Activity of ETX0462 toward Some Burkholderia spp.

Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic human pathogens that are inherently multidrug resistant, limiting treatment options for infections. Here, a novel diazabicyclooctane, ETX0462, was evaluated for activity against Bcc and B. gladioli. Ninety-eight percent of the isolates examined in this study were susceptible. ETX0462 was found to demonstrate in vitro activity superior to that of currently available treatment options (e.g., trimethoprim-sulfamethoxazole and ceftazidime).

Examining the activity of cefepime-taniborbactam against Burkholderia cepacia complex and Burkholderia gladioli isolated from cystic fibrosis patients in the United States.

The novel clinical-stage ß-lactam-ß-lactamase inhibitor combination, cefepime-taniborbactam, demonstrates promising activity toward many Gram-negative bacteria producing class A, B, C, and/or D ß-lactamases. We tested this combination against a panel of 150 Burkholderia cepacia complex (Bcc) and Burkholderia gladioli strains. The addition of taniborbactam to cefepime shifted cefepime minimum inhibitory concentrations toward the provisionally susceptible range in 59% of the isolates tested. Therefore, cefepime-taniborbactam possessed similar activity as first-line agents, ceftazidime and trimethoprim-sulfamethoxazole, supporting further development.

Activity of Imipenem-Relebactam against Multidrug- and Extensively Drug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli.

The Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic pathogens that most commonly infect persons with cystic fibrosis or compromised immune systems. Members of the Burkholderia genus are intrinsically multidrug resistant (MDR), possessing both a PenA carbapenemase and an AmpC ß-lactamase, rendering treatment of infections due to these species problematic. Here, we tested the ß-lactam-ß-lactamase inhibitor combination imipenem-relebactam against a panel of MDR Bcc and B. gladioli strains. The addition of relebactam to imipenem dramatically lowered the MICs for Bcc and B. gladioli: only 16% of isolates tested susceptible to imipenem, while 71.3% were susceptible to the imipenem-relebactam combination. While ceftazidime-avibactam remained the most potent combination drug against this panel of Bcc and B. gladioli strains, imipenem-relebactam was active against 71.4% of the ceftazidime-avibactam-resistant isolates. Relebactam demonstrated potent inactivation of Burkholderia multivorans PenA1, with an apparent Ki (Kiapp) value of 3.2 µM. Timed mass spectrometry revealed that PenA1 formed a very stable adduct with relebactam, without any detectable desulfation for as long as 24 h. Based on our results, imipenem-relebactam may represent an alternative salvage therapy for Bcc and B. gladioli infections, especially in cases where the isolates are resistant to ceftazidime-avibactam.

In Vitro Antibacterial Activity and In Vivo Efficacy of Sulbactam-Durlobactam against Pathogenic Burkholderia Species.

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen ß-lactamase, which confers resistance to ß-lactams. The ß-lactam-ß-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli (n = 150), B. mallei (n = 30), and B. pseudomallei (n = 28), studied the kinetics of inhibition of the PenA1 ß-lactamase from B. multivorans and the PenI ß-lactamase from B. pseudomallei by durlobactam, tested for blaPenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 µg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation (k2/K) values of 3.9 × 106 M-1 s-1 and 2.6 × 103 M-1 s-1 and apparent Ki (Kiapp) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections.

Ceftazidime-Avibactam in Combination With Fosfomycin: A Novel Therapeutic Strategy Against Multidrug-Resistant Pseudomonas aeruginosa.

Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to ß-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-ß-lactamases.

Resurrecting Old ß-Lactams: Potent Inhibitory Activity of Temocillin against Multidrug-Resistant Burkholderia Species Isolates from the United States.

Burkholderia spp. are opportunistic human pathogens that infect persons with cystic fibrosis and the immunocompromised. Burkholderia spp. express class A and C ß-lactamases, which are transcriptionally regulated by PenRA through linkage to cell wall metabolism and ß-lactam exposure. The potency of temocillin, a 6-methoxy-ß-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 ß-lactamase and AmpC1 class C ß-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 (kcat/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 ß-lactam with potent activity against Burkholderia spp., as it does not induce bla expression and is poorly hydrolyzed by endogenous ß-lactamases.

"Switching Partners": Piperacillin-Avibactam Is a Highly Potent Combination against Multidrug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates.

