Mohamed S. Sarhan

Background: The Iceman mummy, a 5300-year-old natural alpine glacier mummy, provides a unique opportunity to study ancient microbial ecosystems. However, disentangling the mummy’s endogenous microbiome from modern environmental contaminants introduced during three decades of conservation remains a significant challenge. Results: By integrating culture-dependent and culture-independent approaches, including amplicon sequencing, shotgun metagenomics and de novo metagenomic assembly, as well as isolate-level genomics, we performed a comprehensive characterization of the Iceman’s microbial landscape. We identified three distinct microbial drivers: endogenous post-mortem succession, ancient glacier-derived relicts, and modern anthropogenic introduction. Metagenomic analysis of internal tissues revealed anaerobic bacteria, including ancient gut taxa, including such as Romboutsia hominis , Clostridium moniliforme , Eubacterium sp., Ruminococcus bromii , Kineothrix sp., Treponema succinifaciens , Enterousia sp., and Huintestinicola butyrica . These taxa, characterized by ancient DNA (aDNA) damage profiles (C to T deamination frequency), show high similarity to ancestral, non-Westernized human gut communities, providing a rare baseline for Copper Age intestinal ecosystems. Conversely, we identified a shift in the external mycobiome, marked by the recent proliferation of psychrophilic yeasts, including Glaciozyma watsonii , Mrakia robertii , Phenoliferia glacialis , and Goffeauzyma sp. While internal bacterial communities remained stable, these external yeast populations showed increased relative abundance and reduced DNA damage signatures between 2010 and 2019, indicating active, modern colonization. Furthermore, strain-level analysis of Pseudomonas sp. 5C2 confirmed that specific environmental strains have successfully colonized the mummy, persisting across multiple tissue sites with minimal genetic divergence. Conclusions: Our study demonstrates that the Iceman is not a static relic but a dynamic biological interface. The coexistence of ancient, endogenous gut microbes and modern, psychrophilic colonizers highlights the potential for ongoing microbial activity even at sub-zero temperatures. These findings underscore that maintaining strict environmental parameters is essential to prevent these specialized microbial communities from transitioning from latent persistence to active microorganisms.

Streptococcus pyogenes , or Group A Streptococcus (GAS), is a human pathogen responsible for a range of diseases, from mild infections to severe illnesses. Despite its significance in modern clinical settings, little is known about the pathogen’s evolutionary history or its presence in ancient human populations. Here, we present genomic evidence of S. pyogenes in the pre-Columbian Americas. We analysed a tooth from a naturally mummified individual dating to the Late Intermediate Period (1283–1383 cal AD), housed in the National Museum of Archeology (MUNARQ) in La Paz, Bolivia. Mitochondrial DNA analysis confirmed the host’s Native American ancestry. Shotgun metagenomic sequencing and de-novo assembly enabled the near-complete reconstruction of an ancient S. pyogenes genome displaying close similarity to contemporary strains linked to pharyngitis. The genome contains core virulence genes, but prophages lack streptococcal pyrogenic exotoxins. Phylogenetic analyses place the strain at the base of modern S. pyogenes diversity, and Bayesian analyses indicate that most extant lineages diversified globally within the past ~5,500 years. Our results push back the confirmed presence of S. pyogenes in the Americas by several centuries and suggest that the pathogen circulated among Indigenous populations prior to the European contact.

