title: Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli.
abstract: OBJECTIVE
  The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of β-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of β-lactamases.
  
  
  METHODS
  We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for ∼60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured.
  
  
  RESULTS
  High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure].
  
  
  CONCLUSIONS
  Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. β-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, Husargatan 3, SE-751 23 Uppsala, Sweden.
authors: Marlen
authors: M
authors: Adler
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, Husargatan 3, SE-751 23 Uppsala, Sweden.
authors: Mehreen
authors: M
authors: Anjum
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, Husargatan 3, SE-751 23 Uppsala, Sweden.
authors: Dan I
authors: DI
authors: Andersson
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, Husargatan 3, SE-751 23 Uppsala, Sweden linus.sandegren@imbim.uu.se.
authors: Linus
authors: L
authors: Sandegren
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015780:Carbapenems / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D016680:Genome, Bacterial; D009154:Mutation; D017422:Sequence Analysis, DNA; D012692:Serial Passage; D018440:beta-Lactam Resistance
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D029968:Escherichia coli Proteins