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Mycobacterium abscessus is a highly drug-resistant nontuberculous mycobacterium (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating, with a focus on cell wall synthesis proteins (M. abscessusl,d-transpeptidases 1 to 5 [LdtMab1 to LdtMab5] and d,d-carboxypeptidase) that are targeted by β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase (BlaMab). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of M. abscessus isolates (MIC50 ≤ 0.25 μg/ml; MIC90 ≤ 0.5 μg/ml). Combining ceftaroline and imipenem with a β-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the β-lactams alone. In steady-state kinetic assays, BlaMab exhibited a lower Ki app (0.30 ± 0.03 μM for avibactam and 136 ± 14 μM for relebactam) and a higher acylation rate for avibactam (k2/K = 3.4 × 105 ± 0.4 × 105 M−1 s−1 for avibactam and 6 × 102 ± 0.6 × 102 M−1 s−1 for relebactam). The kcat/Km was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM−1 s−1) than for imipenem (0.056 ± 0.006 μM−1 s−1). Timed mass spectrometry captured complexes of avibactam and BlaMab, LdtMab1, LdtMab2, LdtMab4, and d,d-carboxypeptidase, whereas relebactam bound only BlaMab, LdtMab1, and LdtMab2. Interestingly, LdtMab1, LdtMab2, LdtMab4, LdtMab5, and d,d-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil for LdtMab1, LdtMab2, LdtMab4, and LdtMab5 and showed that imipenem bound >100-fold more avidly than ceftaroline fosamil to LdtMab1 and LdtMab2 (e.g., Ki app or Km of LdtMab1 = 0.01 ± 0.01 μM for imipenem versus 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modeling indicates that LdtMab2 readily accommodates imipenem, but the active site must widen to ≥8 A for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (l,d-transpeptidases and d,d-carboxypeptidase) and provides a mechanistic rationale for the effectiveness of this dual β-lactam combination in M. abscessus infections.

Cefiderocol is a novel catechol siderophore cephalosporin antibiotic developed to treat resistant gram-negative infections. We describe its successful use as rescue therapy, combined with surgical debridement, to treat a patient with osteomyelitis due to extensively drug-resistant Acinetobacter baumannii. Bacterial whole-genome sequencing identified the strain and antibiotic resistance determinants., Cefiderocol is a novel catechol siderophore cephalosporin developed to treat resistant gram-negative infections. We report its successful use in conjunction with surgical debridement to treat osteomyelitis due to extensively drug-resistant Acinetobacter baumannii. Bacterial genome sequencing revealed the determinants of resistance.

OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ∼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of β-lactam drugs.

β-lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β-lactams is the expression of β-lactamase enzymes. To overcome β-lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β-lactamases also contain the characteristic β-lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non-β-lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem-hydrolyzing class D β-lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β-lactamase inhibitors, nor are they effectively inhibited by the newest, non-β-lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β-lactamases, and are not extensively characterized as inhibitors of class D β-lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β-lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA-24/40. Several compounds were identified as effective inhibitors of OXA-24/40, with Ki values as low as 5 μM. The X-ray crystal structures of OXA-24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β-lactamases.

