title: Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline.
abstract: OBJECTIVE
  The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo.
  
  
  METHODS
  Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants.
  
  
  RESULTS
  The LPS mutants were 48-85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%-13% for ERN mutants and ∼24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs.
  
  
  CONCLUSIONS
  Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system.
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden.
authors: Marius
authors: M
authors: Linkevicius
authors: Statens Serum Institut, Ørestads Boulevard 5, 2300 Copenhagen S, Denmark.
authors: Jytte Mark
authors: JM
authors: Anderssen
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden.
authors: Linus
authors: L
authors: Sandegren
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden dan.andersson@imbim.uu.se.
authors: Dan I
authors: DI
authors: Andersson
mesh_terms: D000818:Animals; D021461:Animals, Outbred Strains; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001693:Biological Transport, Active; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000502:physiology*; D004927:Escherichia coli Infections / Q000382:microbiology / Q000473:pathology; D005260:Female; D026901:Membrane Transport Proteins / Q000096:biosynthesis; D051379:Mice; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008911:Minocycline / Q000031:analogs & derivatives* / Q000494:pharmacology; D013312:Stress, Physiological; D000078304:Tigecycline
chemical_list: D000900:Anti-Bacterial Agents; D026901:Membrane Transport Proteins; D000078304:Tigecycline; D008911:Minocycline