Modification and Matrix Extension of the Bio-Rad iQ-Check E. coli O157:H7, STEC VirX, and STEC SerO Test Kits for the Detection of Shiga Toxin-Producing Escherichia coli (STEC) and Escherichia coli O157 From a Single Enrichment.

BACKGROUND: The iQ-Check Real-Time PCR kits use PCR technology based on gene amplification and detection by a real-time PCR thermalcycler for the detection of target analytes in select food matrices. The iQ-Check E. coli O157:H7 [Performance Tested MethodSM (PTM) 020801] and STEC VirX and STEC SerO (combined PTM 121203) methods were previously validated for different matrices under different enrichment schemes. OBJECTIVE: To modify the current iQ-Check E. coli O157:H7 Kit for the detection of Escherichia coli O157:H7 from 25 to 375 g for raw ground beef (17% fat), raw beef trim, and fresh spinach. In addition, a matrix extension was validated for iQ-Check E. coli O157:H7 for raw chicken breast without skin (25 g), raw chicken thigh with skin (25 g), mechanically separated chicken (25 g), and raw ground pork (25 g). The study also included the modification of the iQ-Check STEC VirX and SerO Kits for the detection of non-O157 Shiga toxin-producing E. coli (STEC) for raw ground beef (375 g), raw beef trim (375 g), and fresh spinach (375 g) from STEC Enrichment Broth to buffered peptone water (BPW). All tests were carried out at 8-22 h (10-22 h for fresh spinach). METHODS: Ground beef, beef trim, and spinach were co-inoculated with E. coli O157:H7, non-O157 STECs, and Salmonella spp. and analyzed for E. coli O157:H7 and non-O157 STECs after an 8-22 h enrichment in BPW for the beef matrices and after a 10-22 h enrichment in BPW for spinach. The chicken matrices were inoculated with E. coli O157:H7 only and analyzed after an 8-22 h enrichment in BPW. The iQ-Check Free DNA Removal Solution workflow was utilized for all matrices. Confirmations at the 22 h time point and method comparisons were conducted with the appropriate reference method as outlined in the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A or the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapters 5.09 and 5B.05. For the iQ-Check STEC VirX and STEC SerO Kits, inclusivity and exclusivity were also performed. RESULTS: The two inclusivity and exclusivity evaluations indicated that the test methods can accurately detect the target analytes and correctly excluded nontarget organisms after 8 h of enrichment. In the method comparison study, the iQ-Check E. coli O157:H7 and STEC VirX and STEC SerO test kits demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for all food matrices analyzed and the two time points (8 or 10 and 22 h). Both time points produced the same results, with no discrepancies. CONCLUSIONS: The iQ-Check real-time PCR kits are effective methods for the detection of E. coli O157 and non-O157 STECs (both the virulence factors and the O groups) from raw ground beef, raw beef trim, and fresh spinach in 375 g samples enriched in BPW for 8-22 h (10-22 h for fresh spinach). In addition, the iQ-Check E. coli O157 Kit is effective in detecting E. coli O157 in 25 g samples of raw chicken breast without skin, raw chicken thigh with skin, mechanically separated chicken, and raw ground pork. The iQ-Check test kits allow the end user to pair enrichments for multiple target analytes, allowing the user to prepare a single enrichment and perform a single DNA extraction. The Free DNA Removal Solution removes free DNA from samples prior to PCR analysis, protecting DNA from intact and living cells. HIGHLIGHTS: The method modifications were granted based on the data collected.

Modification of the Bio-Rad iQ-Check Listeria spp. Kit for the Detection of Listeria Species in Environmental Surfaces.