Background Antimicrobial resistance is a major global health threat. Pseudomonas aeruginosa is a leading cause of nosocomial infections and a key opportunistic pathogen in cystic fibrosis. Multidrug resistant strains are classified as a “serious threat” by the CDC. Pseudomonas-derived cephalosporinase (PDC) is largely responsible for β-lactam antibiotic resistance in P. aeruginosa. Single amino acid substitutions in the essential Ω-loop region (e.g. Y221H by structural alignment-based numbering of class C β-lactamases) have been shown to enhance hydrolysis of ceftazidime (CAZ) and ceftolozane (TOL), limiting therapeutic options for P. aeruginosa. Methods We undertook detailed studies to explore the mechanisms by which Y221H enhances CAZ and TOL MICs. MIC measurements were performed per CLSI guidelines using MH Agar. Thermal stability was determined by circular dichroism. Enzyme kinetic properties were determined using spectrophotometric techniques. Molecular dynamics techniques were used to predict structural changes. Results E. coli expressing blaPDC-3-Y221H is less susceptible to CAZ (MIC 0.5 mg/L WT → 8 mg/L Y221H) and TOL (MIC 2 mg/L WT → 16 mg/L Y221H). Using steady-state kinetic analysis, Y221H was found to hydrolyze CAZ with a KM = 585 µM, a kcat = 3.4 sec-1, and kcat/KM = 0.0058 µM-1s-1. With cephalothin, a good PDC substrate, we observed KM = 26.6 µM, kcat = 70.1 s-1, and kcat/KM = 2.6 µM-1 s-1 for Y221H. Using Electrospray ionization mass spectrometry (ESI-MS), CAZ was detected covalently bound to WT, but not Y221H when incubated at 1000-fold molar excess. Avibactam (AVI) inhibited Y223H (Ki = 70 nM vs. 19 nM for WT). Y221H thermal stability decreased by 5°C (Tm = 47°C vs 52°C WT). AVI at 10-fold molar excess does not increase Tm in Y221H or WT. WT-MetaDynamics (WT MDS) predicts the opening of a hidden pocket by repositioning residue 221 (Figure 1).). Figure 1: (Left) We carried out enhanced sampling metadynamics simulations to generate free-energy landscapes as a function of the dihedral angles of residue 221. This identifies the differences in the dynamics of the tyrosyl side chains in the wild type Y221 and the imidazole ring of the H221 variant. (Right) The rotation of the side chain in H221 opens a cryptic pocket (green mesh), which is occluded in the wild type. The Ω-loop is colored red. Conclusion PDC-3 Y221H increases CAZ & TOL MICs and alters catalytic activity, primarily by a change in kcat. Our modelling analyses suggest altered conformational flexibility and structure-function relationships in the Ω-loop. These results help to advance our understanding of PDC and will inform development of novel antibiotics and inhibitors. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Multiple antibiotic resistance mechanisms, including two β-lactamases, L1, a metallo-β-lactamase, and L2, a class A cephalosporinase, make S. maltophilia naturally multidrug resistant. Thus, infections caused by S. maltophilia pose a big therapeutic challenge. Our study aims to understand the microbiological and molecular characteristics of S. maltophilia isolates recovered from human sources. A highlight of the resistance profile of this collection is the excellent activity of the ceftazidime-avibactam and aztreonam combination. We hope this result prompts controlled and observational studies to add clinical data on the utility and safety of this therapy. We also identify 35 and 43 novel variants of L1 and L2, respectively, some of which harbor novel substitutions that could potentially affect substrate and/or inhibitor binding. We believe our results provide valuable knowledge to understand the epidemiology of this species and to advance mechanism-based inhibitor design to add to the limited arsenal of antibiotics active against this pathogen., Stenotrophomonas maltophilia is a Gram-negative, nonfermenting, environmental bacillus that is an important cause of nosocomial infections, primarily associated with the respiratory tract in the immunocompromised population. Aiming to understand the population structure, microbiological characteristics and impact of allelic variation on β-lactamase structure and function, we collected 130 clinical isolates from across the United States. Identification of 90 different sequence types (STs), of which 63 are new allelic combinations, demonstrates the high diversity of this species. The majority of the isolates (45%) belong to genomic group 6. We also report excellent activity of the ceftazidime-avibactam and aztreonam combination, especially against strains recovered from blood and respiratory infections for which the susceptibility is higher than the susceptibility to trimethoprim-sulfamethoxazole, considered the “first-line” antibiotic to treat S. maltophilia. Analysis of 73 blaL1 and 116 blaL2 genes identified 35 and 43 novel variants of L1 and L2 β-lactamases, respectively. Investigation of the derived amino acid sequences showed that substitutions are mostly conservative and scattered throughout the protein, preferentially affecting positions that do not compromise enzyme function but that may have an impact on substrate and inhibitor binding. Interestingly, we detected a probable association between a specific type of L1 and L2 and genomic group 6. Taken together, our results provide an overview of the molecular epidemiology of S. maltophilia clinical strains from the United States. In particular, the discovery of new L1 and L2 variants warrants further study to fully understand the relationship between them and the β-lactam resistance phenotype in this pathogen.

