title: Fitness costs and stability of a high-level ciprofloxacin resistance phenotype in Salmonella enterica serotype enteritidis: reduced infectivity associated with decreased expression of Salmonella pathogenicity island 1 genes.
abstract: The fitness costs associated with high-level fluoroquinolone resistance were examined for phenotypically and genotypically characterized ciprofloxacin-resistant Salmonella enterica serotype Enteritidis mutants (104-cip and 5408-cip; MIC, >32 microg/ml). The stability of the fluoroquinolone resistance phenotype in both mutants was investigated to assess whether clones with better fitness could emerge in the absence of antibiotic selective pressure. Mutants 104-cip and 5408-cip displayed altered morphology on agar and by electron microscopy, reduced growth rates, motility and invasiveness in Caco-2 cells, and increased sensitivity to environmental stresses. Microarray data revealed decreased expression of virulence and motility genes in both mutants. Two clones, 104-revert and 1A-revertC2, with ciprofloxacin MICs of 3 and 2 microg/ml, respectively, were recovered from separate lineages of 104-cip after 20 and 70 passages, respectively, on antibiotic-free agar. All fitness costs, except motility, were reversed in 104-revert. Potential mechanisms associated with reversal of the resistance phenotype were examined. Compared to 104-cip, both 104-revert and 1A-revertC2 showed decreased expression of acrB and soxS but still overexpressed marA. Both acquired additional mutations in SoxR and ParC, and 1A-revertC2 acquired two mutations in MarA. The altered porin and lipopolysaccharide (LPS) profiles observed in 104-cip were reversed. In contrast, 5408-cip showed no reversal in fitness costs and maintained its high-level ciprofloxacin resistance for 200 passages on antibiotic-free agar. In conclusion, high-level ciprofloxacin resistance in S. Enteritidis is associated with fitness costs. In the absence of antibiotic selection pressure, isolates may acquire mutations enabling reversion to an intermediate-level ciprofloxacin resistance phenotype associated with less significant fitness costs.
authors.affiliation: Centre for Food Safety, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Edel
authors.initials: E
authors.lastname: O'Regan
authors.forename: Teresa
authors.initials: T
authors.lastname: Quinn
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Frye
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Pagès
authors.forename: Steffen
authors.initials: S
authors.lastname: Porwollik
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Fedorka-Cray
authors.forename: Michael
authors.initials: M
authors.lastname: McClelland
authors.forename: Séamus
authors.initials: S
authors.lastname: Fanning
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001422:Bacterial Adhesion; D018938:Caco-2 Cells; D002939:Ciprofloxacin / Q000494:pharmacology*; D003227:Conjugation, Genetic; D027081:DNA Gyrase / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D018272:Porins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012480:Salmonella Infections / Q000382:microbiology; D012477:Salmonella enteritidis / Q000187:drug effects* / Q000235:genetics* / Q000472:pathogenicity
chemical_list: D000900:Anti-Bacterial Agents; D008070:Lipopolysaccharides; D018272:Porins; D002939:Ciprofloxacin; D027081:DNA Gyrase