Khashef, M. T. | bioRxiv Science

bioRxiv2026

The first digital twin of Enterococcus faecium metabolism reproduces high-throughput phenotyping data

Enterococci are Gram-positive opportunistic pathogens responsible for a wide range of nosocomial infections. One enterococcocal species, Enterococcus faecium, is steadily increasing in prevalence and has been listed among major multidrug-resistant ESKAPE pathogens. To gain systems-level insights into its metabolism and support discovery of potential therapeutic targets, we constructed iDR478, a comprehensive manually curated genome-scale metabolic model (GEM) to serve as a digital twin for E. faecium TX0016 (strain DO). The reconstruction was based on extensive homology searches and literature evidence, and further refined and gap-filled through experimental validation. Phenotypic profiling using Biolog microarrays enabled assessment of carbon source utilization, while amino acid leave-out growth assays allowed the evaluation of auxotrophies. The final refined model accurately predicted 100% of amino acid auxotrophies and 85% of growth on sole carbon sources. Discrepancies between model predictions and experimental phenotypes identified specific knowledge gaps across metabolic pathways, including unresolved carbon source utilization phenotypes, e.g., psicose, sorbitol, and palatinose utilization. Those gaps will guide future experimental characterization. Additionally, we conducted gene essentiality analysis to evaluate the predictive capacity of iDR478. Since no experimental gene essentiality data are currently available for E. faecium, model predictions were compared against Tn-seq experimental results from E. faecalis MMH594. Under simulated unconstrained uptake conditions, iDR478 achieved 84.2% concordance with the experimental essentiality results of E. faecalis MMH594. iDR478 thus provides a framework for studying E. faecium, offers insights into its metabolic network, and serves as a source for guiding future research and identification of therapeutic targets.

systems biology↗

bioRxiv2023

Diversity and community structure of anaerobic gut fungi in the rumen of wild and domesticated herbivores

The rumen houses a diverse community that plays a major role in the digestion process in ruminants. Anaerobic gut fungi (AGF) are key contributors to plant digestion in the rumen. Here, we present a global amplicon-based survey of the rumen mycobiome by examining 206 samples from 15 animal species, 15 countries and six continents. The rumen mycobiome was highly diverse, with 81 out of 88 currently recognized AGF genera or candidate genera identified. However, only six genera (Neocallimastix, Orpinomyces, Caecomyces, Cyllamyces, NY9, and Piromyces) were present at > 4% relative abundance. AGF diversity was higher in members of the families Antilocapridae and Cervidae compared to Bovidae. Community structure analysis identified a pattern of phylosymbiosis, where host family (10% of total variance) and species (13.5%) partially explained the rumen mycobiome composition. Domestication (11.14%) and biogeography (14.1%) also partially explained AGF community structure, although sampling limitation, geographic range restrictions, and direct association between domestication status and host species hindered accurate elucidation of the relative contribution of each factor. Pairwise comparison of rumen versus fecal samples obtained from the same subject (n=13) demonstrated greater diversity and inter-sample variability in rumen over fecal samples. The genera Neocallimastix and Orpinomyces were present in higher abundance in rumen samples, while Cyllamyces and Caecomyces were enriched in fecal samples. Comparative analysis of global rumen and feces datasets revealed a similar pattern. Our results provide a global view of AGF community in the rumen and identify patterns of AGF variability between rumen and feces in herbivores tract. ImportanceRuminants are highly successful and economically important mammalian suborder. Ruminants are herbivores that digest plant material with the aid of microorganisms residing in their GI tract. The rumen compartment represents the most important location where microbially-mediated plant digestion occurs in ruminants, and is known to house a bewildering array of microbial diversity. An important component of the rumen microbiome is the anaerobic gut fungi, members of the phylum Neocallimastigomycota. So far, studies examining AGF diversity have mostly employed fecal samples, and little is currently known regarding the identity of AGF residing in the rumen compartment, factors that impact the observed patterns of diversity and community structure of AGF in the rumen, and how AGF communities in the rumen compare to AGF communities in feces. Here, we examined the rumen AGF diversity using amplicon-based surveys targeting a wide range of wild and domesticated ruminants (n=206, 15 different animal species) obtained from 15 different countries. Our results demonstrate that while highly diverse, no new AGF genera were identified in the rumen mycobiome samples examined. Our analysis also indicate that animal host phylogeny plays a more important role in shaping AGF diversity in the rumen, compared to biogeography and domestication status. Finally, we demonstrate that a greater level of diversity and higher inter-sample variability was observed in rumen compared to fecal samples, with two genera (Neocallimastix and Orpinomyces) present in higher abundance in rumen samples, and two others (Cyllamyces and Caecomyces) enriched in fecal samples. Our results provide a global view of the identity, diversity, and community structure of AGF in ruminants, elucidate factors impacting diversity and community structure of the rumen mycobiome, and identify patterns of AGF community variability between the rumen and feces in the herbivorous GIT tract.

microbiology↗