Molecular and Phylogenetic Characterization of Carbapenemase-Producing Gram- Negative Bacteria from VAP Patients: With Structural Insights into OXA-48

Background: Carbapenemase-producing Gram-negative bacteria are emerging as a significant threat to critically ill patients, particularly those with ventilator-associated pneumonia (VAP). The increasing resistance to last-resort antimicrobials—including novel β-lactam/β-lactamase inhibitor combinations and cefiderocol—warrants urgent molecular and epidemiological investigations. Objective: To investigate the antimicrobial resistance patterns, gene prevalence, and clonal relationships of Acinetobacter baumannii , Klebsiella pneumoniae , and Elizabethkingia spp. isolated from VAP patients, with a focus on resistance to cefiderocol, imipenem/Relebactam, Meropenem/vaborbactam, and Ceftazidime/avibactam, and the presence of key Carbapenemase genes (OXA-48, NDM, VIM, and IMP). Methods: A total of 67 carbapenem-resistant isolates were obtained from 104 tracheal aspirate samples collected from ICU VAP patients between January 2022 and December 2023. Antimicrobial susceptibility testing was performed using broth microdilution and MIC strips. PCR and Sanger sequencing were used for genotypic detection of Carbapenemase genes. Phylogenetic analysis was conducted via maximum-likelihood trees, and structural modeling of OXA-48 enzymes was performed using SWISS-MODEL and PyMOL. Results: All A. baumannii isolates (n = 10) exhibited high-level resistance to cefiderocol and β-lactam/β-lactamase inhibitor combinations. PCR revealed blaOXA-48 in 3 isolates, blaNDM in 1, and blaVIM in 1, and co-carriage of blaOXA-48 + blaIMP in another. K. pneumoniae isolates (n = 7) showed partial susceptibility to Ceftazidime/avibactam but complete resistance to cefiderocol; blaOXA-48 and blaNDM were the predominant genes. Elizabethkingia spp. (n = 12) showed universal resistance to all tested antibiotics and harbored blaOXA-48 (100%) and blaNDM (33%). Phylogenetic analysis revealed global clonal relationships and interspecies gene transfer, with zoonotic and environmental links. Structural modeling confirmed functional conservation of OXA-48 active sites across isolates. Conclusion: This study highlights the extensive spread of Carbapenemase genes across clinical and environmental strains of Gram-negative bacteria, with a worrying level of resistance to novel antibiotics. Our findings underscore the urgent need for genomic surveillance, rational antimicrobial use, and One Health-based infection control strategies to combat this escalating resistance crisis.