Impeding, as well as reducing, the burden of antimicrobial resistance in Gram-negative pathogens is an urgent public health endeavor. Our current antibiotic armamentarium is dwindling, while major resistance determinants (e.g., extended-spectrum β-lactamases [ESBLs]) continue to evolve and disseminate around the world. One approach to attack this problem is to develop novel therapies that will protect our current agents. AAI101 is a novel penicillanic acid sulfone β-lactamase inhibitor similar in structure to tazobactam, with one important difference. AAI101 possesses a strategically placed methyl group that gives the inhibitor a net neutral charge, enhancing bacterial cell penetration. AAI101 paired with cefepime, also a zwitterion, is in phase III of clinical development for the treatment of serious Gram-negative infections. Here, AAI101 was found to restore the activity of cefepime against class A ESBLs (e.g., CTX-M-15) and demonstrated increased potency compared to that of piperacillin-tazobactam when tested against an established isogenic panel. The enzymological properties of AAI101 further revealed that AAI101 possessed a unique mechanism of β-lactamase inhibition compared to that of tazobactam. Additionally, upon reaction with AAI101, CTX-M-15 was modified to an inactive state. Notably, the in vivo efficacy of cefepime-AAI101 was demonstrated using a mouse septicemia model, indicating the ability of AAI101 to bolster significantly the therapeutic efficacy of cefepime in vivo. The combination of AAI101 with cefepime represents a potential carbapenem-sparing treatment regimen for infections suspected to be caused by Enterobacteriaceae expressing ESBLs.

Burkholderia spp. are opportunistic human pathogens that infect persons with cystic fibrosis and the immunocompromised. Burkholderia spp. express class A and C β-lactamases, which are transcriptionally regulated by PenRA through linkage to cell wall metabolism and β-lactam exposure. The potency of temocillin, a 6-methoxy-β-lactam, was tested against a panel of multidrug-resistant (MDR) Burkholderia spp. In addition, the mechanistic basis of temocillin activity was assessed and compared to that of ticarcillin. Susceptibility testing with temocillin and ticarcillin was conducted, as was biochemical analysis of the PenA1 class A β-lactamase and AmpC1 class C β-lactamase. Molecular dynamics simulations (MDS) were performed using PenA1 with temocillin and ticarcillin. The majority (86.7%) of 150 MDR Burkholderia strains were susceptible to temocillin, while only 4% of the strains were susceptible to ticarcillin. Neither temocillin nor ticarcillin induced bla expression. Ticarcillin was hydrolyzed by PenA1 (k cat/Km = 1.7 ± 0.2 μM-1 s-1), while temocillin was slow to form a favorable complex (apparent Ki [Ki app] = ∼2 mM). Ticarcillin and temocillin were both potent inhibitors of AmpC1, with Ki app values of 4.9 ± 1.0 μM and 4.3 ± 0.4 μM, respectively. MDS of PenA revealed that ticarcillin is in an advantageous position for acylation and deacylation. Conversely, with temocillin, active-site residues K73 and S130 are rotated and the catalytic water molecule is displaced, thereby slowing acylation and allowing the 6-methoxy of temocillin to block deacylation. Temocillin is a β-lactam with potent activity against Burkholderia spp., as it does not induce bla expression and is poorly hydrolyzed by endogenous β-lactamases.

Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) β-lactamase inhibitor that inactivates class A and C β-lactamases and exhibits intrinsic antibiotic and β-lactam “enhancer” activity against Enterobacteriaceae. In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying bla(KPC-2) or bla(KPC-3); meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent K(i) [K(i)( app)] = 31 ± 3 μM) more efficiently than the K234R variant (K(i)( app) = 270 ± 27 μM) and displayed a faster acylation rate (k(2)/K), which was 5,815 ± 582 M(−1) s(−1) for KPC-2 versus 247 ± 25 M(−1) s(−1) for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam’s aminoethoxy tail formed unproductive interactions with the K234R variant’s active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae. Moreover, our data suggest that β-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

Successful treatment of Acinetobacter baumannii infections require early and appropriate antimicrobial therapy. One of the first steps in this process is understanding which β-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.

