Insights into the activity of cefiderocol against PER-2 producing Enterobacterales.

The PER-2 ß-lactamase is a unique class A enzyme conferring broad spectrum cephalosporin resistance. In this study, we explored the stability of cefiderocol (FDC) against PER-2 ß-lactamase to gain insights into structure activity relationships (SAR) of this synthetic siderophore-conjugated antibiotic. Herein, we show that the MICs of FDC for PER-2 producing isolates and transformants ranged between 0.125 and 64 µg/mL; diazabicyclooctanes (DBOs) reduced the MIC values. In PER-2 mutants, MIC values decreased up to 10-12 dilutions in agreement with previous observations especially in the case of Arg220 substitutions. Catalytic efficiency for PER-2 was 0.072 µM-1 s-1, comparable with PER-1 (0.046 µM-1 s-1) and NDM-1 (0.067 µM-1 s-1). In silico models revealed that FDC within the active site of PER-2 demonstrates unique interactions as a result of the inverted Ω loop fold and extension of the ß3-ß4 connecting loop.

Structural Insights into the Inhibition of the Extended-Spectrum ß-Lactamase PER-2 by Avibactam.

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-ß-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D ß-lactamases (i.e., k2/K of ≈103 M-1 s-1) but lower than other class A ß-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 ß-lactamases (ESBLs). Comparatively, relebactam combined with a ß-lactam is more potent against Escherichia coli producing PER-2 variants than ß-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.

Complete Sequence of the IncA/C1 Plasmid pCf587 Carrying blaPER-2 from Citrobacter freundii.

The blaPER-2-harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C1 and is closely related to pRA1. It contains a large resistance island including the blaPER-2 gene between two copies of ISKox2-like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn2 transposon, a Tn21-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.

Impact of Mutations at Arg220 and Thr237 in PER-2 ß-Lactamase on Conformation, Activity, and Susceptibility to Inhibitors.

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 (kcat/Km ). 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 ß-lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER ß-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 ∼300- and 500-fold reductions in the rate constant of inactivation (kinact)/Ki 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.

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 ß-Lactamase.

PER ß-lactamases are an emerging family of extended-spectrum ß-lactamases (ESBL) found in Gram-negative bacteria. PER ß-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 ß-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-ß-lactam ß-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ± 0.1 × 103 M-1 s-1, where k2 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 ß-lactamases (i.e., k2/K range of ≈103 M-1 s-1) and not class A ß-lactamases (i.e., k2/K range, 104 to 105 M-1 s-1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 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 ß-lactamase and that the sulfate of AVI formed interactions with the ß-lactam carboxylate binding site of the PER-2 ß-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 ß-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/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 ß-lactamase, we tested different ß-lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing blaPER-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.

Crystal structure of the extended-spectrum ß-lactamase PER-2 and insights into the role of specific residues in the interaction with ß-lactams and ß-lactamase inhibitors.

PER-2 belongs to a small (7 members to date) group of extended-spectrum ß-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most ß-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 ß-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 Ω 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 ß-lactamases. PER ß-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 ß-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 ß-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.

Diversity of genetic platforms harboring the blaPER-2 gene in Enterobacterales and insights into the role of ISPa12 in its mobilization and dissemination.

The production of PER-like extended-spectrum ß-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring blaPER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with blaPER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the blaPER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the blaPER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of blaPER genes), indicates a role of ISPa12 in blaPER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the blaPER-2 gene was carried by a novel ISPa12-composite transposon, Tn7390. In addition, its association with ISKox2-like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of blaPER-2 genes.

Whole-Genome Analysis of a High-Risk Clone of Klebsiella pneumoniae ST147 Carrying Both mcr-1 and blaNDM-1 Genes in Peru.

The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other ß-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

Characterisation of OXA-258 enzymes and AxyABM efflux pump in Achromobacter ruhlandii.

OBJECTIVES: The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii. METHODS: Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine ß-naphthylamide (PAßN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward ß-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAßN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles. CONCLUSIONS: The contribution of OXA-258 enzymes to the final ß-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.

Biochemical Characterization of ß-Lactamases from Mycobacterium abscessus Complex and Genetic Environment of the ß-Lactamase-Encoding Gene.

The objectives of this study were to determine the kinetic parameters of purified recombinant BlaMab and BlaMmas by spectrophotometry, analyze the genetic environment of the blaMab and blaMmas 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 BlaMab and BlaMmas have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. BlaMmas hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. BlaMab and BlaMmas 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 ß-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 blaMab and blaMmas promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of ß-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.