Molecular Mechanisms of Carbapenem Resistance in Clinical Enterobacterales Isolates: The Role of Carbapenemases and Porin Loss

Background and objective Carbapenem-resistant Enterobacterales (CRE) have emerged as a major global public health threat, driven by the rapid dissemination of carbapenemase genes that compromise the efficacy of last-line antimicrobial agents. Comprehensive characterization of these mechanisms is essential for informing antimicrobial stewardship and infection-control strategies. The objective of this study was to investigate the molecular mechanisms underlying carbapenem resistance among clinical Enterobacterales isolates, with particular emphasis on carbapenemase genes and outer membrane porin expression alterations. Methods A retrospective cross-sectional study was conducted on 120 non-duplicate CRE isolates recovered from clinical specimens at a tertiary care hospital in Rajasthan, India, between January 2024 and December 2025. Species identification was performed using MALDI-TOF mass spectrometry, while antimicrobial susceptibility testing and carbapenem minimum inhibitory concentration (MIC) determination were carried out according to CLSI guidelines. Carbapenemase production was assessed using the Carba NP test. PCR was used to detect major carbapenemase and extended-spectrum β-lactamase (ESBL) genes. Outer membrane porin expression was evaluated by SDS-PAGE with densitometric analysis. Results Klebsiella pneumoniae accounted for the majority of isolates (75.0%), and most originated from intensive care units (54.2%). Carbapenemase activity was detected in 85.0% of isolates, while 87.5% harbored at least one carbapenemase gene. The most prevalent genes were bla_NDM (50.0%), followed by bla_KPC (16.7%) and bla_OXA-48-like (16.7%). ESBL genes were highly prevalent, particularly bla_CTX-M (79.2%). A marked reduction in OmpK36 expression was observed in 62.5% of isolates, and concurrent OmpK35/OmpK36 reduction occurred in 25.0%. Isolates with both carbapenemase genes and porin expression reduction demonstrated higher imipenem and meropenem MICs compared with carbapenemase-positive isolates without porin reduction (Mann-Whitney U test: for imipenem, U = 1420, Z = -5.02, p < 0.001; for meropenem, U = 1385, Z = -5.18, p < 0.001). Conclusions Carbapenem resistance in clinical Enterobacterales is strongly associated with carbapenemase production, particularly bla_NDM and bla_KPC, and is augmented by reduced outer membrane porin expression. These findings highlight the importance of integrating molecular diagnostics with routine susceptibility testing to guide antimicrobial stewardship and infection-control interventions.