J. N. Martin

Use of Metagenomic Shotgun Sequencing Technology To Detect Foodborne Pathogens within the Microbiome of the Beef Production Chain

ABSTRACT Foodborne illnesses associated with pathogenic bacteria are a global public health and economic challenge. The diversity of microorganisms (pathogenic and nonpathogenic) that exists within the food and meat industries complicates efforts to understand pathogen ecology. Further, little is known about the interaction of pathogens within the microbiome throughout the meat production chain. Here, a metagenomic approach and shotgun sequencing technology were used as tools to detect pathogenic bacteria in environmental samples collected from the same groups of cattle at different longitudinal processing steps of the beef production chain: cattle entry to feedlot, exit from feedlot, cattle transport trucks, abattoir holding pens, and the end of the fabrication system. The log read counts classified as pathogens per million reads for Salmonella enterica, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Clostridium spp. (C. botulinum and C. perfringens), and Campylobacter spp. (C. jejuni, C. coli, and C. fetus) decreased over subsequential processing steps. Furthermore, the normalized read counts for S. enterica, E. coli, and C. botulinum were greater in the final product than at the feedlots, indicating that the proportion of these bacteria increased (the effect on absolute numbers was unknown) within the remaining microbiome. From an ecological perspective, data indicated that shotgun metagenomics can be used to evaluate not only the microbiome but also shifts in pathogen populations during beef production. Nonetheless, there were several challenges in this analysis approach, one of the main ones being the identification of the specific pathogen from which the sequence reads originated, which makes this approach impractical for use in pathogen identification for regulatory and confirmation purposes.

National Beef Quality Audit–2016: In-plant survey of carcass characteristics related to quality, quantity, and value of fed steers and heifers

The National Beef Quality Audit (NBQA)-2016 used in-plant cooler assessments to benchmark the current status of the fed steer and heifer beef industry in the United States. In-plant cooler assessments ( = 9,106 carcasses) were conducted at 30 facilities, where approximately 10% of a single days production were evaluated for USDA quality grade (QG) and yield grade (YG) factors. Frequencies of evaluated traits were 66.5% steer and 33.4% heifer sex classes and 82.9% native, 15.9% dairy-type, and 1.2% estimated breed types. Mean USDA YG factors were 1.42 cm for adjusted fat thickness, 89.5 cm for LM area, 390.3 kg for HCW, and 1.9% for KPH. Mean USDA YG was 3.1, with a frequency distribution of 9.6% YG 1, 36.7% YG 2, 39.2% YG 3, 12.0% YG 4, and 2.5% YG 5. Mean USDA QG traits were Small for marbling score, A for overall maturity, A55 for lean maturity, and A for skeletal maturity. Mean USDA QG was Select with a frequency distribution of QG of 3.8% Prime, 67.3% Choice, 23.2% Select, and 5.6% lower score. Lower score included dark cutter (1.9%), blood splash (0.1%), and hard bone, which are USDA overall maturity scores of C or older (1.8%). Marbling score distributions were 0.85% Slightly Abundant or greater, 7.63% Moderate, 23.54% Modest, 39.63% Small, 23.62% Slight, and 0.83% Traces or less. Carcasses that were Choice or Select and USDA YG 2 or 3 accounted for 70.7% of the carcasses evaluated. Compared with the previous NBQA, we found a numerical increase in mean USDA YG, USDA QG, adjusted fat thickness, HCW, LM area, and marbling score with an increase in dairy-type carcasses and percentage of carcasses grading USDA Prime and Choice as well as frequency of USDA YG 4 and 5. The findings from this study will be used by all segments of the industry to understand and improve the quality of fed steer and heifer beef that is being produced.

