Research article

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis

Karen L Stirrett¹ , Julian A Ferreras¹ , Sebastian M Rossi^1,2 , Richard L Moy¹ , Fabio V Fonseca^1,3 and Luis EN Quadri¹

¹  Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA

²  Universidad Nacional de Misiones, Facultad de Ciencias Exactas, Quimicas y Naturales, Felix de Azara 1552, C.P. N3300LQH, Posadas, Argentina

³  Medical College of Georgia, Vascular Biology Center, 1459 Laney Walker Boulevard, CB 3201B, Augusta, Georgia 30912, USA

author email corresponding author email

BMC Microbiology 2008, 8:122doi:10.1186/1471-2180-8-122

Published:	21 July 2008

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 (robA_Yp) that increased the MIC₉₉ of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robA_Yp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robA_Yp also upregulated the expression of several efflux pumps in Y. pestis.

Conclusion

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.