A-039. Effect of MexXY Overexpression on Ceftobiprole Susceptibility in Pseudomonas aeruginosa

S. M. Crespo-Carbone, T. Davies, B. Foleno, B. Morrow, A. M. Queenan, K. Bush, E. Z. Baum;
Johnson & Johnson PRD, L.L.C., Raritan, NJ.

Background: Ceftobiprole (BPR), a broad spectrum cephalosporin with anti-MRSA activity, has MICs ≤ 4 μg/ml against many P. aeruginosa strains. A common resistance mechanism in P. aeruginosa to β-lactams including cefepime (FEP) and ceftazidime (CAZ) is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing FEP but not CAZ. In BPR clinical studies, paired isolates of P. aeruginosa from 3 subjects demonstrated a 4 to 8 fold increase in BPR MICs while on treatment, unrelated to β-lactamase levels. For these strains, expression of Mex genes was examined. Methods: Paired P. aeruginosa isolates (baseline and post-treatment) from a BPR clinical study were analyzed by pulsed-field gel electrophoresis (PFGE) to confirm relatedness. MICs were determined by CLSI broth microdilution. β-lactamase activity was determined by nitrocefin hydrolysis. For expression studies, total RNA was isolated from late log cultures, and first strand cDNA was synthesized using random primers. Real Time-PCR was performed on a LightCycler using primers for mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY. The expression level of each gene was normalized to rpsL. The DNA sequence of mexZ (repressor of mexXY transcription) was determined from PCR-amplified genomic DNA. Results: Three pairs of P. aeruginosa isolates were identified with (i) BPR MICs of 2 - 4 μg/ml at baseline but 16 μg/ml post-treatment, (ii) identical or closely related PFGE profiles, and (iii) no increase in β-lactamase activity. The MICs of FEP, but not CAZ, also increased (2 - 4X) in the post-treatment isolates. Within each pair, the level of mexXY RNA increased by >50-fold in comparing baseline to post-treatment isolates. In contrast, mexA, B, C, D, E, F and oprM and N RNA levels were similar within each pair. Sequencing of the mexZ gene indicated that the post-treatment isolates contained mutation(s) not present in the baseline strains. Conclusion: Increased expression of mexXY, due to mutation of the gene encoding the MexZ repressor, correlated with increased ceftobiprole MICs in P. aeruginosa. Thus, ceftobiprole, like cefepime, appears to be a substrate for the MexXY efflux pump in these isolates.