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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.

SUBMITTER: Hujer KM

PROVIDER: S-EPMC1168656 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes.

Hujer Kristine M KM Hamza Nashaat S NS Hujer Andrea M AM Perez Federico F Helfand Marion S MS Bethel Christopher R CR Thomson Jodi M JM Anderson Vernon E VE Barlow Miriam M Rice Louis B LB Tenover Fred C FC Bonomo Robert A RA

Antimicrobial agents and chemotherapy 20050701 7

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. P ...[more]

PMID: 15980372

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Project description:The ADC (AmpC) β-lactamase is universally present in the Acinetobacter baumannii chromosome, suggesting it may have a yet-to-be-identified cellular function. Using peptidoglycan composition analysis, we show that overexpressing the ADC-7 β-lactamase in A. baumannii drives changes consistent with altered l,d-transpeptidase activity. Based on this, we tested whether cells overexpressing ADC-7 would exhibit new vulnerabilities. As proof of principle, a screen of transposon insertions revealed that an insertion in the distal 3' end of canB, encoding carbonic anhydrase, resulted in a significant loss of viability when the adc-7 gene was overexpressed. A canB deletion mutant exhibited a more pronounced loss of viability than the transposon insertion, and this became amplified when cells overexpressed ADC-7. Interestingly, overexpression of the OXA-23 or TEM-1 β-lactamases also led to a pronounced loss of viability in cells with reduced carbonic anhydrase activity. In addition, we demonstrate that reduced CanB activity led to increased sensitivity to peptidoglycan synthesis inhibitors and to the carbonic anhydrase inhibitor ethoxzolamide. Furthermore, this strain exhibited a synergistic interaction with the peptidoglycan inhibitor fosfomycin and ethoxzolamide. Our results highlight the impact of ADC-7 overexpression on cell physiology and reveal that the essential carbonic anhydrase CanB may represent a novel target for antimicrobial agents that would exhibit increased potency against β-lactamase-overexpressing A. baumannii. IMPORTANCE Acinetobacter baumannii has become resistant to all classes of antibiotics, with β-lactam resistance responsible for the majority of treatment failures. New classes of antimicrobials are needed to treat this high-priority pathogen. This study had uncovered a new genetic vulnerability in β-lactamase-expressing A. baumannii, where reduced carbonic anhydrase activity becomes lethal. Inhibitors of carbonic anhydrase could represent a new method for treating A. baumannii infections.

Dataset Information

Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes.

Publications

Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 beta-lactamase: defining a unique family of class C enzymes.

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