title: Impact of AAC(6')-Ib-cr in combination with chromosomal-mediated mechanisms on clinical quinolone resistance in Escherichia coli.
abstract: aac(6')-Ib-cr is the most prevalent plasmid-mediated fluoroquinolone (FQ) resistance mechanism in Enterobacteriaceae. We aimed to analyse the interplay between this plasmid-mediated gene and chromosomal-mediated quinolone resistance mechanisms on both FQ resistance and bacterial fitness in Escherichia coli.
  
  
  
  E. coli ATCC 25922 and derived isogenic strains carrying chromosomal-mediated quinolone resistance modifications (Ser83Leu-Asp87Asn in GyrA, Ser80Arg in ParC and/or a marR gene deletion) were electroporated with a pBK-CMV vector encoding AAC(6')-Ib-cr. The MICs of FQs were determined by microdilution and bactericidal activity was determined using time-kill curves. A peritoneal sepsis murine model was used to evaluate the in vivo impact. Bacterial fitness was analysed using growth curves and competition assays.
  
  
  
  The presence of the aac(6')-Ib-cr gene increased the MICs of ciprofloxacin and norfloxacin 4-8-fold for all E. coli genotypes, independently of the initial resistance level. Combination of the aac(6')-Ib-cr gene with three or four chromosomal mechanisms was necessary to reach MIC values above the susceptible category. Killing curve assays showed a clear selective advantage for survival in strains harbouring the aac(6')-Ib-cr gene (up to 7 log10cfu/mL after 24 h). AAC(6')-Ib-cr significantly reduced the ciprofloxacin efficacy in vivo. In terms of bacterial fitness cost, maximal OD was significantly lower for all strains harbouring the aac(6')-Ib-cr gene, independently of chromosomal mutations associated.
  
  
  
  The aac(6')-Ib-cr gene, in spite of producing low-level resistance by itself, plays a relevant role in acquisition of a clinical level of ciprofloxacin and norfloxacin resistance, when combined with three or four chromosomal mutations, both in vitro and in vivo.
authors: Unidad Intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena y Virgen del Rocío, Sevilla, Spain.
authors: Jesús
authors: J
authors: Machuca
authors: Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain.
authors: Miriam
authors: M
authors: Ortiz
authors: Unidad Intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena y Virgen del Rocío, Sevilla, Spain.
authors: Esther
authors: E
authors: Recacha
authors: Unidad Intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena y Virgen del Rocío, Sevilla, Spain.
authors: Paula
authors: P
authors: Díaz-De-Alba
authors: Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain.
authors: Fernando
authors: F
authors: Docobo-Perez
authors: Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain jmrodriguez@us.es.
authors: José-Manuel
authors: JM
authors: Rodríguez-Martínez
authors: Unidad Intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena y Virgen del Rocío, Sevilla, Spain.|Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain.
authors: Álvaro
authors: Á
authors: Pascual
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002876:Chromosomes, Bacterial; D002939:Ciprofloxacin / Q000494:pharmacology; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000382:microbiology / Q000473:pathology; D005798:Genes, Bacterial; D008297:Male; D008810:Mice, Inbred C57BL; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D009643:Norfloxacin / Q000494:pharmacology; D010538:Peritonitis / Q000150:complications / Q000382:microbiology / Q000473:pathology; D010957:Plasmids; D015363:Quinolones / Q000494:pharmacology*; D018805:Sepsis / Q000382:microbiology / Q000473:pathology; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents; D015363:Quinolones; D002939:Ciprofloxacin; D009643:Norfloxacin