Extended-Spectrum Cephalosporinase in
            <i>Acinetobacter baumannii</i>

Rodríguez-Martínez, José Manuel; Nordmann, Patrice; Ronco, E; Poirel, Laurent

doi:10.1128/aac.00050-10

Cited by 73 publications

(67 citation statements)

References 22 publications

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“…Extended-spectrum ampC-type b-lactamases (ESAC) have also been identified in A. baumannii, possessing activity toward expanded-spectrum cephalosporins and monobactams due to a Pro210Arg substitution and a duplication of an Ala residue at position 215 (inside the O-loop). These enzymes hydrolyze all cephalosporins, but do not compromise the efficacy of carbapenems (4).…”

Section: Naturally-occurring B-lactamasesmentioning

confidence: 99%

Genetic basis of antibiotic resistance in pathogenic Acinetobacter species

2011

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SummaryAntibiotic resistance in Acinetobacter spp., particularly Acinetobacter baumannii, is increasing rapidly. A. baumannii possesses two intrinsic b-lactamase genes, in addition to weak permeability and efflux systems, that together confer a natural reduced susceptibility to antibiotics. In addition, numerous acquired mechanisms of resistance have been identified in A. baumannii. The very high genetic plasticity of A. baumannii allows an accumulation of resistance determinants that give rise to multidrug resistance at an alarming rate. The role of novel genetic elements, such as resistance islands, in concentrating antibiotic resistance genes in A. baumannii requires detailed investigation in the near future.

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Section: Naturally-occurring B-lactamasesmentioning

confidence: 99%

Genetic basis of antibiotic resistance in pathogenic Acinetobacter species

2011

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“…When combined with other resistance mechanisms, such as porin loss or efflux (2)(3)(4)(5), or when increased expression occurs in derepressed strains (1,6), organisms expressing class C ␤-lactamases become resistant to cefepime and carbapenems. Point mutations and deletions in the omega loop or helix H2 or H10 and near the C terminus of the AmpC ␤-lactamases that cause an extended-spectrum AmpC (ESAC) phenotype have been described (1,7). Of the CMY enzymes, 97 unique types have been described to date (see http://www.lahey.org/Studies), including enzymes such as CMY-30, CMY-32, CMY-33, CMY-37, and CMY-44 (8)(9)(10)(11)(12), with alterations of the omega loop or the H10 helix.…”

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confidence: 99%

N152G, -S, and -T Substitutions in CMY-2 β-Lactamase Increase Catalytic Efficiency for Cefoxitin and Inactivation Rates for Tazobactam

Skalweit

Conklin

et al. 2013

Antimicrob Agents Chemother

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Class C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in the Escherichia coli AmpC and P99 enzymes and have suggested that interactions between C-6= or C-7= substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A ␤-lactamase inhibitor with in vitro activity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC 50 ) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C ␤-lactamase inhibitors are developed. C lass C ␤-lactamases such as CMY-2, found in Gram-negative pathogens, confer resistance to a wide variety of ␤-lactam antibiotics, including narrow-and extended-spectrum cephalosporins and penicillins (1). When combined with other resistance mechanisms, such as porin loss or efflux (2-5), or when increased expression occurs in derepressed strains (1, 6), organisms expressing class C ␤-lactamases become resistant to cefepime and carbapenems. Point mutations and deletions in the omega loop or helix H2 or H10 and near the C terminus of the AmpC ␤-lactamases that cause an extended-spectrum AmpC (ESAC) phenotype have been described (1, 7). Of the CMY enzymes, 97 unique types have been described to date (see http://www.lahey.org/Studies), including enzymes such as , with alterations of the omega loop or the H10 helix. Recently, Dahyot and Mammeri described a ceftazidime-and cefepime-hydrolyzing CMY-2 laboratory variant based on a clinical mutant in which a Y-X-N loop mutation (R148H) accounted for the ESAC phenotype (13). Little has been written about the behavior of the ESAC variants in regard to ␤-lactamase inhibitors, although a tazobactam-susceptible H-10 helix variant of Escherichia coli AmpC has been described (14).Previous studies on the Y-X-N loop of class C ␤-lactamases explored the role of N152 in the E. coli AmpC (15) and P99 (16) enzymes and suggested that interactions between C-6= or C-7= substituents of penicillins or cephalosporins and N152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of N152 in the cli...

