Vesicle-Mediated Transfer of CTX-M β-Lactamase Genes and Proteins Confers Ampicillin Resistance in Escherichia coli

TL;DR

This study shows that Escherichia coli can spread antibiotic resistance through tiny vesicles that carry both resistance genes and enzymes that inactivate antibiotics.

Contribution

The study uniquely demonstrates that OMVs from CTX-M-15–producing E. coli contain both resistance genes and active β-lactamase enzymes.

Findings

OMVs from E. coli contain CTX-M-15 genes protected from digestion.

OMVs exhibit β-lactamase activity primarily within their interior.

OMVs inactivate ampicillin and protect susceptible bacteria from antibiotic effects.

Abstract

The global rise of antimicrobial resistance represents a critical challenge to public health, with Escherichia coli emerging as one of the most significant contributors due to its high adaptability and prevalence of extended-spectrum β-lactamase (ESBL) production. Outer membrane vesicles (OMVs), nanoscale structures released by Gram-negative bacteria, have recently been implicated in the dissemination of resistance determinants and direct antibiotic inactivation. This study investigated the role of OMVs derived from ESBL-producing E. coli in mediating resistance to ampicillin. Clinical strains harboring CTX-M-15 resistance genes were cultured under selective pressure, and OMVs were purified via size-exclusion chromatography. Characterization using tunable resistive pulse sensing (TRPS) and cryo-transmission electron microscopy confirmed vesicle integrity, with sizes ranging from 80 to 150 nm. DNA quantification and PCR analysis revealed the presence of CTX-M-15 genes within vesicles, which remained protected from DNase digestion, confirming encapsulation. Functional assays demonstrated β-lactamase activity within OMVs, with proteinase K treatment indicating localization primarily within vesicles rather than on their surface. Importantly, OMVs inactivated ampicillin in a dose-dependent manner, significantly reducing its efficacy against susceptible E. coli. Disc diffusion and microtiter plate assays confirmed that β-lactamase-positive OMVs protected susceptible strains from antibiotic killing, promoting bacterial survival and growth. This study uniquely demonstrates that OMVs from CTX-M-15–producing Escherichia coli carry both resistance genes and active β-lactamase enzymes, thereby facilitating both genetic dissemination and direct antibiotic inactivation. Targeting OMV biogenesis may represent a novel strategy to combat antimicrobial resistance.

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