U.S. Department Of Agriculture-Food Safety And Inspection Service Research Articles

Combined Quantification and Deep Serotyping for Salmonella Risk Profiling in Broiler Flocks.

Despite a reduction of Salmonella contamination on final poultry products, the level of human salmonellosis cases attributed to poultry has remained unchanged over the last few years. There needs to be improved effort to target serovars which may survive antimicrobial interventions and cause illness, as well as to focus on lessening the amount of contamination entering the processing plant. Advances in molecular enumeration approaches allow for the rapid detection and quantification of Salmonella in pre- and postharvest samples, which can be combined with deep serotyping to properly assess the risk affiliated with a poultry flock. In this study, we collected a total of 160 boot sock samples from 20 broiler farms across four different integrators with different antibiotic management programs. Overall, Salmonella was found in 85% (68/80) of the houses, with each farm having at least one Salmonella-positive house. The average Salmonella quantity across all four complexes was 3.6 log10 CFU/sample. Eleven different serovars were identified through deep serotyping, including all three key performance indicators (KPIs; serovars Enteritidis, Infantis, and Typhimurium) defined by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS). There were eight multidrug resistant isolates identified in this study, and seven which were serovar Infantis. We generated risk scores for each flock based on the presence or absence of KPIs, the relative abundance of each serovar as calculated with CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), and the quantity of Salmonella organisms detected. The work presented here provides a framework to develop directed processing approaches and highlights the limitations of conventional Salmonella sampling and culturing methods. IMPORTANCE Nearly one in five foodborne Salmonella illnesses are derived from chicken, making it the largest single food category to cause salmonellosis and indicating a need for effective pathogen mitigation. Although industry has successfully reduced Salmonella incidence in poultry products, there has not been a concurrent reduction in human salmonellosis linked to chicken consumption. New efforts are focused on improved control at preharvest, which requires improved Salmonella surveillance. Here, we present a high-resolution surveillance approach that combines quantity and identity of Salmonella in broiler flocks prior to processing which will further support improved Salmonella controls in poultry. We developed a framework for this approach, indicating that it is possible and important to harness deep serotyping and molecular enumeration to inform on-farm management practices and to minimize risk of cross-contamination between flocks at processing. Additionally, this framework could be adapted to Salmonella surveillance in other food animal production systems.

Decreased Incidence of Infections Caused by Pathogens Transmitted Commonly Through Food During the COVID-19 Pandemic - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2017-2020.

Foodborne illnesses are a substantial and largely preventable public health problem; before 2020 the incidence of most infections transmitted commonly through food had not declined for many years. To evaluate progress toward prevention of foodborne illnesses in the United States, the Foodborne Diseases Active Surveillance Network (FoodNet) of CDC's Emerging Infections Program monitors the incidence of laboratory-diagnosed infections caused by eight pathogens transmitted commonly through food reported by 10 U.S. sites.* FoodNet is a collaboration among CDC, 10 state health departments, the U.S. Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS), and the Food and Drug Administration. This report summarizes preliminary 2020 data and describes changes in incidence with those during 2017-2019. During 2020, observed incidences of infections caused by enteric pathogens decreased 26% compared with 2017-2019; infections associated with international travel decreased markedly. The extent to which these reductions reflect actual decreases in illness or decreases in case detection is unknown. On March 13, 2020, the United States declared a national emergency in response to the COVID-19 pandemic. After the declaration, state and local officials implemented stay-at-home orders, restaurant closures, school and child care center closures, and other public health interventions to slow the spread of SARS-CoV-2, the virus that causes COVID-19 (1). Federal travel restrictions were declared (1). These widespread interventions as well as other changes to daily life and hygiene behaviors, including increased handwashing, have likely changed exposures to foodborne pathogens. Other factors, such as changes in health care delivery, health care-seeking behaviors, and laboratory testing practices, might have decreased the detection of enteric infections. As the pandemic continues, surveillance of illness combined with data from other sources might help to elucidate the factors that led to the large changes in 2020; this understanding could lead to improved strategies to prevent illness. To reduce the incidence of these infections concerted efforts are needed, from farm to processing plant to restaurants and homes. Consumers can reduce their risk of foodborne illness by following safe food-handling and preparation recommendations.