Background Mycobacterium abscessus (Mab) is a highly drug-resistant nontuberculous mycobacteria (NTM). Efforts to discover new treatments for Mab infections are accelerating with a focus on cell wall synthesis proteins (L, D-transpeptidases, LdtMab1-5, and D, D-carboxypeptidase) that are targeted by combination β-lactam antibiotics. The US Food and Drug Administration (FDA) has granted Qualified Infectious Disease Product (QIDP) to the oral and intravenous (IV) formulations of Sulopenem (SUL). Data on SUL in vitro activity against Mab is currently unavailable. Here, we evaluated activity of SUL alone and in combination with Cefuroxime salt (CEF) against representative clinical isolates belonging to the Mab complex. Both CEF and SUL are available in oral formulation and can be considered as oral step-down therapy. Methods Minimum inhibitory concentrations (MICs) of SUL and CEF alone and in combination were determined using microdilution. Approximately 5 x 105 colony-forming units (CFU) per milliliter were inoculated into Middlebrook 7H9 Broth supplemented with 10% (vol/vol) oleic albumin dextrose catalase and 0.05% (vol/vol) Tween 80. CEF was added at fixed concentration of 4 µg/ml to serial dilutions of SUL. Mab isolates were incubated with test agents at 30 °C for 48 h, and MIC was defined as lowest antibiotic concentration that prevented visible bacterial growth. Results Fifty-five clinically derived and previously characterized isolates were tested in these assays. MIC50 and MIC90 of CEF is 16 and 32 ug/ml; MIC50 and MIC90 of SUL is 2 and 4 ug/ml, the range of MICs are as follows: CEF (8 → 64 ug/ml); SUL (1→8 ug/ml); and SUL and CEF at fixed 4 ug/ml (< 0.25 → 4 ug/ml). Combination SUL and CEF lowered MIC to < 0.25 ug/ml in 52 clinical isolate (Figure). Fig. MIC distributions of cefuroxime salt, sulopenem, sulopenem with 4 μg/ml cefuroxime monohydrate against 55 Mab clinical strains Conclusion Our results support the emerging hypothesis that dual β-lactam therapy is a promising strategy in the treatment of serious Mab infections. Investigating the biochemical rationale for this combination will support the application to clinical trials. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Background Stenotrophomonas maltophilia is a Gram-negative, non-fermenting opportunistic pathogen. Two β-lactamases provide intrinsic resistance to β-lactams: a class B Metallo- β-lactamase L1, and a class A serine β-lactamase (SβL) L2. Recently, we described novel variants of the L1 and L2 in a collection of clinical S. maltophilia isolates collected in the US, and showed through analyses of the amino acid sequences that L1 and L2 grouped into 4 (A-D, B, C, and E) and 2 (A and D) clades, respectively. We aimed to characterize the new L1 and L2 clinical variants biochemically. Methods Representative blaL1 and blaL2 genes from each of the identified clades were cloned into pBC-SK and pET24 vectors and transformed into E. coli DH10B and BL21 (DE3) cells, respectively. Minimal inhibitory concentrations (MICs) were determined using CLSI approved methods. Cell-based assays and biochemical characterization performed on purified enzymes, including circular dichroism (CD), thermal stability, and steady-state kinetics assays, were performed. Results Susceptibility testing results using DH10-B E. coli strains expressing the L1 and L2 variants are shown in Table 1. Remarkably, while all L1 variants confer the same level of resistance to carbapenems, L2B conferred higher MICs to 3rd gen cephalosporins and aztreonam than L2D. Kinetics assays confirmed differences in the kcat of both enzymes to ceftazidime (32s-1 for L2B vs. 7s-1 for L2D) and avibactam inhibition constant Ki (1.7 μM for L2B vs. 4.5 μM for L2D). Structurally, L2B and L2D present distinctive CD spectra and thermal stabilities (ΔTm 5°C). Table 1 Conclusion As opposed to the L2 variants, our results suggest that the L1 variants may not be functionally nor structurally different. Differences between L2B and L2D might have arisen due to the use of cephalosporins and SβL inhibitors. Further experiments are on the way to determine the structural basis of these observations and the implication of these for the design of novel β-lactamase inhibitors. Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli . Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose “a new twist” for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei , a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a β-lactamase profoundly affects β-lactam resistance.

