Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, <4 g/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to -lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P. aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 g/ml at baseline but 16 g/ml posttreatment, unrelated to -lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB, mexCD, and mexEF, increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ. Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB, -CD, and -EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical -lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.Ceftobiprole is a broad-spectrum, anti-methicillin-resistant Staphylococcus aureus cephalosporin with in vitro microbiological activity against many strains of Pseudomonas aeruginosa (MIC for 50% of strains tested, Յ4 g/ml) (4, 22). Resistance to antipseudomonal cephalosporins can be due to overexpression of the chromosomal AmpC cephalosporinase (17); however, decreased susceptibility of P. aeruginosa to a wide variety of antibiotics can also be attributed to drug efflux (17, 21). In particular, basal expression of the Mex pumps of the resistance nodulation division family appears to be responsible for the intrinsic resistance of P. aeruginosa to multiple antibiotics and for enhanced resistance upon overexpression (21, 23). The efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM have each been shown to be clinically relevant factors for decreased antibiotic susceptibility, with expression of the operons encoding these pumps subject to regulation (21). Pump overexpression may be due to an assortment of mutations that affect regulation of transcription from the efflux operon, such as mutations in the cognate repressor or in a global regulatory gene, or upstream of the operon, including but not limited to its promoter (21).The MexAB-OprM efflux pump is constitutively expressed in P. aerugi...