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“…Point mutations and deletions in the omega-loop or helices H2 or H10 and near the C terminus of the AmpC ␤-lactamases cause an expanded-spectrum AmpC (ESAC) phenotype (2, 3). At least 65 unique types of the ADC enzymes (4), including enzymes such as ADC-7, ADC-8, 3,5,6), have been described. The latter two enzymes have an ESAC phenotype.…”

mentioning

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Effect of Asparagine Substitutions in the YXN Loop of a Class C β-Lactamase of Acinetobacter baumannii on Substrate and Inhibitor Kinetics

Skalweit

Taracila

2015

Antimicrob Agents Chemother

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Class C cephalosporinases are a growing threat, and inhibitors of these enzymes are currently unavailable. Studies exploring the YXN loop asparagine in the Escherichia coli AmpC, P99, and CMY-2 enzymes have suggested that interactions between C6= or C7= substituents on penicillins or cephalosporins and this Asn are important in determining substrate specificity and enzymatic stability. We sought to characterize the YXN loop asparagine in the clinically important ADC-7 class C ␤-lactamase of Acinetobacter baumannii. Mutagenesis at the N148 position in ADC-7 yields functional mutants (N152G, -S, -T, -Q, -A, and -C) that retain cephalosporinase activity. Using standard assays, we show that N148G, -S, and -T variants possess good catalytic activity toward cefoxitin and ceftaroline but that cefepime is a poor substrate. Because N152 variants of CMY-2, another class C ␤-lactamase, are more readily inhibited by tazobactam due to higher rates of inactivation, we also tested if the N148 substitutions in ADC-7 would affect inactivation by sulfone inhibitors, sulbactam and tazobactam, class A ␤-lactamase, and A. baumannii penicillin-binding protein (PBP) inhibitors with in vitro activity against ADC-7. The 50% inhibitory concentrations (IC 50 s) for tazobactam and sulbactam were improved, with 7-fold and 2-fold reductions, respectively, for the N148S variant. A homology model of the N148S ADC-7 enzyme in a Michaelis-Menten complex with tazobactam showed a loss of interaction between N148 and the sulfone moiety of the inhibitor. We postulate that this may result in more-rapid secondary ring opening of the inhibitor, as the unbound sulfone is an excellent leaving group, leading to more-rapid formation of the stable linearized inhibitor. C lass C ␤-lactamases such as ADC-7, found in Acinetobacter baumannii, confer resistance to a wide variety of ␤-lactam antibiotics, including narrow-spectrum and extended-spectrum cephalosporins and penicillins (1, 2). Point mutations and deletions in the omega-loop or helices H2 or H10 and near the C terminus of the AmpC ␤-lactamases cause an expanded-spectrum AmpC (ESAC) phenotype (2, 3). At least 65 unique types of the ADC enzymes (4), including enzymes such as ADC-7, ADC-8, 3,5,6), have been described. The latter two enzymes have an ESAC phenotype. The inhibition behavior of ADC enzymes has been explored for boronate inhibitors and carbapenems (7) and for 7-alkylidenecephalosporin sulfones (8). We previously described the inhibition behavior of CMY-2 variants at the N152 position, with the N152G, -S, and -T variants showing enhanced k inact values for tazobactam (9) and increased 50% inhibitory concentrations (IC 50 s) for avibactam (10) (Fig. 1). Although not yet studied in the CMY-2 or ADC enzymes, the complex of avibactam with the AmpC of Pseudomonas aeruginosa, PDC-3, was recently obtained, revealing the key interactions of this novel, first-in-class diazabicyclooctane inhibitor with the YXN loop asparagine 152 in a class C active site (11,12). A more detailed study of the inhibition ...

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Extended-Spectrum Cephalosporinase in Acinetobacter baumannii

Cited by 73 publications

References 22 publications

Genetic basis of antibiotic resistance in pathogenic Acinetobacter species

Genetic basis of antibiotic resistance in pathogenic Acinetobacter species

N152G, -S, and -T Substitutions in CMY-2 β-Lactamase Increase Catalytic Efficiency for Cefoxitin and Inactivation Rates for Tazobactam

Effect of Asparagine Substitutions in the YXN Loop of a Class C β-Lactamase of Acinetobacter baumannii on Substrate and Inhibitor Kinetics

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