Resistance to Cefepime and Cefpirome Due to a 4-Amino-Acid Deletion in the Chromosome-Encoded AmpC β-Lactamase of a Serratia marcescens Clinical Isolate

Abstract

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...

AmpC β-Lactamases

“…In either case, the k cat /K m ratio or catalytic efficiency for ceftazidime and related substrates increased compared to that of the wild-type enzyme with the result that the ceftazidime MICs for a strain carrying such enzymes were in the resistance range (MIC Ն 32 g/ml), while the MICs for cefotaxime and cefepime usually reflected only reduced susceptibility, such as a cefepime MIC of 8 g/ml for E. coli with the AmpC enzymes from E. cloacae CHE or Enterobacter aerogenes Ear2. The enzyme from S. marcescens HD, however, when expressed in E. coli, conferred a cefepime MIC of 512 g/ml (196), and those from E. coli strains EC14, EC18, and BER were associated with cefepime MICs of 16 g/ml (197,199). MICs for aztreonam and imipenem were usually little affected except that an aztreonam MIC of 128 g/ml was produced by CMY-10 (172).…”

Antibacterial-ResistantPseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms

“…Extended-spectrum AmpCs were first identified in Serratia marcescens and Enterobacter spp. (10,143,153) and, most recently, in E. coli (141,142). Amino acid modifications near the active sites of these enzymes can lead to FIG.…”

Extended-spectrumβ-Lactamases: Implications for the Clinical Laboratory and Therapy

“…Although it has been determined that such mutants were mainly located on bacterial chromosome [14][15][16], two plasmid-mediated extended-spectrum cephalosporinases, CMY-19 [17] and CMY-10 [18], have been reported. Some specialists may insist on regarding such enzymes as ESBLs because of their wide spectrum of activity.…”