Abstract
Escherichia coli, a leading cause of both community- and hospital-acquired infections, has shown a marked increase in antimicrobial resistance driven by the acquisition of Extended-Spectrum β-Lactamases (ESBLs) and carbapenemases. However, β-lactam resistance in E. coli is often multifactorial, arising from different mechanisms such as the combined effects of β-lactamase production, alterations in outer membrane permeability, and regulatory mechanisms influencing gene expression. In particular, the major embedded outer membrane porins OmpC and OmpF play a critical role in modulating antibiotic influx and can significantly impact β-lactam MICs, especially in the presence of hydrolysing enzymes. Despite extensive focus on ESBLs and carbapenemases, non-carbapenemase OXA-type β-lactamases (e.g., blaOXA-1, blaOXA-2, blaOXA-9, blaOXA-10) remain underrepresented in molecular diagnostics, even though they can contribute to reduced susceptibility to β-lactam/β-lactamase inhibitor (BL/BLIs) combinations and broader resistance phenotypes. Their detection is further complicated by high sequence similarity and variable expression levels, highlighting the need for improved molecular surveillance strategies that account for both gene presence and regulatory elements such as promoter regions.To address these gaps, this study evaluates the combined impact of porin loss and β-lactamase expression on β-lactam susceptibility in E. coli. Isogenic derivatives of E. coli BW25113, including ΔOmpC, ΔOmpF, and ΔOmpC/ΔOmpF knockout strains, were utilized to assess the individual and combined effects of porin deletions. These strains were transformed with plasmids encoding clinically relevant β-lactamases, including blaCMY-2, blaDHA-1, blaSHV-5, and blaKPC-2. Antimicrobial susceptibility was determined using Minimum Inhibitory Concentration (MIC) assays, while bacterial growth was assessed through Optical Density (OD600) measurements and Colony-Forming Unit (CFU/mL) quantification to account for potential fitness costs associated with porin loss. Additionally, a multiplex Polymerase Chain Reaction (PCR) assay was developed and optimized for the simultaneous detection of OXA-type β-lactamase genes, and preliminary analysis of the blaOXA-1 promoter region was conducted to investigate potential regulatory influences on gene expressions.
Our findings demonstrate that porin loss modulates β-lactam susceptibility in a β-lactamase-dependent manner, with distinct and, in some cases, unexpected changes in MICs observed across different genetic backgrounds. Notably, the combined loss of OmpC and OmpF in the presence of blaCMY-2 resulted in carbapenem resistance, suggesting a synergistic effect contributing to higher MICs. In contrast, certain combinations of porin deletions and β-lactamase expression led to decreases in MICs, highlighting the complexity of resistance mechanisms and the potential influence of altered membrane permeability, enzyme dynamics, and bacterial fitness. Furthermore, optimization of multiplex PCR conditions enabled improved detection of OXA-type non-carbapenemase β-lactamases, supporting their inclusion in broader surveillance and diagnostic panels. Overall, this study underscores the importance of considering both structural and enzymatic mechanisms of resistance in the interpretation of antimicrobial susceptibility. The findings highlight potential limitations in current diagnostic techniques that rely heavily on β-lactamase detection without accounting for porin presence or gene regulation. By interpreting the interplay between porin loss and β-lactamase expression, this work contributes to a more comprehensive understanding of resistance phenotypes in E. coli and supports the development of more accurate and clinically relevant diagnostic approaches.