Decoding the ICU resistome: integron-mediated spread of blaIMP-85 and novel gene cassettes in XDR pathogens

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Healthcare-associated infections (HAIs) in intensive care units (ICUs) are critically compounded by multidrug-resistant (MDR) bacterial pathogens. Mobile integrons act as pivotal site-specific recombination systems that facilitate the capture and horizontal gene transfer (HGT) of antimicrobial resistance determinants. This study aimed to characterize the molecular epidemiology, cassette array diversity, and clinical impact of plasmid-borne integrons ( intI1 , intI2 , and intI3 ) among bacterial isolates recovered from ICU patients in Egypt. A total of 153 non-duplicate clinical bacterial isolates were recovered from 117 ICU patients (50 monomicrobial and 67 polymicrobial specimens). Isolates were stratified into Gram-negative bacilli and Gram-positive cocci. Species identification and phenotypic antimicrobial susceptibility testing (AST) were performed using the VITEK® 2 system, Kirby-Bauer disk diffusion, and CLSI-compliant broth microdilution assays. Extrachromosomal plasmid DNA was extracted and screened for integrase genes and variable cassette regions via PCR, followed by bidirectional Sanger sequencing, restriction fragment length polymorphism (RFLP) validation, and bioinformatic analysis. Statistical associations and Benjamini-Hochberg FDR-adjusted p -values were evaluated using R software (v4.3.3). Gram-negative isolates (n=116) comprised Klebsiella pneumoniae (49.1%, n= 57), Escherichia coli (37.1%, n= 43), Pseudomonas aeruginosa (11.2%, n= 13), Proteus mirabilis (1.7%, n= 2), and Serratia fonticola (0.9%, n=1). All Gram-positive isolates were identified as Staphylococcus aureus (n =37), with 100% exhibiting resistance to cefoxitin and oxacillin ( mecA -positive MRSA status). Isolates displayed elevated rates of MDR (49.2%) and extensive drug resistance (XDR; 27.0%), with high-level vancomycin resistance (VRSA) confirmed in 70.3% of S. aureus strains. Integron prevalence was significantly higher in Gram-negative isolates (91.3%) than in S. aureus (44.0%). Class 1 integrons ( intI1 ) predominated (52.1%), whereas intI2 (13.0%) and intI3 (17.4%) were restricted exclusively to Gram-negative species. Sanger sequencing confirmed the metallo- β -lactamase allele bla IMP-85 (GenBank: PP663295) in P. aeruginosa , a novel tandem dfrA27 – aadA2 cassette structure (PP663294) in E. coli , and an extrachromosomal sat2 – aadA1 array (PP663297) harbored by S. aureus , confirming cross-gram-class interspecies HGT. Non-synonymous point mutations within aadA1 , sat2 , and intI3 indicated active genetic drift. Correlation analysis demonstrated that total Gram-negative integron carriage strongly predicted XDR phenotypes ( p = 0.69, p < 0.01) and resistance to levofloxacin, trimethoprim-sulfamethoxazole, gentamicin, and imipenem. Plasmid-borne integrons are strongly associated with MDR and XDR profiles in critical care pathogens. The identification of functional, plasmid-located intI1 arrays bridging Gram-negative species and S. aureus underscores the urgent necessity of integrating molecular integron surveillance alongside routine AST to combat plasmid-mediated resistance dissemination in ICUs.