BACKGROUND: The Bio-Rad iQ-Check Listeria spp. Kit uses real-time PCR technology for detection of Listeria species in select food matrixes and environmental surfaces. OBJECTIVE: The iQ-Check Listeria spp. method was modified to reduce the enrichment medium volume for environmental sponges from 225 and 100 to 60 mL and to reduce the enrichment time for sponges and swabs from 25 ± 1 to as short as 18 h. The modified method was validated with stainless steel, polystyrene plastic, and sealed concrete using sponges or swabs with two different neutralizing buffers (Letheen Broth and HiCap™ Neutralizing Broth). In addition, the Bio-Rad Free DNA Removal Solution was used for all environmental samples. METHODS: The iQ-Check Listeria spp. modified method was compared with the reference culture method in the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 8.10 using an unpaired study design. RESULTS: In the method comparison study, the iQ-Check Listeria spp. modified method demonstrated no statistical difference in performance between candidate and reference method results or between presumptive and confirmed results for all environmental surfaces analyzed using HiCap Neutralizing Broth (World Bioproducts LLC) and Letheen broth. CONCLUSIONS: The modified iQ-Check Listeria spp. method is an effective method for the detection of Listeria species in environmental surfaces using both types of neutralizing buffer. HIGHLIGHTS: The method modification was granted based on the data collected.

Validation of Solus One Salmonella from Select Food Matrixes and Stainless-Steel and Plastic Environmental Surfaces.

Background: Solus One Salmonella is designed to accurately detect Salmonella species (Salmonella enterica subspecies enterica, salamae, arizonae, diarizonae, houtenae, indica, and Salmonella bongori) from select food matrixes and stainless-steel and plastic environmental surfaces. Solus One Salmonella uses an antibody-based technology test system that is paired with media and our proprietary media supplement, the Solus One Salmonella supplement combined with a manual or automated sample preparation method. Objective: Solus One Salmonella was evaluated for inclusivity and exclusivity, and a matrix comparison study was done for six food matrixes (raw beef trim, pasteurized liquid egg, raw salmon, cheddar cheese, Romaine lettuce, nonfat dry milk) and two environmental surfaces (stainless steel and polystyrene). Methods: Solus One Salmonella was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5: Salmonella (July 2018) and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Manual, 4.09 (January 2017) in the matrix study. Both the manual and automated sample preparation methods were performed for cheddar cheese and stainless-steel environmental surfaces. Results: For the inclusivity and exclusivity evaluation, Solus One Salmonella correctly detected all 108 target organism isolates and correctly excluded all 35 nontarget strains that were analyzed. Conclusions: In the method comparison study, both Solus One Salmonella manual and automated sample preparation methods demonstrated no significant differences based on probability of detection (POD) statistical analysis between presumptive and confirmed results or between candidate and reference method results for the six food matrixes after 20-22 h and two environmental surfaces after 16-20 h of enrichment time. POD analysis of Solus One Salmonella method robustness, product consistency, and stability studies using the automated sample preparation method demonstrated no statistically significant differences.

The Validation of the VereBeef™ Detection Kit.

VereBeef™ Detection Kit, incorporating both multiplex PCR and microarray technologies on a lab-on-chip platform, is intended for qualitative detection and differentiation of Escherichia coli O157:H7, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145, Shiga toxin-producing E. coli (STEC) virulence factors (stx1A, stx2A, eae), and Salmonella species in one test using raw beef trim samples. This product underwent extensive evaluations, including inclusivity-exclusivity, method comparison, robustness, lot-to-lot variability, and stability studies. The inclusivity/exclusivity study demonstrated that VereBeef Detection Kit specifically detects and identifies target analytes without occurrence of false-positive and false-negative detection. In the method comparison study, the performance of the VereBeef Detection Kit was compared with U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook's methods for target organism detection in raw beef trim using E. coli O157:H7 single inoculation and Salmonella and non-O157 STEC dual inoculation. Data demonstrated equivalence in both methods. The robustness study showed that changes in the test parameters do not impact assay performance. Collectively, VereBeef Detection Kit is able to detect target pathogens in raw beef trim with a minimum enrichment time of 8 h for E. coli O157:H7 detection and 10 h for Salmonella and non-O157 STEC detection.

Evaluation of the Solus One Listeria Method for the Detection of Listeria Species on Environmental Surfaces.

