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.

Deciphering the Coevolutionary Dynamics of L2 ß-Lactamases via Deep Learning.

L2 ß-lactamases, serine-based class A ß-lactamases expressed by Stenotrophomonas maltophilia, play a pivotal role in antimicrobial resistance (AMR). However, limited studies have been conducted on these important enzymes. To understand the coevolutionary dynamics of L2 ß-lactamase, innovative computational methodologies, including adaptive sampling molecular dynamics simulations, and deep learning methods (convolutional variational autoencoders and BindSiteS-CNN) explored conformational changes and correlations within the L2 ß-lactamase family together with other representative class A enzymes including SME-1 and KPC-2. This work also investigated the potential role of hydrophobic nodes and binding site residues in facilitating the functional mechanisms. The convergence of analytical approaches utilized in this effort yielded comprehensive insights into the dynamic behavior of the ß-lactamases, specifically from an evolutionary standpoint. In addition, this analysis presents a promising approach for understanding how the class A ß-lactamases evolve in response to environmental pressure and establishes a theoretical foundation for forthcoming endeavors in drug development aimed at combating AMR.

AMPLON: Amplifying DNA with Multiarm Priming and Looping Optimization of Nucleic Acid.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL-1. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.

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.

The Structural Role of N170 in Substrate-Assisted Deacylation in KPC-2 ß-Lactamase.

The amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC-2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. Specific substitutions in the omega loop (R164-D179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop - more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and an altered spectrum of enzymatic activity.

Utilizing Ceftazidime/Avibactam Therapeutic Drug Monitoring in the Treatment of Neurosurgical Meningitis Caused by Difficult-to-Treat Resistant Pseudomonas aeruginosa and KPC-Producing Enterobacterales.

Background: Central nervous system (CNS) infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales and difficult-to-treat resistant (DTR) Pseudomonas aeruginosa represent a formidable clinical challenge. Antimicrobial regimens that efficiently penetrate the cerebrospinal fluid (CSF) and achieve sufficient concentrations associated with microbiologic and clinical cure are limited. We evaluated therapy with ceftazidime-avibactam (CAZ-AVI) in order to guide precise dosing in the treatment of CNS infections. Methods: Therapeutic drug monitoring (TDM) was performed in 3 patients with health care-associated ventriculitis and meningitis (HAVM) using CAZ-AVI 2.5 g infused intravenously every 8 hours as standard and extended infusion. Simultaneous CSF and plasma samples were obtained throughout the dosing interval in each patient. Concentrations of CAZ and AVI were determined by liquid chromatography/mass spectrometry. Results: Bacterial identification revealed KPC-producing Klebsiella pneumoniae (KPC-Kp), DTR Pseudomonas aeruginosa, and KPC-producing Enterobacter cloacae (KPC-Ent.c). All isolates were resistant to carbapenems. The minimum inhibitory concentrations (MICs) of CAZ-AVI were 0.25/4, 4/4, and 0.25/4 µg/mL, respectively. CAZ and AVI concentrations were determined in CSF samples ranging from 29.0 to 15.0 µg/mL (CAZ component) and 4.20 to 0.92 µg/mL (AVI component), respectively. AVI achieved concentrations ≥1 µg/mL in 11 out of 12 CSF samples collected throughout the dosing interval. Clinical and microbiologic cure were attained in all patients. Conclusions: Postinfusion concentrations of CAZ-AVI were measured in plasma and CSF samples obtained from 3 patients with complicated CNS infections caused by antimicrobial-resistant isolates. The measured concentrations revealed that standard CAZ and AVI exposures sufficiently attained values correlating to 50% fT > MIC, which are associated with efficient bacterial killing.

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.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

The role of the plasmid-mediated fluoroquinolone resistance genes as resistance mechanisms in pediatric infections due to Enterobacterales.

Introduction: Fluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies. Methods: A case-control study of children (0-18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, ß-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed. Results: Of 170 ß-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were bla SHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were bla CTX-M-1-group ESBL (79%) and aac(6')-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found. Conclusions: Paradoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

3D Printed Materials for Combating Antimicrobial Resistance.

