Effect of beef long-storage under different temperatures and vacuum-packaging conditions on meat quality, oxidation processes and microbial growth.

BACKGROUND: The global beef market demands the meat industry to ensure product quality and safety in markets that are often very distant. The present study aimed to evaluate the effects of chilled (CH, 120 d) and chilled-then-frozen (CHF, 28 d + 92 d) storage conditions of beef vacuum packaged (VP) and vacuum packaged with antimicrobial (VPAM) on meat quality, oxidative status and microbial loads. Treatments resulted from the combination of storage condition and packaging type: VP + CH, VP + CHF, VPAM + CH and VPAM + CHF. RESULTS: Warner-Bratzler shear force values decreased in all treatments after 28 d of chilling. Except for VP + CH, L* values (lightness) of meat color did not differ in each treatment as the storage time increased. Meat from VP + CH had greater a* values than CHF treatments on day 120 of storage. A consumer panel did not detect differences in tenderness, flavor and overall liking between VP and VPAM beef, but they preferred CHF steaks rather than CH beef. TBARS values did not differ between VP and VPAM and between CH and CHF at any time during the storage period. At the end of storage time, all treatments except VP + CHF presented a greater concentration of thiols than at 48 h post-mortem. On day 120 of storage, VP + CH had greater catalase enzyme activity than CHF treatments while VP + CH and VP + CHF showed a greater superoxide dismutase activity than VPAM + CHF. Storage condition (CH or CHF) had a greater impact on microbial counts than the type of packaging. CONCLUSION: Freezing meat after an ageing period represents a suitable strategy to extend beef storage life without a detrimental impact on its quality. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Supplement feed efficiency of growing beef cattle grazing native Campos grasslands during winter: a collated analysis.

Supplementing growing cattle grazing native subtropical Campos grasslands during winter improves the low, even negative, average daily weight gain (ADG) typical of extensive animal production systems in Uruguay. Nonetheless, to render the practice profitable, it is crucial to control supplement feed efficiency (SFE), that is, the difference in ADG between supplemented and control animals (ADGchng) per unit of supplement dry matter (DM) intake. Little has been studied specifically on how SFE varies in these systems. The objective of this study was to quantify the magnitude and variation in SFE of growing beef cattle grazing stockpiled native Campos grasslands during winter and assess putative associations with herbage, animals, supplements, and climatic variables. We compiled data from supplementation trials carried out in Uruguay between 1993 and 2018, each evaluating between one and six supplementation treatments. The average ADG of unsupplemented and supplemented animals were 0.13 ±â€…0.174 and 0.49 ±â€…0.220 kg/animal/day, respectively. In both cases, ADG decreased linearly as the proportion of green herbage in the grazed grassland was lower, but the ADG of unsupplemented animals was further reduced when winter frosts were numerous. Estimated SFE were moderately high, with an average of 0.21 ±â€…0.076 ADGchng/kg DM, resulting from average ADGchng of 0.38 ±â€…0.180 kg/animal/day in response to an average supplementation rate of 1.84 ±â€…0.68 kg supplement DM intake/animal/day (0.86%  ±â€…0.27% body weight). No association was found between SFE and supplementation rate or type (protein vs. energy-based; P > 0.05), but forage allowance negatively affected it, and herbage mass positively affected it, yet in a smaller magnitude, suggesting that a balance is needed between the two to maximize SFE. Weather conditions during trials affected SFE (P < 0.05), with greater SFE in winters with lower temperatures and more frosts. Daytime grazing time was consistently lower in supplemented animals compared to their unsupplemented counterparts, whereas ruminating time during the day was similar, increasing as the proportion of green herbage decreased. Herbage intake estimated from energy balance suggested the existence of some substitution effect. This agrees with the moderately high SFE and with the total digestible nutrients-to-protein ratio of these subtropical humid grasslands being higher than in semi-arid rangelands and dry-season tropical pastures but lower than in sown pastures.

Short-Term Impact of Oxytetracycline Administration on the Fecal Microbiome, Resistome and Virulome of Grazing Cattle.

Antimicrobial resistance (AMR) is an important public health concern around the world. Limited information exists about AMR in grasslands-based systems where antibiotics are seldom used in beef cattle. The present study investigated the impacts of oxytetracycline (OTC) on the microbiome, antibiotic resistance genes (ARGs), and virulence factor genes (VFGs) in grazing steers with no previous exposure to antibiotic treatments. Four steers were injected with a single dose of OTC (TREAT), and four steers were kept as control (CONT). The effects of OTC on fecal microbiome, ARGs, and VFGs were assessed for 14 days using 16S rRNA sequencing and shotgun metagenomics. Alpha and beta microbiome diversities were significantly affected by OTC. Following treatment, less than 8% of bacterial genera had differential abundance between CONT and TREAT samples. Seven ARGs conferring resistance to tetracycline (tet32, tet40, tet44, tetO, tetQ, tetW, and tetW/N/W) increased their abundance in the post-TREAT samples compared to CONT samples. In addition, OTC use was associated with the enrichment of macrolide and lincosamide ARGs (mel and lnuC, respectively). The use of OTC had no significant effect on VFGs. In conclusion, OTC induced short-term alterations of the fecal microbiome and enrichment of ARGs in the feces of grazing beef cattle.

