Ω-Loop mutations control the dynamics of the active site by modulating a network of hydrogen bonds in PDC-3 ß-lactamase.

The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. To explore the relationships between structural dynamics and altered substrate specificity as a result of amino acid substitutions in PDC-3, innovative computational methods like machine learning driven adaptive bandit molecular dynamics simulations and markov state modeling of the wild-type PDC-3 and nine clinically identified variants were conducted. Our analysis reveals that structural changes in the Ω loop controls the dynamics of the active site. The E219K and Y221A substitutions have the most pronounced effects. The modulation of three key hydrogen bonds K67(sc)-G220(bb), Y150(bb)-A292(bb) and N287(sc)-N314(sc) were found to result in an expansion of the active site, which could have implications for the binding and inactivation of cephalosporins. Overall, the findings highlight the importance of understanding the structural dynamics of PDC-3 in the development of new treatments for antibiotic-resistant infections.

Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase.

A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C ß-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.

Transcending the challenge of evolving resistance mechanisms in Pseudomonas aeruginosa through ß-lactam-enhancer-mechanism-based cefepime/zidebactam.

Multi-drug resistant (MDR) Pseudomonas aeruginosa harbor a complex array of ß-lactamases and non-enzymatic resistance mechanisms. In this study, the activity of a ß-lactam/ß-lactam-enhancer, cefepime/zidebactam, and novel ß-lactam/ß-lactamase inhibitor combinations was determined against an MDR phenotype-enriched, challenge panel of P. aeruginosa (n = 108). Isolates were multi-clonal as they belonged to at least 29 distinct sequence types (STs) and harbored metallo-ß-lactamases, serine ß-lactamases, penicillin binding protein (PBP) mutations, and other non-enzymatic resistance mechanisms. Ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam, and cefepime/taniborbactam demonstrated MIC90s of >128 mg/L, while cefepime/zidebactam MIC90 was 16 mg/L. In a neutropenic-murine lung infection model, a cefepime/zidebactam human epithelial-lining fluid-simulated regimen achieved or exceeded a translational end point of 1-log10 kill for the isolates with elevated cefepime/zidebactam MICs (16-32 mg/L), harboring VIM-2 or KPC-2 and alterations in PBP2 and PBP3. In the same model, to assess the impact of zidebactam on the pharmacodynamic (PD) requirement of cefepime, dose-fractionation studies were undertaken employing cefepime-susceptible P. aeruginosa isolates. Administered alone, cefepime required 47%-68% fT >MIC for stasis to ~1 log10 kill effect, while cefepime in the presence of zidebactam required just 8%-16% for >2 log10 kill effect, thus, providing the pharmacokinetic/PD basis for in vivo efficacy of cefepime/zidebactam against isolates with MICs up to 32 mg/L. Unlike ß-lactam/ß-lactamase inhibitors, ß-lactam enhancer mechanism-based cefepime/zidebactam shows a potential to transcend the challenge of ever-evolving resistance mechanisms by targeting multiple PBPs and overcoming diverse ß-lactamases including carbapenemases in P. aeruginosa.IMPORTANCECompared to other genera of Gram-negative pathogens, Pseudomonas is adept in acquiring complex non-enzymatic and enzymatic resistance mechanisms thus remaining a challenge to even novel antibiotics including recently developed ß-lactam and ß-lactamase inhibitor combinations. This study shows that the novel ß-lactam enhancer approach enables cefepime/zidebactam to overcome both non-enzymatic and enzymatic resistance mechanisms associated with a challenging panel of P. aeruginosa. This study highlights that the ß-lactam enhancer mechanism is a promising alternative to the conventional ß-lactam/ß-lactamase inhibitor approach in combating ever-evolving MDR P. aeruginosa.

Imipenem/Relebactam Resistance in Clinical Isolates of Extensively Drug Resistant Pseudomonas aeruginosa: Inhibitor-Resistant ß-Lactamases and Their Increasing Importance.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.

Genomic heterogeneity underlies multidrug resistance in Pseudomonas aeruginosa: A population-level analysis beyond susceptibility testing.

BACKGROUND: Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. METHODS: Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. RESULTS: The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. CONCLUSIONS: The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

Allosteric communication in class A ß-lactamases occurs via cooperative coupling of loop dynamics.

