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Recent work has demonstrated that cancer-specific microbial communities often colonize tumor tissues. However, untangling low-biomass signals from environmental contamination makes this research technically challenging. We utilize pseudomyxoma peritonei (PMP), a cancer characterized by the spread of mucus-secreting cells throughout the peritoneal cavity, to develop a robust workflow for identifying reproducible tumor microbiomes. Typically originating from the rupture of an appendiceal tumor into the peritoneal cavity, metastasized tumors have been previously shown to harbor a core set of microbes. However, that work did not control for the potential contamination of these low microbial biomass samples. We expand upon these prior findings by characterizing the microbiome of 70 additional PMP tumors and six normal peritoneal control tissues along with appropriate laboratory controls. Additionally, DNA from a subset of 25 tissues was extracted and sequenced at an independent laboratory. We found evidence of reproducible microbial signatures between the replicates of six different PMP tumors that include a set of core taxa that may be introduced from surgical contamination, as well as patient-specific taxa that are also commonly implicated in colorectal cancer. In addition, preoperative chemotherapy treatment was found to reduce tumor microbiome diversity. Our findings demonstrate how independent sample replication can be a powerful approach to investigate low-biomass microbial communities associated with tumor tissues that will improve low microbial biomass research.IMPORTANCERecent work has demonstrated that microbial communities colonize over 30 different types of tumor tissues. The origin of these communities and their possible involvement in carcinogenesis or cancer treatment outcomes remains an unclear, yet important area of research. A current major challenge in characterizing low-biomass, tumor-associated microbiomes is the introduction of environmental contamination during collection, handling, DNA extraction, PCR, and sequencing. Here, we provide a framework for replicating low-biomass tumor microbiome samples to help identify tumors with robust microbial signals and low background contamination. Using this replication approach, we show that pseudomyxoma peritonei (PMP) tumors host reproducible microbial communities, including organisms that have previously been associated with colorectal cancer. Incorporating sample replication into future tumor microbiome studies is a promising approach that will help identify robust signals and increase reproducibility in the field.

The microbiome of dairy calves undergoes extensive change due to various forces during the first weeks of life. Importantly, diseases such as bovine respiratory disease (BRD) and calf diarrhea can have profound impacts on the early-life microbiome. Therefore, a longitudinal, repeated-measures pilot study was designed to characterize the establishment of nasal and fecal microbiomes of dairy calves, assess the governing forces of microbial assembly, and evaluate how disease states impact these microbial ecologies. Dairy calves ( n = 19) were clinically evaluated for gastrointestinal and respiratory disease across three weeks beginning at age ≤ seven days old. Fecal ( n = 57) and nasal ( n = 57) microbial samples were taken for paired-end 16S rRNA gene amplicon sequencing. Taxonomy and diversity analyses were used to characterize early-life nasal and fecal microbiomes. Stochasticity and determinism were measured using normalized stochasticity testing (NST) and Dirichlet multinomial model (DMM). All analyses were tested for statistical significance. Clinical diarrhea was observed in 11 of the 19 calves. Clinical BRD was not independently observed among the cohort; however, two calves presented clinical signs of both BRD and diarrhea. Taxonomic analysis revealed that fecal samples were highlighted by Bacteroidaceae (40%; relative abundance), Ruminococcaceae (13%), and Lachnospiraceae (10%), with changes in diversity (Kruskal-Wallis; p < 0.05) and composition (PERMANOVA; p < 0.05). Clinical diarrhea reduced diversity in the fecal microbiome but did not impact composition. Nasal samples featured Moraxellaceae (49%), Mycoplasmataceae (16%), and Pasteurellaceae (3%). While no diversity changes were seen in nasal samples, compositional changes were observed ( p < 0.05). NST metrics (Kruskal-Wallis; p > 0.01) and DMM (PERMANOVA; p < 0.01) revealed that stochastic, neutral theory-based assembly dynamics govern early-life microbial composition and that distinct microbial populations drive community composition in healthy and diarrheic calves.

In 2020, we identified cancer-specific microbial signals in The Cancer Genome Atlas (TCGA) [1]. Multiple peer-reviewed papers independently verified or extended our findings [2-12]. Given this impact, we carefully considered concerns by Gihawi et al. [13] that batch correction and database contamination with host sequences artificially created the appearance of cancer type-specific microbiomes. (1) We tested batch correction by comparing raw and Voom-SNM-corrected data per-batch, finding predictive equivalence and significantly similar features. We found consistent results with a modern microbiome-specific method (ConQuR [14]), and when restricting to taxa found in an independent, highly-decontaminated cohort. (2) Using Conterminator [15], we found low levels of human contamination in our original databases (~1% of genomes). We demonstrated that the increased detection of human reads in Gihawi et al. [13] was due to using a newer human genome reference. (3) We developed Exhaustive, a method twice as sensitive as Conterminator, to clean RefSeq. We comprehensively host-deplete TCGA with many human (pan)genome references. We repeated all analyses with this and the Gihawi et al. [13] pipeline, and found cancer type-specific microbiomes. These extensive re-analyses and updated methods validate our original conclusion that cancer type-specific microbial signatures exist in TCGA, and show they are robust to methodology., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Obesity and metabolic dysfunction have been shown to be associated with overproduction of reactive oxygen species (ROS) in the gastrointestinal (GI) tract, which contributes to dysbiosis or imbalances in the gut microbiota. Recently, the reversal of dysbiosis has been observed as a result of dietary supplementation with antioxidative compounds including polyphenols. Likewise, dietary polyphenols have been associated with scavenging of GI ROS, leading to the hypothesis that radical scavenging in the GI tract is a potential mechanism for the reversal of dysbiosis. The objective of this study was to investigate the relationship between GI ROS, dietary antioxidants and beneficial gut bacterium Akkermansia muciniphila . The results of this study demonstrated A. muciniphila to be a discriminant microorganism between lean ( n = 7) and obese ( n = 7) mice. The relative abundance of A. muciniphila was also found to have a significant negative correlation with extracellular ROS in the GI tract as measured using fluorescent probe hydroindocyanine green. The ability of the dietary antioxidants ascorbic acid, β-carotene and grape polyphenols to scavenge GI ROS was evaluated in tandem with their ability to support A. muciniphila bloom in lean mice ( n = 20). While the relationship between GI ROS and relative abundance of A. muciniphila was conserved in lean mice, only grape polyphenols stimulated the bloom of A. muciniphila . Analysis of fecal antioxidant capacity and differences in the bioavailability of the antioxidants of interest suggested that the poor bioavailability of grape polyphenols contributes to their superior radical scavenging activity and support of A. muciniphila in comparison to the other compounds tested. These findings demonstrate the utility of the GI redox environment as a modifiable therapeutic target in the treatment of chronic inflammatory diseases like metabolic syndrome.