Antimicrobial interventions for O157:H7 and non-O157 Shiga toxin-producing Escherichia coli on beef subprimal and mechanically tenderized steaks.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) is an emerging risk for food safety. Although numerous postharvest antimicrobial interventions have been effectively used to control E. coli O157:H7 during beef harvesting, research regarding their effectiveness against non-O157 STEC is scarce. The objectives of this study were (i) to evaluate effects of the spray treatments-ambient water, 5% lactic acid (LA), 200 ppm of hypobromous acid (HA), and 200 ppm of peroxyacetic acid (PA)-on the reduction of O157:H7 or non-O157 STEC (O26, O103, O111, and O145) with high (10(6) log CFU/50 cm(2)) or low (10(2) log CFU/50 cm(2)) levels on beef subprimals after vacuum storage for 14 days and (ii) to evaluate the association of the antimicrobial treatments and cooking (50 or 70°C) on the reduction of the pathogens in blade-tenderized steaks. The treatment effects were only observed (P = 0.012) on samples taken immediately after spray intervention treatment following inoculation with a high level of O157:H7. The LA and PA treatments significantly reduced low-inoculated non-O157 STEC after spray intervention; further, the LA and HA treatments resulted in significant reductions of non-O157 STEC on the low-inoculated samples after storage. Although cooking effectively reduced the detection of pathogens in internal steak samples, internalized E. coli O157:H7 and non-O157 STEC were able to survive in steaks cooked to a medium degree of doneness (70°C). This study indicated that the reduction on surface populations was not sufficient enough to eliminate the pathogens detection following vacuum storage, mechanical tenderization, and cooking. Nevertheless, the findings of this study emphasize the necessity for a multihurdle approach and further investigations of factors that may influence thermal tolerance of internalized pathogenic STEC.

National Beef Quality Audit–2016: Transportation, mobility, live cattle, and carcass assessments of targeted producer-related characteristics that affect value of market cows and bulls, their carcasses, and associated by-products

Abstract The National Beef Quality Audit–2016 marks the fourth iteration in a series assessing the quality of live beef and dairy cows and bulls and their carcass counterparts. The objective was to determine the incidence of producer-related defects, and report cattle and carcass traits associated with producer management. Conducted from March through December of 2016, trailers (n = 154), live animals (n = 5,470), hide-on carcasses (n = 5,278), and hide-off hot carcasses (n = 5,510) were surveyed in 18 commercial packing facilities throughout the United States. Cattle were allowed 2.3 m2 of trailer space on average during transit indicating some haulers are adhering to industry handling guidelines for trailer space requirements. Of the mixed gender loads arriving at processing facilities, cows and bulls were not segregated on 64.4% of the trailers surveyed. When assessed for mobility, the greatest majority of cattle surveyed were sound. Since the inception of the quality audit series, beef cows have shown substantial improvements in muscle. Today over 90.0% of dairy cows are too light muscled. The mean body condition score for beef animals was 4.7 and for dairy cows and bulls was 2.6 and 3.3, respectively. Dairy cattle were lighter muscled, yet fatter than the dairy cattle surveyed in 2007. Of cattle surveyed, most did not have horns, nor any visible live animal defects. Unbranded hides were observed on 77.3% of cattle. Carcass bruising was seen on 64.1% of cow carcasses and 42.9% of bull carcasses. However, over half of all bruises were identified to only be minor in severity. Nearly all cattle (98.4%) were free of visible injection-site lesions. Current results suggest improvements have been made in cattle and meat quality in the cow and bull sector. Furthermore, the results provide guidance for continued educational and research efforts for improving market cow and bull beef quality.

National Beef Quality Audit-2016: Transportation, mobility, and harvest-floor assessments of targeted characteristics that affect quality and value of cattle, carcasses, and by-products

Abstract The National Beef Quality Audit-2016 (NBQA-2016) was conducted to assess current transportation, mobility, and quality characteristics of U.S. fed steers and heifers. Data were collected at 17 beef processing facilities between March and November 2016. About 8,000 live cattle were evaluated for transportation and mobility, and about 25,000 carcasses were evaluated on the slaughter floor. Cattle were in transit to the slaughter facility for a mean duration of 2.7 h from a mean distance of 218.5 km using trailers with dimensions ranging from 17.84 m2 to 59.09 m2. Area allotted per animal averaged 1.13 m2 and ranged from 0.85 m2 to 2.28 m2. A total of 96.8% of cattle received a mobility score of 1 (walks easily, no apparent lameness). Identification types (35.1% had multiple) were lot visual tags (61.5%), individual tags (55.0%), electronic tags (16.9%), metal-clip tags (9.2%), bar-coded tags (0.05%), wattles (0.01%), and other (2.6%). Cattle were black-hided (57.8%), Holstein (20.4%), red-hided (10.5%), yellow-hided (4.8%), gray-hided (2.9%), brown-hided (1.3%), and white-hided (1.1%). Unbranded hides were observed on 74.3% of cattle; 18.6% had brands located on the butt, 6.3% on the side, and 1.3% on the shoulder (values exceed 100% due to multiple brands). For hide-on carcasses, 37.7% displayed no mud or manure; specific locations for mud or manure were legs (40.8%), belly (33.0%), tail region (15.5%), side (6.8%), and top-line (3.9%). Cattle without horns represented 83.3% of the sample, and cattle that did have horns measured: < 2.54 cm (5.5%), 2.54 to 12.7 cm (8.3%), and > 12.7 cm (2.9%). Carcasses without bruises represented 61.1% of those sampled, whereas 28.2% had 1, 8.2% had 2, 2.1% had 3, and 0.3% had 4 bruises. Of those carcasses with a bruise, the bruise was located on the loin (29.7%), round (27.8%), chuck (16.4%), rib (14.4%), and brisket/plate/flank (11.6%). Frequencies of offal condemnations were livers (30.8%), lungs (18.2%), viscera (16.3%), hearts (11.1%), heads (2.7%), and tongues (2.0%). Compared to NBQA-2011, fewer cattle were identified for traceability, fewer were black-hided, a greater number were Holstein cattle, more with no brand and no horns, fewer without bruises, more liver, lung, and viscera condemnations, and fewer heads and tongues were condemned. The NBQA remains an influential survey for the U.S. beef industry to provide benchmarks and strategic plans for continued improvement of beef quality and consistency.

Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing

BackgroundShotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for <u20091% of all sequenced DNA, leading to limitations in detection of low-abundance resistome-virulome elements. This study describes the extent and composition of the low-abundance portion of the resistome-virulome, using a bait-capture and enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias.ResultsThe use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins.ConclusionsThese results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

National Beef Quality Audit – 2016: Survey of carcass characteristics through instrument grading assessments

The instrument grading assessment portion of the National Beef Quality Audit (NBQA) - 2016 allows the unique opportunity to evaluate beef carcass traits over the course of a year. One week of instrument grading data was collected each month from 5 beef processing corporations encompassing 18 facilities from January 2016 through December 2016 ( = 4,544,635 carcasses). Mean USDA yield grade (YG) was 3.1 with 1.37 cm fat thickness (FT), 88.9 cm LM area, 393.6 kg HCW, and 2.1% KPH. Frequency distribution of USDA YG was 9.5% YG 1, 34.6% YG 2, 38.8% YG 3, 14.6% YG 4, and 2.5% YG 5. Increases in HCW and FT since the NBQA-2011 were major contributors to differences in mean YG and the (numerically) increased frequency of YG 3, 4, and 5 carcasses found in the current audit. Mean marbling score was Small, and the distribution of USDA quality grades was 4.2% Prime, 71.4% Choice, 21.7% Select, and 2.7% other. Frequency of carcasses grading Prime on Monday (6.43%) was numerically higher than the average frequency of carcasses grading Prime overall (4.2%). Monthly HCW means were 397.6 kg in January, 397.2 kg in February, 396.5 kg in March, 389.3 kg in April, 384.8 kg in May, 385.0 kg in June, 386.1 kg in July, 394.1 kg in August, 399.1 kg in September, 403.9 kg in October, 406.5 kg in November, and 401.9 kg in December. Monthly mean marbling scores were Small in January, Small in February, Small in March, Small in April, Small in May, Small in June, Small in July, Small in August, Small in September, Small in October, Small in November, and Small in December. Both mean HCW and mean marbling score declined in the months of May and June. The month with the greatest numerical frequency of dark cutters was October (0.74%). Comparison of overall data from in-plant carcass and instrument grading assessments revealed close alignment of information, especially for YG (3.1 for in-plant assessment versus 3.1 for instrument grading) and marbling (Small for in-plant assessment versus Small for instrument grading). These findings allow the beef industry access to the greatest volume of beef value-determining characteristics for the U.S. fed steer and heifer population than ever reported, resulting in potentially more precise targeting of future quality and consistency efforts.

National Beef Quality Audit–2016: assessment of cattle hide characteristics, offal condemnations, and carcass traits to determine the quality status of the market cow and bull beef industry