Background: Solus One Listeria is designed to accurately detect Listeria species (Listeria grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. seeligeri, and L. welshimeri) from stainless steel and plastic environmental surface matrixes using an antibody-based technology test system paired with proprietary SOLO+ media and combined with manual or automated sample preparation method. Objective: Solus One Listeria was evaluated for inclusivity and exclusivity and a matrix comparison study for two environmental surfaces. Methods: Solus One Listeria was compared with the following reference method for the method comparison study: the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 10 from stainless steel and plastic environmental surfaces. Both the manual and automated preparation methods were performed for stainless steel and plastic environmental surfaces. Results: For the inclusivity and exclusivity evaluation, Solus One Listeria correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains that were analyzed. In the method comparison study, both Solus One Listeria manual and automated preparation methods demonstrated no significant differences based on probability of detection statistical analysis between presumptive and confirmed results or between candidate and reference method results for two environmental surfaces after 22-30 h of enrichment time. Probability of detection analysis of Solus One Listeria method robustness, product consistency (lot-to-lot), and stability studies using the automated preparation method demonstrated no statistically significant differences. Conclusions: The data from the study support the product claims of Solus One Listeria for the accurate detection of Listeria species, using both the manual and automated methods (using the Dynex DS2 instrument), on both environmental surfaces analyzed.

Validation of the TadpoleTM Campylobacter jejuni Real-Time PCR Identification Kit.

Background: The gene-based real-time PCR method for identification of Campylobacter jejuni is more simple, rapid and accurate than the traditional biochemical method. Objective: A performance validation of the TadpoleTMCampylobacter jejuni Real-Time PCR Identification Kit was performed. Method: The assay uses TaqMan Real-time PCR technology to amplify target genes from isolated colonies. Bacterial deoxyribonucleic acid (DNA) from inclusivity and exclusivity organisms cultured on Columbia Blood Agar, Campy-Cefex agar and modified Charcoal Cefoperazone Deoxycholate was extracted and analyzed on three instruments: Applied Biosystems (ABI) 7500 Fast, ABI StepOne Plus and Bio-Rad CFX96. Results: When 57 distinct strains of C. jejuni were tested for inclusivity, all 57 strains produced positive results on the three instruments. In exclusivity testing, all 35 strains of related organisms, including 7 non-target Campylobacter strains and other common species, produced negative results on the three instruments. The Independent Laboratory validation consisting of an inclusivity and exclusivity evaluation for 10 C. jejuni isolates and 10 nontarget Campylobacter isolates also showed 100% expected results on the three instruments. In addition, in robustness testing, small, deliberate changes to the assay parameters, including cell suspension turbidity, heat lysis time, and DNA template volume in the PCR reaction, did not affect the kit performance. Finally, the combined lot-to-lot and stability study on both the ABI 7500 Fast and the ABI StepOne Plus showed that the 11 C. jejuni strains and 5 nontarget Campylobacter strains can be correctly identified by the three independently manufactured, lots and it supported a shelf life of 9 months when stored at -20°C. Conclusions: The Tadpole method offers a rapid, accurate, and robust alternative for C. jejuni identification. Highlights: Rapid and accurate method to identify C. jejuni, which has a good robustness and high stability. It is flexible and offers the advantages of reduced labor and time saving.

Validation of Workflow Changes, Phage Concentration and Reformatted Detection Threshold for the Sample6 DETECT/L™ Test: Level 3 Modification.

The AOAC Research Institute Performance Tested MethodsSM Program certified Sample6 DETECT/L™ in April 2014 (Certification No. 041401) for the detection of Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. marthii, L. welshimeri) on stainless steel environmental surfaces. A modification was approved in January 2016, increasing the concentration of sanitizer-neutralizing reagents in detection reagents, increasing the number of phage in the detection solution, and increasing the sample test volume. Moreover, changes to reduce the number of negative controls and add compatibility with polyurethane sponges were also approved. In this modification, to ensure that DETECT/L continues to meet performance expectations, Sample6 evaluated workflow changes to enhance sensitivity and the ease-of-use of the assay. Changes to the phage concentration and detection threshold, plus the inclusion of a confirmation step (DETECT Check), were validated to obtain better accuracy and optimize assay performance. Inclusivity, exclusivity, and robustness testing were conducted by Sample6 to evaluate the changes. A third-party laboratory compared the DETECT/L assay and the U.S. Department of Agriculture reference method in a stainless steel environmental surface matrix study. The data presented in this report demonstrate that the changes proposed to the DETECT/L assay meet or exceed the performance in the current configuration.

MC-Media Pad SA (Sanita-kun SA) for the Enumeration of Staphylococcus aureus in a Variety of Foods.