Three-dimensional (3D) printing is a rapidly growing technology with a significant capacity for translational applications in both biology and medicine. 3D-printed living and non-living materials are being widely tested as a potential replacement for conventional solutions for testing and combating antimicrobial resistance (AMR). The precise control of cells and their microenvironment, while simulating the complexity and dynamics of an in vivo environment, provides an excellent opportunity to advance the modeling and treatment of challenging infections and other health conditions. 3D-printing models the complicated niches of microbes and host-pathogen interactions, and most importantly, how microbes develop resistance to antibiotics. In addition, 3D-printed materials can be applied to testing and delivering antibiotics. Here, we provide an overview of 3D printed materials and biosystems and their biomedical applications, focusing on ever increasing AMR. Recent applications of 3D printing to alleviate the impact of AMR, including developed bioprinted systems, targeted bacterial infections, and tested antibiotics are presented.

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.

Insights into dynamic changes in ADC-7 and P99 cephalosporinases using small angle x-ray scattering (SAXS).

To counter the emergence of ß-lactamase (BL) mediated resistance, design of new ß-lactamase inhibitors (BLIs) is critical. Many high-resolution crystallographic structures of BL complexed with BLIs are available. However, their impact on BLI design is struggling to keep pace with novel and emerging variants. Small angle x-ray scattering (SAXS) in combination with molecular modeling is a useful tool to determine dynamic structures of macromolecules in solution. An important application of SAXS is to determine the conformational changes that occur when BLI bind to BL. To probe if conformational dynamics occur in class C cephalosporinases, we studied SAXS profiles of two clinically relevant class C ß-lactamases, Acinetobacter baumannii ADC-7 and Enterobacter cloacae P99 in apo format complexed with BLIs. Importantly, SAXS data analysis demonstrated that in solution, these representative class C enzymes remain monomeric and did not show the associated assemblies that were seen in various crystal structures. SAXS data acquired for ADC-7 and P99, in apo and inhibitor bound states, clearly showed that these enzymes undergo detectable conformational changes, and these class C ß-lactamases also close upon binding inhibitors as does BlaC. Further analysis revealed that addition of inhibitor led to the compacting of a range of residues around the active site, indicating that the conformational changes that both P99 and ADC-7 undergo are central to inhibitor recognition and efficacy. Our findings support the importance of exploring conformational changes using SAXS analysis in the design of future BLIs.Communicated by Ramaswamy H. Sarma.

Synthesis of a Novel Boronic Acid Transition State Inhibitor, MB076: A Heterocyclic Triazole Effectively Inhibits Acinetobacter-Derived Cephalosporinase Variants with an Expanded-Substrate Spectrum.

Class C Acinetobacter-derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii. Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC ß-lactamase variants with Ki values <1 µM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Sulfonamidoboronic Acids as "Cross-Class" Inhibitors of an Expanded-Spectrum Class C Cephalosporinase, ADC-33, and a Class D Carbapenemase, OXA-24/40: Strategic Compound Design to Combat Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to ß-lactams. One of the most important mechanisms is the production of ß-lactamase enzymes capable of hydrolyzing ß-lactam antibiotics. Co-expression of multiple classes of ß-lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical ß-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C ß-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other ß-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum ß-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

Characterization of a Novel Pathogen in Immunocompromised Patients: Elizabethkingia anophelis-Exploring the Scope of Resistance to Contemporary Antimicrobial Agents and ß-lactamase Inhibitors.

Background: Elizabethkingia anophelis is an emerging Gram-negative nonlactose fermenter in the health care setting, where it causes life-threatening infections in immunocompromised patients. We aimed to characterize the molecular mechanisms of antimicrobial resistance and evaluate the utility of contemporary antibiotics with the intent to offer targeted therapy against an uncommonly encountered pathogen. Methods: Whole-genome sequencing (WGS) was conducted to accurately identify isolate species and elucidate the determinants of ß-lactam resistance. Antimicrobial susceptibility testing was performed using broth microdilution and disk diffusion assays. To assess the functional contribution of the major metallo-ß-lactamase (MBL) encoding genes to the resistance profile, bla BlaB was cloned into pBCSK(-) phagemid vector and transformed into Escherichia coli DH10B. Results: WGS identified the organism as E. anophelis. MBL genes bla BlaB-1 and bla GOB-26 were identified, in addition to bla CME-2, which encodes for an extended-spectrum ß-lactamase (ESBL). Plasmids were not detected. The isolate was nonsusceptible to all commonly available ß-lactams, carbapenems, newer ß-lactam ß-lactamase inhibitor combinations, and to the combination of aztreonam (ATM) with ceftazidime-avibactam (CAZ-AVI). Susceptibility to the novel siderophore cephalosporin cefiderocol was determined. A BlaB-1 transformant E. coli DH10B isolate was obtained and demonstrated increased minimum inhibitory concentrations to cephalosporins, carbapenems, and CAZ-AVI, but not ATM. Conclusions: Using WGS, we accurately identified and characterized an extensively drug-resistant E. anophelis in an immunocompromised patient. Rapid evaluation of the genetic background can guide accurate susceptibility testing to better inform antimicrobial therapy selection.

