Influence of acquired -lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli

Abstract

To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.