Although multiple antimicrobial resistance (AMR) determinants can confer the same in vitro antimicrobial susceptibility testing (AST) phenotype, their differing effect on optimal therapeutic choices is uncertain. Using a large population-based collection of clinical strains spanning a 3.5-year period, we applied WGS to detect inhibitor resistant (IR), extended-spectrum β-lactamase (ESBL), and carbapenem resistant (CR) β-lactamase (bla) genes and compared the genotype to the AST phenotype in select isolates. All blaNDM-1 (9/9) and the majority of blaNDM-1/OXA-48 (3/4) containing isolates were resistant to CAZ/AVI as predicted by WGS. The combination of ATM and CAZ/AVI restored susceptibility by disk diffusion assay. Unexpectedly, clinical Kp isolates bearing blaKPC-8 (V240G) and blaKPC-14 (G242 and T243 deletion) did not test fully resistant to CAZ/AVI. Lastly, despite the complexity of the β-lactamase background, CAZ/AVI retained potency. Presumed phenotypes conferred by AMR determinants need to be tested if therapeutic decisions are being guided by their presence or absence.

Background Multidrug-resistant (MDR) A. baumannii presents a critical need for innovative antibacterial development. We have identified a new series of γ-lactam (oxopyrazole) antibiotics that target penicillin binding proteins (PBPs) and incorporate a siderophore moiety to facilitate periplasmic uptake. YU253911, an advanced iteration of this class shows potent in vitro activity against clinically relevant Gram-negative organisms including Acinetobacter spp. Methods Minimum inhibitory concentrations (MICs) for YU253911 were determined using broth microdilution against a 198-member panel of clinical isolates of Acinetobacter spp. Resistant strains were further evaluated for susceptibility to YU253911 in combination with sulbactam. The antibiotic’s target protein was evaluated by binding studies with Bocillin™, a fluorescent penicillin analogue, and modeled in the PBP active site. YU253911 was evaluated in vivo in a mouse soft tissue infection model. Results MIC testing for YU253911 revealed an MIC50 of 0.5 μg/mL and an MIC90 of 16 μg/mL, which compared favorably to all tested β-lactam antibiotics including penicillins, cephalosporins, monobactams and carbapenems (MIC50 = 2 to > 16 μg/mL). Combination with sulbactam augmented the activity of the agent. There was no apparent correlation between YU253911-resistance and the presence of specific β-lactamase genes, and incubation with representative β-lactamase proteins (KPC-2, OXA-23, OXA-24, PER-2, PDC-3, NDM-1, VIM-2, and IMP-1) showed negligible hydrolysis of the agent. YU253911 showed promising preclinical pharmacokinetics in mice with a 15 h half-life from intravenous administration and demonstrated a dose-dependent reduction in colony forming units from 50 and 100 mg/kg q6h dosing in a mouse thigh infection model using P. aeruginosa. Conclusion YU253911, a new generation γ-lactam antibiotic effective against MDR A. baumannii demonstrated promising in in vitro potency and favorable pharmacokinetics which correlated with in vivo efficacy. Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

