Biology subjects

Trachsel, J. M.

Publications and source records attributed to Trachsel, J. M..

8 recordsLinked to original sources

bioRxiv2026

High reproducibility demonstrated in a direct-to-consumer microbiome testing workflow under real-world conditions using fecal reference material

Recent studies have documented substantial variation in microbiome profiles recovered from identical biological samples across direct-to-consumer (DTC) gut microbiome testing providers and, in some cases, within individual providers. These reports underscore the need for transparent, published assessments of analytical performance from the companies offering these services. To assess analytical reproducibility in our own testing workflow, we processed 10 replicates of a homogenized National Institute of Standards and Technology (NIST) human fecal reference material (RM 8048) through our full sample processing pipeline, from sample collection to data generation. Replicates were shipped from five U.S. sites over a staggered 35-day window. They were processed amongst over 6,000 customer samples, batched for DNA extraction and sequencing based on their arrival in the lab in nine separate sequencing runs, and analyzed using our standard pipeline. The 10 reference material replicates yielded highly concordant community profiles regardless of shipping time or temperature. Technical variability was minimal (median Bray-Curtis dissimilarity = 0.04) compared with the variation observed in samples from the same Tiny Health customer collected the same day (median Bray-Curtis: 0.08), within one week (0.25), one to two weeks apart (0.28), and between unrelated individuals (0.88). When evaluated using NIST's Coefficient of Disagreement (COD) metric, our workflow yielded a maximum pairwise COD of 0.045, well below NIST's published upper bound of 0.073 for this reference material. All three spike-in control strains were successfully recovered from every sample. These results demonstrate the reproducibility of our DTC microbiome testing workflow under real-world conditions, with robust consistency across sample collection, shipping, laboratory processing, and bioinformatics analysis. They also highlight the value of RM 8048 and similar reference materials for evaluating and transparently reporting analytical performance in DTC microbiome testing.

microbiology↗

bioRxiv2026

Integrating stool chemistry measurements with metagenomics in adults reveals distinct gut health profiles

Stool chemistry metrics, including calprotectin, lactoferrin, and secretory IgA, are used to assess gut inflammation in clinical or research settings. However, these measures in isolation provide a limited view of gut physiology and lack microbial context. Shotgun metagenomics offers a comprehensive assessment of the gut microbiome's taxonomic composition and functional potential, yet its integration with stool chemistry metrics remains underexplored. We analyzed 877 adult stool samples with paired shotgun metagenomic sequencing and chemistry measurements to integrate the microbiome with 12 stool chemistry metrics. Calprotectin, lactoferrin, and secretory IgA were associated with shifts in microbiome composition (beta diversity), while deviations from typical levels of short-chain fatty acids (SCFAs), including butyrate, were associated with both changes in alpha diversity and microbiome composition. Network analysis revealed two distinct microbial neighborhoods: one associated with higher butyrate and lower calprotectin and lactoferrin and another associated with lower butyrate and higher calprotectin and lactoferrin. Machine learning models trained on taxonomic and functional data showed modest discrimination of stool chemistry states, performing best for SCFA deficiency and less well for host-derived inflammatory metrics. Together, these findings systematically characterize microbiome-stool chemistry relationships across a large paired dataset, providing a framework for interpreting stool chemistry metrics in the context of shotgun metagenomic profiles.

microbiology↗

bioRxiv2026

Robust taxonomic classification in gut and vaginal microbiomes demonstrated through benchmarking with age-specific synthetic communities