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.

The Oft Forgotten Part of Nutrition: Lessons from an Integrated Approach to Understand Consumer Food Safety Behaviors

Abstract Objectives A multi-year, integrated methods study showed comprehensive consumer food handling practices when preparing a meal, compared to prevalence of self-reported behaviors. Experimental conditions provided insights on the effectiveness of current and future food safety messages. Methods Demographically-diverse participants were recruited from various communities in one state. Year 1: Participants prepared raw turkey burgers in a simulated home kitchen. Half of participants viewed an educational video on thermometer use before meal preparation; the other half served as a control. Year 2: Participants who self-reported washing poultry prepared raw chicken products. Half of participants received mock social media messages about not washing poultry; the other half served as a control. Food handling behaviors were coded and compared to self-reported actions and attitudes towards food safety, collected via written surveys and in-depth interviews. Results At least one contributing factor to foodborne illness was examined during each observation; cross-contamination actions were always observed. Year 1: Over 60% (n = 383) of participants self-reported owning and using a food thermometer when cooking raw meat and poultry products; however, only 35% of unprompted participants measured final internal temperature with a food thermometer. Year 2: Nearly 25% (n = 300) of participants cross-contaminated a ready-to-eat side; over 50% cross-contaminated due to poor sink sanitation. Differences between self-reported and observed food safety behaviors were seen across participant groups. Lack of sufficient handwashing leading to bacterial cross-contamination was a notable secondary outcome. Conclusions Observations more accurately reflect consumer food handling behaviors and allow for targeted message development to decrease risk of foodborne illness through safe at-home food handling. Findings and messages are shared with health educators, news and social media outlets, and directly to consumers through various resources. Funding Sources This work was funded by the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS). All views expressed here are those of the authors and not necessarily of USDA FSIS. USDA is an equal opportunity provider, employer, and lender.

Surface pH of Fresh Beef as a Parameter To Validate Effectiveness of Lactic Acid Treatment against Escherichia coli O157:H7 and Salmonella

The U.S. beef industry must provide documentation to the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) that the antimicrobial interventions implemented or any subsequent changes in the process are effective under the actual conditions that apply in its operation. The main objective of this study was to determine whether surface pH after application of diluted lactic acid solution on surfaces of fresh meat can be used as a control measure indicator for the reduction of E. coli O157:H7 and Salmonella. Samples (240 each) of lean and adipose beef tissues were inoculated with cocktail mixtures of Escherichia coli O157:H7 and Salmonella. Application parameters were varied such that lean and adipose tissues were spray treated with either 2 or 4.5% lactic acid solution at either 38 or 60°C for 1 to 10 s. Lean and adipose tissues were collected before and after spray treatments for enumeration of the pathogens. Based on the conditions of this study, there was no difference between spray treatments at 38 or 60°C, but 4.5% lactic acid solution reduced pathogens more effectively ( P ≤ 0.05) than did 2% lactic acid solution. Spray treatment with lactic acid solution for 1 to 10 s reduced surface pH values of lean tissues (3 to 3.8) and adipose tissues (2.75 to 3.65). At surface pH values of 3.0 and 2.75, lactic acid reduced E. coli O157:H7 on surfaces of lean and adipose tissues by approximately 1.60 and 1.54 log CFU/cm2, respectively. At surface pH values of 3.8 and 3.65, lactic acid reduced E. coli O157:H7 on lean and adipose tissues by approximately 0.3 and 0.42 log CFU/cm2, respectively. The surface pH values after lactic acid treatment and the reductions of both pathogens showed a strong linear relationship; this indicates that a surface pH of 3.1 would provide at least 1-log reduction of E. coli O157:H7 and Salmonella, regardless of lactic acid application parameters. Therefore, surface pH after spray treatment with lactic acid could be used to validate pathogen reduction.

