Histological and metagenomic analysis of microbial communities in archaeological human bones

RESEARCH ARTICLE Histological and metagenomic analysis of microbial communities in archaeological human bones Damla Kaptan 1*, Anne Cecilie Flemming Elvers2, Anna Kjær Knudsen Hannes Schroeder2, Hege Ingjerd Hollund 1 , 2 1 Museum of Archaeology, University of Stavanger, Stavanger, Norway, 2 Faculty of Health and Medical Sciences, Globe Institute, University of Copenhagen, Copenhagen, Denmark * Abstract OPEN ACCESS Citation: Kaptan D, Flemming Elvers AC, Kjær Knudsen A, Schroeder H, Hollund HI (2026) Histological and metagenomic analysis of microbial communities in archaeological human bones. PLoS One 21(5): e0340244. https://doi.org/10.1371/journal.pone.0340244 Editor: Furqan A. Shah, University of Gothenburg: Goteborgs Universitet, SWEDEN Received: December 18, 2025 Accepted: April 14, 2026 Published: May 27, 2026 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal. pone.0340244 Copyright: © 2026 Kaptan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, Buried archaeological bones tend to be heavily degraded by microorganisms. This type of biodegradation was already identified in the 19th century and remains a subject of continuous investigation. However, the underlying processes are still not fully understood, and the organisms responsible for the decay have not been clearly identified. Technological advances in genetic sequencing now allow detailed study of the bone microbiome. And yet, identifying the species causing the observed bioerosion has proven challenging. Relatively few studies have combined the investigation of bone degradation by microscopy, so-called histotaphonomy, with metagenomic analyses. This study aims to bridge this gap. We utilize a large set of human bone samples from medieval cemeteries in south-western Norway. Detailed microscopic analyses have been carried out, showing diverse levels of preservation. The extent of bioerosion is correlated with the results from metagenomic analyses as well as environmental factors. Microbiome diversity is greater and more evenly distributed in well-preserved bones with limited bioerosion, particularly those recovered from burials beneath church floors, contrasting with outdoor cemeteries. Fungal taxa were detected in only a single sample in the metagenomic data despite histological evidence of fungal structures, and their role in bone bioerosion remains unclear. Our findings show that preservation state is strongly associated with microbiome composition. The most prevalent genus found was Streptomyces, supporting previous research suggesting that bacteria within this group could be involved in bone bioerosion. Introduction Bone degradation by microorganisms occurs through enzymatic activities and demineralisation, a process known as bioerosion. After decades of research into bone diagenesis, the organisms causing deterioration of bone remain elusive. The PLOS One | https://doi.org/10.1371/journal.pone.0340244 May 27, 2026 1 / 18 and reproduction in any medium, provided the original author and source are credited. Data availability statement: All archaeological human remains sampled in this study are stored within the Museum of Archaeology, University of Stavanger. We have deposited all raw sequencing data (FASTQ files) in the European Nucleotide Archive (ENA) under the study accession PRJEB104929. The dataset is currently under controlled access and will be made publicly available upon publication of the manuscript, in accordance with journal and ENA data-sharing policies. Funding: This work is part of an ongoing research project funded by the Research Council of Norway (project number 301877). Competing interests: The authors have declared that no competing interests exist. result of the deterioration, on the other hand, is well studied. Microscopy [1,2], porosity measures [3–7], and chemical analyses [8,9] show how microbial action alter the structure and chemistry of buried bones. Understanding bioerosion is crucial for comprehending bone decay due to microbial activity. Already in Child’s article on bioerosion in archaeological bones from 1995, micro-organisms likely involved in bone decomposition were suggested based partially on the characteristics they are expected to have, and partially on cultivation experiments. However, his statement, that ‘...the organisms involved, have not yet been comprehensively defined’ [10] still rings true today. Child (1995) also suggested that work on identification of the organisms involved should be combined with histological analyses of the bone samples [10]. Despite the recent interest in metagenomic analyses and environmental DNA, histological investigation of bone has rarely been combined with analyses of the bone microbiome [11–17]. This study aims to bridge this knowledge gap by investigating how the bone bioerosion and microbiome are correlated, and how the microbiome varies based on the preservation status and the environmental conditions of the b iological remains. The ultimate goal is to provide new insights into the types of microorganisms that may be involved in the bone decay process. To address these questions, we analyzed bone samples from medieval cemeteries on the southwest coast of Norway, using light microscopy and scanning electron microscopy to describe and score the bioerosion patterns, and applied metagenome analysis to identify microorganisms. To our knowledge, this is the first time such detailed histotaphonomic analysis of bioeroded bone samples from archaeological contexts are combined with metagenomic analyses of the bone microbiome. Bioerosion of skeletal remains has been known since the 19th century, when Wedl made his first observations of tunnels in teeth and bone [18]. Later scholars have suggested that these were caused by fungi [19,20], however this has been contested and there is still debate on whether some of the tunnelling observed in bone can be connected to fungal activity or not [6,21,22]. The spongiform destructive foci frequently observed in archaeological bones is generally agreed to be caused by bacteria, being made up of remineralized bone matrix and fine sub-micron sized tunnels [6,21,23–25]. So far, however, despite decades of research and new, improved techniques, it has not been possible to confidently identify the microorganisms doing the actual bone bioeroding. To be able to directly connect observed bioerosion in bone with the bone microbiome of the exact same sample, we combined microscopic analyses with metagenomic profiling of 83 medieval to post-medieval human bone samples of variable preservation level and assessed the result in rela (...truncated)