title: Interplay between plasmid-mediated and chromosomal-mediated fluoroquinolone resistance and bacterial fitness in Escherichia coli.
abstract: OBJECTIVE
  The aim of this study was to analyse the interplay among plasmid-mediated qnr genes, alone or in combination with multiple chromosomal-mediated fluoroquinolone (FQ) resistance determinants, susceptibility to FQs and bacterial fitness in an isogenic Escherichia coli collection.
  
  
  METHODS
  E. coli ATCC 25922 was used to modify or delete chromosomal genes. qnr genes were cloned into the pBK-CMV vector. The MICs of FQs were determined by microdilution. Mutant prevention concentration and frequency of mutants were evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays using in vitro and in vivo models.
  
  
  RESULTS
  The relationships between the number of resistance mutations and bacterial fitness were complex. With specific combinations of resistance mechanisms the addition of a new resistance mutation was shown to improve bacterial fitness. qnrA1 caused a decrease in fitness (7%-21%) while qnrS1 caused an increase in fitness (9%-21%) when combined with chromosomal mutations. We identified susceptible triple mutants in which the acquisition of a fourth resistance mutation significantly increased fitness and at the same time reached the clinical resistance level (the acquisition of qnrS1 in a S83L + D87N + ΔmarR genetic background). A strong correlation with the production of reactive oxygen species, as well as changes in susceptibility, was observed following treatment with ciprofloxacin.
  
  
  CONCLUSIONS
  Our data indicate that there may be critical stages (depending on the genotype) in resistance development, including chromosomal- and plasmid-mediated mechanisms, at which some low-fitness mutants below the resistance breakpoint are able to evolve clinical resistance with just one or two mutations, and show increased fitness.
authors.affiliation: Infectious Diseases and Clinical Microbiology Unit, University Hospital Virgen Macarena, Seville, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Machuca
authors.affiliation: Department of Microbiology, University of Seville, Seville, Spain Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Briales
authors.affiliation: Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.
authors.forename: Gema
authors.initials: G
authors.lastname: Labrador
authors.affiliation: Department of Microbiology, University of Seville, Seville, Spain.
authors.forename: Paula
authors.initials: P
authors.lastname: Díaz-de-Alba
authors.affiliation: Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: López-Rojas
authors.affiliation: Infectious Diseases and Clinical Microbiology Unit, University Hospital Virgen Macarena, Seville, Spain Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Docobo-Pérez
authors.affiliation: Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain University Hospital Marques de Valdecilla and Valdecilla Biomedical Research Institute (IDIVAL), Santander, Spain Department of Molecular Biology, University of Cantabria, Santander, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Martínez-Martínez
authors.affiliation: Infectious Diseases and Clinical Microbiology Unit, University Hospital Virgen Macarena, Seville, Spain Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain Medicine Department, University of Seville, Seville, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Rodríguez-Baño
authors.affiliation: Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Pachón
authors.affiliation: Infectious Diseases and Clinical Microbiology Unit, University Hospital Virgen Macarena, Seville, Spain Department of Microbiology, University of Seville, Seville, Spain Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Pascual
authors.affiliation: Department of Microbiology, University of Seville, Seville, Spain Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain jmrodriguez@us.es.
authors.forename: José-Manuel
authors.initials: JM
authors.lastname: Rodríguez-Martínez
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D058491:Bacterial Load; D002876:Chromosomes, Bacterial; D004195:Disease Models, Animal; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development / Q000502:physiology*; D004927:Escherichia coli Infections / Q000382:microbiology; D005260:Female; D024841:Fluoroquinolones / Q000494:pharmacology*; D005798:Genes, Bacterial; D008810:Mice, Inbred C57BL; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones
keywords: bacterial fitness; qnr; quinolones