Microbiological Changes during Long-Storage of Beef Meat under Different Temperature and Vacuum-Packaging Conditions.

We evaluated a combination of two temperatures and two packaging materials for long-term storage of vacuum-packaged (VP) beef striploins. Microbial populations and microbiome composition were monitored during refrigerated storage (120 days between 0-1.5 °C) and refrigerated-then-frozen storage (28 days between 0-1.5 °C then 92 days at -20 °C) under low-O2 permeability VP and high-O2 permeability VP with an antimicrobial (VPAM). Pseudomonas (PSE) and Enterobacteriaceae (EB) counts in VPAM samples were significantly higher (p < 0.05) than in VP samples at 28, 45, 90, and 120 days of storage. Microbiome data showed that bacteria of the genera Serratia and Brochothrix were more abundant in VPAM samples at 120 days, while lactic acid bacteria (LAB) dominated in VP samples. Frozen temperatures inhibited microbial growth and maintained a relatively stable microbiome. Refrigerated and frozen VPAM samples showed the greatest difference in the predicted metabolic functions at the end of storage driven by the microbiome composition, dominated by PSE and LAB, respectively. Although no signs of visible meat deterioration were observed in any sample, this study suggests that VP meat refrigerated and then frozen achieved better microbiological indicators at the end of the storage period.

Key traits for ruminant livestock across diverse production systems in the context of climate change: perspectives from a global platform of research farms.

Ruminant livestock are raised under diverse cultural and environmental production systems around the globe. Ruminant livestock can play a critical role in food security by supplying high-quality, nutrient-dense food with little or no competition for arable land while simultaneously improving soil health through vital returns of organic matter. However, in the context of climate change and limited land resources, the role of ruminant-based systems is uncertain because of their reputed low efficiency of feed conversion (kilogram of feed required per kilogram of product) and the production of methane as a by-product of enteric fermentation. A growing human population will demand more animal protein, which will put greater pressure on the Earth's planetary boundaries and contribute further to climate change. Therefore, livestock production globally faces the dual challenges of mitigating emissions and adapting to a changing climate. This requires research-led animal and plant breeding and feeding strategies to optimise ruminant systems. This study collated information from a global network of research farms reflecting a variety of ruminant production systems in diverse regions of the globe. Using this information, key changes in the genetic and nutritional approaches relevant to each system were drawn that, if implemented, would help shape more sustainable future ruminant livestock systems.

A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; A Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification.

This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI's nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.

Characterization of the Microbial Resistome in Conventional and "Raised Without Antibiotics" Beef and Dairy Production Systems.

Metagenomic investigations have the potential to provide unprecedented insights into microbial ecologies, such as those relating to antimicrobial resistance (AMR). We characterized the microbial resistome in livestock operations raising cattle conventionally (CONV) or without antibiotic exposures (RWA) using shotgun metagenomics. Samples of feces, wastewater from catchment basins, and soil where wastewater was applied were collected from CONV and RWA feedlot and dairy farms. After DNA extraction and sequencing, shotgun metagenomic reads were aligned to reference databases for identification of bacteria (Kraken) and antibiotic resistance genes (ARGs) accessions (MEGARes). Differences in microbial resistomes were found across farms with different production practices (CONV vs. RWA), types of cattle (beef vs. dairy), and types of sample (feces vs. wastewater vs. soil). Feces had the greatest number of ARGs per sample (mean = 118 and 79 in CONV and RWA, respectively), with tetracycline efflux pumps, macrolide phosphotransferases, and aminoglycoside nucleotidyltransferases mechanisms of resistance more abundant in CONV than in RWA feces. Tetracycline and macrolide-lincosamide-streptogramin classes of resistance were more abundant in feedlot cattle than in dairy cow feces, whereas the ß-lactam class was more abundant in dairy cow feces. Lack of congruence between ARGs and microbial communities (procrustes analysis) suggested that other factors (e.g., location of farms, cattle source, management practices, diet, horizontal ARGs transfer, and co-selection of resistance), in addition to antimicrobial use, could have impacted resistome profiles. For that reason, we could not establish a cause-effect relationship between antimicrobial use and AMR, although ARGs in feces and effluents were associated with drug classes used to treat animals according to farms' records (tetracyclines and macrolides in feedlots, ß-lactams in dairies), whereas ARGs in soil were dominated by multidrug resistance. Characterization of the "resistance potential" of animal-derived and environmental samples is the first step toward incorporating metagenomic approaches into AMR surveillance in agricultural systems. Further research is needed to assess the public-health risk associated with different microbial resistomes.

Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period.

The objective was to examine effects of treating commercial beef feedlot cattle with therapeutic doses of tulathromycin, a macrolide antimicrobial drug, on changes in the fecal resistome and microbiome using shotgun metagenomic sequencing. Two pens of cattle were used, with all cattle in one pen receiving metaphylaxis treatment (800 mg subcutaneous tulathromycin) at arrival to the feedlot, and all cattle in the other pen remaining unexposed to parenteral antibiotics throughout the study period. Fecal samples were collected from 15 selected cattle in each group just prior to treatment (Day 1), and again 11 days later (Day 11). Shotgun sequencing was performed on isolated metagenomic DNA, and reads were aligned to a resistance and a taxonomic database to identify alignments to antimicrobial resistance (AMR) gene accessions and microbiome content. Overall, we identified AMR genes accessions encompassing 9 classes of AMR drugs and encoding 24 unique AMR mechanisms. Statistical analysis was used to identify differences in the resistome and microbiome between the untreated and treated groups at both timepoints, as well as over time. Based on composition and ordination analyses, the resistome and microbiome were not significantly different between the two groups on Day 1 or on Day 11. However, both the resistome and microbiome changed significantly between these two sampling dates. These results indicate that the transition into the feedlot-and associated changes in diet, geography, conspecific exposure, and environment-may exert a greater influence over the fecal resistome and microbiome of feedlot cattle than common metaphylactic antimicrobial drug treatment.

Effects of Ceftiofur and Chlortetracycline on the Resistomes of Feedlot Cattle.

Treatment of food-producing animals with antimicrobial drugs (AMD) is controversial because of concerns regarding promotion of antimicrobial resistance (AMR). To investigate this concern, resistance genes in metagenomic bovine fecal samples during a clinical trial were analyzed to assess the impacts of treatment on beef feedlot cattle resistomes. Four groups of cattle were exposed, using a 2-by-2 factorial design, to different regimens of antimicrobial treatment. Injections of ceftiofur crystalline-free acid (a third-generation cephalosporin) were used to treat all cattle in treatment pens or only a single animal, and either chlortetracycline was included in the feed of all cattle in a pen or the feed was untreated. On days 0 and 26, respectively, pre- and posttrial fecal samples were collected, and resistance genes were characterized using shotgun metagenomics. Treatment with ceftiofur was not associated with changes to ß-lactam resistance genes. However, cattle fed chlortetracycline had a significant increase in relative abundance of tetracycline resistance genes. There was also an increase of an AMR class not administered during the study, which is a possible indicator of coselection of resistance genes. Samples analyzed in this study had previously been evaluated by culture characterization (Escherichia coli and Salmonella) and quantitative PCR (qPCR) of metagenomic fecal DNA, which allowed comparison of results with this study. In the majority of samples, genes that were selectively enriched through culture and qPCR were not identified through shotgun metagenomic sequencing in this study, suggesting that changes previously documented did not reflect changes affecting the majority of bacterial genetic elements found in the predominant fecal resistome.IMPORTANCE Despite significant concerns about public health implications of AMR in relation to use of AMD in food animals, there are many unknowns about the long- and short-term impact of common uses of AMD for treatment, control, and prevention of disease. Additionally, questions commonly arise regarding how to best measure and quantify AMR genes in relation to public health risks and how to determine which genes are most important. These data provide an introductory view of the utility of using shotgun metagenomic sequencing data as an outcome for clinical trials evaluating the impact of using AMD in food animals.

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

BACKGROUND: Shotgun 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 < 1% 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. RESULTS: The 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. CONCLUSIONS: These 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.

Impact of "Raised without Antibiotics" Beef Cattle Production Practices on Occurrences of Antimicrobial Resistance.

