WGS reveals high-risk clones of Pseudomonas aeruginosa harbouring extensive antimicrobial and predicted anti-phage defense systems recovered from diabetic foot ulcer patients in Egypt

Abstract Diabetic foot ulcers (DFUs) are a major complication of diabetes mellitus and a leading cause of morbidity, amputation, and mortality among affected patients. In this study, five Pseudomonas aeruginosa isolates recovered from DFUs of diabetic patients in Egypt were investigated. Antimicrobial susceptibility was evaluated using the disc diffusion method and broth microdilution assay, while biofilm-forming ability was phenotypically assessed using the crystal violet assay. All isolates exhibited multidrug-resistant (MDR) phenotypes, with two isolates classified as pan-drug resistant (PDR). Moreover, all isolates demonstrated a strong capacity for biofilm formation. Whole-genome sequencing (WGS) and subsequent bioinformatic analysis revealed three sequence types, ST369, ST664, and ST773, corresponding to serotypes O6, O2, and O11, respectively, with ST664 and ST773 representing high-risk clones. These high-risk clones carried horizontally transferable integrative conjugative elements (ICEs) encoding metal resistance and anti-phage defense systems. The isolates also harboured a diverse resistome comprising bla NDM-1 , bla PER-1 , rmtB , qnrVC1 , aac(3) , ant(4')-IIb , aph(3')-VIb , sul1 , tet(G) , and cmlA9 in addition to biocide resistance genes ( qacE , triABC ). Colistin resistance in three isolates was associated with L71R pmrA mutation. Virulence profiling revealed a minimum of 230 genes associated with adhesion, biofilm formation, toxin production, and secretion systems. Notably, high-risk clones (ST773 and ST664) carried an extensive array of anti-phage defense systems, up to 32 per genome, potentially reducing the efficacy of bacteriophage-based therapeutic alternatives. Collectively, these findings highlight the remarkable genomic plasticity and adaptive resilience of P. aeruginosa in chronic diabetic wounds, emphasizing the urgent need for improved infection control practices, genomic surveillance, and the development of novel therapeutic strategies for effective DFU management. To our knowledge, this is the first genomic characterization of MDR P. aeruginosa isolates from DFUs in Egypt.