Associations between age and the human microbiota are robust and reproducible. The microbial composition at several body sites can predict human chronological age relatively accurately. Although it is largely unknown why specific microorganisms are more abundant at certain ages, human microbiota research has elucidated a series of microbial community transformations that occur between birth and death. In this Review, we explore microbial succession in the healthy human microbiota from the cradle to the grave. We discuss the stages from primary succession at birth, to disruptions by disease or antibiotic use, to microbial expansion at death. We address how these successions differ by body site and by domain (bacteria, fungi or viruses). We also review experimental tools that microbiota researchers use to conduct this work. Finally, we discuss future directions for studying the microbiota's relationship with age, including designing consistent, well-powered, longitudinal studies, performing robust statistical analyses and improving characterization of non-bacterial microorganisms., (© 2022. Springer Nature Limited.)

Liver abscess etiology in feedlot steers involves the escape of bacteria from the digestive tract to form a polymicrobial abscess within or on the external surface of the liver. However, little is known about the effects of feedlot finishing systems on the microbial composition of the liver abscess purulent material. Liver abscesses were collected at the time of harvest from steers originating from a single feedlot managed in either a traditional program (which included tylosin phosphate supplementation) or a natural program (without tylosin phosphate supplementation). The purulent material of liver abscesses from traditionally managed steers (N = 53 abscesses) and that of naturally managed steers (N = 62 abscesses) was characterized using the V4 region of the 16S rRNA gene. Two phyla and three genera were found in greater than 1% relative abundance across all abscesses. The genus Fusobacterium was identified in all liver abscess samples and accounted for 64% of sequencing reads. Bacteroides and Porphyromonas genera accounted for 33% and 1% of reads, respectively. Trueperella was more likely to be found in the liver abscesses of naturally managed steers than traditionally managed steers (P = 0.022). Over 99% of the genus-level bacterial sequences observed across all liver abscesses belonged to Gram-negative genera. Bacteria known to colonize both the rumen and hindgut were identified within liver abscesses. No differences in alpha diversity or beta diversity were detected between liver abscess communities (between the two management programs or individual pens) when tested as richness, Shannon Diversity Index, or weighted UniFrac distances (P > 0.05). These results were consistent with previous identification of Fusobacterium necrophorum as the primary bacteriologic agent within liver abscesses and emphasized the relationship between the gastrointestinal microbiota and liver abscess formation. Though the microbiota of the liver abscess purulent material was similar between steers fed an antibiotic-free diet and those fed an antibiotic-containing diet from the same feedlot, divergence was detected in liver abscess communities with some being dominated by Fusobacterium and others being dominated by Bacteroides., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

During decomposition, flies interact with the remains to lay eggs and acquire nutrients, and in the process, they bring their microbes with them. While it is known that flies have their own unique core microbiome, it is not known if flies associated with human cadavers have a different core microbiome. Differences in the fly microbiome may influence the types of microbes transmitted from the flies to the cadaver, therefore potentially affecting assembly of the human decomposer microbiome. The first purpose of this study was to characterize the microbiome of flies associated with human cadavers by fly organ and season. This is because fly interactions with cadavers vary by season, and because it is likely that external fly organs [i.e., the labellum and tarsi] make more direct contact and are likely involved in increased mechanical transmission with the cadaver than internal organs such as the oocyte. The second purpose of this study was to determine if the fly microbes contribute to the human decomposer microbiome. To accomplish these aims, 10 human cadavers were placed outdoors across three seasons and allowed to decompose. A total of 40 flies that landed on the cadaver were collected and dissected by the labellum, tarsi, and oocyte. In addition to fly collections, samples from the cadavers were collected using a sterile swab at sites including the cheek of the face, inner cheek, bicep, torso, and anus. Overall, it was shown that flies associated with human cadavers have a similar microbiome to flies from previous studies that were not associated with human cadavers. However, there are differences in the microbiome between seasons and fly parts. We also show evidence that flies act as a microbial source to the human decomposer microbiome, which is important for understanding the ecological mechanisms of human cadaver microbial community assembly., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Microbial communities which persist in food processing facilities may have a detrimental impact on food safety and spoilage. In meat processing, Listeria monocytogenes is an organism of concern due to its ability to cause significant human illnesses and persist in refrigerated environments. The microbial ecology of Listeria spp. in small meat processing facilities has not been well characterized. Therefore, we collected samples from a newly constructed meat processing facility as an opportunity to investigate several research objectives: (i) to determine whether a stable, consistent microbiome develops in a small meat processing facility during the first 18 months of operation, (ii) to evaluate the environmental factors that drive microbial community formation, and (iii) to elucidate the relationship between microbial communities and the presence of Listeria species. We evaluated microbiomes using 16S rRNA gene sequencing and Listeria presence using quantitative PCR. We demonstrated that microbial communities differentiate by the functional room type, which is representative of several environmental differences such as temperature, sources of microbes, and activity. Temperature was an especially important factor; in rooms with low temperatures, communities were dominated by psychotrophs, especially Pseudomonas, while warmer rooms supported greater diversity. A stable core community formed in facility drains, indicating that mechanisms which cause persistence are present in the communities. The overall presence of Listeria in the facility was low but could be tied to specific organisms within a room, and the species of Listeria could be stratified by room function. IMPORTANCE This study provides critical knowledge to improve meat safety and quality from small meat processing facilities. Principally, it demonstrates the importance of facility design and room condition to the development of important microbial communities; temperature, sanitation regimen, and physical barriers all influence the ability of microorganisms to join the stable core community. It also demonstrates a relationship between the microbial community and Listeria presence in the facility, showing the importance of managing facility sanitation plans for not only pathogens, but also the general facility microbiome.