Protracted Outbreak of Salmonella Newport Infections Linked to Ground Beef: Possible Role of Dairy Cows — 21 States, 2016–2017

In January 2017, CDC identified a cluster of Salmonella enterica serotype Newport infections with isolates sharing an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern, JJPX01.0010 (pattern 10), through PulseNet, the national molecular subtyping network for foodborne disease surveillance. This report summarizes the investigation by CDC, state and local health and agriculture departments, and the U.S. Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) and discusses the possible role of dairy cows as a reservoir for strains of Salmonella that persistently cause human illness. This investigation combined epidemiologic and whole genome sequencing (WGS) data to link the outbreak to contaminated ground beef; dairy cows were hypothesized to be the ultimate source of Salmonella contamination.

Detection and Isolation of the “Top Seven” Shiga Toxin–Producing Escherichia coli in Ground Beef: Comparison of RapidFinder Kits to the U.S. Department of Agriculture Microbiology Laboratory Guidebook Method

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the "top seven" STEC, and these have been declared to be adulterants in beef by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods described in the USDA FSIS Microbiology Laboratory Guidebook (MLG) to a two-stage Applied Biosystems RapidFinder STEC real-time PCR method to test for the top seven STEC in raw ground beef. The specificity of the RapidFinder workflow that targets non-O157 STEC O-antigen genes, stx1, stx2, and eae, and E. coli O157-specific targets was determined with 132 top seven STEC strains and 283 exclusion strains. All inclusion strains were positive, and all exclusion strains gave negative results with the RapidFinder assay. Strains carrying all of the known variants of stx1 and stx2, including stx2f and stx2g, were also detected. For RapidFinder analysis, 375-g ground beef samples spiked with ≥4 CFU of representative STEC strains were enriched in 1 L of tryptic soy broth (TSB) for 10 h at 42 ± 1°C, and for the MLG method, 325-g samples were similarly spiked and enriched in 975 mL of modified TSB for 15 h at 42 ± 1°C. Following DNA extraction, real-time PCR was performed using RapidFinder Express software, and for the MLG method, the BAX Real-Time PCR STEC Suite and the BAX Real-Time E. coli O157:H7 assay were used with the BAX System Q7 software. Following immunomagnetic separation, presumptive colonies from modified Rainbow agar O157 plates were confirmed by the real-time PCR assays. Results of the RapidFinder and BAX assays were similar; all samples were positive after 10 and 15 h of enrichment, respectively. Isolation and confirmation of isolates was possible on all samples, except that two O111:NM strains could not be isolated from a portion of the inoculated samples. Thus, the RapidFinder system can be used for routine and rapid detection of the top seven STEC in beef.

Evaluation of the 3M™ Petrifilm™ Salmonella express system for the detection of Salmonella species in selected foods: collaborative study.

The 3M™ Petriflm™ Salmonella Express (SALX) System is a simple, ready-to-use chromogenic culture medium system for the rapid qualitative detection and biochemical confirmation of Salmonella spp. in food and food process environmental samples. The 3M Petrifilm SALX System was compared using an unpaired study design in a multilaboratory collaborative study to the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) Microbiology Laboratory Guidebook (MLG) 4.07 (2013) Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products and Carcass and Environmental Sponges for raw ground beef and the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) Chapter 5, Salmonella (2011) reference method for dry dog food following the current AOAC validation guidelines. For this study, a total of 17 laboratories located throughout the continental United States evaluated 1872 test portions. For the 3M Petrifilm SALX System, raw ground beef was analyzed using 25 g test portions, and dry dog food was analyzed using 375 g test portions. For the reference methods, 25 g test portions of each inatrix were analyzed. The two matrices were artificially contaminated with Salmonella at three inoculation levels: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). Each inoculation level was statistically analyzed using the probability of detection statistical model. For the raw ground beef and dry dog food test portions, no significant differences at the 95% confidence interval were observed in the number of positive samples detected by the 3M Petrifilm SALX System versus either the USDA/FSIS-MLG or FDA/BAM methods.

