A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.