Group B Streptococcus (GBS) in the vaginal tract is a risk factor for preterm birth and adverse pregnancy outcomes. GBS colonization is also transient in nature, which likely reflects the contributions of pathogen determinants, interactions with commensal flora, and host factors, making this environment particularly challenging to understand. Additionally, dietary zinc deficiency is a health concern on the global scale that is known to be associated with recurrent bacterial infection and increased rate of preterm birth or stillbirth. However, the impact of zinc deficiency on vaginal health has not yet been studied. Here we use a murine model to assess the role of dietary zinc on GBS burden and the impact of GBS colonization on the vaginal microbiome. We show that GBS vaginal colonization is increased in a zinc-deficient host and that the presence of GBS significantly alters the microbial community structure of the vagina. Using machine learning approaches, we show that vaginal community turnover during GBS colonization is driven by computationally predictable changes in key taxa, including several organisms not previously described in the context of the vaginal microbiota, such as Akkermansia muciniphila. We observed that A. muciniphila increases GBS vaginal persistence and, in a cohort of human vaginal microbiome samples collected throughout pregnancy, we observed an increased prevalence of codetection of GBS and A. muciniphila in patients who delivered preterm compared to those who delivered at full term. These findings reveal the importance and complexity of both host zinc availability and native microbiome to GBS vaginal persistence. IMPORTANCE The presence of group B Streptococcus (GBS) in the vaginal tract, perturbations in the vaginal microbiota, and dietary zinc deficiency are three factors that are independently known to be associated with increased risk of adverse pregnancy outcomes. Here, we developed an experimental mouse model to assess the impact of dietary zinc deficiency on GBS vaginal burden and persistence and to determine how changes in GBS colonization impact vaginal microbial structure. We have employed unique animal, in silica metabolic, and machine learning models, paired with analyses of human cohort data, to identify taxonomic biomarkers that contribute to host susceptibility to GBS vaginal persistence. Collectively, the data reported here identify that both dietary zinc deficiency and the presence of A. muciniphila could perpetuate an increased GBS burden and prolonged exposure in the vaginal tract, which potentiate the risk of invasive infection in utero and in the newborn.

Ruminants are a critical human food source and have been implicated as a potentially important source of global methane emissions. Because of their unique digestive physiology, ruminants rely upon a symbiotic relationship with the complex and rich community of microorganism in the foregut to allow digestion of complex carbohydrates. This study used 16S rRNA gene sequencing to investigate the composition of microbial communities from three rumen micro-environments of cattle fed identical diets: (1) free fluid, (2) the fibrous pack, and (3) the mucosa. Community composition analysis revealed that while a phylogenetic core including the most abundant and most common ruminal taxa (members of Bacteroidetes and Firmicutes) existed across micro-environments, the abundances of these taxa differed significantly between fluid- and mucosa-associated communities, and specific lineages were discriminant of individual micro-environments. Members of Firmicutes, specifically Clostridiales, Lachnospiraceae, Mogibacteriaceae, Christenellaceae, and Erysipelotrichaceae were significantly more abundant in fluid communities, while members of Bacteroidetes, namely Muribaculaceae and Prevotellaceae were more abundant in mucosa-associated communities. Additionally, Methanobacteriaceae, a family of methanogenic Archaea, was more abundant in fluid-associated communities. A set of four more diverse lineages were discriminant of pack-associated communities that included Succinivibrionaceae, RFP12 (Verruco-5), Fibrobacteraceae, and Spirochaetaceae. Our findings indicate that different ecological niches within each micro-environment have resulted in significant differences in the diversity and community structure of microbial communities from rumen fluid, pack, and mucosa without the influence of diet that will help contextualize the influence of other environmental factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinnell, Reyes, Wolfe, Weinroth, Metcalf, Delmore, Belk, Morley and Engle.)