Boronic acids are attracting a lot of attention as β-lactamase inhibitors, and in particular, compound S02030 (Ki = 44 nM) proved to be a good lead compound against ADC-7 (Acinetobacter-derived cephalosporinase), one of the most significant resistance determinants in A. baumannii. The atomic structure of the ADC-7/S02030 complex highlighted the importance of critical structural determinants for recognition of the boronic acids. Herein, to elucidate the role in recognition of the R2-carboxylate, which mimics the C3/C4 found in β-lactams, we designed, synthesized, and characterized six derivatives of S02030 (3a). Out of the six compounds, the best inhibitors proved to be those with an explicit negative charge (compounds 3a–c, 3h, and 3j, Ki = 44–115 nM), which is in contrast to the derivatives where the negative charge is omitted, such as the amide derivative 3d (Ki = 224 nM) and the hydroxyamide derivative 3e (Ki = 155 nM). To develop a structural characterization of inhibitor binding in the active site, the X-ray crystal structures of ADC-7 in a complex with compounds 3c, SM23, and EC04 were determined. All three compounds share the same structural features as in S02030 but only differ in the carboxy-R2 side chain, thereby providing the opportunity of exploring the distinct binding mode of the negatively charged R2 side chain. This cephalosporinase demonstrates a high degree of versatility in recognition, employing different residues to directly interact with the carboxylate, thus suggesting the existence of a “carboxylate binding region” rather than a binding site in ADC enzymes. Furthermore, this class of compounds was tested against resistant clinical strains of A. baumannii and are effective at inhibiting bacterial growth in conjunction with a β-lactam antibiotic.

The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement.

Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to β-lactam antibiotics in Acinetobacter spp. is mediated by class C β-lactamases known as Acinetobacter-derived cephalosporinases (ADCs). ADCs are unaffected by clinically used β-lactam-based β-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Ki values ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the β-lactamase–inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 Å). In the ADC-7/CR192 complex, the BATSI with the lowest Ki (0.45 nM) and greatest ΔTm (+9 °C), a trifluoromethyl substituent, interacts with Arg340. Arg340 is unique to ADCs and may play an important role in the inhibition of ADC-7. The ADC-7/BATSI complexes determined in this study shed light into the unique recognition sites in ADC enzymes and also offer insight into further structure-based optimization of these inhibitors.

Stenotrophomonas maltophilia is an emerging opportunistic pathogen, classified by the World Health Organization as one of the leading multidrug-resistant organisms in hospital settings. The need to discover novel compounds and/or combination therapies for S. maltophilia is urgent. We demonstrate the in vitro efficacy of aztreonam-avibactam (ATM-AVI) against S. maltophilia and kinetically characterize the inhibition of the L2 β-lactamase by avibactam. ATM-AVI overcomes aztreonam resistance in selected clinical strains of S. maltophilia, addressing an unmet medical need. Fil: Mojica, Maria F.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Papp-Wallace, Krisztina M.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos Fil: Barnes, Melissa D.. Louis Stokes Veterans Affairs Medical Center; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Rutter, Joseph D.. Louis Stokes Veterans Affairs Medical Center; Estados Unidos Fil: Jacobs, Michael R.. University Hospitals Cleveland Medical Center; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: LiPuma, John J.. University of Michigan; Estados Unidos Fil: Walsh, Thomas J.. Weill Cornell Medical Center; Estados Unidos Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos. CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology; Estados Unidos

PER β -lactamases are an emerging family of extended-spectrum β -lactamases (ESBL) found in Gram-negative bacteria. PER β -lactamases are unique among class A enzymes as they possess an inverted omega (?) loop and extended B3 β -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- β -lactam β -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 β -lactamases (i.e., k2/K range of =103 M-1s-1) and not class A β -lactamases (i.e., k2/K range, 104 to 105 M-1s-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 β -lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β -lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β -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 β-lactamase, we tested different β -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. Fil: Ruggiero, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Papp Wallace, Krisztina M.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Taracila, Magdalena A.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Mojica, Maria F.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos Fil: Bethel, Christopher R.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos Fil: Rudin, Susan D.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Zeiser, Elise T.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Bonomo, Robert A.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying bla CMY , novel β-lactam–β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry bla CMY-2 or bla CMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC 90 , ≤0.5 mg/liter); in comparison, ceftazidime had a MIC 90 of >128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 β-lactamase, as demonstrated by a second-order onset of acylation rate constant ( k 2 / K ) of (4.9 ± 0.5) × 10 4 M −1 s −1 and the off-rate constant ( k off ) of (3.7 ± 0.4) ×10 −4 s −1 . Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2–avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69.

Inhibitor resistant (IR) class A β-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV β-lactamase, from Klebsiella pneumoniae , results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting β-lactamases revealed that only -Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM K(i) for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR β-lactamases.

