PER ␤-lactamases are an emerging family of extended-spectrum ␤-lactamases (ESBL) found in Gram-negative bacteria. PER ␤-lactamases are unique among class A enzymes as they possess an inverted omega (⍀) loop and extended B3 ␤-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-␤-lactam ␤-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 /K ϭ 2 ϫ 10 3 Ϯ 0.1 ϫ 10 3 M Ϫ1 s Ϫ1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D ␤-lactamases (i.e., k 2 /K range of Ϸ10 3 M Ϫ1 s Ϫ1 ) and not class A ␤-lactamases (i.e., k 2 /K range, 10 4 to 10 5 M Ϫ1 s Ϫ1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 Ϯ 3 atomic mass units [amu] ¡ 31,604 Ϯ 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the ␤-lactamase and that the sulfate of AVI formed interactions with the ␤-lactam carboxylate binding site of the PER-2 ␤-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 ␤-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2 /K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 ␤-lactamase, we tested different ␤-lactam-AVI combinations. By lowering MICs to Յ2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2 . Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

PER-2 accounts for up to 10% of oxyimino-cephalosporin resistance in Klebsiella pneumoniae and Escherichia coli in Argentina and hydrolyzes both cefotaxime and ceftazidime with high catalytic efficiencies (k cat /K m ). Through crystallographic analyses, we recently proposed the existence of a hydrogen bond network connecting Ser70-Gln69-oxyanion water-Thr237-Arg220 that might be important for the activity and inhibition of the enzyme. Mutations at Arg244 in most class A ␤-lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER ␤-lactamases is equivalent to Arg244. Alterations in the hydrogen bond network of the active site in PER-2, through modifications in key residues such as Arg220 and (to a much lesser extent) Thr237, dramatically impact the overall susceptibility to inactivation, with up to ϳ300-and 500-fold reductions in the rate constant of inactivation (k inact )/K i values for clavulanic acid and tazobactam, respectively. Hydrolysis on cephalosporins and aztreonam was also affected, although to different extents compared to with wild-type PER-2; for cefepime, only an Arg220Gly mutation resulted in a strong reduction in the catalytic efficiency. Mutations at Arg220 entail modifications in the catalytic activity of PER-2 and probably local perturbations in the protein, but not global conformational changes. Therefore, the apparent structural stability of the mutants suggests that these enzymes could be possibly selected in vivo.

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-β-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D β-lactamases (i.e., k2/K of ≈103 M−1 s−1) but lower than other class A β-lactamases (i.e., k2/K = 104 to 105 M−1 s−1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K.) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum β-lactamases (ESBLs). Comparatively, relebactam combined with a β-lactam is more potent against Escherichia coli producing PER-2 variants than β-lactam–AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

The objectives of this study were to determine the kinetic parameters of purified recombinant Bla and Bla by spectrophotometry, analyze the genetic environment of the bla and bla genes in both species by polymerase chain reaction and sequencing, furthermore, in silico models of both enzymes in complex with imipenem were obtained by modeling tools. Our results showed that Bla and Bla have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. Bla hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. Bla and Bla showed that their closest structural homologs are KPC-2 and SFC-1, which correlate to the mild carbapenemase activity toward imipenem observed at least for BlaMmas. They also seem to differ from other class A β-lactamases by the presence of a more flexible Ω loop, which could impact in the hydrolysis efficiency against some antibiotics. A -35 consensus sequence (TCGACA) and embedded at the 3' end of MAB_2874, which may constitute the bla and bla promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of β-lactamases that have improved catalytic efficiencies against some of the drugs commonly used for the treatment of mycobacterial infections, endangering the use of important drugs like the carbapenems.

b PER-2 belongs to a small (7 members to date) group of extended-spectrum ␤-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most ␤-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward ␤-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted ⍀ loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A ␤-lactamases. PER ␤-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A ("A" indicates an insertion according to Ambler's scheme for residue numbering in PER ␤-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different ␤-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

The contribution of OXA-258 enzymes to the final β-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii.

The -harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C and is closely related to pRA1. It contains a large resistance island including the gene between two copies of IS-like elements, the toxin-antitoxin module , several other resistance genes inserted within a Tn transposon, a Tn-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.

No abstract