Accurate taxonomic profiling of human microbiomes is essential for advancing research and understanding the complex role microbial communities play in human health. When using shotgun metagenomics, the sequencing data is analyzed through metagenomic pipelines, which incorporate various open-source tools and classify microbes based on matched paired-end DNA reads. However, differences in sequencing and computational approaches can produce substantially different microbiome profiles from the same sample, making validation critical. One approach for validation is benchmarking with realistic mock communities, but this remains relatively rare. Additionally, existing benchmarks often overlook microbiome variability across life stages and body sites, limiting their clinical and research utility. Here, we developed age- and body site-stratified synthetic metagenomes, enabling context-aware benchmarking of microbiome pipelines. Using novelty-based sampling to prioritize microbial diversity and minimize redundancy among selected samples, we selected 300 representative, real biological samples spanning six categories: adult, child, toddler, and infant (>6 months and <6 months) gut samples, as well as adult vaginal samples. We validated three pipelines, Tiny Health's proprietary Metagenomic Classifier v2 (THMCv2), MetaPhlAn4, and Kraken2+Bracken, using precision, recall, F1 score, and area under the precision-recall curve (AUPR) across age groups and sample types. THMCv2 demonstrated higher recall and F1 scores, detecting more taxa across sample types and ages, while MetaPhlAn4 achieved the highest precision. THMCv2 also achieved the highest area under the precision-recall curve, reflecting peak performance across both abundant and rare species. When analyses were weighted by abundance, THMCv2 and MetaPhlAn4 each characterized the mock community nearly perfectly. Errors for THMCv2 were largely restricted to very low-abundance taxa (<0.001%), whereas MetaPhlAn4 occasionally produced false positives for higher-abundance taxa. Species-level analyses of clinically relevant microbes confirmed these patterns, with THMCv2 demonstrating higher sensitivity, MetaPhlAn4 higher specificity, and Kraken2 lower overall performance. These results demonstrate clear precision-recall trade-offs in metagenomic profiling. This benchmarking framework provides a reproducible approach for evaluating pipeline performance across diverse microbiome contexts and life stages.

microbiology↗

bioRxiv2025

In-feed bacitracin methylene disalicylate alters microbiota function and increases antibiotic resistance in a dose-dependent manner

Antibiotics are commonly used in turkey production to prevent and treat infection which can improve animal growth and efficiency, but the mechanism by which antibiotics improve animal performance, and the resistance risks associated with the antibiotic inclusion levels remain unclear, particularly in turkey production. Therefore, we investigated the longitudinal effect of subtherapeutic and therapeutic doses of bacitracin methylene disalicylate (BMD) on antibiotic resistance genes, mobile genetic elements, and metabolism genes by analyzing the turkey cecal metagenome. The therapeutic dose of BMD increased a vast array of antibiotic resistant genes (ARGs), conjugation-related genes of type IV secretion system and transduction-related genes for the length of the experiment (78 days), while a smaller, transient effect was observed due to the subtherapeutic dose. Estimated bacterial growth rate, estimated by metagenome assembled genome sequence coverage, decreased after 7 days of in-feed BMD, but increased in the therapeutic group over time. Tryptophan synthesis from chorismate increased in a dose-dependent manner between days 7 - 35. Overall, the effects of subtherapeutic BMD on the turkey cecal microbiota was temporary while those of a therapeutic dose were longer lasting. This study shows that antimicrobial resistance genes belonging to multiple antibiotic classes, and mobile genetic elements (MGEs) were increased after BMD administration. The enrichment these genes by BMD shows the risk associated with antimicrobial feed additives. BMDs effect on tryptophan synthesis provides a potential metabolic target for developing non-antibiotic microbiome modulatory growth promoters for turkey production. ImportanceAntibiotic use in agricultural animals remains a hotly debated and important topic to human, animal, and environmental health. The dose-dependent responses to BMD, an antibiotic fed additive allowed at both therapeutic and subtherapeutic doses, are not well understood. This study highlights that therapeutic use of BMD is a stronger selective pressure than the subtherapeutic dose for antibiotic resistance genes and genes related to horizontal gene transfer. This indicates that use of BMD could select for antibiotic resistant bacteria that may pose a risk to animal, human and environmental health. Additionally, this study highlighted that BMD decreased the activity of beneficially bacteria, and therefore may be associated with a decreased concentration of bacterial metabolites (especially tryptophan related) in the cecum. This may indicate that growth promoting antibiotics suppress bacterial activity generally, rather than allowing beneficial bacteria to generate beneficial metabolites.

microbiology↗

bioRxiv2024

Growth dynamics and protein-expression of Escherichia coli serotypes O26:H11, O111:H8 and O145:NM in the bovine rumen.

