Journal of Antimicrobial Chemotherapy (1999) 43, 23-29
© 1999 The British Society for Antimicrobial Chemotherapy
Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 ß-lactamases
a Department of Bacteriology, Hellenic Pasteur Institute; b Laboratory of Antimicrobial Agents, Department of Microbiology, Medical School, University of Athens, M. Asias 75, 11527, Athens, Greece
In SHV-type ß-lactamases, position 276 (in Ambler's numbering scheme) is
occupied by an asparagine (Asn) residue. The effect on SHV-1 ß-lactamase and its
extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276
was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure.
Wild-type SHV-1 and -5 ß-lactamases and their respective Asn276
Asp mutants were
expressed under isogenic conditions by cloning the respective bla genes into the
pBCSK(+) plasmid and transforming Escherichia coliDH5
.
Determination of IC50 showed that SHV-1(Asn276Asp), compared with
SHV-1, was inhibited by
8-
and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of
Asn276 by Asp in SHV-5 ß-lactamase caused a ten-fold increase in the IC50 of
clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and
5.5-fold, respectively. ß-Lactam
susceptibility testing showed that both Asn276Asp mutant enzymes, compared with the
parental ß-lactamases, conferred slightly lower levels of resistance to penicillins
(amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of
the expanded-spectrum oxyimino ß-lactams tested (cefotaxime, ceftriaxone and aztreonam).
The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were
slightly elevated in the clones producing the mutant ß-lactamases. The latter clones were
also less susceptible to penicillin-inhibitor combinations. Asn276Asp mutation was
associated
with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of
TEM-1 ß-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5
are discussed.
* Corresponding author. Tel:+33-(1)778-5638; Fax+33-(1)770-9180; Email: Lstbact{at}hotmail.com
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