Quantification, Serovars, and Antibiotic Resistance of Salmonella Isolated from Retail Raw Chicken Meat in Vietnam

The objectives of this study were to quantify Salmonella counts on retail raw poultry meat in Vietnam and to phenotypically characterize (serovars and antibiotic resistance) the isolates. A total of 300 chicken carcasses were collected from two cities and two provinces in Vietnam. Salmonella counts on the samples were determined according to the most-probable-number (MPN) method of the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). A total of 457 isolates were serotyped and tested for antibiotic susceptibility. Overall, 48.7% of chicken samples were Salmonella positive with a count of 2.0 log MPN per carcass. There were no significant differences (P > 0.05) in log MPN per carcass by the study variables (market type, storage condition, and chicken production system). There was a significant difference (P < 0.05) in Salmonella-positive prevalence by chicken production system. Among the 22 Salmonella serovars identified, Albany was the most frequent (34.1%), followed by Agona (15.5%) and Dabou (8.8%). Resistance to at least one antibiotic was common (i.e., 73.3%), with high resistance to tetracycline (59.1%) and ampicillin (41.6%). Resistance to three antibiotics was the most frequently found multidrug resistance profile (17.7%, n = 81); the profile that was resistant to the highest number of drugs was resistant to nine antibiotics (0.7%, n = 3). Only Salmonella Albany posed phenotypic resistance to ceftriaxone (a drug of choice to treat severe cases of salmonellosis). The data revealed that, whereas Salmonella prevalence on raw poultry was high (48.7%), counts were low, which suggests that the exposure risk to Salmonella is low. However, improper storage of raw chicken meat and cross-contamination may increase Salmonella cell counts and pose a greater risk for infection. These data may be helpful in developing risk assessment models and preventing the transmission of foodborne Salmonella from poultry to humans in Vietnam.

Arsenic Levels in Chicken: Nachman et al. Respond

In our paper (Nachman et al. 2013) we focused on dietary arsenic exposure from the use of arsenic-based drugs in food animal production, specifically chicken. We thank Lasky for broadening the discussion on arsenic regulations for food to include federal agencies beyond the Food and Drug Administration (FDA). The results of our study (Nachman et al. 2013) indicate that the use of arsenic-based drugs increases the levels of inorganic arsenic in chicken meat. Based on these findings, we recommend banning the use of arsenic-based drugs in food animal production, which are under the jurisdiction of the FDA. It is important, however, to recognize the potential role that could be played by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) under its National Residue Program (NRP). Under its mandate, the NRP facilitates the monitoring of arsenic levels in poultry products and supports enforcement actions for animal products in violation of arsenic standards (USDA FSIS 2012). Unfortunately, the NRP faces constraints (in addition to those noted by Lasky) that limit its effectiveness (Silbergeld and Nachman 2008). The most important of these constraints is the current arsenic standard for meat, which was set before 1963 (FDA 1963) and does not account for recent epidemiologic research. In addition, the standard applies to total arsenic concentrations rather than to inorganic arsenic, the species of greatest health relevance. Because arsenic can be present in food in various forms that have widely varying toxicity, standards might need to be species specific. The U.S. Environmental Protection Agency (EPA) is currently revising its toxicological assessment for inorganic arsenic (U.S. EPA 2010) as part of its Integrated Risk Information System (IRIS) program. The purpose of this revision is to produce health-based guidance that can be useful in setting arsenic standards in different media (including foods) that reflect our current understanding of dose–response relationships between arsenic exposures and adverse health outcomes. To achieve this goal, coordination between the FDA, U.S. EPA, and NRP is essential. By applying appropriate standards and methods with adequate sensitivity for the arsenic species of interest, the NRP could play a central role in minimizing dietary exposure to arsenic through animal products. Although sale of roxarsone remains suspended in the United States, nitarsone, a chemically similar arsenical drug, continues to be sold (Zoetis 2013). Industry statements in the media have confirmed nitarsone use in the turkey industry (Aubrey 2013), and the USDA estimates of per capita turkey consumption are increasing (USDA 2013). Research is needed to characterize potential contributions of nitarsone to inorganic arsenic concentrations in turkey meat. For these reasons, monitoring efforts remain relevant. In the absence of regulations that limit inorganic arsenic in our foods, the banning of arsenic-based drugs would minimize dietary arsenic exposures in poultry consumers.