In persons with cystic fibrosis (CF), airway infection with Burkholderia cepacia complex (Bcc) species or Burkholderia gladioli presents a significant challenge due to inherent resistance to multiple antibiotics. Two chromosomally encoded inducible ß-lactamases, a Pen-like class A and AmpC are produced in Bcc and B. gladioli Previously, ceftazidime-avibactam demonstrated significant potency against Bcc and B. gladioli isolated from the sputum of individuals with CF; however, 10% of the isolates tested resistant to ceftazidime-avibactam. Here, we describe an alternative antibiotic combination to overcome ceftazidime-avibactam resistance. Antimicrobial susceptibility testing was performed on Bcc and B. gladioli clinical and control isolates. Biochemical analysis was conducted on purified PenA1 and AmpC1 ß-lactamases from Burkholderia multivorans ATCC 17616. Analytic isoelectric focusing and immunoblotting were conducted on cellular extracts of B. multivorans induced by various ß-lactams or ß-lactam-ß-lactamase inhibitor combinations. Combinations of piperacillin-avibactam, as well as piperacillin-tazobactam plus ceftazidime-avibactam (the clinically available counterpart), were tested against a panel of ceftazidime-avibactam nonsusceptible Bcc and B. gladioli The piperacillin-avibactam and piperacillin-tazobactam-ceftazidime-avibactam combinations restored susceptibility to 99% of the isolates tested. Avibactam is a potent inhibitor of PenA1 (apparent inhibitory constant [Kiapp] = 0.5 µM), while piperacillin was found to inhibit AmpC1 (Kiapp = 2.6 µM). Moreover, piperacillin, tazobactam, ceftazidime, and avibactam, as well as combinations thereof, did not induce expression of blapenA1 and blaampC1 in the B. multivorans ATCC 17616 background. When ceftazidime-avibactam is combined with piperacillin-tazobactam, the susceptibility of Bcc and B. gladioli to ceftazidime and piperacillin is restored in vitro Both the lack of blapenA1 induction and potent inactivation of PenA1 by avibactam likely provide the major contributions toward susceptibility. With in vivo validation, piperacillin-tazobactam-ceftazidime-avibactam may represent salvage therapy for individuals with CF and highly drug-resistant Bcc and B. gladioli infections.

Characterization of the AmpC ß-Lactamase from Burkholderia multivorans.

Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. ß-Lactam antibiotics are recommended as therapy for infections due to Bmultivorans, which possesses two ß-lactamase genes, blapenA and blaAmpC PenA is a carbapenemase with a substrate profile similar to that of the Klebsiella pneumoniae carbapenemase (KPC); in addition, expression of PenA is inducible by ß-lactams in Bmultivorans Here, we characterize AmpC from Bmultivorans ATCC 17616. AmpC possesses only 38 to 46% protein identity with non-Burkholderia AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of Bmultivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward ß-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (Kiapp > 600 µM), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible in Bmultivorans ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 µg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in Bmultivorans compared to PenA, which is the dominant ß-lactamase in Bmultivorans ATCC 17616.

Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam.

Ceftazidime-avibactam is a "second-generation" ß-lactam-ß-lactamase inhibitor combination that is effective against Enterobacteriaceae expressing class A extended-spectrum ß-lactamases, class A carbapenemases, and/or class C cephalosporinases. Knowledge of the interactions of avibactam, a diazabicyclooctane with different ß-lactamases, is required to anticipate future resistance threats. FOX family ß-lactamases possess unique hydrolytic properties with a broadened substrate profile to include cephamycins, partly as a result of an isoleucine at position 346, instead of the conserved asparagine found in most AmpCs. Interestingly, a single amino acid substitution at N346 in the Citrobacter AmpC is implicated in resistance to the aztreonam-avibactam combination. In order to understand how diverse active-site topologies affect avibactam inhibition, we tested a panel of clinical Enterobacteriaceae isolates producing blaFOX using ceftazidime-avibactam, determined the biochemical parameters for inhibition using the FOX-4 variant, and probed the atomic structure of avibactam with FOX-4. Avibactam restored susceptibility to ceftazidime for most isolates producing blaFOX; two isolates, one expressing blaFOX-4 and the other producing blaFOX-5, displayed an MIC of 16 µg/ml for the combination. FOX-4 possessed a k2/K value of 1,800 ± 100 M-1 · s-1 and an off rate (koff) of 0.0013 ± 0.0003 s-1 Mass spectrometry showed that the FOX-4-avibactam complex did not undergo chemical modification for 24 h. Analysis of the crystal structure of FOX-4 with avibactam at a 1.5-Å resolution revealed a unique characteristic of this AmpC ß-lactamase. Unlike in the Pseudomonas-derived cephalosporinase 1 (PDC-1)-avibactam crystal structure, interactions (e.g., hydrogen bonding) between avibactam and position I346 in FOX-4 are not evident. Furthermore, another residue is not observed to be close enough to compensate for the loss of these critical hydrogen-bonding interactions. This observation supports findings from the inhibition analysis of FOX-4; FOX-4 possessed the highest Kd (dissociation constant) value (1,600 nM) for avibactam compared to other AmpCs (7 to 660 nM). Medicinal chemists must consider the properties of extended-spectrum AmpCs, such as the FOX ß-lactamases, for the design of future diazabicyclooctanes.

