title: Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Biochemistry and Microbiology,  Uppsala University, SE-751 23 Uppsala,  Sweden.
authors.forename: Marlen
authors.initials: M
authors.lastname: Adler
authors.forename: Mehreen
authors.initials: M
authors.lastname: Anjum
authors.forename: Dan I
authors.initials: DI
authors.lastname: Andersson
authors.forename: Linus
authors.initials: L
authors.lastname: Sandegren
mesh_terms: D015780:Carbapenems / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D019143:Evolution, Molecular; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation / Q000235:genetics; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D015780:Carbapenems; D001618:beta-Lactamases