Background: The potential to distribute bacteria resistant to antimicrobial drugs in the meat supply is a public health concern. Market cows make up a fifth of the U.S. beef produced but little is known about the entire population of bacteria (the microbiome) and entirety of all resistance genes (the resistome) that are found in this population. The objective of this study was to characterize and compare the resistomes and microbiome of beef, dairy, and organic dairy market cows at slaughter., Methods: Fifty-four (N = 54) composite samples of both colon content and meat trimmings rinsate samples were collected over six visits to two harvest facilities from cows raised in three different production systems: conventional beef, conventional dairy, and organic dairy (n = 3 samples per visit per production system). Metagenomic DNA obtained from samples were analyzed using target-enriched sequencing (resistome) and 16S rRNA gene sequencing (microbiome)., Results: All colon content samples had at least one identifiable antimicrobial resistance gene (ARG), while 21 of the 54 meat trimmings samples harbored at least one identifiable ARGs. Tetracycline ARGs were the most abundant class in both colon content and carcass meat trimmings. The resistome found on carcass meat trimmings was not significantly different by production system (P = 0.84, R 2  = 0.00) or harvest facility (P = 0.10, R 2  = 0.09). However, the resistome of colon content differed (P = 0.01; R 2  = 0.05) among production systems, but not among the harvest facilities (P = 0.41; R 2  = 0.00). Amplicon sequencing revealed differences (P < 0.05) in microbial populations in both meat trimmings and colon content between harvest facilities but not production systems (P > 0.05)., Conclusions: These data provide a baseline characterization of an important segment of the beef industry and highlight the effect that the production system where cattle are raised and the harvest facilities where an animal is processed can impact associated microbiome and resistomes., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The bones of decomposing vertebrates are colonized by a succession of diverse microbial communities. If this succession is similar across individuals, microbes may provide clues about the postmortem interval (PMI) during forensic investigations in which human skeletal remains are discovered. Here, we characterize the human bone microbial decomposer community to determine whether microbial succession is a marker for PMI. Six human donor subjects were placed outdoors to decompose on the soil surface at the Southeast Texas Applied Forensic Science facility. To also assess the effect of seasons, three decedents were placed each in the spring and summer. Once ribs were exposed through natural decomposition, a rib was collected from each body for eight time points at 3 weeks apart. We discovered a core bone decomposer microbiome dominated by taxa in the phylum Proteobacteria and evidence that these bone-invading microbes are likely sourced from the surrounding decomposition environment, including skin of the cadaver and soils. Additionally, we found significant overall differences in bone microbial community composition between seasons. Finally, we used the microbial community data to develop random forest models that predict PMI with an accuracy of approximately ±34 days over a 1- to 9-month time frame of decomposition. Typically, anthropologists provide PMI estimates based on qualitative information, giving PMI errors ranging from several months to years. Previous work has focused on only the characterization of the bone microbiome decomposer community, and this is the first known data-driven, quantitative PMI estimate of terrestrially decomposed human skeletal remains using microbial abundance information. IMPORTANCE Microbes are known to facilitate vertebrate decomposition, and they can do so in a repeatable, predictable manner. The succession of microbes in the skin and associated soil can be used to predict time since death during the first few weeks of decomposition. However, when remains are discovered after months or years, often the only evidence are skeletal remains. To determine if microbial succession in bone would be useful for estimating time since death after several months, human subjects were placed to decompose in the spring and summer seasons. Ribs were collected after 1 to 9 months of decomposition, and the bone microbial communities were characterized. Analysis revealed a core bone decomposer microbial community with some differences in microbial assembly occurring between seasons. These data provided time since death estimates of approximately ±34 days over 9 months. This may provide forensic investigators with a tool for estimating time since death of skeletal remains, for which there are few current methods.

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., Competing Interests: The authors have declared that no competing interests exist.

Vertebrate decomposition leads to an efflux of fluids rich with biochemicals and microbes from the carcass into the surrounding soil affecting the endogenous soil bacterial community. These perturbations are detectable in soils associated with carcasses (gravesoil) and influence soil bacterial ecology for years after the decomposition event, but it is unknown for how long. Measuring these impacts over extended timescales is critical to expanding vertebrate decomposition's role in the ecosystem and may provide useful information to forensic science. Using 16S rRNA gene amplicon data, this study surveyed bacterial composition in terrestrial soils associated with surface-exposed swine decomposition for 10 years after carcass placement. This pilot study utilizes the increased statistical power associated with repeated measure/within-subjects sampling to analyze bacterial diversity trends over time. Our results demonstrate that the soil bacterial diversity was significantly impacted by decomposition, with this impact being localized to the area underneath the carcass. Bacterial community dissimilarity was greatest 12 months postmortem before beginning recovery. Additionally, random forest regressions were utilized to determine 10 important genera for distinguishing decomposition timepoints, an important component of forensic investigations. Of these 10 genera, four were further analyzed for their significant relative abundance shifts underneath the carcass. This pilot study helps expand the current knowledge of long-term effects of carcass decomposition on soil bacterial communities, and is the first to our knowledge to characterize these communities temporally from placement through a decade of decomposition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Belk et al.)

Oral microbiome dysbiosis has been associated with various local and systemic human diseases such as dental caries, periodontal disease, obesity, and cardiovascular disease. Bacterial composition may be affected by age, oral health, diet, and geography, although information about the natural variation found in the general public is still lacking. In this study, citizen-scientists used a crowdsourcing model to obtain oral bacterial composition data from guests at the Denver Museum of Nature & Science to determine if previously suspected oral microbiome associations with an individual's demographics, lifestyle, and/or genetics are robust and generalizable enough to be detected within a general population. Consistent with past research, we found bacterial composition to be more diverse in youth microbiomes when compared to adults. Adult oral microbiomes were predominantly impacted by oral health habits, while youth microbiomes were impacted by biological sex and weight status. The oral pathogen Treponema was detected more commonly in adults without recent dentist visits and in obese youth. Additionally, oral microbiomes from participants of the same family were more similar to each other than to oral microbiomes from non-related individuals. These results suggest that previously reported oral microbiome associations are observable in a human population containing the natural variation commonly found in the general public. Furthermore, these results support the use of crowdsourced data as a valid methodology to obtain community-based microbiome data.