VIDAS® Listeria species Xpress (LSX)

The AOAC GovVal study compared the VIDAS Listeria species Xpress (LSX) to the Health Products and Food Branch MFHPB-30 reference method for detection of Listeria on stainless steel. The LSX method utilizes a novel and proprietary enrichment media, Listeria Xpress broth, enabling detection of Listeria species in environmental samples with the automated VIDAS in a minimum of 26 h. The LSX method also includes the use of the chromogenic media, chromID Ottaviani Agosti Agar (OAA) and chromID Lmono for confirmation of LSX presumptive results. In previous AOAC validation studies comparing VIDAS LSX to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference methods, the LSX method was approved as AOAC Official Method 2010.02 for the detection of Listeria species in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry, and as AOAC Performance Tested Method 100501 in a variety of foods and on environmental surfaces. The GovVal comparative study included 20 replicate test portions each at two contamination levels for stainless steel where fractionally positive results (5-15 positive results/20 replicate portions tested) were obtained by at least one method at one level. Five uncontaminated controls were included. In the stainless steel artificially contaminated surface study, there were 25 confirmed positives by the VIDAS LSX assay and 22 confirmed positives by the standard culture methods. Chi-square analysis indicated no statistical differences between the VIDAS LSX method and the MFHPB-30 standard methods at the 5% level of significance. Confirmation of presumptive LSX results with the chromogenic OAA and Lmono media was shown to be equivalent to the appropriate reference method agars. The data in this study demonstrate that the VIDAS LSX method is an acceptable alternative method to the MFHPB-30 standard culture method for the detection of Listeria species on stainless steel.

Growth Potential of Clostridium perfringens from Spores in Acidified Beef, Pork, and Poultry Products during Chilling

The ability of Clostridium perfringens to germinate and grow in acidified ground beef as well as in 10 commercially prepared acidified beef, pork, and poultry products was assessed. The pH of ground beef was adjusted with organic vinegar to achieve various pH values between 5.0 and 5.6; the pH of the commercial products ranged from 4.74 to 6.35. Products were inoculated with a three-strain cocktail of C. perfringens spores to achieve ca. 2-log (low) or 4-log (high) inoculum levels, vacuum packaged, and cooled exponentially from 54.4 to 7.2°C for 6, 9, 12, 15, 18, or 21 h to simulate abusive cooling; the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) recommends a cooling time of 6.5 h. Total germinated C. perfringens populations were determined after plating on tryptose-sulfite-cycloserine agar and incubating the plates anaerobically at 37°C for 48 h. In addition, C. perfringens growth from spores was assessed at an isothermal temperature of 44°C. Growth from spores was inhibited in ground beef with a pH of 5.5 or below, even during extended cooling from 54.4 to 7.2°C in 21 h. In ground beef with a pH of 5.6, the growth was >1 log after 18 h of cooling from 54.4 to 7.2°C. However, 15 h of cooling controlled the growth to <1 log, regardless of the inoculum level. In addition, no growth was observed in any product with a pH ranging from 4.74 to 5.17, both during exponential abusive cooling periods of up to 21 h and during storage for 21 h at 44°C. While <1-log growth of C. perfringens from spores was observed in the pH 5.63 product cooled exponentially from 54.4 to 7.2°C in 15 h or less, the pH 6.35 product supported growth, even after 6 h of cooling from 54.4 to 7.2°C. These challenge tests demonstrate that adjustment of ground beef to pH of 5.5 or less and of barbeque products to pH of 5.63 or less inhibits C. perfringens spore germination and outgrowth during extended cooling periods from 54.4 to 7.2°C up to 15 h. Therefore, safe cooling periods for products with homogeneous, lower pHs can be substantially longer.

