Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes.
ABSTRACT Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a beta-lactamase with a pI of > or = 9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this beta-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 microg/ml versus 0.06 microg/ml). The kinetic characteristics of this beta-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K(i) < 40 microM). The amino acid compositions of this novel enzyme and other class C beta-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 beta-lactamase. The coalescence of Acinetobacter ampC beta-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.
Article: Inactivation of CMY-2 beta-lactamase by tazobactam: initial mass spectroscopic characterization.[show abstract] [hide abstract]
ABSTRACT: The CMY-2 beta-lactamase, a plasmid determined class C cephalosporinase, was shown to be susceptible to inhibition by tazobactam (K(i)=40 microM). The reaction product(s) of CMY-2 beta-lactamase with the beta-lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI/MS) to characterize the prominent intermediates of the inactivation pathway. The ESI/MS determined mass of CMY-2 beta-lactamase was 39851+/-3 Da. After inactivating CMY-2 beta-lactamase with excess tazobactam, a single species, M(r)=39931+/-3.0, was detected. Comparison of the peptide maps from tryptic digestion of the native enzyme and the inactivated beta-lactamase followed by LC/MS identified two 22 amino acid peptides containing the active site Ser64 modified by a fragment of tazobactam. These two peptides were increased in mass by 70 and 88 Da, respectively. UV difference spectra following inactivation revealed the presence of a new species with a 302 nm lambda(max). Based upon the increase in molecular mass of the tazobactam inactivated CMY-2 beta-lactamase, we propose that during the inactivation of this beta-lactamase by tazobactam an imine is formed. Tautomerization forms the spectrally observed enamine. Hydrolysis generates the covalently attached malonyl semialdehyde, its hydrate, or an enol. This work provides information on the mass of a stable enzyme intermediate of a class C beta-lactamase inactivated by tazobactam and, for the first time, unequivocal evidence that a cross-linked species is not required for apparent inactivation.Biochimica et Biophysica Acta 07/2001; 1547(2):196-205. · 4.66 Impact Factor