A multiresistant Serratia marcescens strain, HD, isolated from a patient with a urinary tract infection, was resistant to amino-, carboxy-, and ureidopenicillins, ceftazidime, and cefepime and was susceptible to cefotaxime and ceftriaxone, according to the guidelines of the NCCLS. No synergy was found between expanded-spectrum cephalosporins and clavulanic acid, according to the double-disk synergy test. The bla AmpC gene of the strain was amplified by PCR and cloned into Escherichia coli DH10B, giving rise to high-level resistance to ceftazidime, cefepime, and cefpirome. Sequencing analysis revealed that the bla AmpC gene from S. marcescens HD had a 12-nucleotide deletion compared to the bla AmpC gene from reference strain S. marcescens S3, leading to a 4-amino-acid deletion located in the H-10 helix of the ␤-lactamase. Kinetic analysis showed that this enzyme significantly hydrolyzed ceftazidime, cefepime, and cefpirome. This work underlined that resistance to the latest expanded-spectrum cephalosporins may be mediated by structurally modified AmpC-type ␤-lactamases.Serratia marcescens is a saprophytic, water-borne gram-negative rod (7) that possesses an inducible, chromosomally encoded AmpC-type ␤-lactamase (11) and is often involved in nosocomial infections (5,8,16,19). Infections caused by cephalosporinase-producing Enterobacteriaceae may be difficult to treat due to their ability to confer resistance to a variety of ␤-lactams, including the expanded-spectrum cephalosporins cefotaxime and ceftazidime. This resistance may arise from at least three different mechanisms in S. marcescens: high-level production of the chromosomal AmpC-type cephalosporinase (9), acquisition of an Ambler class A extended-spectrum ␤-lactamase (21), and acquisition of metallo-␤-lactamases (21). Cefepime and cefpirome, which contain quaternary nitrogen atoms at three different positions, remain active against overexpressed AmpC-producing enterobacteria. This activity results from both the stability of these molecules to attack by cephalosporinase (2, 6) and their rapid penetration through the outer membrane (15, 18).We analyzed an S. marcescens clinical isolate harboring a chromosomally encoded ␤-lactamase with ␤-lactam hydrolysis extended to a cefepime that possessed a 4-amino-acid deletion compared to a wild-type cephalosporinase. MATERIALS AND METHODSBacterial strains and plasmids. Clinical isolate S. marcescens HD was identified by using the API 20E system (bioMérieux, Marcy l'Etoile, France). Escherichia coli DH10B was used for the experiments (3). S. marcescens strain S3 was previously characterized as a wild-type AmpC producer (gift from H. Nikaido) (17).Cloning of ␤-lactamase genes. Whole-cell DNAs of S. marcescens isolates HD and S3 were extracted as described previously (3). On the basis of the ampC gene sequence of S. marcescens S3 (17), primers PRESM1 (5Ј-ATACCCTGCAACC TAAGAGC-3Ј) and PRESM2 (5Ј-ATCGCTGGTAGGGGCGCCTC-3Ј) were designed to amplify a 1,195-bp fragment corresponding to ampC genes without their own promoter seq...