Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 17 5 2025 10 14 761 772 Zeb Hussain Department of Microbiology, University of Karachi, Karachi, Pakistan; Department of Clinical Laboratory Sciences, Dow Institute of Medical Technology, Dow University of Health Sciences, Karachi, Pakistan Asma Naim Department of Microbiology, University of Karachi, Karachi, Pakistan Ambreen Fatima Department of Pathology, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan Asad Karim Department of Molecular Medicine, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Fakhur Uddin Department of Microbiology, Basic Medical Sciences Institute (BMSI), Jinnah Postgraduate Medical Centre (JPMC), Karachi, Pakistan Surraya Shams Department of Clinical Laboratory Sciences, Dow Institute of Medical Technology, Dow University of Health Sciences, Karachi, Pakistan 2025 05 20 2025 08 25 Background and Objectives: Ventilator-associated pneumonia (VAP) caused by carbapenem-resistant Gram-negative bacteria is a serious ICU challenge. This study determined the prevalence, antimicrobial susceptibility profiles, and phenotypic carbapenemase resistance mechanisms of Gram-negative isolates from VAP patients in two tertiary hospitals in Karachi, Pakistan. Materials and Methods: We included 104 consecutive cases of VAP (July 2021–January 2023). A total of 67 carbapenem-resistant Gram-negative isolates were identified and tested. Antibiotic susceptibility was assessed by disk diffusion and broth micro dilution, according to CLSI and EUCAST guidelines. Modified and enhanced carbapenem inactivation methods (mCIM/eCIM) were used to distinguish metallo-β-lactamase (MBL) and serine carbapenemase production. Results: The mean age was 44.6 ± 18.3 years; 52.2% were male. Early-onset VAP accounted for 37.3% and late-onset for 62.7%. The most frequent pathogens were Acinetobacter baumannii (49.3%, 33/67) and Klebsiella pneumoniae (20.9%, 14/67). Notably, 67% of isolates produced MBLs, and 33% produced serine carbapenemases (phenotypically). The prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) phenotypes was 42.6%, 31.3%, and 19.4%, respectively. Conclusion: VAP in our ICUs was dominated by A. baumannii and K. pneumoniae with high levels of MBL-mediated resistance. These findings highlight the urgent need for surveillance, stewardship, and new therapeutic options. https://ijm.tums.ac.ir/index.php/ijm/article/view/5505 https://ijm.tums.ac.ir/index.php/ijm/article/download/5505/1822