A new extended-spectrum beta-lactamase was detected in a lactose-positive Salmonella enterica subsp. enterica strain that caused a nosocomial outbreak involving eight patients in a pediatric cardiology unit. This strain showed high levels of resistance to ceftazidime and aztreonam and relatively low levels of resistance to cefotaxime and ceftriaxone. Resistance was associated with a conjugative plasmid of 59 kb, which encoded a new beta-lactamase with an isoelectric point of 5.9 that strongly hydrolyzed ceftazidime and to a much lesser extent hydrolyzed cefotaxime. The enzyme activity was inhibited by clavulanate. The corresponding bla gene was cloned and sequenced. The deduced amino acid sequence showed three significant amino acid replacements with respect to the TEM-1 sequence: Arg-164-->His, Glu-240-->Lys, and Thr-265-->Met. This combination is unique among extended-spectrum beta-lactamases and served to characterize the new enzyme, TEM-27.