The rOmpA vaccine has been shown to protect mice from lethal infection caused by extreme-drug-resistant (XDR) Acinetobacter baumannii. The role of dose in immunology of the rOmpA vaccine was explored.Mice were vaccinated with various doses of rOmpA plus aluminum hydroxide (Al(OH)(3)) adjuvant. The impact of dose on antibody titers, cytokine production, and immunodominant epitopes was defined.Anti-rOmpA IgG and IgG subtype titers were higher at larger vaccine doses (30 and 100 μg vs. 3 μg). The 3 μg dose induced a balanced IFN-γ-IL-4 immune response while the 100 μg dose induced a polarized IL-4/Type 2 response. Epitope mapping revealed distinct T cell epitopes that activated IFN-γ-, IL-4-, and IL-17-producing splenocytes. Vaccination with the 100 μg dose caused epitope spreading among IL-4-producing splenocytes, while it induced fewer reactive epitopes among IFN-γ-producing splenocytes.Vaccine dose escalation resulted in an enhanced Type 2 immune response, accompanied by substantial IL-4-inducing T cell epitope spreading and restricted IFN-γ-inducing epitopes. These results inform continued development of the rOmpA vaccine against A. baumannii, and also are of general importance in that they indicate that immune polarization and epitope selectivity can be modulated by altering vaccine dose.

The most common mechanism responsible for β-lactam resistance in Gram-negative bacteria is the production of β-lactamases (EC 3.5.2.6) (1). At present, there are more than 850 different β-lactamases described in nature (http://www.lahey.org/Studies/). As a family of proteins, β-lactamases are grouped into four main classes (i.e., A to D) based upon amino acid sequence homology (2). Classes A, C, and D employ serine in the active site as the reactive nucleophile, while class B β-lactamases use a single or pair of metal ions (Zn2+) to catalyze the opening of the β-lactam ring. Both serine and metallo-β-lactamases make use of a strategically positioned water molecule to hydrolyze the lactam bond (3-5). In general, class A, C, and D β-lactamases follow a three-step reaction mechanism that is represented as follows: E+S⇌k−1k1E:S→k2E-S→H2Ok3E+P (Eq. 1) Here, E is the β-lactamase enzyme, S is the β-lactam substrate, E:S is the Henri-Michaelis complex, E-S is the acyl enzyme, and P is the inactivated β-lactam. The rate constants for each step are designated by k1, k-1, k2, and k3. Usually, class A and C β-lactamases are found in the Enterobacteriaceae. Acquired class A enzymes, often carried on mobile plasmids, are common in Escherichia coli and Klebsiella pneumoniae, are constitutively expressed and confer resistance to penicillins and narrow-spectrum cephalosporins (1). Class C, or AmpC-type β-lactamases (AmpCs), are encoded by chromosomal genes in many Gram-negative pathogens (e.g., Citrobacter freundii, Enterobacter spp., and Pseudomonas aeruginosa), are inducible, and confer resistance to narrow and extended-spectrum cephalosporins. Most recently, an increasing number of AmpC β-lactamase genes are being discovered on plasmids that also spread by horizontal transfer (6-8). Unlike class A enzymes, AmpCs are poorly inhibited by the commercially available β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) (7). Among class C enzymes, CMY-type β-lactamases represent the most frequently detected plasmid encoded AmpCs (pAmpCs) (8-13). Unfortunately, we know little regarding the ability of clinically important CMY enzymes to hydrolyze different classes of β-lactam antibiotics (14-16). Furthermore, the activity of novel β-lactamase inhibitors against the CMY type pAmpCs has not yet been fully studied (17). In class A β-lactamases, single amino acid substitutions (primarily, but not exclusively, at Ambler positions Gly238 on the b3 β-strand and Arg164 or Asp179 in the Ω loop) remodel a “broad-spectrum” β-lactamase into an extended-spectrum β-lactamase (ESBL) (18-20). The bacterium possessing a class A ESBL becomes resistant to ceftazidime and/or cefotaxime. However, these changes come at a “price” (18, 21). Among class A ESBLs, one uniformly detects a decrease in penicillin resistance (lower MICs) and an increase in the susceptibility to β-lactamase inhibitors and carbapenems (18, 21). Herein, we analyzed the biochemical properties of a novel CMY-2-like pAmpC (i.e., CMY-32) cloned from an E. coli isolate found in the clinic. In comparison with CMY-2 (wild type, WT), we determined that CMY-32 possesses a single amino acid substitution in the Ω loop (i.e., Gly214Glu) resulting in significant changes in the resistance phenotype and hydrolytic profile (making this β-lactamase an “extended-spectrum type”). We also evaluated the mechanisms by which carbapenems, a monobactam antibiotic aztreonam, and the novel monobactam derivative, {"type":"entrez-protein","attrs":{"text":"BAL29880","term_id":"359272361"}}BAL29880, react and inhibit CMY-2 and CMY-32 enzymes. Our data reveal the impact of single amino acid substitutions on protein evolution in this emerging family of resistance enzymes and highlight the manner in which carbapenems and monobactams inactivate class C enzymes.