Liver abscesses in feedlot cattle are detrimental to animal performance and economic return. Tylosin, a macrolide antibiotic, is used to reduce prevalence of liver abscesses, though there is variable efficacy among different groups of cattle. There is an increased importance in better understanding the etiology and pathogenesis of this condition because of growing concern over antibiotic resistance and increased scrutiny regarding use of antibiotics in food animal production. The objective of this study was to compare the microbiomes and antimicrobial resistance genes (resistomes) of feces of feedlot cattle administered or not administered tylosin and in their pen soil in 3 geographical regions with differing liver abscess prevalences. Cattle (total of 2,256) from 3 geographical regions were selected for inclusion based on dietary supplementation with tylosin (yes/no). Feces and pen soil samples were collected before harvest, and liver abscesses were identified at harvest. Shotgun and 16S rRNA amplicon sequencing were used to evaluate the soil and feces. Microbiome and resistome composition of feces (as compared by UniFrac distances and Euclidian distances, respectively) did not differ (P > 0.05) among tylosin or no tylosin-administered cattle. However, feedlot location was associated with differences (P ≤ 0.05) of resistomes and microbiomes. Using LASSO, a statistical model identified both fecal and soil microbial communities as predictive of liver abscess prevalence in pens. This model explained 75% of the variation in liver abscess prevalence, though a larger sample size would be needed to increase robustness of the model. These data suggest that tylosin exposure does not have a large impact on cattle resistomes or microbiomes, but instead, location of cattle production may be a stronger driver of both the resistome and microbiome composition of feces., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Comparative data from non-human primates provide insight into the processes that shaped the evolution of the human gut microbiome and highlight microbiome traits that differentiate humans from other primates. Here, in an effort to improve our understanding of the human microbiome, we compare gut microbiome composition and functional potential in 14 populations of humans from ten nations and 18 species of wild, non-human primates., Results: Contrary to expectations from host phylogenetics, we find that human gut microbiome composition and functional potential are more similar to those of cercopithecines, a subfamily of Old World monkey, particularly baboons, than to those of African apes. Additionally, our data reveal more inter-individual variation in gut microbiome functional potential within the human species than across other primate species, suggesting that the human gut microbiome may exhibit more plasticity in response to environmental variation compared to that of other primates., Conclusions: Given similarities of ancestral human habitats and dietary strategies to those of baboons, these findings suggest that convergent ecologies shaped the gut microbiomes of both humans and cercopithecines, perhaps through environmental exposure to microbes, diet, and/or associated physiological adaptations. Increased inter-individual variation in the human microbiome may be associated with human dietary diversity or the ability of humans to inhabit novel environments. Overall, these findings show that diet, ecology, and physiological adaptations are more important than host-microbe co-diversification in shaping the human microbiome, providing a key foundation for comparative analyses of the role of the microbiome in human biology and health.

Pseudomyxoma peritonei (PMP) is a subtype of peritoneal carcinomatosis that is traditionally treated by cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC). A growing body of evidence suggests that microbes are associated with various tumor types and have been found in organs and cavities that were once considered sterile. Prior and ongoing research from our consortium of PMP researchers strongly suggests that bacteria are associated with PMP tumors. While the significance of this association is unclear, in our opinion, further research is warranted to understand whether these bacteria contribute to the development, maintenance and/or progression of PMP. Elucidation of a possible causal role for bacteria in PMP could suggest a benefit for supplementation of antibiotics to current treatment protocols., (Published by Elsevier Ltd.)

Animal microbiomes play an important role in dietary adaptation, yet the extent to which microbiome changes exhibit parallel evolution is unclear. Of particular interest is an adaptation to extreme diets, such as blood, which poses special challenges in its content of proteins and lack of essential nutrients. In this study, we assessed taxonomic signatures (by 16S rRNA amplicon profiling) and potential functional signatures (inferred by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt)) of haematophagy in birds and bats. Our goal was to test three alternative hypotheses: no convergence of microbiomes, convergence in taxonomy and convergence in function. We find a statistically significant effect of haematophagy in terms of microbial taxonomic convergence across the blood-feeding bats and birds, although this effect is small compared to the differences found between haematophagous and non-haematophagous species within the two host clades. We also find some evidence of convergence at the predicted functional level, although it is possible that the lack of metagenomic data and the poor representation of microbial lineages adapted to haematophagy in genome databases limit the power of this approach. The results provide a paradigm for exploring convergent microbiome evolution replicated with independent contrasts in different host lineages. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Ground beef makes up more than half of the beef consumed in the U.S. market. Although numerous studies have been conducted on microbial safety and shelf life of ground beef limited work has been done using a culture-independent approach. While past studies have allowed for the evaluation of a few organisms of interest, there is limited work on the microbial community associated with fresh ground beef. In order to have a more complete picture of the microbial ecology of the product, a culture-independent approach utilizing 16S rRNA gene amplicon sequencing was used. The objectives of this study were to characterize the fresh ground beef microbiome and the effect that antimicrobial interventions and antioxidants, applied to beef trim before grinding, and product storage have on community composition using 16S rRNA gene amplicon sequencing. Beef trimmings were treated with antimicrobials and an antioxidant. Samples were ground, loafed, and overwrapped before being packaged in modified-atmosphere packaging. Samples were in dark storage for 21 days followed by five days in retail display. Periodically during storage, samples were collected for microbiological analysis and DNA isolation. Due to low microbial biomass, only 52 of 210 samples were included in the final analysis. These samples represented two antimicrobial treatments (peroxyacetic acid, and a sulfuric acid and sodium sulfate blend) and a control, from day-15 of dark storage and day-5 of retail display. As sample age increased, so did the number of raw reads (P < 0.001) and aerobic plate counts (P < 0.001), which were correlated (r = 0.94, P = 0.017). Across all samples, lactic acid bacteria were most abundant followed by Enterobacteriaceae; several rare taxa were also identified (namely Geobacillus, Thermus, and Sporosarcina). Antimicrobial treatment altered the bacterial alpha (P < 0.001) and beta (P = 0.001) diversity, while storage day altered alpha (P = 0.001) diversity. Enterobacteriaceae relative abundance differed (P < 0.05) among treatments and was highest in control samples. In addition to confirming previously described dominant microbial differences in culture-dependent results, these data identified genera not typically associated with ground beef and allowed for study of shifts in the entire microbiome and not just a subset of indicator organisms., Competing Interests: The authors have declared that no competing interests exist.

