title: A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis.
abstract: BACKGROUND
  Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis.
  
  
  RESULTS
  We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.
  
  
  CONCLUSIONS
  Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA. kls2002@med.cornell.edu
authors.forename: Karen L
authors.initials: KL
authors.lastname: Stirrett
authors.forename: Julian A
authors.initials: JA
authors.lastname: Ferreras
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Rossi
authors.forename: Richard L
authors.initials: RL
authors.lastname: Moy
authors.forename: Fabio V
authors.initials: FV
authors.lastname: Fonseca
authors.forename: Luis En
authors.initials: LE
authors.lastname: Quadri
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D015698:Genomic Library; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D015242:Ofloxacin / Q000494:pharmacology*; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012997:Solvents / Q000494:pharmacology; D015010:Yersinia pestis / Q000187:drug effects* / Q000235:genetics*
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012997:Solvents; D015242:Ofloxacin