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical Escherichia coli isolates obtained from a patient. The CMY-2-producing E. coli isolate (CMY-2- Ec ) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli isolate (CMY-33- Ec ) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33- Ec , the patient received cefepime. CMY-33- Ec and CMY-2- Ec were identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤0.5 μg/ml for CMY-33- Ec and CMY-2- Ec , respectively). Identical CMY-2- Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli strain DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). The k cat / K m or inhibitor complex inactivation ( k inact )/ K i app (μM −1 s −1 ) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2- Ec isolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.

β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki∗ values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E∗I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution. Fil: Mojica, Maria F.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Mahler, S. Graciela. Universidad de la República; Uruguay Fil: Bethel, Christopher R.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Kosmopoulou, Magda. University of Bristol; Reino Unido Fil: Papp Wallace, Krisztina M.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Wilson, Brigid M.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Marshall, Steven H.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Wallace, Christopher J.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Villegas, Maria V.. Centro Internacional de Entrenamiento e Investigaciones Medicas; Colombia Fil: Harris, Michael E.. Case Western Reserve University; Estados Unidos Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Spencer, James. University of Bristol; Reino Unido Fil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with kcat values ranging from 0.38 ± 0.04 to 460 ± 46 s−1 and possesses high kcat/kinact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s−1. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower kcat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens. Background: Resistance to β-lactams in Burkholderia is mediated by different β-lactamases (e.g. PenA and PenI). Results: PenA from B. multivorans is a carbapenemase, and PenI from B. pseudomallei is an extended-spectrum enzyme. Conclusion: Subtle changes within the active site of β-lactamases result in major phenotypic changes. Significance: Future antibiotic design must consider the distinctive phenotypes of PenA and PenI β-lactamases.

Acinetobacter baumannii is an increasingly problematic pathogen in United States hospitals. Antibiotics that can treat A. baumannii are becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of β-lactam antibiotics, to β-lactam–sulbactam susceptibility and β-lactam resistance in A. baumannii . Decreased expression of PBPs as well as loss of binding of β-lactams to PBPs was previously shown to promote β-lactam resistance in A. baumannii . Using an in vitro assay with a reporter β-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC 50 s) for PBP1a from A. baumannii and PBP3 from Acinetobacter sp. ranged from 1 to 5 μM for a series of β-lactams. In contrast, PBP3 demonstrated a narrower range of IC 50 s against β-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 μM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC 50 s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective against A. baumannii . Unraveling the contribution of PBPs to β-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel β-lactams.

ADC-56, a novel extended-spectrum AmpC (ESAC) β-lactamase, was identified in an Acinetobacter baumannii clinical isolate. ADC-56 possessed an R148Q change compared with its putative progenitor, ADC-30, which enabled it to hydrolyze cefepime. Molecular modeling suggested that R148 interacted with Q267, E272, and I291 through a hydrogen bond network which constrained the H-10 helix. This permitted cefepime to undergo conformational changes in the active site, with the carboxyl interacting with R340, likely allowing for better binding and turnover.