MC-Media Pad SA (formerly known as Sanita-kun SA) is a dry rehydratable film medium for the enumeration of Staphylococcus aureus. The performance of the method in a variety of foods was compared with that of ISO 6888-1:1999, Microbiology of Food and Animal Feeding Stuffs - Horizontal Method for the Enumeration of Coagulase-Positive Staphylococci (Staphylococcus aureus and Other Species) - Part 1: Technique Using Baird-Parker Agar Medium. The validated matrixes included pastrami, a sliced cooked chicken roll, cooked prawns, cold-smoked salmon, pasta salad, sandwich spread, fresh uncooked pasta, infant cereal, custard, and raw-milk Brie cheese. In the matrix study, five replicates at each of three contamination levels were tested as paired test portions. Across all matrixes, the difference in mean log10 values ranged from -0.32 to 0.10, which was within the acceptable range of -0.50 to 0.50. Thus, all 10 matrixes met the acceptance criterion at all concentration levels. Further, only two matrixes, cooked prawns and raw-milk Brie cheese, had 95% confidence limits outside the -0.50 to 0.50 criterion, and these were at the lowest concentration level for each matrix. The candidate method sr varied from 0.03 to 0.22 log10 CFU/g. This compares favorably with the reference method SD, which ranged from 0.06 to 0.30 log10 CFU/g. The candidate and reference methods detected 51 of 53 inclusivity strains, with both methods not detecting the same two strains. The candidate method did not detect any of the 32 exclusivity strains, whereas the reference method did not detect 30 of the 32 exclusivity strains; the 2 strains detected by the reference method were S. delphini and S. hyicus, both developing atypical colonies on Baird-Parker plates. The product consistency study demonstrated no significant difference between lots of product and supported the 1 year shelf life. Robustness testing yielded no significant differences when small variations were made in sample volume, incubation temperature, and incubation time. Thus, the data show equivalent or better performance of the Sanita-kun SA/MC-Media Pad SA method compared with the International Organization for Standardization reference method, in support of AOAC Performance Tested MethodSM certification.

MC-Media Pad ACplus™ for Enumeration of Aerobic Counts in a Variety of Foods.

The MC-Media Pad ACplus™ is a dry, rehydratable film medium for the enumeration of aerobic bacterial colonies. The performance of the method in a variety of foods was compared to that of U.S. reference methods: U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Chapter 3.02 "Quantitative Analysis of Bacteria in Foods as Sanitary Indicators" (USDA/FSIS MLG 3.02); Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6 "Microbiological Count Methods, Standard Plate Count Method" (SMEDP 6); AOAC Official MethodSM 966.23 Microbiological Methods; and ISO 4833-1:2013 "Microbiology of the food chain-Horizontal method for the enumeration of microorganisms-Part 1: Colony count at 30 degrees C by the pour plate technique." The validated matrixes included raw chicken breast and raw ground pork for USDA/FSIS MLG 3.02; cream cheese and yogurt drink for SMEDP 6; parsley, vegetable juice, prawns, tuna pate, sandwiches, and pasta salad for AOAC Method 966.23, and raw chicken breast, raw ground pork, cream cheese, yogurt drink, parsley, vegetable juice, prawns, tuna pate, sandwiches, and pasta salad for ISO 4833-1:2013. In each matrix study, five replicates at each of three contamination levels were tested as paired test portions. All 10 matrixes were compared to the appropriate U.S. reference methods under MC-Media Pad ACplus standard-usage conditions (35 ± 1°C for 48 ± 2 h). Across all matrixes, the difference of mean log10 values ranged from -0.43 to 0.44, within the acceptable range of -0.50 to 0.50. The candidate method repeatability SD (sr) varied from 0.03 to 0.23 log10 CFU/g, comparing favorably to the reference method SD, which ranged from 0.06 to 0.30 log10 CFU/g. Seven matrixes were compared to the appropriate U.S. reference methods under MC-Media Pad ACplus rapid-usage conditions (35 ± 1°C for 24 ± 2 h). Of the 21 matrix/concentration combinations, only three instances of difference of mean >0.5 log were observed. The ranges of sr values of the rapid-usage candidate method (0.023-0.324) and the reference method (0.013-0.236) were similar for the seven matrixes tested. All 10 matrixes were compared to the International Organization for Standardization (ISO) reference method under MC-Media Pad ACplus alternate-method conditions (30 ± 1°C for 72 ± 3 h). All 10 matrixes yielded a mean difference between methods of <0.5 log, and the ranges of sr values were similar between the candidate alternate method (0.037-0.378) and the ISO reference method (0.037-0.437). The product consistency study demonstrated no significant difference between lots of product and supported the 2-year shelf life. Robustness testing yielded no significant differences when small variations were made in sample volume, incubation temperature, and incubation time. Thus, the data show equivalent or better performance of the MC-Media Pad ACplus method compared to the relevant reference methods in support of AOAC Performance Tested MethodSM certification.