Abstract To continue the series that began in 1994, the National Beef Quality Audit (NBQA) – 2016 was conducted to quantify the quality status of the market cow and bull beef sector, as well as determine improvements made in the beef and dairy industry since 2007. The NBQA-2016 was conducted from March through December of 2016, and assessed hide-on carcasses (n = 5,278), chilled carcasses (n = 4,285), heads (n = 5,720), and offal items (n = 4,800) in 18 commercial processing facilities throughout the United States. Beef cattle were predominantly black-hided; 68.0% of beef cows and 67.2% of beef bulls possessed a black hide. Holstein was the predominant type of dairy animal observed. Just over half (56.0%) of the cattle surveyed had no mud contamination on the hide, and when mud was present, 34.1% of cattle only had small amounts. Harvest floor assessments found 44.6% of livers, 23.1% of lungs, 22.3% of hearts, 20.0% of viscera, 8.2% of heads, and 5.9% of tongues were condemned. Liver condemnations were most frequently due to abscess presence. In contrast, contamination was the primary reason for condemnation of all other offal items. Of the cow carcasses surveyed, 17.4% carried a fetus at the time of harvest. As expected, mean carcass weight and loin muscle area values observed for bulls were heavier and larger than cows. The marbling scores represented by cull animal carcasses were most frequently slight and traces amounts. Cow carcasses manifested a greater amount of marbling on average than bull carcasses. The predominant fat color score showed all carcasses surveyed had some level of yellow fat. Only 1.3% of carcasses exhibited signs of arthritic joints. Results of the NBQA-2016 indicate there are areas in which the beef and dairy industries have improved and areas that still need attention to prevent value loss in market cows and bulls.

Salmonella Contamination in Broiler Synovial Fluid: Are We Missing a Potential Reservoir?

The objective of this study was to assess the presence and characteristics of Salmonella enterica found in the synovial fluid of broiler carcasses. The synovial fluid of three individual joints from 500 broiler carcasses was individually sampled from five broiler processing facilities located in the Southeast and West regions of the United States (1,500 total samples). The external surface of broiler carcass was decontaminated before sampling of the shoulder, coxofemoral, and tibiofemoral joints. Individual samples were enriched, composited, and subjected to rapid PCR-based detection of Salmonella. Individual samples from any positive composites were also enriched before determination of Salmonella presence in the same manner. Positive individual samples were subjected to secondary enrichment before plating onto selective agar for isolation of Salmonella. Salmonella isolates were serotyped before determination of antimicrobial susceptibility. Overall, 1.00% (5 of 500 broiler carcasses) of composite samples and 0.47% (7 of 1,500 samples) of individual samples were positive for Salmonella. Five of the seven isolates were susceptible to all drugs tested and determined to be Salmonella Enteritidis. The remaining two isolates, identified as Salmonella Typhimurium, were resistant to streptomycin. To our knowledge, no previous assessments of Salmonella in the synovial fluid of broilers has been reported; however, results of the present study suggested that the synovial fluid may be a reservoir for Salmonella in broilers. Although the prevalence of Salmonella is low, this information provides valuable insight into potential poultry contamination pathways and warrants further exploration.

National Beef Quality Audit-2016: Phase 1, Face-to-face interviews

Abstract The National Beef Quality Audit (NBQA) is conducted every 5 yr and was most recently again conducted in 2016. Face-to-face interviews gauged progress in quality associated with live cattle production using procedures first utilized in NBQA 2011. The 2016 NBQA was the first in which interviews concerning fed steers and heifers were combined with an audit of market cow and bull beef. Face-to-face interviews were designed to illicit definitions for beef quality, estimate willingness to pay (WTP) for quality attributes, establish relative importance rankings for important quality factors, and assess images, strengths, weaknesses, potential threats, and shifting trends in the beef industry since the 2011 audit. Individuals making purchasing decisions in 5 market sectors of the steer/heifer and cow/bull beef supply chain were interviewed, including packers (n = 36), retailers (including large and small supermarket companies and warehouse food sales companies; n = 35), food service operators (including quick-serve, full-service, and institutional establishments; n = 29), further processors (n = 64), and peripherally-related government and trade organizations (GTO; n = 30). Face-to-face interviews were conducted between January and November of 2016 using a designed dynamic routing system. Definitions (as described by interviewees) for 7 pre-determined quality factors, including: (1) How and where the cattle were raised, (2) Lean, fat, and bone, (3) Weight and size, (4) Visual characteristics, (5) Food safety, (6) Eating satisfaction, and (7) Cattle genetics were recorded verbatim and categorized into similar responses for analysis. Compared to NBQA-2011, a higher percentage of companies were willing to pay premiums for guaranteed quality attributes, but overall were willing to pay lower average premiums than the companies interviewed in 2011. Food safety had the highest share of preference among all interviewees, generating a double-digit advantage over any other quality factor. The 2 beef industries have an overall positive image among interviewees, and despite lingering weaknesses, product quality continued to be at the forefront of the strengths category for both steer and heifer beef and market cow and bull beef.