Microbiome surrounding death and decay, The: microbial ecology of food processing, meat spoilage, and human decomposition environments

2021 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

Comparison of Salmonella Presence in Bovine Lymph Nodes across Feeding Stages

Peripheral lymph nodes (LNs) located in the fatty tissues of beef carcasses have been shown to harbor Salmonella, and thus are a potential source of contamination in beef trimmings. Salmonella prevalence within LNs differs among feedlots, although the sources and routes of transmission have yet to be confirmed. The objective of this study was to determine if Salmonella prevalence in bovine LNs varies across feeding stages and feedlot environments in South Texas. Two feedlots with historically different levels of Salmonella prevalence within bovine LNs were selected. Twenty steers were to be harvested at each of 4 feeding stages: (1) post-weaning, (2) stocker, (3) 60 d on feed, and (4) 120 d on feed. Four steers did not complete the study. Left and right subiliac and superficial cervical LNs were collected from each carcass (n = 304), and similar node types were pooled by animal (n = 152). Salmonella, if present, was isolated from the LNs following the USDA-FSIS Microbiological Laboratory Guidebook (MLG) 4.08. Presumptive positive isolates were confirmed using PCR, and isolates were submitted for serotyping. Results showed a difference (P < 0.05) in prevalence of Salmonella in bovine lymph nodes between feedlots and among feeding stages. There was no Salmonella isolated from LN samples taken after feeding stage 1 (weaning) or from Feedlot A. Within feedlot B, there was an increase in Salmonella prevalence as cattle moved into later stages of feeding, at 22.2% (4/18), 77.8% (14/18), and 94.4% (17/18) for feeding stages 2, 3, and 4, respectively. There was a difference (P < 0.05) in LN Salmonella prevalence between stages 2 and 3, and a numerical difference between stages 3 and 4. It appears there is an environmental effect that influences the prevalence of Salmonella in LNs. The cause of these differences is unknown, and provides opportunity for future investigation into pre-harvest environmental conditions relating to Salmonella exposures

Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies

Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics—in addition to the traditional considerations when conducting an animal science study—makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type

Microbiome Data Accurately Predicts the Postmortem Interval Using Random Forest Regression Models

Death investigations often include an effort to establish the postmortem interval (PMI) in cases in which the time of death is uncertain. The postmortem interval can lead to the identification of the deceased and the validation of witness statements and suspect alibis. Recent research has demonstrated that microbes provide an accurate clock that starts at death and relies on ecological change in the microbial communities that normally inhabit a body and its surrounding environment. Here, we explore how to build the most robust Random Forest regression models for prediction of PMI by testing models built on different sample types (gravesoil, skin of the torso, skin of the head), gene markers (16S ribosomal RNA (rRNA), 18S rRNA, internal transcribed spacer regions (ITS)), and taxonomic levels (sequence variants, species, genus, etc.). We also tested whether particular suites of indicator microbes were informative across different datasets. Generally, results indicate that the most accurate models for predicting PMI were built using gravesoil and skin data using the 16S rRNA genetic marker at the taxonomic level of phyla. Additionally, several phyla consistently contributed highly to model accuracy and may be candidate indicators of PMI

Air versus Water Chilling of Chicken: a Pilot Study of Quality, Shelf-Life, Microbial Ecology, and Economics.

