Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate

Enterobacter cloacae CHE. a clinical strain with overproduced cephalosporin ase was found to be highly resistant to the new cephalosporins, cefepime an d cefpirome (MICs greater than or equal to 128 mug ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic effic iency of the purified enzyme with the third-generation cephalosporins, cefe pime and cefpirome, was 10 times higher than that with the E. cloacae P99 e nzyme. This was mostly due to a decrease in K-m for these p-lactams. The cl inical isolate produced large amounts of the cephalosporinase because intro duction of the (ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA ( residues 289-294). According to the crystal structure of P99 B-lactamase. t his deletion was located in the H-10 helix. The ampR-ampC genes from the cl inical isolates CHE and OUDhyp were cloned and expressed in Escherichia col i JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and a mpR genes from CHE were 100-200 times higher than those of E. Lull harborin g ampC and ampR genes from OUDhyp. This suggests that the deletion. confirm ed by sequencing of the ampC gene, is involved in resistance to cefepime an d cefpirome. However. the high level of resistance to cefepime and cefpirom e observed in the E. cloacae clinical isolate was due to a combination of h yperproduction of the AmpC beta -lactamase and structural modification of t he enzyme. This is the first example of an AmpC variant conferring resistan ce to cefepirne and cefpirome, isolated as a clinical strain. (C) 2001 Fede ration of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.