Metagenomics provides broad detection of pathogens, antimicrobial resistance, and virulence genes in pig diarrhoea and complement conventional methods

Abstract Background: Post-weaning diarrhoea (PWD) remains a major cause of morbidity in pig production and is commonly associated with enterotoxigenic Escherichia coli (ETEC). Conventional diagnostics rely on culturing and targeted qPCR, which provide limited resolution of pathogen diversity, virulence and antimicrobial resistance. Here, we evaluated Oxford Nanopore Technologies (ONT) metagenomic sequencing as a diagnostic tool for direct detection of pathogens, virulence factors and antimicrobial resistance genes (ARGs) from diarrhoeal pig faeces. Results: Twenty-six diarrhoeal and six healthy pig faecal samples were analysed using culture, qPCR and ONT metagenomics with both high-output and rapid workflows. Culturing recovered 26 haemolytic E. coli and nine Clostridium perfringens isolates. PromethION metagenomics detected a significantly higher diversity of bacterial species, virulence factors and ARGs compared with GridION. Direct read mapping achieved 71–96% genome coverage for six E. coli isolates. Fourteen high- and medium-quality E. coli metagenome-assembled genomes (MAGs) were reconstructed, of which seven clustered closely with corresponding cultured isolates. All virulence factors detected in isolates were captured by metagenomics, while metagenomics identified additional fimbrial and enterotoxin genes not recovered by culture. Metagenomic ARG profiling identified resistance to 16 antibiotic classes, compared to eight classes in cultured isolates. No ESBL, carbapenemase or mcr genes were detected. Conclusions: Long-read ONT metagenomics enables culture-independent, strain-resolved characterisation of the pig gut microbiome during PWD, capturing pathogen diversity together with virulence and antimicrobial resistance profiles. This approach reveals within-sample strain heterogeneity and functional potential that are not resolved by conventional culturing, supporting its value for studying microbial ecology and dysbiosis in diseased animal microbiomes.