The United States' large-scale poultry meat industry is energy and water intensive, and opportunities may exist to improve sustainability during the broiler chilling process. By USDA regulation, after harvest the internal temperature of the chicken must be reduced to 40°F or less within 16 h to inhibit bacterial growth that would otherwise compromise the safety of the product. This step is accomplished most commonly by water immersion chilling in the United States, while air chilling methods dominate other global markets. A comprehensive understanding of the differences between these chilling methods is lacking. Therefore, we assessed the meat quality, shelf-life, microbial ecology, and techno-economic impacts of chilling methods on chicken broilers in a university meat laboratory setting. We discovered that air chilling methods resulted in superior chicken odor and shelf-life, especially prior to 14 days of dark storage. Moreover, we demonstrated that air chilling resulted in a more diverse microbiome that we hypothesize may delay the dominance of the spoilage organism Pseudomonas Finally, a techno-economic analysis highlighted potential economic advantages to air chilling compared to water chilling in facility locations where water costs are a more significant factor than energy costs.IMPORTANCE As the poultry industry works to become more sustainable and to reduce the volume of food waste, it is critical to consider points in the processing system that can be altered to make the process more efficient. In this study, we demonstrate that the method used during chilling (air versus water chilling) influences the final product microbial community, quality, and physiochemistry. Notably, the use of air chilling appears to delay the bloom of Pseudomonas spp. that are the primary spoilers in packaged meat products. By using air chilling to reduce carcass temperatures instead of water chilling, producers may extend the time until spoilage of the products and, depending on the cost of water in the area, may have economic and sustainability advantages. As a next step, a similar experiment should be done in an industrial setting to confirm these results generated in a small-scale university lab facility

Vacuum Packaging Maintains Fresh Characteristics of Previously Frozen Beef Steaks during Simulated Retail Display

The impact of frozen storage on beef steaks prior to the retail setting may result in changes to the quality and safety of the packaged meat. Therefore, the objective of the current study was to evaluate fresh characteristics on previously frozen steaks during a simulated retail display. Steaks were allocated to one of three packaging treatments (MB, MDF, MFS) and stored frozen (−13 °C) for 25 days in the absence of light. After thawing, steaks were stored in a lighted retail case at 3 °C and evaluated for instrumental surface color, pH, purge loss, lipid oxidation, and microbial spoilage organisms throughout the 25-day fresh display period. There was an increase (p p p < 0.05) throughout the entire display for steaks packaged in MFS and MDF. It is evident that barrier properties of MB limiting oxygen exposure of the steak preserved fresh meat characteristics after frozen storage. Results from the current study suggest that vacuum packaging films can aid in retarding detrimental effects caused by frozen storage after placing the steaks in fresh retail conditions

A conserved interdomain microbial network underpins cadaver decomposition despite environmental variables.

Microbial breakdown of organic matter is one of the most important processes on Earth, yet the controls of decomposition are poorly understood. Here we track 36 terrestrial human cadavers in three locations and show that a phylogenetically distinct, interdomain microbial network assembles during decomposition despite selection effects of location, climate and season. We generated a metagenome-assembled genome library from cadaver-associated soils and integrated it with metabolomics data to identify links between taxonomy and function. This universal network of microbial decomposers is characterized by cross-feeding to metabolize labile decomposition products. The key bacterial and fungal decomposers are rare across non-decomposition environments and appear unique to the breakdown of terrestrial decaying flesh, including humans, swine, mice and cattle, with insects as likely important vectors for dispersal. The observed lockstep of microbial interactions further underlies a robust microbial forensic tool with the potential to aid predictions of the time since death

Experiences and lessons learned from two virtual, hands-on microbiome bioinformatics workshops.

In October of 2020, in response to the Coronavirus Disease 2019 (COVID-19) pandemic, our team hosted our first fully online workshop teaching the QIIME 2 microbiome bioinformatics platform. We had 75 enrolled participants who joined from at least 25 different countries on 6 continents, and we had 22 instructors on 4 continents. In the 5-day workshop, participants worked hands-on with a cloud-based shared compute cluster that we deployed for this course. The event was well received, and participants provided feedback and suggestions in a postworkshop questionnaire. In January of 2021, we followed this workshop with a second fully online workshop, incorporating lessons from the first. Here, we present details on the technology and protocols that we used to run these workshops, focusing on the first workshop and then introducing changes made for the second workshop. We discuss what worked well, what didn't work well, and what we plan to do differently in future workshops