Fungal mitochondrial genomes are critical for understanding phylogenetics, evolution, and ecology of the Kingdom Fungi, yet they remain underrepresented in public databases. To address this, we developed a workflow to recover mitochondrial genomes from 12,902 fungal short read sequencing data housed in the Sequence Read Archive (SRA) records, assembling complete circular genomes from 2,695 species. This effort expanded fungal mitochondrial genome diversity by nearly 2.3X particularly in understudied phyla such as Mucoromycota (11X increase) and Zoopagomycota (8X increase). The new dataset contains novel yet undescribed mitochondrial genomes at numerous taxonomic levels, including 15 classes, 64 orders, 178 families, and 544 genera. Taxonomic analysis revealed broad ecological representation among the top-assembled species, including human pathogens (e.g., Cryptococcus tetragattii), plant pathogens (e.g., Melampsora larici-populina), edible mushrooms (e.g., Suillus luteus), and industrial fungi. By leveraging the not yet fully exploited SRA sequencing data, this study fills critical gaps in fungal mitochondrial genomics, tripling the currently known mitochondrial genome diversity of the Kingdom Fungi, and provides an extensive resource for phylogenetic and evolutionary research.

Chronic kidney disease (CKD) is a complex disease affecting >10% of the global population, with large between- and within-continent variability reflecting major environmental determinants. To identify molecular targets for treatment and prevention, genome-wide association study meta-analyses (GWAMAs) of CKD-defining traits have identified hundreds of genetic loci in aggregated population samples. However, while GWAMAs estimate the average allelic effect across studies, single population studies may be relevant to unravel specific mechanisms. To assess whether a study sample from a specific population could extend existing knowledge on kidney function genetics, we selected 147 kidney function relevant loci identified by a large GWAMA, assessing their association with the glomerular filtration rate estimated from serum creatinine (eGFRcrea) in 10,146 participants to the Cooperative Health Research In South Tyrol (CHRIS) study, conducted in an Alpine region where thyroid dysfunction is common. We identified associations with single nucleotide polymorphisms (SNPs) at 11 loci, showing up-to-5.4 times larger effect sizes than in the corresponding GWAMA, not explainable by allele frequency differences. Systematic mediation analysis across 70 quantitative traits identified serum magnesium and the activated partial thromboplastin time as partial mediators of the eGFRcrea associations at SHROOM3 and SLC34A1, respectively. Given that free triiodothyronine and thyroxine acted as effect modifiers across all loci, we conducted SNP-by-thyroid stimulating hormone (TSH) interaction analyses, identifying significant interactions at STC1: SNPs had larger effects on eGFRcrea at higher TSH levels, possibly reflecting stanniocalcin-1 autocrine and paracrine role. Individual population studies can help characterize genetic associations. The interplay between phenotypes at SHROOM3 and SLC34A1 and the role of thyroid function as a genetic effect modifier warrant further investigations.

Background Access to sample-level metadata is important when selecting public metagenomic sequencing datasets for reuse in new biological analyses. The Standards, Precautions, and Advances in Ancient Metagenomics community (SPAAM, https://spaam-community.org) has previously published AncientMetagenomeDir, a collection of curated and standardised sample metadata tables for metagenomic and microbial genome datasets generated from ancient samples. However, while sample-level information is useful for identifying relevant samples for inclusion in new projects, Next Generation Sequencing (NGS) library construction and sequencing metadata are also essential for appropriately reprocessing ancient metagenomic data. Currently, recovering information for downloading and preparing such data is difficult when laboratory and bioinformatic metadata is heterogeneously recorded in prose-based publications. Methods Through a series of community-based hackathon events, AncientMetagenomeDir was updated to provide standardised library-level metadata of existing and new ancient metagenomic samples. In tandem, the companion tool 'AMDirT' was developed to facilitate rapid data filtering and downloading of ancient metagenomic data, as well as improving automated metadata curation and validation for AncientMetagenomeDir. Results AncientMetagenomeDir was extended to include standardised metadata of over 6000 ancient metagenomic libraries. The companion tool 'AMDirT' provides both graphical- and command-line interface based access to such metadata for users from a wide range of computational backgrounds. We also report on errors with metadata reporting that appear to commonly occur during data upload and provide suggestions on how to improve the quality of data sharing by the community. Conclusions Together, both standardised metadata reporting and tooling will help towards easier incorporation and reuse of public ancient metagenomic datasets into future analyses.