Survival of O157:H7 and Non-O157 Serogroups ofEscherichia coliin Bovine Rumen Fluid and Bile Salts

While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

SDIX RapidChek™ Listeria F.A.S.T.™ Environmental Test System for the Detection of Listeria species on Environmental Surfaces

The SDIX RapidChek Listeria F.A.S.T. test system was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) cultural reference method for the detection of Listeria species on stainless steel, plastic, rubber, and painted concrete. The SDIX method uses a proprietary RapidChek Listeria enrichment media for a one-step, 24-40 h enrichment at 30 degrees C, and detects Listeria on an immunochromatographic lateral flow device in 10 min. Different Listeria species were used to spike each of the environmental surfaces. Environmental surfaces were spiked at levels ranging from 50 to 400 CFU/surface (1 in.2 swabs for painted concrete, 4 in.(2) for sponge). A total of 120 spiked samples were tested by the SDIX method at 24 and 40 h and the cultural reference method. Total confirmed positives were 49, 54, and 48 for the SDIX 24 h method, the SDIX 40 h method, and the USDA-FSIS cultural reference method, respectively. Nonspiked samples from all environmental surfaces were reported as negative for Listeria spp. by all methods. The overall Chi square was 0.017 (P = 0.104) and 0.611 (P= 0.566) after a 24 and 40 h enrichment, respectively, indicating that the test method was equivalent in performance to the reference method at both enrichment times. The SDIX method was evaluated for the detection of 50 Listeria and 35 non-Listeria bacterial strains. All 50 Listeria strains were detected by the method (100% sensitivity). Five out of 35 non-Listeria species gave light test signals when grown in nonselective broth culture and tested undiluted. However, when grown in the RapidChek Listeria F.A.S.T. proprietary media, only one bacterial strain (Staphylococcus aureus) was detected, giving a very low test signal (97% specificity). The method was shown to be robust toward several alterations in testing and storage conditions.

Reveal®Listeria 2.0 Test for Detection of Listeria spp. in Foods and Environmental Samples

A Performance Tested Method validation study was conducted for a new lateral flow immunoassay (Reveal Listeria 2.0) for detection of Listeria spp. in foods and environmental samples. Results of inclusivity testing showed that the test detects all species of Listeria, with the exception of L. grayi. In exclusivity testing conducted under nonselective growth conditions, all non-listeriae tested produced negative Reveal assay results, except for three strains of Lactobacillus spp. However, these lactobacilli are inhibited by the selective Listeria Enrichment Single Step broth enrichment medium used with the Reveal method. Six foods were tested in parallel by the Reveal method and the U.S. Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) reference culture procedure. Considering data from both internal and independent laboratory trials, overall sensitivity of the Reveal method relative to that of the FDA/BAM procedure was 101%. Four foods were tested in parallel by the Reveal method and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedure. Overall sensitivity of the Reveal method relative to that of the USDA-FSIS procedure was 98.2%. There were no statistically significant differences in the number of positives obtained by the Reveal and reference culture procedures in any food trials. In testing of swab or sponge samples from four types of environmental surfaces, sensitivity of Reveal relative to that of the USDA-FSIS reference culture procedure was 127%. For two surface types, differences in the number of positives obtained by the Reveal and reference methods were statistically significant, with more positives by the Reveal method in both cases. Specificity of the Reveal assay was 100%, as there were no unconfirmed positive results obtained in any phase of the testing. Results of ruggedness experiments showed that the Reveal assay is tolerant of modest deviations in test sample volume and device incubation time.