Exploring the Role of the Ω-Loop in the Evolution of Ceftazidime Resistance in the PenA ß-Lactamase from Burkholderia multivorans, an Important Cystic Fibrosis Pathogen.

The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ≥ 8 µg/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the Ω-loop in mediating ceftazidime resistance in the PenA ß-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA ß-lactamase. Cell-based susceptibility testing revealed that 21 of 75 PenA variants engineered in this study were resistant to ceftazidime, with MICs of >8 µg/ml. Under steady-state conditions, each of the selected variants (R164S, T167G, L169A, and D179N) demonstrated a substrate preference for ceftazidime compared to wild-type PenA (32- to 320-fold difference). Notably, the L169A variant hydrolyzed ceftazidime significantly faster than PenA and possessed an ∼65-fold-lower apparent Ki (Kiapp) than that of PenA. To understand why these amino acid substitutions result in enhanced ceftazidime binding and/or turnover, we employed molecular dynamics simulation (MDS). The MDS suggested that the L169A variant starts with the most energetically favorable conformation (-28.1 kcal/mol), whereas PenA possessed the most unfavorable initial conformation (136.07 kcal/mol). In addition, we observed that the spatial arrangement of E166, N170, and the hydrolytic water molecules may be critical for enhanced ceftazidime hydrolysis by the L169A variant. Importantly, we found that two clinical isolates of B. multivorans possessed L169 amino acid substitutions (L169F and L169P) in PenA and were highly resistant to ceftazidime (MICs ≥ 512 µg/ml). In conclusion, substitutions in the Ω-loop alter the positioning of the hydrolytic machinery as well as allow for a larger opening of the active site to accommodate the bulky R1 and R2 side chains of ceftazidime, resulting in resistance. This analysis provides insights into the emerging phenotype of ceftazidime-resistant BCC and explains the evolution of amino acid substitutions in the Ω-loop of PenA of this significant clinical pathogen.

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

PER ß-lactamases are an emerging family of extended-spectrum ß-lactamases (ESBL) found in Gram-negative bacteria. PER ß-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 ß-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-ß-lactam ß-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ± 0.1 × 103 M-1 s-1, where k2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D ß-lactamases (i.e., k2/K range of ≈103 M-1 s-1) and not class A ß-lactamases (i.e., k2/K range, 104 to 105 M-1 s-1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the ß-lactamase and that the sulfate of AVI formed interactions with the ß-lactam carboxylate binding site of the PER-2 ß-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 ß-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 ß-lactamase, we tested different ß-lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing blaPER-2 Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

The Effectiveness of Imipenem-Relebactam against Ceftazidime-Avibactam Resistant Variants of the KPC-2 ß-Lactamase.

BACKGROUND: Ceftazidime-avibactam was approved by the FDA to treat infections caused by Enterobacterales carrying blaKPC-2. However, variants of KPC-2 with amino acid substitutions at position 179 have emerged and confer resistance to ceftazidime-avibactam. METHODS: The activity of imipenem-relebactam was assessed against a panel of 19 KPC-2 D179 variants. KPC-2 and the D179N and D179Y variants were purified for biochemical analyses. Molecular models were constructed with imipenem to assess differences in kinetic profiles. RESULTS: All strains were susceptible to imipenem-relebactam, but resistant to ceftazidime (19/19) and ceftazidime-avibactam (18/19). KPC-2 and the D179N variant hydrolyzed imipenem, but the D179N variant's rate was much slower. The D179Y variant was unable to turnover imipenem. All three ß-lactamases hydrolyzed ceftazidime at varying rates. The acylation rate of relebactam for the D179N variant was ~2.5× lower than KPC-2. Poor catalytic turnover by the D179Y variant precluded the determination of inhibitory kinetic parameters. Acyl-complexes with imipenem and ceftazidime were less prevalent with the D179N variant compared to the D179Y variant, supporting the kinetic observations that the D179Y variant was not as active as the D179N variant. Relebactam was slower to form an acyl-complex with the D179Y variant compared to avibactam. The D179Y model with imipenem revealed that the catalytic water molecule was shifted, and the carbonyl of imipenem was not within the oxyanion hole. Conversely in the D179N model, imipenem was oriented favorably for deacylation. CONCLUSIONS: Imipenem-relebactam overcame the resistance of the D179 variants, suggesting that this combination will be active against clinical isolates harboring these derivatives of KPC-2.

The Class A ß-Lactamase Produced by Burkholderia Species Compromises the Potency of Tebipenem against a Panel of Isolates from the United States.

