Abstract
BackgroundYersinia 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.ResultsWe 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.ConclusionOur 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.
Highlights
Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare
The screen permitted the identification of a gene that reduced the susceptibility of Yersinia pestis (Yp) to fluoroquinolones and other antibiotic classes when overexpressed from a multicopy plasmid
A multicopy suppressor screen led to the isolation of a Y. pestis genomic fragment involved in ofloxacin resistance We constructed a plasmid-based expression library of the Yp genome comprised of 15,648 E. coli (Ec) clones and screened the library for strains with reduced OFX susceptibility
Summary
Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. 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. Yersinia pestis (Yp) is one of the most virulent known bacteria [1] and a potential agent of bioterrorism and biowarfare [2,3] included in the Category A of biological agents for public health preparedness against bioterrorism [4]. Patients with plague need prompt antibiotic treatment or else death may be unavoidable. The aminoglycosides streptomycin (STR) and gentamicin (GEN) are the preferred antibiotics for treatment, but a number of other drugs are effective [9,10]. Fluoroquinolones have been suggested for treatment and prophylaxis and are noted as a chemotherapeutic alternative against strains resistant to the first line anti-plague drugs [2,10]
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