In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.

The molecular basis of resistance to b-lactams and b-lactam-b-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective b-lactam therapy. Recent crystal structures of KPC-2 and other class A b-lactamases suggest that Ambler position Trp105 may be of importance in binding b-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe b-lactamase displayed improved catalytic efficiencies, k cat /K m , toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k cat /K m toward cefotaxime and imipenem when compared to WT b-lactamase. The Trp105Asn variant exhibited increased K m s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k cat /K m ) for all substrates. With respect to clavulanic acid, the K i s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the b-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in b-lactam and b-lactamase inhibitor discrimination.

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β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki* values of 40–640 nM) through a two-step process in which an initial enzyme (E)–inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E*I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2–BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.