Antimicrobial resistance and virulence in polymicrobial chronic wound infections: A metagenomic perspective

Chronic wound infections represent a significant clinical and public health challenge due to their polymicrobial nature and the increasing burden of antimicrobial resistance (AMR). Conventional culture-based diagnostics often fail to capture the full microbial diversity and resistance potential associated with these infections.

Chronic wound samples persisting for more than 15 days were collected from patients at a tertiary-care hospital in Pakistan. Samples are categorized into five groups: lower leg (ll-H1), upper leg (ul-H2), foot (ft-H3), chest (ct-H4) and catheter (ca-H5). Shotgun metagenomic sequencing was employed alongside routine culture-based methods to characterize microbial communities, antimicrobial resistance genes, and virulence determinants. Taxonomic and functional profiling were performed to assess microbial diversity and resistance patterns across wound subgroups.

Metagenomic analysis revealed a predominance of Proteobacteria, Bacteroidetes, and Actinobacteria. Clinically relevant pathogens, including Achromobacter xylosoxidans, Staphylococcus aureus, and Pseudomonas aeruginosa, were frequently detected, along with less commonly reported taxa such as Achromobacter insolitus and Stenotrophomonas maltophilia. Multiple antimicrobial resistance gene clusters and biofilm-associated virulence factors were identified, indicating substantial multidrug resistance potential. Site-specific analysis showed that Pseudomonas aeruginosa dominated ul-H2 (∼32%), while Enterobacter hormaechei was most abundant in ft-H3 (∼40%). Culture-based methods primarily recovered common aerobic pathogens, whereas metagenomics detected additional opportunistic and unculturable taxa, highlighting the limitations of routine diagnostics. Resistome analysis identified ARGs conferring resistance to β-lactams, aminoglycosides, fluoroquinolones, tetracyclines, and macrolides.

Chronic wound infections in Pakistan harbor diverse polymicrobial communities with substantial antimicrobial resistance and virulence potential. Shotgun metagenomics provides a more comprehensive characterization than culture-based methods by detecting additional pathogens and resistance determinants across wound sites. These findings support the integration of metagenomic diagnostics to improve clinical decision-making, strengthen antimicrobial stewardship, and guide infection control strategies in resource-limited healthcare settings.