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M ß-Lactamases: Biochemical and Structural Analyses.

Design of novel ß-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two α-amido-ß-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum ß-lactamase [ESBL]) ß-lactamases were evaluated. The 50% inhibitory concentrations (IC50s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k2/K for CTX-M-96 (24,000 M-1 s-1) was twice the reported value for KPC-2 (12,000 M-1 s-1); for MB_076, the k2/K values ranged from 1,200 M-1 s-1 (KPC-2) to 3,900 M-1 s-1 (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-Å resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to ß-lactamase inhibitor design.

Multidrug-resistant Acinetobacter pittii is adapting to and exhibiting potential succession aboard the International Space Station.

BACKGROUND: Monitoring the adaptation of microorganisms to the extreme environment of the International Space Station (ISS) is crucial to understanding microbial evolution and infection prevention. Acinetobacter pittii is an opportunistic nosocomial pathogen, primarily impacting immunocompromised patients, that was recently isolated from two missions aboard the ISS. RESULTS: Here, we report how ISS-associated A. pittii (n = 20 genomes) has formed its own genetically and functionally discrete clade distinct from most Earth-bound isolates (n = 291 genomes). The antimicrobial susceptibility testing of ISS strains and two related clinical isolates demonstrated that ISS strains acquired more resistance, specifically with regard to expanded-spectrum cephalosporins, despite no prediction of increased resistance based on genomic analysis of resistance genes. By investigating 402 longitudinal environmental and host-associated ISS metagenomes, we observed that viable A. pittii is increasing in relative abundance and therefore potentially exhibiting succession, being identified in >2X more metagenomic samples in back-to-back missions. ISS strains additionally contain functions that enable them to survive in harsh environments, including the transcriptional regulator LexA. Via a genome-wide association study, we identified a high level of mutational burden in methionine sulfoxide reductase genes relative to the most closely related Earth strains. CONCLUSIONS: Overall, these results indicated a step forward in understanding how microorganisms might evolve and alter their antibiotic resistance phenotype in extreme, resource-limited, human-built environments. Video Abstract.

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.

Different Conformations Revealed by NMR Underlie Resistance to Ceftazidime/Avibactam and Susceptibility to Meropenem and Imipenem among D179Y Variants of KPC ß-Lactamase.

ß-Lactamase-mediated resistance to ceftazidime-avibactam (CZA) is a serious limitation in the treatment of Gram-negative bacteria harboring Klebsiella pneumoniae carbapenemase (KPC). Herein, the basis of susceptibility to carbapenems and resistance to ceftazidime (CAZ) and CZA of the D179Y variant of KPC-2 and -3 was explored. First, we determined that resistance to CZA in a laboratory strain of Escherichia coli DH10B was not due to increased expression levels of the variant enzymes, as demonstrated by reverse transcription PCR (RT-PCR). Using timed mass spectrometry, the D179Y variant formed prolonged acyl-enzyme complexes with imipenem (IMI) and meropenem (MEM) in KPC-2 and KPC-3, which could be detected up to 24 h, suggesting that IMI and MEM act as covalent ß-lactamase inhibitors more than as substrates for D179Y KPC-2 and -3. This prolonged acyl-enzyme complex of IMI and MEM by D179Y variants was not observed with wild-type (WT) KPCs. CAZ was studied and the D179Y variants also formed acyl-enzyme complexes (1 to 2 h). Thermal denaturation and differential scanning fluorimetry showed that the tyrosine substitution at position 179 destabilized the KPC ß-lactamases (KPC-2/3 melting temperature [Tm] of 54 to 55°C versus D179Y Tm of 47.5 to 51°C), and the D179Y protein was 3% disordered compared to KPC-2 at 318 K. Heteronuclear 1H/15N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy also revealed that the D179Y variant, compared to KPC-2, is partially disordered. Based upon these observations, we discuss the impact of disordering of the Ω loop as a consequence of the D179Y substitution. These conformational changes and disorder in the overall structure as a result of D179Y contribute to this unanticipated phenotype.