Creating Best Practices for the Submission of Actionable Food and Feed Testing Data Generated in State and Local Laboratories.

Laboratory accreditation provides a level of standardization in laboratories and confidence in generated food and feed testing results. For some laboratories, ISO/IEC 17025:2005 accreditation may not be fiscally viable, or a requested test method may be out of the scope of the laboratory's accreditation. To assist laboratories for whom accreditation is not feasible, the Association of Public Health Laboratories Data Acceptance Work Group developed a white paper entitled "Best Practices for Submission of Actionable Food and Feed Testing Data Generated in State and Local Laboratories." The basic elements of a quality management system, along with other best practices that state and local food and feed testing laboratories should follow, are included in the white paper. It also covers program-specific requirements that may need to be addressed. Communication with programs and end data users is regarded as essential for establishing the reliability and accuracy of laboratory data. Following these suggested best practices can facilitate the acceptance of laboratory data, which can result in swift regulatory action and the quick removal of contaminated product from the food supply, improving public health nationally.

Validation of the Applied Biosystems RapidFinder Shiga Toxin-Producing E. coli (STEC) Detection Workflow.

The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant differences were observed between the reference method and the individual or combined kits forming the candidate assay using either of the DNA preparation kits (manual or automated extraction). For the inclusivity and exclusivity evaluation, the RapidFinder STEC Detection Workflow, comprising both RapidFinder STEC screening and confirmation kits, correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains for both of the assays evaluated. The results of these studies demonstrate the sensitivity and selectivity of the RapidFinder STEC Detection Workflow for the detection of E. coli O157:H7 and the "Big 6" STEC serotypes in both raw ground beef and beef trim. The robustness testing demonstrated that minor variations in the method parameters did not impact the accuracy of the assay and highlighted the importance of following the correct incubation temperatures.

Matrix Extension Study: Validation of the Compact Dry CF Method for Enumeration of Total Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" CF method, Performance Tested Method(SM) 110401, was originally certified for enumeration of coliform bacteria by the AOAC Research Institute Performance Tested Methods(SM) program for raw meat products. Compact Dry CF is a ready-to-use dry media sheet, containing a cold-soluble gelling agent, a chromogenic medium, and selective agents, which are rehydrated by adding 1 mL of diluted sample. Coliform bacteria produce blue/blue-green colonies on the Compact Dry CF, allowing a coliform colony count to be determined in the sample after 24 ± 2 h incubation. A validation study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom, to extend the method's claim to include cooked chicken, fresh bagged prewashed shredded iceberg lettuce, frozen fish, milk powder, and pasteurized 2% milk. Campden BRI collected single-laboratory data for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Thirteen laboratories participated in the interlaboratory study. The Compact Dry CF method was compared to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current version at the time this study was conducted. Each matrix was evaluated at either four or five contamination levels of coliform bacteria (including an uncontaminated level). After logarithmic transformation of counts at each level, the data for pasteurized whole milk were analyzed for sr, sR, RSDr, and RSDR. Regression analysis was also performed and r(2) was reported. Mean difference between methods with 95% confidence interval (CI) was calculated. A log10 range of -0.5 to 0.5 for the CI was used as the acceptance criterion to establish significant statistical difference between methods. In the single-laboratory evaluation (for cooked chicken, lettuce, frozen fish, and milk powder), sr and RSDr values were analyzed and r(2) was reported. Statistical differences were indicated between the Compact Dry CF and ISO 4832 methods in two of five contamination levels tested for lettuce, and in the low contamination levels for cooked chicken, frozen fish, and dry milk powder. For the low levels of cooked chicken, frozen fish, and milk powder, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. In most cases, mean differences between the Compact Dry CF and ISO 4832 methods were small (<0.5 log10), with CIs within the acceptance criterion. The sr and RSDr values were similar for both methods, and r(2) values were >0.94 for all matrixes. In the multilaboratory study, no statistical differences were indicated between methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller for the Compact Dry CF. The r(2) value was 0.99. The Compact Dry CF method offers comparable results to ISO 4832 in a space saving, easy-to-use format.