The gastrointestinal microbiota (GIM) plays an essential role in maintaining intestinal homeostasis with disruptions having profound effects on the wellbeing of the host animal. Parasitic infection is a long-standing issue for the equine industry, and the use of anthelmintic drugs for parasite control has long been standard practice. The impact of anthelmintic treatment on the GIM in healthy horses is not well known. This study evaluated the hypothesis that anthelmintic administration will alter the equine fecal microbiota in horses without an observed helminth infection. Ten horses were treated with a single dose of QUEST PLUS (active ingredients: Moxidectin and Praziquantel) (Zoetis), and fecal samples were collected before and after treatment. Amplicon sequencing data were quality filtered, processed, and analyzed using QIIME2. Anthelmintic treatment corresponded with a small but significant decrease in alpha diversity (P-value < .05). Analysis of taxonomic abundances before and after treatment with DESeq2 identified 21 features that were significantly different after treatment (P adj -value < .05). Differences in beta diversity associated with treatment were not significant and potentially suggest factors unique to the individual may play an essential role in the specific responses observed. Overall, the present study does not indicate a broad, large-scale impact on the GIM after anthelmintic treatment. The results do, however, suggest the potential of individualized responses that are based instead on host factors. Identification of these factors and investigation of their impact on the host/microbiota relationship will contribute significantly to our understanding of the role of the microbiome in horse health., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Microbes can be used effectively as trace evidence, at least in research settings. However, it is unknown whether skin microbiomes change prior to autopsy and, if so, whether these changes interfere with linking objects to decedents. The current study included microbiomes from 16 scenes of death in the City and County of Honolulu and tested whether objects at the scenes can be linked to individual decedents. Postmortem skin microbiomes were stable during repeated sampling up to 60 h postmortem and were similar to microbiomes of an antemortem population. Objects could be traced to decedents approximately 75% of the time, with smoking pipes and medical devices being especially accurate (100% match), house and car keys being poor (0%), and other objects like phones intermediate (~80%). These results show that microbes from objects at death scenes can be matched to individual decedents, opening up a new method of establishing associations and identifications., (© 2018 American Academy of Forensic Sciences.)

A global online register of women scientists, ready to share their science, was established by a cohort of volunteer women from the grassroots organization 500 Women Scientists on January 17th, 2018. In less than one year, the database "Request a Woman Scientist" comprised over 7,500 women from 174 scientific disciplines and 133 countries. The database is built upon a voluntary questionnaire regarding career stage, degree, scientific discipline, geographic location, and other self-identifying dimensions of representation. The information was visualized using the software platform Tableau, with dropdown menus that help query the database and output a list of names, email addresses, and websites. The biological sciences and women scientists from the United States of America were best represented in the database. A survey of women in the database conducted in November 2018 showed that of 1,278 respondents, 11% had been contacted since signing up for a variety of engagements, including media, peer review, panel participation, educational outreach, and professional/research connections. These engagements resulted in consultations for articles, video chats with students, and speaking opportunities at conferences and events. With improved functionality and marketing, outreach in the global south, and future translation in other languages, this database will further promote the profile and participation of women scientists across society, which in turn will benefit the advancement of science., Competing Interests: The authors have declared that no competing interests exist.

Over the past decade several studies have reported that the gut microbiomes of mammals with similar dietary niches exhibit similar compositional and functional traits. However, these studies rely heavily on samples from captive individuals and often confound host phylogeny, gut morphology, and diet. To more explicitly test the influence of host dietary niche on the mammalian gut microbiome we use 16S rRNA gene amplicon sequencing and shotgun metagenomics to compare the gut microbiota of 18 species of wild non-human primates classified as either folivores or closely related non-folivores, evenly distributed throughout the primate order and representing a range of gut morphological specializations. While folivory results in some convergent microbial traits, collectively we show that the influence of host phylogeny on both gut microbial composition and function is much stronger than that of host dietary niche. This pattern does not result from differences in host geographic location or actual dietary intake at the time of sampling, but instead appears to result from differences in host physiology. These findings indicate that mammalian gut microbiome plasticity in response to dietary shifts over both the lifespan of an individual host and the evolutionary history of a given host species is constrained by host physiological evolution. Therefore, the gut microbiome cannot be considered separately from host physiology when describing host nutritional strategies and the emergence of host dietary niches.