The specific antimicrobial resistance (AMR) decreases that can be expected from reducing antimicrobial (AM) use in U.S. beef production have not been defined. To address this data gap, feces were recovered from 36 lots of "raised without antibiotics" (RWA) and 36 lots of "conventional" (CONV) beef cattle. Samples (n = 719) were collected during harvest and distributed over a year. AMR was assessed by (i) the culture of six AM-resistant bacteria (ARB), (ii) quantitative PCR (qPCR) for 10 AMR genes (ARGs), (iii) a qPCR array of 84 ARGs, and (iv) metagenomic sequencing. Generally, AMR levels were similar, but some were higher in CONV beef cattle. The prevalence of third-generation cephalosporin-resistant (3GCr) Escherichia coli was marginally different between production systems (CONV, 47.5%; RWA, 34.8%; P = 0.04), but the seasonal effect (summer, 92.8%; winter, 48.3%; P < 0.01) was greater. Erythromycin-resistant (ERYr) Enterococcus sp. concentrations significantly differed between production systems (CONV, 1.91 log10 CFU/g; RWA, 0.73 log10 CFU/g; P < 0.01). Levels of aadA1, ant(6)-I, bla ACI, erm(A), erm(B), erm(C), erm(F), erm(Q), tet(A), tet(B), tet(M), and tet(X) ARGs were higher (P < 0.05) in the CONV system. Aggregate abundances of all 43 ARGs detected by metagenomic sequencing and the aggregate abundances of ARGs in the aminoglycoside, ß-lactam, macrolide-lincosamide-streptogramin B (MLS), and tetracycline AM classes did not differ (log2 fold change < 1.0) between CONV and RWA systems. These results suggest that further reductions of AM use in U.S. beef cattle production may not yield significant AMR reductions beyond MLS and tetracycline resistance.IMPORTANCE The majority of antimicrobial (AM) use in the United States is for food-animal production, leading to concerns that typical AM use patterns during "conventional" (CONV) beef cattle production in the United States contribute broadly to antimicrobial resistance (AMR) occurrence. In the present study, levels of AMR were generally similar between CONV and "raised without antibiotics" (RWA) cattle. Only a limited number of modest AMR increases was observed in CONV cattle, primarily involving macrolide-lincosamide-streptogramin B (MLS) and tetracycline resistance. Macrolides (tylosin) and tetracyclines (chlortetracycline) are administered in-feed for relatively long durations to reduce liver abscesses. To ensure judicious AM use, the animal health, economic, and AMR impacts of shorter duration in-feed administration of these AMs should be examined. However, given the modest AMR reductions observed, further reductions of AM use in U.S. beef cattle production may not yield significant AMR reductions beyond MLS and tetracycline resistance.

MEGARes: an antimicrobial resistance database for high throughput sequencing.

Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database.

Microbial populations on hides of grazing steers in a forage-based production system in Uruguay.

The objective of the study was to evaluate the microbiological status of hides of grazing steers in a typical forage-based system in Uruguay. The study was conducted on a single farm with samples taken on 3 days during the spring of 2007. Four anatomical hide sites (perineum area, flank, back, and shoulder) of 10 steers were individually swabbed each sampling day at the farm environment (n = 120). Each sample was analyzed by the Laboratorio Tecnológico del Uruguay for aerobic plate counts (APC), total coliform counts (TCC), and Escherichia coli counts (ECC). Mean log values for APC, TCC, and ECC on external animal hide surfaces, across all sampling sites, were 5.52, 1.89, and 1.70 log CFU/cm2, respectively. There were no significant differences among bacterial counts from the four hide surface locations. Mean log values for APC, TCC, and ECC were 1.49, 1.15, and 1.12 log CFU/cm2 lower, respectively, on sampling day 2 than on sampling day 3. Microbial populations on hides of grazing steers are highly variable and dependent on climatic and environmental conditions. To our knowledge this is the first study published evaluating the hygienic conditions of grazing livestock operations in Uruguay and their potential implications on the red meat chain.

Laparoscopía estadificadora en cáncer gástrico / Laparoscopic staging for gastric cancer

Antecedentes: Los métodos de estadificación como la Ecografía y la Tomografía presentan una limitada exactitud diagnóstica. El advenimiento de la laparoscopia y su rol en la estadificación tumoral ha cambiado el algoritmo de estudio en muchos centros. Objetivo: evaluar la estatificación laparoscopica y su incidencia en los cambios terapéuticos. Lugar de aplicación: Servicio de Cirugía Hospital Escuela Diseño: Prospectivo Población: pacientes con diagnóstico histopatológico de cáncer gástrico. Método: Se correlacionó la clínica, estatificación preoperatoria, laparoscopia estadificadora y la cirugía realizada. Se agrupó a los pacientes en: potencialmente resecables y con alta sospecha de irresecabilidad. A todos se les realizó laparoscopia estadificadora. Resultados: Los 11 pacientes potencialmente resecables se consideraron un estadio II mientras que, en los pacientes con alta sospecha de irresecabilidad, dos fueron un estadio III y uno un estadio IV. La estatificación laparoscopica, cambió de un estadio II a un estadío IV en 50%. La laparoscopia estadificadora modificó la estrategia terapéutica y evito la laparotomía innecesaria en 6 de 15 pacientes (40 %). Conclusión: La laparoscopia estadificadora permite el cambio de conducta terapéutica en un impor tante porcentaje de casos. Este cambio en la estadificación, lograda con la exploración laparoscópica, evita laparotomías innecesarias y sus evidentes consecuencias.