title: Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model.
abstract: OBJECTIVE
  To determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM).
  
  
  METHODS
  Canine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105 CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing.
  
  
  RESULTS
  Bacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5×10(-3). All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10×WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants.
  
  
  CONCLUSIONS
  Intracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge.
authors.affiliation: Departamento de Microbiología, Complejo Asistencial Universitario de Salamanca, Spain.
authors.forename: Elisa
authors.initials: E
authors.lastname: Rodríguez Tarazona
authors.affiliation: Catedrático emérito Universidad de Salamanca, Spain.
authors.forename: José Ángel
authors.initials: JÁ
authors.lastname: García Rodríguez
authors.affiliation: Departamento de Microbiología, Complejo Asistencial Universitario de Salamanca, Spain; Departamento de Medicina Preventiva, Salud Pública y Microbiología Médica, Universidad de Salamanca, Spain; Grupo de Investigación Reconocido MICRAPE, Universidad de Salamanca, Spain; Instituto de Investigación Biomédica (IBSAL), Salamanca, Spain. Electronic address: jlmubel@usal.es.
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Muñoz Bellido
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D017347:Brucella melitensis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D027081:DNA Gyrase / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004285:Dogs; D024841:Fluoroquinolones / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000382:microbiology*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D016415:Sequence Alignment
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D027081:DNA Gyrase
keywords: Brucella; Fluoroquinolonas; Fluoroquinolone; GyrA; Infección experimental en macrófagos; Macrophage experimental infection; gyrA