Liver abscesses in feedlot cattle form secondary to high concentrate feeds and rumen acidosis. Antimicrobial drugs are commonly included in cattle feed for prevention of liver abscesses, but concerns regarding antimicrobial resistance have increased the need for alternative treatments. A block randomized clinical trial was conducted to evaluate the effects of a Saccharomyces cerevisiae fermentation product (SCFP) on liver abscesses, fecal microbiomes, and resistomes in cattle raised without antibiotics in a Colorado feedlot. At enrollment, steers (n = 4,689) were sorted, by weight and source, into 2 pens comprising a block (n = 14 blocks, 28 pens); pens were randomly allocated to either the control group or the treatment group, where the diet was supplemented with SCFP. Prior to harvest, composited feces were collected for characterization of the microbiome and resistome using 16S rRNA gene and shotgun sequencing. At harvest, liver abscess severity was quantified for individual cattle. There were no statistical differences detected by treatment group in animal health, liver abscess prevalence or severity. Organisms classified to phylum, Elusimicrobia were more abundant in the feces of treated cattle, however, there were no differences in the resistome by treatment group. Both microbiome and resistome varied significantly among enrollment blocks.

Microbes have potential to be used as physical evidence for forensic science because they are ubiquitous and have predictable ecologies. With the advent of next generation sequencing technology and the subsequent boost to microbiome science (study of the genes and molecules of microbial communities), it has become possible to develop new microbial-based tools for forensic science. One promising approach is the use of microbial succession during the ecological process of decomposition to estimate the time since death, or postmortem interval (PMI). This microbial clock of death is developed by building a regression model using microbiome data collected from postmortem samples (e.g. swab of skin) with known PMIs. In a death investigation, a similar sample type (e.g. swab of skin) would be collected, the microbes profiled using DNA sequencing, and the microbes would be matched to a point on the clock (i.e. the regression model). Recent research by several independent scientific teams has provided a proof of concept for this new microbiome forensic tool. However, developing and transitioning new forensic science technologies into the justice system requires overcoming scientific, investigative, and legal hurdles. In this article, I address the apparent knowledge gaps in the science of microbiome technology to estimate PMI, and discuss a path for bringing this technology into the justice system., (Copyright © 2018 The Author. Published by Elsevier B.V. All rights reserved.)

Although much work has linked the human microbiome to specific phenotypes and lifestyle variables, data from different projects have been challenging to integrate and the extent of microbial and molecular diversity in human stool remains unknown. Using standardized protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-scientists, together with an open research network, we compare human microbiome specimens primarily from the United States, United Kingdom, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes compared to environmental samples; demonstrate the utility of procedures for removing the effects of overgrowth during room-temperature shipping for revealing phenotype correlations; uncover new molecules and kinds of molecular communities in the human stool metabolome; and examine emergent associations among the microbiome, metabolome, and the diversity of plants that are consumed (rather than relying on reductive categorical variables such as veganism, which have little or no explanatory power). We also demonstrate the utility of the living data resource and cross-cohort comparison to confirm existing associations between the microbiome and psychiatric illness and to reveal the extent of microbiome change within one individual during surgery, providing a paradigm for open microbiome research and education. IMPORTANCE We show that a citizen science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n = 1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet, which we confirm with untargeted metabolomics on hundreds of samples.

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., Competing Interests: The authors declare no conflict of interest.

Over the past 50,000 y, biotic extinctions and declines have left a legacy of vacant niches and broken ecological interactions across global terrestrial ecosystems. Reconstructing the natural, unmodified ecosystems that preceded these events relies on high-resolution analyses of paleoecological deposits. Coprolites are a source of uniquely detailed information about trophic interactions and the behaviors, gut parasite communities, and microbiotas of prehistoric animal species. Such insights are critical for understanding the legacy effects of extinctions on ecosystems, and can help guide contemporary conservation and ecosystem restoration efforts. Here we use high-throughput sequencing (HTS) of ancient eukaryotic DNA from coprolites to reconstruct aspects of the biology and ecology of four species of extinct moa and the critically endangered kakapo parrot from New Zealand (NZ). Importantly, we provide evidence that moa and prehistoric kakapo consumed ectomycorrhizal fungi, suggesting these birds played a role in dispersing fungi that are key to NZ's natural forest ecosystems. We also provide the first DNA-based evidence that moa frequently supplemented their broad diets with ferns and mosses. Finally, we also find parasite taxa that provide insight into moa behavior, and present data supporting the hypothesis of coextinction between moa and several parasite species. Our study demonstrates that HTS sequencing of coprolites provides a powerful tool for resolving key aspects of ancient ecosystems and may rapidly provide information not obtainable by conventional paleoecological techniques, such as fossil analyses., Competing Interests: The authors declare no conflict of interest.

[This corrects the article DOI: 10.1128/mSystems.00166-16.].

The mammal gut microbiome, which includes host microbes and their respective genes, is now recognized as an essential second genome that provides critical functions to the host. In humans, studies have revealed that lifestyle strongly influences the composition and diversity of the gastrointestinal microbiome. We hypothesized that these trends in humans may be paralleled in mammals subjected to anthropogenic forces such as domestication and captivity, in which diets and natural life histories are often greatly modified. We investigated fecal microbiomes of Przewalski's horse (PH; Equus ferus przewalskii), the only horses alive today not successfully domesticated by humans, and herded, domestic horse (E. f. caballus) living in adjacent natural grasslands. We discovered PH fecal microbiomes hosted a distinct and more diverse community of bacteria compared to domestic horses, which is likely partly explained by different plant diets as revealed by trnL maker data. Within the PH population, four individuals were born in captivity in European zoos and hosted a strikingly low diversity of fecal microbiota compared to individuals born in natural reserves in France and Mongolia. These results suggest that anthropogenic forces can dramatically reshape equid gastrointestinal microbiomes, which has broader implications for the conservation management of endangered mammals.