Validation of a quantitative and confirmatory method for residue analysis of aminoglycoside antibiotics in poultry, bovine, equine and swine kidney through liquid chromatography-tandem mass spectrometry

The use of aminoglycoside antibiotics in food animals is approved in Brazil. Accordingly, Brazilian food safety legislation sets maximum levels for these drugs in tissues from these animals in an effort to guarantee that food safety is not compromised. Aiming to monitor the levels of these drugs in tissues from food animals, the validation of a quantitative, confirmatory method for the detection of residues of 10 aminoglycosides antibiotics in poultry, swine, equine and bovine kidney, with extraction using a solid phase and detection and quantification by LC-MS/MS was performed. The procedure is an adaptation of the US Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) qualitative method, with the inclusion of additional clean-up and quantification at lower levels, which proved more efficient. Extraction was performed using a phosphate buffer containing trifluoroacetic acid followed by neutralization, purification on a cationic exchange SPE cartridge, with elution with methanol/acetic acid, evaporation, and dilution in ion-pair solvent. The method was validated according to the criteria and requirements of the European Commission Decision 2002/657/EC, showing selectivity with no matrix interference. Linearity was established for all analytes using the method of weighted minimum squares. CCα and CCβ varied between 1036 and 12,293 µg kg−1, and between 1073 and 14,588 µg kg−1, respectively. The limits of quantification varied between 27 and 688 µg kg−1. The values of recovery for all analytes in poultry kidney, fortified in the range of 500–1500 µg kg−1, were higher than 90%, and the relative standard deviations were lower than 15%, except spectinomycin (21.8%). Uncertainty was estimated using a simplified methodology of ‘bottom-up’ and ‘top-down’ strategies. The results showed that this method is effective for the quantification and confirmation of aminoglycoside residues and could be used by the Brazilian programme of residue control.

Alternative cooling procedures for large, intact meat products to achieve stabilization microbiological performance standards.

Achieving the U. S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) stabilization microbiological performance standards for cooling procedures proves to be challenging for processors of large, whole-muscle meat products. This study was conducted to determine if slower cooling times than those provided by USDA-FSIS guidance will comply with the performance standard for Clostridium perfringens. Large (9 to 12 kg) cured bone-in hams (n = 110) and large (8 to 13 kg) uncured beef inside rounds (n = 100) were used. Stabilization treatments extended times to reduce internal product temperature from 54.4 to 26.7°C (hams and rounds) and from 26.7 to 7.2°C (for hams) and 26.7 to 4.4°C (for rounds). Control treatments, defined by current USDA-FSIS Appendix B guidelines, and a "worst-case scenario" treatment, in which products were cooled at room temperature (approximately 22.8°C) until internal product temperature equilibrated, were used. For both hams and rounds, stabilization showed less than 1-log growth of C. perfringens for all treatments, with the exception of the worst-case scenario for rounds. As expected for products cooled at room temperature, there was >1-log growth of C. perfringens reported for rounds, and the addition of curing ingredients to hams had an inhibitory effect on the growth of C. perfringens. The results demonstrate that industry may have increased flexibility associated with cooling large, whole-muscle cuts while still complying with the required stabilization microbiological performance standards.