To adapt to the ruminal environment, Shiga toxin-producing Escherichia coli (STEC) O157:H7 (O157) expresses proteins involved in survival rather than virulence. Additionally, STEC O157 strains exhibit distinct in vitro but shared in vivo survival patterns in rumen fluids that sets them apart from non-STEC, commensal E. coli. To determine if similar responses would be observed with other STEC, we evaluated three non-O157 serotypes, O26:H11, O111:H8 and O145:NM, along with a non-STEC E. coli, under the growth conditions used for STEC O157: (i) anaerobically, in vitro, in rumen fluid from cattle on a lactation (low fiber, high protein) or maintenance (high fiber, low protein) diet, at 39{degrees}C for 48 hr and (ii) in vivo for 48 hr within the rumen of cattle on the same diets using a non-terminal, rumen-fistulated animal model that allows for introduction of bacteria without ruminal contamination. On the lactation diet, the ruminal pH was acidic ranging from 5.2 - 6.0 and the total volatile fatty acids (VFA) concentration, 141 - 230 M/ml. On the maintenance diet, the ruminal fluid pH was close to neutral ranging from 6.0 - 7.0, with a total VFA of 87 - 190 M/ml. Unlike STEC O157, the three non-O157 serotypes demonstrated survival patterns similar to each other and the control non-STEC E. coli. A greater reduction in viable STEC counts was observed in vitro in rumen fluid from cattle fed the lactation diet than in vivo, corroborating previous reports that in vitro conditions cannot mimic those observed in vivo. Like STEC O157, the non-O157 serotypes mainly expressed proteins supporting ruminal adaptation although not all proteins matched those expressed by STEC O157, and included the virulence protein, intimin. Explorative studies such as this could provide insights into common conditions/ targets that may have application in broader STEC control in cattle. Importance of this studyThis study demonstrates that non-O157 serotypes O26:H11, O111:H8, O145:NM have similar survival and protein expression patterns in rumen fluid with variations being influenced primarily by rumen fluid composition associated with diet. Unlike STEC O157, under in vivo conditions, the growth dynamics of the non-O157 serotypes were comparable to that of non-STEC, commensal E. coli. Hence, exploring bacterial protein expression within the host is critical in discerning therapeutic targets, unique to/shared between STEC, for broad control strategies. In addition, this study further validates the value of using a non-terminal animal model for rumen studies that reduces number of animals used for an experiment.

microbiology↗

bioRxiv2023

Metabolic and genomic characterization of two novel butyrate producing Lachnospiraceae isolated from swine feces

Commensal bacteria from the swine gut microbiome that can be isolated have numerous potential applications in the animal production industry, including mitigation of disease, improving performance, and promoting colonization resistance to human foodborne pathogens. Butyrate-producing bacteria are targets for next-generation probiotics and microbiome-engineering strategies because butyrate is a metabolite of central importance in large intestinal homeostasis and may augment colonization resistance to enteric pathogens. However, relatively few butyrate-producers from swine have been cultured and extensively characterized. Here, we describe the substrate utilization, metabolic profiles, and genomic features of two novel species that produce high concentration of butyrate in vitro, Roseburia sp. 831b and Petralouisia sp. 499, isolated from swine feces. The complete genomes illustrated versatility in carbon metabolism and unique carbohydrate-active enzymes not observed in other species of Roseburia and Petralouisia that encode a combination of glycosidic hydrolases and carbohydrate-binding modules involved in starch and pectin utilization. Roseburia sp. 831b fermented a broader range and more complex mono- and polysaccharides than Petralouisia sp. 499. Fecal and cecal metagenomes from eight-week-old pigs challenged with Salmonella revealed that Roseburia sp. 831b increased to detectable abundances in the swine hindgut in most animals at [~]63-70 days of age. Additionally, the abundance of Roseburia sp. 831b in fecal metagenomes correlated with fecal butyrate concentrations in the pigs fed a diet supplemented with a prebiotic resistant potato starch. Together, these findings highlight the probiotic potential and ecological niche in the swine gastrointestinal tract for two novel butyrate-producers. ImportanceAntibiotics have been important for swine production and management of enteric pathogens; however, the Veterinary Feed Directive limits the use of medically important in-feed antibiotics for production purposes. As a result, there is a need for alternatives to antibiotics. Butyrate-producing bacteria can improve colonization resistance to human pathogens within the swine gastrointestinal tract by reinforcing the intestinal barrier, increasing mucus production, and reducing local oxygen and pH levels. Here, we demonstrate the versatile substrate utilization and metabolic potential of two novel species isolated from swine that produce high butyrate concentrations in vitro. These findings will help develop strategies that increase the abundance of these species and other butyrate producers in the swine gut. Further, isolating and characterizing swine butyrate producers is necessary for controlled studies that provide a mechanistic understanding of how this functional group of bacteria promotes swine gut health and colonization resistance to bacteria of public health concern.

