Shotgun metagenomic characterization of human adenovirus F40 and a non-cpe Clostridium perfringens strain in pediatric prolonged diarrhea: a genome-resolved case study

Abstract Background Viral–bacterial co-infections contribute to disease severity in pediatric acute gastroenteritis but are frequently underdiagnosed using routine methods. Enteric viruses, including human adenoviruses (HAdVs), may coexist with bacterial pathogens that influence intestinal pathology through virulence and antimicrobial resistance determinants. This study applied shotgun metagenomic sequencing to characterize concurrent viral and bacterial pathogens in a pediatric prolonged diarrhea. Methods Shotgun metagenomic sequencing was performed on a pediatric diarrheic stool sample, followed by quality filtering, de novo assembly, taxonomic classification, phylogenetic reconstruction, and integrated virulome-resistome profiling, including plasmid-associated resistance elements. Results Routine diagnostics identified adenovirus as the sole pathogen. Metagenomic analysis confirmed human adenovirus F40, showing close phylogenetic relatedness to globally distributed HAdV-F40 reference strains and a clear distinction from HAdV-F41. In addition, metagenomics uniquely detected a co-occurring Clostridium perfringens strain (SAM_A8, ST-5) that was not recovered by culture. The strain lacked the classical enterotoxin gene ( cpe ) but harbored a conserved histotoxic virulence profile, including plc , pfoA , colA , hyaluronidases, and sialidases. Plasmid-associated tetracycline resistance genes ( tetA(P) and tetB(P) ) were also identified. Conclusion Shotgun metagenomic sequencing enabled genomic characterization of a viral–bacterial co-infection missed by conventional diagnostics, highlighting its value for pathogen identification and resistance surveillance.