Multiscale Simulations Identify Origins of Differential Carbapenem Hydrolysis by the OXA-48 ß-Lactamase.

OXA-48 ß-lactamases are frequently encountered in bacterial infections caused by carbapenem-resistant Gram-negative bacteria. Due to the importance of carbapenems in the treatment of healthcare-associated infections and the increasingly wide dissemination of OXA-48-like enzymes on plasmids, these ß-lactamases are of high clinical significance. Notably, OXA-48 hydrolyzes imipenem more efficiently than other commonly used carbapenems, such as meropenem. Here, we use extensive multiscale simulations of imipenem and meropenem hydrolysis by OXA-48 to dissect the dynamics and to explore differences in the reactivity of the possible conformational substates of the respective acylenzymes. Quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reaction for both substrates demonstrate that deacylation is favored when the 6α-hydroxyethyl group is able to hydrogen bond to the water molecule responsible for deacylation but disfavored by the increasing hydration of either oxygen of the carboxylated Lys73 general base. Differences in free energy barriers calculated from the QM/MM simulations correlate well with the experimentally observed differences in hydrolytic efficiency between meropenem and imipenem. We conclude that the impaired breakdown of meropenem, compared to imipenem, which arises from a subtle change in the hydrogen bonding pattern between the deacylating water molecule and the antibiotic, is most likely induced by the meropenem 1ß-methyl group. In addition to increased insights into carbapenem breakdown by OXA ß-lactamases, which may aid in future efforts to design antibiotics or inhibitors, our approach exemplifies the combined use of atomistic simulations in determining the possible different enzyme-substrate substates and their influence on enzyme reaction kinetics.

Cryptic ß-Lactamase Evolution Is Driven by Low ß-Lactam Concentrations.

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary ß-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and ß5-ß6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of ß-lactams can drive the evolution of ß-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance.IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of ß-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low ß-lactam concentrations on the widespread ß-lactamase OXA-48. Our data indicate that the exposure to ß-lactams at very low concentrations enhances ß-lactamase diversity and drives the evolution of ß-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.

Antimicrobial Resistance Conferred by OXA-48 ß-Lactamases: Towards a Detailed Mechanistic Understanding.

OXA-48-type ß-lactamases are now routinely encountered in bacterial infections caused by carbapenem-resistant Enterobacterales These enzymes are of high and growing clinical significance due to the importance of carbapenems in treatment of health care-associated infections by Gram-negative bacteria, the wide and increasing dissemination of OXA-48 enzymes on plasmids, and the challenges posed by their detection. OXA-48 confers resistance to penicillin (which is efficiently hydrolyzed) and carbapenem antibiotics (which is more slowly broken down). In addition to the parent enzyme, a growing array of variants of OXA-48 is now emerging. The spectrum of activity of these variants varies, with some hydrolyzing expanded-spectrum oxyimino-cephalosporins. The growth in importance and diversity of the OXA-48 group has motivated increasing numbers of studies that aim to elucidate the relationship between structure and specificity and establish the mechanistic basis for ß-lactam turnover in this enzyme family. In this review, we collate recently published structural, kinetic, and mechanistic information on the interactions between clinically relevant ß-lactam antibiotics and inhibitors and OXA-48 ß-lactamases. Collectively, these studies are starting to form a detailed picture of the underlying bases for the differences in ß-lactam specificity between OXA-48 variants and the consequent differences in resistance phenotype. We focus specifically on aspects of carbapenemase and cephalosporinase activities of OXA-48 ß-lactamases and discuss ß-lactamase inhibitor development in this context. Throughout the review, we also outline key open research questions for future investigation.

An Efficient Computational Assay for ß-Lactam Antibiotic Breakdown by Class A ß-Lactamases.

Class A ß-lactamases cause clinically relevant resistance to ß-lactam antibiotics. Carbapenem degradation is a particular concern. We present an efficient QM/MM molecular simulation protocol that accurately predicts the activity of ß-lactamases against carbapenems. Simulations take less than 24 CPU hours, a greater than 99% reduction, and do not require fitting against experimental data or significant parametrization. This computational assay also reveals mechanistic details of ß-lactam breakdown and should assist in evaluating emerging ß-lactamase variants and developing new antibiotics.

ß-Lactamases and ß-Lactamase Inhibitors in the 21st Century.

The ß-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, ß-lactamase enzymes that hydrolyze the amide bond of the four-membered ß-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). ß-Lactamases divide into four classes; the active-site serine ß-lactamases (classes A, C and D) and the zinc-dependent or metallo-ß-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for ß-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of ß-lactam breakdown. A second focus is ß-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of ß-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of ß-lactams with diazabicyclooctanone and cyclic boronate serine ß-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of ß-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new ß-lactam:inhibitor combinations and the continuing clinical importance of ß-lactams mean that this remains a rewarding research area.