The synergistic action of reduced membrane permeability and antibiotic sequestration as a novel mechanism for carbapenem resistance in Campylobacter

Carbapenems are last-resort antibiotics against gram-negative pathogens, including multidrug-resistant Campylobacter . However, carbapenem-resistant Campylobacter strains are emerging and have been isolated in patients treated with carbapenems. This emerging resistance mechanism may be underpinned by multiple genetic determinants, yet it has not been systematically characterized or elucidated until now. In this study, two candidate mutations identified via resistance evolution and whole-genome sequencing were validated through genetic and functional assays. De novo structural modeling and molecular dynamics simulations elucidated the impact of mutations on protein dynamics. Furthermore, carbapenem hydrolysis and binding-pocket interactions were characterized using ultra-high-performance liquid chromatography, MM/PBSA calculations, and alanine substitution experiments. Our findings reveal that carbapenem resistance in Campylobacter is mediated by synergistic mutations in porA , encoding the major outer membrane protein (MOMP), and bla OXA-61 , encoding a non-carbapenemase β-lactamase OXA-61. Specifically, the D157H substitution in MOMP induces structural and functional remodeling, thereby hindering meropenem translocation across the outer membrane, while a G → T transversion in the bla OXA-61 promoter leads to overexpression of the enzyme that sequesters meropenem instead of hydrolyzing it. Notably, either porA alteration or bla OXA-61 overexpression alone confers only a modest increase in carbapenem resistance (2- to 8-fold), whereas their synergy yields a high-level resistance (16- to 128-fold). Together, these results define a novel carbapenem resistance mechanism mediated by reduced porin permeability coupled with antibiotic sequestration acted by a non-carbapenemase β-lactamase. This synergistic mechanism explains clinical carbapenem resistance phenotypes in Campylobacter , facilitates diagnosis and surveillance, and likely represents a general carbapenem resistance strategy across other bacterial species. IMPORTANCE Campylobacter ranks among the leading causes of bacterial gastroenteritis worldwide. In recent years, carbapenem-resistant Campylobacter strains have been emerging in clinical settings and have been increasingly reported from patients following carbapenem treatment. Despite the importance of carbapenems in therapeutic treatment of multidrug-resistant Campylobacter , the molecular basis of this resistance phenotype remains poorly understood. Additionally, non-carbapenemase β-lactamases have been implicated in carbapenem resistance in many bacterial species, but how they contribute to the resistance without hydrolyzing the antibiotic is unknown. Our study defines a new mechanism for carbapenem resistance and accounts for the carbapenem-resistant phenotype observed in clinical isolates. They also provide timely information useful for the diagnosis and treatment of infections caused by antibiotic-resistant Campylobacter . Equally important, this study provides a mechanistic explanation for carbapenem resistance mediated by non-carbapenemase β-lactamases, which exist in many gram-negative pathogens.