Understanding allostery in enzymes and tools to identify it offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways in two prototypical class A ß-lactamases, TEM-1 and KPC-2, which are important determinants of antibiotic resistance. The nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. Propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more of clinically relevant amino acid substitutions map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover, and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

Antibiotics are crucial drugs for treating and preventing bacterial infections, but some bacteria are evolving ways to resist their effects. This 'antibiotic resistance' threatens lives and livelihoods worldwide. ß-lactam antibiotics, like penicillin, are some of the most commonly used, but some bacteria can now make enzymes called ß-lactamases, which destroy these antibiotics. Dozens of different types of ß-lactamases now exist, each with different properties. Two of the most medically important are TEM-1 and KPC-2. One way to counteract ß-lactamases is with drugs called inhibitors that stop the activity of these enzymes. The approved ß-lactamase inhibitors work by blocking the part of the enzyme that binds and destroys antibiotics, known as the 'active site'. The ß-lactamases have evolved, some of which have the ability to resist the effects of known inhibitors. It is possible that targeting parts of ß-lactamases far from the active site, known as 'allosteric sites', might get around these new bacterial defences. A molecule that binds to an allosteric site might alter the enzyme's shape, or restrict its movement, making it unable to do its job. Galdadas, Qu et al. used simulations to understand how molecules binding at allosteric sites affect enzyme movement. The experiments examined the structures of both TEM-1 and KPC-2, looking at how their shapes changed as molecules were removed from the allosteric site. This revealed how the allosteric sites and the active site are linked together. When molecules were taken out of the allosteric sites, they triggered ripples of shape change that travelled via loop-like structures across the surface of the enzyme. These loops contain over half of the known differences between the different types of ß-lactamases, suggesting mutations here may be responsible for changing which antibiotics each enzyme can destroy. In other words, changes in the 'ripples' may be related to the ability of the enzymes to resist particular antibiotics. Understanding how changes in one part of a ß-lactamase enzyme reach the active site could help in the design of new inhibitors. It might also help to explain how ß-lactamases evolve new properties. Further work could show why different enzymes are more or less active against different antibiotics.

A comprehensive and contemporary "snapshot" of ß-lactamases in carbapenem resistant Acinetobacter baumannii.

Successful treatment of Acinetobacter baumannii infections require early and appropriate antimicrobial therapy. One of the first steps in this process is understanding which ß-lactamase (bla) alleles are present and in what combinations. Thus, we performed WGS on 98 carbapenem-resistant A. baumannii (CR Ab). In most isolates, an acquired blaOXA carbapenemase was found in addition to the intrinsic blaOXA allele. The most commonly found allele was blaOXA-23 (n = 78/98). In some isolates, blaOXA-23 was found in addition to other carbapenemase alleles: blaOXA-82 (n = 12/78), blaOXA-72 (n = 2/78) and blaOXA-24/40 (n = 1/78). Surprisingly, 20% of isolates carried carbapenemases not routinely assayed for by rapid molecular diagnostic platforms, i.e., blaOXA-82 and blaOXA-172; all had ISAba1 elements. In 8 CR Ab, blaOXA-82 or blaOXA-172 was the only carbapenemase. Both blaOXA-24/40 and its variant blaOXA-72 were each found in 6/98 isolates. The most prevalent ADC variants were blaADC-30 (21%), blaADC-162 (21%), and blaADC-212 (26%). Complete combinations are reported.

Resistance to Novel ß-Lactam-ß-Lactamase Inhibitor Combinations: The "Price of Progress".

Significant advances were made in antibiotic development during the past 5 years. Novel agents were added to the arsenal that target critical priority pathogens, including multidrug-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacterales. Of these, 4 novel ß-lactam-ß-lactamase inhibitor combinations (ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-cilastatin-relebactam) reached clinical approval in the United States. With these additions comes a significant responsibility to reduce the possibility of emergence of resistance. Reports in the rise of resistance toward ceftolozane-tazobactam and ceftazidime-avibactam are alarming. Clinicians and scientists must make every attempt to reverse or halt these setbacks.

A Standard Numbering Scheme for Class C ß-Lactamases.

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) ß-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.