A-061. Investigation on the Mode of Action of Oritavancin against Staphylococcus aureus through Transcriptional Profiling

H. Moisan, M. Pruneau, M. Gattuso, F. Malouin;
Univ. de Sherbrooke, Sherbrooke, QC, CANADA.

Background: Oritavancin (ORI) is a lipoglycopeptide with bactericidal activity against S. aureus including glycopeptide-intermediate (GISA) and vancomycin-resistant (VRSA) strains. ORI inhibits cell wall synthesis and dissipates transmembrane potential; however, the molecular basis for its superior in vitro activity against S. aureus compared to that of vancomycin (VAN), chloroeremomycin (CEM) and daptomycin (DAP) is poorly understood. We studied the mode of action of ORI by comparing transcriptional responses of methicillin-sensitive S. aureus (MSSA), GISA and VRSA strains to ORI and other drugs. Methods: The minimal inhibitory concentrations (MICs) of ORI, VAN, CEM, DAP and nisin (NIS) against MSSA ATCC 29213, GISA Mu50, and VRSA VRS5 were determined by the CLSI broth microdilution method. Fluorescent cDNA probes from cultures exposed for 1h to test antibiotics (Cy5) and from non-treated controls (Cy3) were co-hybridized on DNA arrays to detect gene expression changes. Concentrations of test drugs that gave a killing rate of ca. 0.6 to 2 log10 cfu/ml over 4h in time-kill experiments were used in transcription studies. Results: MICs of ORI and VAN for MSSA, GISA and VRSA were 0.03-0.06, 2 and 0.12-0.25 µg/ml and 1, 2-4 and 256 µg/ml, respectively. All compounds down-regulated Agr, RNAIII, and some related toxins (hla, nuc) whereas only DAP and NIS down-regulated the virulence regulator sae. Transcriptional analyses and qPCR showed an induction of the cell-wall stress stimulon that included a ≥2-fold up-regulation of genes like vraR/S, sgtB, and msrA in all strains by ORI, CEM, VAN and DAP, or by ORI, CEM, and VAN only for genes like tcaA and murZ. ORI and CEM, but not VAN, distinctively provoked an up-regulation of lytM, encoding a peptidoglycan hydrolase. Also unlike VAN, ORI strongly down-regulated dltA, involved in the D-alanylation of the cell wall, in VRSA. Conclusion: The transcriptional signatures collected here can be associated to the different drug classes and also to individual compounds. The observed overexpression of the peptidoglycan hydrolase LytM and alteration of D-alanylation may contribute to the rapid killing of S. aureus by ORI.