventional culture method for detection of salmonellae in naturally contaminated meat and poultry products and inoculated ground beef samples. All samples which were Salmonella -positive by the culture method were also positive by the DNA probe assay. There were no false-negative or false-positive results by the colorimetric DNA hybridization test, which was slightly more sensitive than the culture method.

A Screening Method for the Isolation of Escherichia coli O157:H7 from Ground Beef.

A screening method was developed for the isolation of Escherichia coli O157:H7 from raw ground beef. Suspensions at a 1:10 dilution of beef were made in a modified EC broth with novobiocin (mEC+n; EC broth with 1.12 g/L instead of 1.5 g/L Bile salts #3 and novobiocin at 20 mg/L). The samples were macerated in a Stomacher for 2 min and either shaken at 37°C (100 RPM) for 6 h, or incubated static at 35°C for 24 h. Appropriate dilutions of the cultures were then spread plated on 150×15 mm plates of MacConkey sorbitol agar (MSA). The MSA plates were incubated at 42°C overnight. A set of two plates consisting of a deep (40 ml/plate) phenol red sorbitol agar plate with 4-methylumbelliferyl ß-D-glucuronide (PRS-MUG), and a Levine EMB agar plate with added agar for a final concentration of 3%, were gridded into 12 numbered sections. Sorbitol negative colonies were picked from the MSA plates, spread on the appropriate section of the EMB, and stabbed into the corresponding section on the PRS-MUG plate. Those cultures that were sorbitol negative and MUG negative on PRS-MUG and were typically E. coli on EMB were confirmed biochemically and serologically. By this procedure O157:H7 was isolated from 5 of 10 meat samples inoculated at 0.6 organisms/g, and 10 of 10 samples at the 5/g level using the 6 h shaken method. With the 24 h static incubation method, O157:H7 was isolated from 8 of 10 samples at the 0.6/g level and 10 of 10 at the 5/g level. Thirteen strains of O157:H7 inoculated at levels between 0.4 and 0.6/g were tested and 9 of the 13 were isolated with the 6 h method, and 13 of the 13 with the 24 h method. The method is reliable and simple enough to be used in large screening programs.

Use of 5-Bromo-4-Chloro-3-lndoxyl-ß-D-Glucuronide in MacConkey Sorbitol Agar to Aid in the Isolation of Escherichia coli 0157:H7 from Ground Beef.

The addition of 5-bromo-4-chloro-3-indoxyl-ß-D-glucuronide (BCIG) at the 0.1 g/L level, to MacConkey sorbitol agar (MSA) plates aided in the isolation of Escherichia coli 0157:H7 from raw ground beef samples by differentiating ß-glucuronidase positive from ß-glucuronidase negative colonies. E. coli 0157:H7 colonies, being sorbitol negative, ß-glucuronidase negative, remained white, while sorbitol negative, ß-glucuronidase positive colonies turned green to blue. Addition of BCIG to the MSA agar reduced the number of false suspect colonies picked from the primary plating medium by 36% when compared to MSA. E. coli 0157:H7 was isolated from 11 out of 12 inoculated meat samples (0.7 E. coli 0157:H7/g) using MSA-BCIG as compared to 8 out of 12 samples using MSA without BCIG.

An Improved Screening Method for the Detection and Isolation of Escherichia coli 0157:H7 From Meat, Incorporating the 3M Petrifilm™ Test Kit - HEC - for Hemorrhagic Escherichia coli 0157:H7.

A screening method was devised incorporating a commercially available reactive disc blot ELISA for Escherichia coli 0157 antigen, into a cultural screening program for the isolation of E. coli 0157:H7 from meat and poultry products. The method includes the inoculation of a raw or cooked meat sample into an enrichment broth, incubation with shaking at 37°C for 6 to 8 h, followed by inoculation of 3M Petrifilm™ E. coli Count plates with dilutions of the enrichment culture. The Petrifilm plates were incubated at 42°C for 18 h and tested for the presence of the 0157 antigen. The enrichment cultures were reincubated static at 35°C after the initial shaken incubation. Isolation was attempted from the positive Petrifilm plates by both a direct picking and streaking method and by the 3M Prompt™ isolation method. Isolation also was attempted from the 24-h enrichment cultures by spread plating serial dilutions on 150 × 15 mm MacConkey sorbitol agar (MSA) and MSA with 5-bromo-4-chloro-3-indoxyl-ß-D-glucuronic acid cyclohexylammonium salt (BCIG). This fast and efficient screening procedure identifies negative and presumptive positive samples in 26-28 h. Isolation and confirmation of the presumptive positive isolates require an additional 3 to 4 d.

Antibacterial Properties of Retail Sponges.

Seven different brands of cellulose sponges and one polyurethane variety were evaluated for inhibitory properties on twelve strains of gram positive and gram negative bacteria. Sponges were cut in 13 mm or 17 mm discs, autoclaved and aseptically placed on inoculated Tryptic Soy agar plates. The inhibitory effects of sterile sponges, unrinsed, and rinsed in distilled water, were measured. The zone of inhibition values were based on the average of the diameters of the clear zones on the inoculated plates. Polyurethane and EXPANDING CELLULOSE SPONGES were the only varieties which did not exhibit antimicrobial properties with any of the selected bacterial strains. A thorough rinsing procedure was often insufficient to remove the inhibitory agents from the sponges. Listeria monocytogenes strain Scott A and Staphylococcus aureus , both gram positive, were strongly inhibited.

Incidence and Toxigenicity of Aeromonas Species in Retail Poultry, Beef and Pork.

Five enrichment broths and five selective and differentia] plating media were tested for efficiency of isolation of Aeromonas spp. from chicken, beef and pork. An overnight incubation of sample in Trypticase soy broth containing 10 µg of ampicillin/ml which was spread on starch ampicillin agar or on MacConkey mannitol ampicillin agar, gave the best results. A small survey was conducted on 10 samples each of chicken thigh-meat, ground beef, and pork sausage or ground unseasoned pork purchased from local food stores. Aeromonads were found in all of the samples in numbers ranging from 4.44 × 10-2->4.44× 103/g except for two of the pork products from which the organisms could not be isolated. Fifty-eight isolates from this survey were tested for hemolysin production and cytotoxin production; 36 isolates were tested for production of cholera-like toxin. Cytotoxin, as detected by mouse adrenyl Y1 cells and Chinese hamster ovary cells, was produced by 92.8% of the Aeromonas hydrophila isolates, by 84.6% of the Aeromonas sobria isolates and by 17.6% of the Aeromonas caviae isolates. Hemolysin production paralleled cytotoxin production in A. hydrophila and A. caviae . Of the A. sobria isolates, 69.2% were hemolysin producers. None of the isolates tested produced cholera-like toxin. It is not known whether the presence of cytotoxin- and hemolysin-producing Aeromonas species in retail meat and poultry has any public health significance, since to date there have been no reported outbreaks of Aeromonas -caused gastroenteritis traced to meat or poultry.