Matrix Extension Study: Validation of the Compact Dry EC Method for Enumeration of Escherichia coli and non-E. coli Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" EC method, originally certified by the AOAC Research Institute Performance Test Method(SM) program for enumeration of Escherichia coli and non-E. coli coliforms in raw meat products (Performance Tested Method(SM) 110402), has undergone an evaluation to extend the method's claim to cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, instant nonfat dry milk powder, and pasteurized milk (2% fat). Compact Dry EC is a ready-to-use dry media sheet containing a cold-soluble gelling agent, selective agents, and a chromogenic medium, which are rehydrated by adding 1 mL diluted sample. E. coli form blue/blue-purple colonies, whereas other coliform bacteria form red/pink colonies. Users can obtain an E. coli count (blue/blue-purple colonies only) and a total coliform count (red/pink plus blue/blue-purple colonies) after 24 ± 2 h of incubation at 37 ± 1°C. The matrix extension study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom. Method comparison data for cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, and instant nonfat dry milk powder were collected in a single-laboratory evaluation by Campden BRI. A multilaboratory study was conducted on pasteurized milk (2% fat), with 13 laboratories participating. The Compact Dry EC method was compared to ISO 16649-2:2001 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli-Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide" and to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current standards at the time of this study. Each matrix was evaluated separately for E. coli and non-E. coli coliforms at each contamination level (including an uncontaminated level). In the single-laboratory evaluation (cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, and instant nonfat dry milk powder), colony counts were logarithmically transformed, and then the data were analyzed at each level for sr, RSDr, and mean difference between methods with 95% confidence intervals (CIs). A CI outside a range of -0.5 to 0.5 on the log10 mean difference between methods was used as the criterion to establish a significant statistical difference. In the multilaboratory study on pasteurized milk, after logarithmic transformation, the data were analyzed for sR and RSDR in addition to sr, RSDr, and mean difference with 95% CIs. Regression analysis was performed on all matrixes and reported as r(2). In the single-laboratory evaluation, statistical differences were indicated between the Compact Dry EC and ISO 16649-2 methods for the enumeration of E. coli in two of five contamination levels tested for lettuce, and in the low contamination level for cooked chicken. For the cooked chicken and lettuce at the low level, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. For the high contamination level of lettuce, counts varied within the sets of five replicates more than 10-fold for each method, which may have contributed to the significant difference. Statistical differences were also indicated between the Compact Dry EC and ISO 4832 methods for the enumeration of coliforms in two of five contamination levels tested for lettuce, two of five contamination levels of milk powder, and in the low contamination level for frozen fish. For the lowest levels of frozen fish and milk powder, only a few colonies were recovered for each method. For the lettuce and the other level of milk powder, counts varied within the sets of five replicates more than 10-fold for each method, which may have contributed to the significant differences indicated in the those contamination levels. In most cases, mean differences between the Compact Dry EC and International Organization of Standardization (ISO) methods were well below 0.5 log10, and the CIs were within the acceptance criterion (-0.5 to 0.5). The sr and RSDr values were similar for both methods, and r(2) values were >0.92 for all comparisons. In the multilaboratory study, no statistical differences were indicated between the methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller in most cases for the Compact Dry EC. The r(2) value was 0.97 in comparison to ISO 16649-2, and 0.99 in comparison to ISO 4832. The Compact Dry EC offers comparable results to the ISO standard plating methods in a space saving, easy-to-use format.

Matrix Extension Study: Validation of the Compact Dry TC Method for Enumeration of Total Aerobic Bacteria in Selected Foods.