microbiology↗

bioRxiv2022

Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing

Intestinal lymphocytes are crucial members of the mucosal immune system with impact over outcomes of intestinal health versus dysbiosis. Resolving intestinal lymphocyte complexity and function is a challenge, as the intestine provides cellular snapshots of a diverse spectrum of immune states. In pigs, intestinal lymphocytes are poorly described relative to humans or traditional model species. Enhanced understanding of porcine intestinal lymphocytes will promote food security and improve utility of pigs as a biomedical model for intestinal research. Single-cell RNA sequencing (scRNA-seq) was performed to provide transcriptomic profiles of lymphocytes in porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine intestine, including SELLhi{gamma}{delta} T cells, group 1 and group 3 innate lymphoid cells (ILCs), and four subsets of B cells. Single-cell transcriptomes in ileum were compared to those in porcine blood, and subsets of activated lymphocytes were detected in ileum but not periphery. Comparison to scRNA-seq human and murine ileum data revealed a general consensus of ileal lymphocytes across species. Lymphocyte spatial context in porcine ileum was conferred through differential tissue dissection prior to scRNA-seq. Antibody-secreting cells, B cells, follicular CD4 {beta} T cells, and cycling T/ILCs were enriched in ileum with Peyers patches, while non-cycling{gamma}{delta} T, CD8 {beta} T, and group 1 ILCs were enriched in ileum without Peyers patches. scRNA-seq findings were leveraged to develop advanced toolsets for further identification of ILCs in porcine ileum via flow cytometry and in situ staining. Porcine ileal ILCs identified via scRNA-seq did not transcriptionally mirror peripheral porcine ILCs (corresponding to natural killer cells) but instead had gene signatures indicative of tissue- and activation-specific functions, indicating potentially similar roles to intestinal ILCs identified in humans. Overall, the data serve as a highly-resolved transcriptomic atlas of the porcine intestinal immune landscape and will be useful in further understanding intestinal immune cell function.

immunology↗

bioRxiv2020

Intraepithelial T cells diverge by intestinal location as pigs age

T cells resident within the intestinal epithelium play a central role in barrier integrity and provide a first line of immune defense. Intraepithelial T cells (IETs) are among the earliest immune cells to populate and protect intestinal tissues, thereby giving them an important role in shaping gut health early in life. In pigs, IETs are poorly defined, and their maturation in young pigs has not been well studied. Given the importance of IETs in contributing to early life and long-term intestinal health through interactions with epithelial cells, the microbiota, and additional environmental factors, a deeper characterization of IETs in pigs is warranted. The objective of this study was to analyze age- and intestinal location-dependent changes in IETs across multiple sites of the small and large intestine in pigs between 4 and 8 weeks of age. IETs increased in abundance over time and belonged to both {gamma}{delta} and {beta} T cell lineages. Similar compositions of IETs were identified across intestinal sites in 4-week-old pigs, but compositions diverged between intestinal sites as pigs aged. CD2+CD8+ {gamma}{delta} T cells and CD4-CD8+ {beta} T cells comprised >78% of total IETs at all intestinal locations and ages examined. Greater percentages of {gamma}{delta} IETs were present in large intestine compared to small intestine in older pigs. Small intestinal tissues had greater percentages of CD2+CD8- {gamma}{delta} IETs, while CD2+CD8+ {gamma}{delta} IET percentages were greater in the large intestine. Percentages of CD4-CD8+ {beta} IETs increased over time across all intestinal sites. Moreover, percentages of CD27+ cells decreased in ileum and large intestine over time, indicating increased IET activation as pigs aged. Percentages of CD27+ cells were also higher in small intestine compared to large intestine at later timepoints. Results herein emphasize 4 to 8 weeks of age as a critical window of IET maturation and suggest strong associations between intestinal location and age with IET heterogeneity in pigs.

immunology↗