The biochemical properties of CMY-32, a class C enzyme possessing a single amino acid substitution in the Ω loop (Gly214Glu) were compared to the parent enzyme, CMY-2, a widespread class C β-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (kcat/Km) by 50-70% against “good” substrates (i.e., cephalothin) while increasing kcat/Km against “poor” substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone β-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both β-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is β-lactamase-catalyzed elimination of the C6 hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new β-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of -SO3-2. Molecular modeling and dynamics simulations suggest that the Ω loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining increased cefotaxime hydrolysis. When docked in the active site, we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight: i) the impact of single amino acid substitutions on protein evolution in clinically important AmpC enzymes; and ii) the novel insights into the mechanisms by which carbapenems and monobactams interact with CMY-2 and CMY-32 β-lactamases.

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Genetic screening of Pseudomonas aeruginosa (PSDA) and Acinetobacter baumannii (ACB) reveals genes that confer increased susceptibility to β-lactams when disrupted, suggesting novel drug targets. One such target is lytic transglycosylase. Bulgecin A (BlgA) is a natural product of Pseudomonas mesoacidophila and a lytic transglycosolase inhibitor that works synergistically with β-lactams targeting PBP3 for Enterobacteriaceae. BlgA also weakly inhibits di-Zn2+ metallo-β-lactamases like L1 of Stenotrophomonas maltophilia. We hypothesized that because of its unique mechanism of action, BlgA could restore susceptibility to carbapenems in carbapenem-resistant PSDA (CR-PSDA) and carbapenem-resistant ACB, as well as ACB resistant to sulbactam. A BlgA-containing extract was prepared using a previously published protocol. CR-PSDA clinical isolates demonstrating a variety of carbapenem resistance mechanisms (VIM-2 carbapenemases, efflux mechanisms, and AmpC producer expression) were characterized with agar dilution minimum inhibitory concentration (MIC) testing and polymerase chain reaction. Growth curves using these strains were prepared using meropenem, BlgA extract, and meropenem plus BlgA extract. A concentrated Blg A extract combined with low concentrations of meropenem, was able to inhibit the growth of clinical strains of CR-PSDA for strains that had meropenem MICs ≥8 mg/L by agar dilution, and a clinical strain of an OXA-24 producing ACB that had a meropenem MIC >32 mg/L and intermediate ampicillin/sulbactam susceptibility. Similar experiments were conducted on a TEM-1 producing ACB strain resistant to sulbactam. BlgA with ampicillin/sulbactam inhibited the growth of this organism. As in Enterobacteriaceae, BlgA appears to restore the efficacy of meropenem in suppressing the growth of CR-PSDA and carbapenem-resistant ACB strains with a variety of common carbapenem resistance mechanisms. BlgA extract also inhibits VIM-2 β-lactamase in vitro. BlgA may prove to be an exciting adjunctive compound to extend the life of carbapenems against these vexing pathogens.

This cohort study compares amputation and mortality rates among patients treated in the Veterans Health Administration for diabetic foot osteomyelitis with and without rifampin.

Drug-resistant pathogens have gained a foothold especially in the most vulnerable patient populations, hospitalized and immunocompromised individuals. Furthermore, extended-spectrum β-lactamase and carbapenemase-producing organisms are finding their way even into the community, with patients presenting to the hospital with established colonization and infection with resistant Enterobacteriaceae in particular. Recently, a novel antipseudomonal cephalosporin in combination with an established Class A β-lactamase inhibitor, ceftolozane/tazobactam has been approved by the FDA for use in the treatment of complicated urinary tract infections and complicated intra-abdominal infections. Ceftolozane is a uniquely potent antipseudomonal cephalosporin because of its high affinity for the penicillin-binding proteins of Pseudomonas aeruginosa, its low affinity for the intrinsic Class C β-lactamases of P. aeruginosa, its ability to enter P. aeruginosa through the outer membrane without the utilization of OprD protein, and the fact that it is not a substrate of the often upregulated MexAB/OprM efflux system of P. aeruginosa. The biological chemistry, pharmacokinetics/pharmacodynamics, microbiologic spectrum, and clinical trials that led to the approval of ceftolozane is reviewed. A discussion regarding its potential role in the treatment of complicated intra-abdominal infections and other infectious disease syndromes associated with drug-resistant pathogens follows.

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...

Ambler position 105 in class A ␤-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine ␤-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed that Escherichia coli DH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all ␤-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, <2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increased K m . Clavulanic acid and penem K i values were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.