A validation study was conducted to extend the matrix claim for the Nissui Compact Dry Total Count (TC), Performance Tested Method(s)(SM) (PTM) Certification No. 010404, to cooked chicken, lettuce, frozen fish, milk powder, and pasteurized whole milk. The method was originally certified by the AOAC Research Institute Performance Tested Method(s)(SM) Program for raw meat products. The Compact Dry TC is a ready-to-use dry media sheet that is rehydrated by adding 1 mL of diluted sample. A total aerobic colony count can be determined in the sample following 48 h of incubation. Matrix extension studies were conducted by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology Limited), Chipping Campden, UK. Single-laboratory data were collected for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Fourteen laboratories participated in the collaborative study. The Compact Dry TC was tested at two time points, 48 ± 3 h and 72 ± 3 h and compared with the current International Organization for Standardization (ISO) method at the time of the study, ISO 4833:2003 (this standard is withdrawn and has been replaced by: ISO 4833-1:2013 and ISO 4833-2:2013), Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of microorganisms-Colony-count technique at 30°C. The data were logarithmically transformed and evaluated for repeatability (plus reproducibility for pasteurized milk), RSD of repeatability (plus RSD of reproducibility for milk), r(2), and mean difference between methods with 95% confidence interval (CI). A CI outside of (-0.5 to 0.5) on the log10 mean difference was used as the criterion to establish significant statistical difference between methods. No significant differences were found between the Compact Dry TC 48 and 72 h time points, with the exception of one contamination level of cooked chicken and one contamination level of dry milk powder. Mean differences were small at these levels (<0.5 log10), but the upper CIs were above 0.5. Statistical differences were indicated between the Compact Dry TC and ISO 4833 in two of five contamination levels tested each for lettuce and frozen fish. In each case, mean differences were >0.5 log10, and the total aerobic colony count was higher for the ISO method. In most cases, mean differences between the Compact Dry and ISO methods were small (<0.5 log10) with CIs within the acceptance criterion. Repeatability, reproducibility, and RSD were similar for both methods, and r(2) values were >0.97 for all matrixes. The Compact Dry TC, at 48 h, offers the advantage of a shorter time to results than ISO 4833 in an easy-to-use format.

Developing a competency framework for U.S. state food and feed testing laboratory personnel.

A competency-based training curriculum framework for U.S. state food and feed testing laboratories personnel is being developed by the International Food Protection Training Institute (IFPTI) and three partners. The framework will help laboratories catalog existing training courses/modules, identify training gaps, inform training curricula, and create career-spanning professional development learning paths, ensuring consistent performance expectations and increasing confidence in shared test results. Ultimately, the framework will aid laboratories in meeting the requirements of ISO/IEC 17025 (2005) international accreditation and the U.S. Food Safety Modernization Act (U.S. Public Law 111-353). In collaboration with the Association of Food and Drug Officials, the Association of Public Health Laboratories, and the Association of American Feed Control Officials, IFPTI is carrying out the project in two phases. In 2013, an expert panel of seven subject matter experts developed competency and curriculum frameworks for five professional levels (entry, mid-level, expert, supervisor/manager, and senior administration) across four competency domains (technical, communication, programmatic, and leadership) including approximately 80 competencies. In 2014 the expert panel will elicit feedback from peers and finalize the framework.

Chicken salad as the source of a case of Listeria monocytogenes infection in Connecticut.

Listeriosis is a severe infection with high morbidity and mortality. We report a fatal case of listeriosis in a patient with a history of Crohn's disease who consumed chicken salad purchased from a retail food establishment before developing listeriosis. As part of the regulatory testing programs, the U.S. Department of Agriculture Food Safety and Inspection Service and the Florida Department of Agriculture and Consumer Affairs found that chicken products from a single food-production establishment were contaminated with Listeria monocytogenes, resulting in a product recall. The case patient's Listeria isolate was subtyped by pulsed-field gel electrophoresis (PFGE) and matched the Listeria isolates from the recalled chicken products. Identification of the source of Listeria involved collaboration among two state public health laboratories and epidemiologists and state and federal regulatory agencies. PFGE typing can be used to reveal correlations between clusters of human illness and contaminated food products and to rapidly identify sources of Listeria infection to allow implementation of corrective actions at both the state and national levels.