Amino acid substitutions at Ambler position Gly238 in the SHV-1 beta-lactamase: exploring sequence requirements for resistance to penicillins and cephalosporins.
Site saturation mutagenesis of the 238 position in the SHV beta-lactamase was performed to identify the complete sequence requirements needed for the extended spectrum beta-lactamase (ESBL) phenotype. MICs (in micrograms per milliliter) in an isogenic background, Escherichia coli DH10B, demonstrated that the Gly238Ala mutation conferred the most resistance to penicillins and cephalosporins. The absolute increase in resistance was greatest against cefotaxime for the Gly238Ala mutant (0.06 to 8 micro g/ml). Except for the strain possessing the Gly238Pro beta-lactamase, ceftazidime MICs were also elevated. None of the mutant SHV beta-lactamases were expressed in as great an amount as the wild-type beta-lactamase. Kinetic analysis of the Gly238Ala mutant revealed that penicillin and cephalosporin substrates have a lower K(m) for the enzyme because of this mutation. Ampicillin and piperacillin MICs were inversely proportional to the side chain volume of the amino acid in cases larger than Ser, suggesting that steric considerations may be a primary requirement for penicillin resistance. Secondary structural effects explain increased resistance to oxyiminocephalosporins. Based upon this study, we anticipate that additional mutations of Gly238 in the SHV beta-lactamase will continue to be discovered with an ESBL (ceftazidime or cefotaxime resistant) phenotype.
Hujer
Andrea M
AM
Research Service. Infectious Diseases Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Ohio 44106, USA.
Hujer
Kristine M
KM
Helfand
Marion S
MS
Anderson
Vernon E
VE
Bonomo
Robert A
RA
D018550:Cephalosporin Resistance / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D016296:Mutagenesis; D010403:Penicillin Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
C054882:beta-lactamase PIT-2; D001618:beta-Lactamases