USDA FSIS, FDA BAM, AOAC, and ISO Culture Methods BD BBL CHROMagar Listeria Media

BBL CHROMagar Listeria Media (CL) was evaluated for detection of Listeria monocytogenes in raw ground beef, smoked salmon, lettuce, and Brie cheese. The recovery of L. monocytogenes on CL was compared to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM), U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS), AOAC, and International Organization for Standardization (ISO) reference-plated media using the recommended pre-enrichments and selective enrichments. Of the 265 food samples tested, 140 were tested using BAM, USDA, or AOAC methods and 125 were tested using ISO methods. CL produced comparable results with the reference methods on all matrixes with a sensitivity of 99.3% and a specificity of 100%. No false negatives were found in testing the food matrixes. There was no statistical difference in recovery based on Chi-square analysis. Known isolates were evaluated, and CL had a sensitivity and specificity of 100%. The results of this study demonstrate that CL is an effective medium for the recovery and detection of L. monocytogenes in raw ground beef, smoked salmon, lettuce, and Brie cheese using FDA BAM, USDA FSIS, AOAC, and ISO culture methods.

USDA FSIS, FDA BAM, and ISO Culture Methods BD BBL CHROMagar O157 Media

BBL CHROMagar O157 media (CO) was evaluated for detection of Escherichia coli O157:H7 in raw ground beef and unpasteurized apple cider. The recovery of E. coli O157:H7 on CO was compared to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM), U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS), and International Organization for Standardization (ISO) reference-plated media using the recommended enrichment broths. Of the 180 food samples tested, 45 were tested using BAM, 45 using the USDA method, and 90 using the ISO method. CO produced comparable results with the reference methods on all matrixes with a sensitivity of 100% and a specificity of 100%. No false negatives were found in testing the food matrixes. There was no statistical difference in recovery based on Chi-square analysis. Method agreement for raw ground beef was 85% for the USDAFSIS method and 95% for the ISO method. Method agreement for unpasteurized apple cider was 100% for the ISO and FDA BAM methods. In all cases where method agreement was <100%, CO detected more positives than the reference method media. Evaluation of known isolates on CO in inclusivity and exclusivity testing had a sensitivity and specificity of 100%. The results of this study demonstrate that CO is an effective medium for the recovery and detection of E. coli O157:H7 in raw ground beef and unpasteurized apple cider using FDA BAM, USDA FSIS, and ISO methods.

Validation of a Modification to Performance-Tested MethodSM 070601: Reveal® Listeria Test for Detection of Listeria spp. in Selected Foods and Selected Environmental Samples

A modification to Performance-Tested Method (PTM) 070601, Reveal Listeria Test (Reveal), is described. The modified method uses a new media formulation, LESS enrichment broth, in single-step enrichment protocols for both foods and environmental sponge and swab samples. Food samples are enriched for 27-30 h at 30 degrees C and environmental samples for 24-48 h at 30 degrees C. Implementation of these abbreviated enrichment procedures allows test results to be obtained on a next-day basis. In testing of 14 food types in internal comparative studies with inoculated samples, there was a statistically significant difference in performance between the Reveal and reference culture [U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM) or U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS)] methods for only a single food in one trial (pasteurized crab meat) at the 27 h enrichment time point, with more positive results obtained with the FDA/BAM reference method. No foods showed statistically significant differences in method performance at the 30 h time point. Independent laboratory testing of 3 foods again produced a statistically significant difference in results for crab meat at the 27 h time point; otherwise results of the Reveal and reference methods were statistically equivalent. Overall, considering both internal and independent laboratory trials, sensitivity of the Reveal method relative to the reference culture procedures in testing of foods was 85.9% at 27 h and 97.1% at 30 h. Results from 5 environmental surfaces inoculated with various strains of Listeria spp. showed that the Reveal method was more productive than the reference USDA-FSIS culture procedure for 3 surfaces (stainless steel, plastic, and cast iron), whereas results were statistically equivalent to the reference method for the other 2 surfaces (ceramic tile and sealed concrete). An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the Reveal method at the 24 h time point. Overall, sensitivity of the Reveal method at 24 h relative to that of the USDA-FSIS method was 153%. The Reveal method exhibited extremely high specificity, with only a single false-positive result in all trials combined for overall specificity of 99.5%.