A-030. Response of the MRSA Strain N315ΔIP to Daptomycin: Why Are Non-Susceptible Clones Emerging?

I. L. B. C. Camargo, L. Cui, H-M. Neoh, K. Hiramatsu;
Juntendo Univ., Tokyo, JAPAN.

Few therapy options are available against multiple-drug resistant Staphylococcus aureus isolated nowadays. Daptomycin studies started 20 years ago, but its action on the cytoplasmic membrane is not completely clear. Despite of being effective during the trials, non-susceptible strains were already isolated. This study was conducted to gain new insights into the mechanism of daptomycin resistance using the MRSA N315ΔIP. We generated 10*3d1, the daptomycin non-susceptible strain (MIC= 3.0 µg/mL), exposing N315ΔIP (MIC = 0.5µg/mL) to daptomycin in vitro in two steps (0.5 µg/mL and 1.0 µg/mL, respectively) and analyzed some phenotypes and gene expression by microarray. Afterwards, we generated 10*3d1-10^th, a daptomycin susceptible strain (MIC = 1.0 µg/mL), by inoculating 10*3d1 in drug free medium for 10 days. 10*3d1 has a very slow growth and presented the highest membrane potential followed by 10*3d1-10^th and N315ΔIP, measured by flow cytometry using DiOC₂(3). 10*3d1 has heterogeneous population resistant to daptomycin, and vancomycin and teicoplanin MICs also increased. Teichoic acid genes, mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were down-regulated in both strains. graS was slightly increased only in 10*3d1, but graR was not altered. Sequence analysis of mprF and graSR did not show any mutation. vraF and vraG encoding for ABC transporter were up-regulated in 10*3d1. The transcription profile of 10*3d1 did not follow exactly that of VISA, but vraRS were up-regulated and cell wall of 10*3d1 was thicker than N315ΔIP, as demonstrated by electron microscopy. We explored and excluded the two components systems SA2151/SA2152 and SA2179/SA2180 by over expressing them in pRIT5 and verifying that daptomycin MIC did not change. Finally, we listed 7 regulatory genes whose expression were up-regulated (SA1748, SA0836, SA1805, and SA0041) and down-regulated (SA2358, SA1998, abd SA0476) only in 10*3d1 and might be involved in the daptomycin resistance mechanism. Even listing possible regulators, the exact mechanism remains unclear and more studies are needed to understand how the MRSA N315ΔIP became daptomycin non-susceptible with similar VISA phenotype.