Assessment of Microbial Genomes Isolated from Ancient City Stratonikeia in Western Anatolia and Speculations on Human Activities and Environmental Interactions

Abstract

Ancient DNA and metagenomic studies provide valuable insights into microbial evolution, host-microbe interactions, and the ecology of ancient microbial communities; however, data from urban archaeological contexts remain limited. This study investigated microbial DNA preserved in paired petrous bone and adjacent sediment samples recovered from the Bath-Gymnasium complex of Stratonikeia, a Hellenistic-Roman city in western Anatolia, to characterize microbial diversity and functional gene profiles in an archaeological context. DNA was extracted under contamination-controlled conditions and analysed using high-throughput shotgun metagenomic sequencing. Bioinformatic analyses included taxonomic profiling, microbial diversity assessment, differential abundance analysis, and characterization of virulence-associated and antimicrobial resistance-related genes. The RH1MK1 (Tepidarium) samples exhibited greater microbial diversity and a higher relative abundance of bacterial taxa with recognized pathogenic potential, including Escherichia coli, Shigella flexneri, Vibrio cholerae, and Clostridioides difficile, compared with the SRH1PPG (Palaestra) samples. Virulence-associated genes, including Shiga toxin-, cholera toxin-, and type III secretion system-related sequences, together with antimicrobial resistance-associated genes, were more abundant in RH1MK1, consistent with differences in depositional and microenvironmental conditions between the two archaeological contexts. Comparative analyses revealed both shared and site-specific microbial profiles relative to other Anatolian archaeological sites. These findings demonstrate the potential of shotgun metagenomics for investigating preserved microbial DNA in archaeological materials while emphasizing that the detected microbial taxa and functional genes should be interpreted within their archaeological and depositional context rather than as direct evidence of ancient infectious diseases or epidemic events.