Liver abscesses are a major economic burden to beef producers. Although a few causative organisms have been cultured from purulent material, the full polymicrobial diversity of liver abscesses has not been reported. The objective of this study was to characterize purulent material collected from liver abscess in beef cattle produced in different production systems in 3 cattle producing states in the United States using 16S rRNA gene sequencing. Differences between purulent material microbial communities among geographic region of feeding and application of a common antimicrobial were also investigated. Cattle included in the study were fed in California (dairy type) and Colorado and Texas (both beef type). Liver abscesses from a cross section of feedlots, geographic areas, and tylosin phosphate-administered groups were collected at harvest; DNA from 34 liver abscess samples was extracted; and the V4 region of the 16S rRNA gene was amplified and sequenced. Sequences were classified into 5 phyla, 13 classes, and 17 orders in the domain Bacteria. The phyla identified included Bacteroidetes (35.2% of reads), Proteobacteria (28.6%), Fusobacteria (18.2%), Firmicutes (12.4%), and Actinobacteria (5.5%). Sequences matching the genera and , which have previously been identified as causative agents in liver abscesses, were both present in the abscess bacterial communities at a relative abundance of 15.1 and 3.2%, respectively, of the overall relative abundance. Furthermore, 3 of the most common phyla were Gram-negative bacteria. An analysis-of-similarities test was conducted on Euclidean distances to assess differences between cattle treated and not treated with tylosin as well as to assess differences between geographic regions. Geographical region and treatment with tylosin did affect the microbiome ( = 0.002 and = 0.026 respectively); however, a more robust sample scheme is needed to explore these differences. To our knowledge, this is the first publication describing the complex community of liver purulent material using next generation sequencing in cattle. These data provide a framework for research on a more targeted approach to liver abscess prevention and treatment.

Microbes are present at every crime scene and have been used as physical evidence for over a century. Advances in DNA sequencing and computational approaches have led to recent breakthroughs in the use of microbiome approaches for forensic science, particularly in the areas of estimating postmortem intervals (PMIs), locating clandestine graves, and obtaining soil and skin trace evidence. Low-cost, high-throughput technologies allow us to accumulate molecular data quickly and to apply sophisticated machine-learning algorithms, building generalizable predictive models that will be useful in the criminal justice system. In particular, integrating microbiome and metabolomic data has excellent potential to advance microbial forensics., (Copyright © 2017. Published by Elsevier Ltd.)

The horseshoe effect is a phenomenon that has long intrigued ecologists. The effect was commonly thought to be an artifact of dimensionality reduction, and multiple techniques were developed to unravel this phenomenon and simplify interpretation. Here, we provide evidence that horseshoes arise as a consequence of distance metrics that saturate-a familiar concept in other fields but new to microbial ecology. This saturation property loses information about community dissimilarity, simply because it cannot discriminate between samples that do not share any common features. The phenomenon illuminates niche differentiation in microbial communities and indicates species turnover along environmental gradients. Here we propose a rationale for the observed horseshoe effect from multiple dimensionality reduction techniques applied to simulations, soil samples, and samples from postmortem mice. An in-depth understanding of this phenomenon allows targeting of niche differentiation patterns from high-level ordination plots, which can guide conventional statistical tools to pinpoint microbial niches along environmental gradients. IMPORTANCE The horseshoe effect is often considered an artifact of dimensionality reduction. We show that this is not true in the case for microbiome data and that, in fact, horseshoes can help analysts discover microbial niches across environments.

Advances in sequencing technologies have enabled novel insights into microbial niche differentiation, from analyzing environmental samples to understanding human diseases and informing dietary studies. However, identifying the microbial taxa that differentiate these samples can be challenging. These issues stem from the compositional nature of 16S rRNA gene data (or, more generally, taxon or functional gene data); the changes in the relative abundance of one taxon influence the apparent abundances of the others. Here we acknowledge that inferring properties of individual bacteria is a difficult problem and instead introduce the concept of balances to infer meaningful properties of subcommunities, rather than properties of individual species. We show that balances can yield insights about niche differentiation across multiple microbial environments, including soil environments and lung sputum. These techniques have the potential to reshape how we carry out future ecological analyses aimed at revealing differences in relative taxonomic abundances across different samples. IMPORTANCE By explicitly accounting for the compositional nature of 16S rRNA gene data through the concept of balances, balance trees yield novel biological insights into niche differentiation. The software to perform this analysis is available under an open-source license and can be obtained at https://github.com/biocore/gneiss. Author Video : An author video summary of this article is available.

Metcalf et al., take a fresh look at the universal question "What happens when we die?", (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The causes of Late Pleistocene megafaunal extinctions (60,000 to 11,650 years ago, hereafter 60 to 11.65 ka) remain contentious, with major phases coinciding with both human arrival and climate change around the world. The Americas provide a unique opportunity to disentangle these factors as human colonization took place over a narrow time frame (~15 to 14.6 ka) but during contrasting temperature trends across each continent. Unfortunately, limited data sets in South America have so far precluded detailed comparison. We analyze genetic and radiocarbon data from 89 and 71 Patagonian megafaunal bones, respectively, more than doubling the high-quality Pleistocene megafaunal radiocarbon data sets from the region. We identify a narrow megafaunal extinction phase 12,280 ± 110 years ago, some 1 to 3 thousand years after initial human presence in the area. Although humans arrived immediately prior to a cold phase, the Antarctic Cold Reversal stadial, megafaunal extinctions did not occur until the stadial finished and the subsequent warming phase commenced some 1 to 3 thousand years later. The increased resolution provided by the Patagonian material reveals that the sequence of climate and extinction events in North and South America were temporally inverted, but in both cases, megafaunal extinctions did not occur until human presence and climate warming coincided. Overall, metapopulation processes involving subpopulation connectivity on a continental scale appear to have been critical for megafaunal species survival of both climate change and human impacts.

Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.