Analyzing taxonomic diversity and identification in diverse ecological samples has become a crucial routine in various research and industrial fields. While DNA barcoding marker-gene approaches were once prevalent, the decreasing costs of next-generation sequencing have made metagenomic shotgun sequencing more popular and feasible. In contrast to DNA-barcoding, metagenomic shotgun sequencing offers possibilities for in-depth characterization of structural and functional diversity. However, analysis of such data is still considered a hurdle due to absence of taxa-specific databases. Here we present taxonize-gb, a command-line software tool to extract GenBank non-redundant nucleotide and protein databases, related to one or more input taxonomy identifier. Our tool allows the creation of taxa-specific reference databases tailored to specific research questions, which reduces search times and therefore represents a practical solution for researchers analyzing large metagenomic data on regular basis. Taxonize-gb is an open-source command-line Python-based tool freely available for installation at https://pypi.org/project/taxonize-gb/ and on GitHub https://github.com/msabrysarhan/taxonize_genbank . It is released under Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Background: Access to sample-level metadata is important when selecting public metagenomic sequencing datasets for reuse in new biological analyses. The Standards, Precautions, and Advances in Ancient Metagenomics community (SPAAM, https://spaam-community.github.io) has previously published AncientMetagenomeDir, a collection of curated and standardised sample metadata tables for metagenomic and microbial genome datasets generated from ancient samples. However, while sample-level information is useful for identifying relevant samples for inclusion in new projects, Next Generation Sequencing (NGS) library construction and sequencing metadata are also essential for appropriately reprocessing ancient metagenomic data. Currently, recovering information for downloading and preparing such data is difficult when laboratory and bioinformatic metadata is heterogeneously recorded in prose-based publications. Methods: Through a series of community-based hackathon events, AncientMetagenomeDir was updated to provide standardised library-level metadata of existing and new ancient metagenomic samples. In tandem, the companion tool 'AMDirT' was developed to facilitate automated metadata curation and data validation, as well as rapid data filtering and downloading. Results: AncientMetagenomeDir was extended to include standardised metadata of over 5000 ancient metagenomic libraries. The companion tool 'AMDirT' provides both graphical- and command-line interface based access to such metadata for users from a wide range of computational backgrounds. We also report on errors with metadata reporting that appear to commonly occur during data upload and provide suggestions on how to improve the quality of data sharing by the community. Conclusions: Together, both standardised metadata and tooling will help towards easier incorporation and reuse of public ancient metagenomic datasets into future analyses.

Background: In 1975, the mummified body of a female has been found in the Franciscan church in Basel, Switzerland. Molecular and genealogic analyses unveiled her identity as Anna Catharina Bischoff (ACB), a member of the upper class of post-reformed Basel, who died at the age of 68 years, in 1787. The reason behind her death is still a mystery, especially that toxicological analyses revealed high levels of mercury, a common treatment against infections at that time, in different body organs. The computed tomography (CT) and histological analysis showed bone lesions in the femurs, the rib cage, and the skull, which refers to a potential syphilis case.Results: Although we could not detect any molecular signs of the syphilis-causing pathogenTreponema pallidumsubsp. pallidum, we realized high prevalence of a nontuberculous mycobacterium (NTM) species in brain tissue sample. The genome analysis of this NTM displayed richness of virulence genes and toxins, and similarity to other infectious NTM, known to infect immunocompromised patients. In addition, it displayed potential resistance to mercury compounds, which might indicate a selective advantage against the applied treatment. This suggests that ACB might have suffered from an atypical mycobacteriosis during her life, which could explain the mummy’s bone lesion and high mercury concentrations.Conclusions: The study of this mummy exemplifies the importance of employing differential diagnostic approaches in paleopathological analysis, by combining classical anthropological, radiological, histological, and toxicological observations with molecular analysis. It represents a proof-of-concept for the discovery of not-yet-described ancient pathogens in well-preserved specimens, using de novo metagenomic assembly.