Tebipenem-pivoxil hydrobromide, an orally bioavailable carbapenem, is currently in clinical development for the treatment of extended-spectrum ß-lactamase- and AmpC-producing Enterobacterales. Previously, tebipenem was found to possess antimicrobial activity against the biothreat pathogens, Burkholderia pseudomallei and Burkholderia mallei. Thus, herein, tebipenem was evaluated against a panel of 150 curated strains of Burkholderia cepacia complex (Bcc) and Burkholderia gladioli, pathogens that infect people who are immunocompromised or have cystic fibrosis. Using the provisional susceptibility breakpoint of 0.12 mg/L for tebipenem, 100% of the Bcc and B. gladioli tested as being provisionally resistant to tebipenem. Bcc and B. gladioli possess two inducible chromosomal ß-lactamases, PenA and AmpC. Using purified PenA1 and AmpC1, model ß-lactamases expressed in Burkholderia multivorans ATCC 17616, PenA1 was found to slowly hydrolyze tebipenem, while AmpC1 was inhibited by tebipenem with a k2/K value of 1.9 ± 0.1 × 103 M-1s-1. In addition, tebipenem was found to be a weak inducer of blaPenA1 expression. The combination of the slow hydrolysis by PenA1 and weak induction of blaPenA1 likely compromises the potency of tebipenem against Bcc and B. gladioli.

Assessing the Potency of ß-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans.

Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including ß-lactams. ß-Lactam resistance in Bcc is conferred largely via PenA-like ß-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using ß-lactamase inhibitors (ß-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on kinact/KI or k2/K values (range: 3.4 × 102 to 2 × 106 M-1 s-1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased Ki-app values and decreased kinact/KI or k2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis-Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k2/K, 3.4 × 102 M-1 s-1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1's Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k2/K for the R220A variant compared to PenA1, acyl-enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favorable interactions compared to PenA1. The former likely impacts the ability of all inhibitors to effectively acylate this variant enzyme. Based on the summation of all evidence herein, the utility of these newer ß-lactamase inhibitors (i.e., relebactam, enmetazobactam, avibactam, and vaborbactam) in combination with a ß-lactam against B. multivorans producing PenA1 and the R220A variant is promising.

Structural Analysis of The OXA-48 Carbapenemase Bound to A "Poor" Carbapenem Substrate, Doripenem.

Carbapenem-resistant Enterobacteriaceae are a significant threat to public health, and a major resistance determinant that promotes this phenotype is the production of the OXA-48 carbapenemase. The activity of OXA-48 towards carbapenems is a puzzling phenotype as its hydrolytic activity against doripenem is non-detectable. To probe the mechanistic basis for this observation, we determined the 1.5 Å resolution crystal structure of the deacylation deficient K73A variant of OXA-48 in complex with doripenem. Doripenem is observed in the Δ1R and Δ1S tautomeric states covalently attached to the catalytic S70 residue. Likely due to positioning of residue Y211, the carboxylate moiety of doripenem is making fewer hydrogen bonding/salt-bridge interactions with R250 compared to previously determined carbapenem OXA structures. Moreover, the hydroxyethyl side chain of doripenem is making van der Waals interactions with a key V120 residue, which likely affects the deacylation rate of doripenem. We hypothesize that positions V120 and Y211 play important roles in the carbapenemase profile of OXA-48. Herein, we provide insights for the further development of the carbapenem class of antibiotics that could render them less effective to hydrolysis by or even inhibit OXA carbapenemases.

Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans.

Multidrug-resistant gram-negative pathogens are a significant health threat. Burkholderia spp. encompass a complex subset of gram-negative bacteria with a wide range of biological functions that include human, animal, and plant pathogens. The treatment of infections caused by Burkholderia spp. is problematic due to their inherent resistance to multiple antibiotics. The major ß-lactam resistance determinant expressed in Burkholderia spp. is a class A ß-lactamase of the PenA family. In this study, significant amino acid sequence heterogeneity was discovered in PenA (37 novel variants) within a panel of 48 different strains of Burkholderia multivorans isolated from individuals with cystic fibrosis. Phylogenetic analysis distributed the 37 variants into 5 groups based on their primary amino acid sequences. Amino acid substitutions were present throughout the entire ß-lactamase and did not congregate to specific regions of the protein. The PenA variants possessed 5 to 17 single amino acid changes. The N189S and S286I substitutions were most prevalent and found in all variants. Due to the sequence heterogeneity in PenA, a highly conserved peptide (18 amino acids) within PenA was chosen as the antigen for polyclonal antibody production in order to measure expression of PenA within the 48 clinical isolates of B. multivorans. Characterization of the anti-PenA peptide antibody, using immunoblotting approaches, exposed several unique features of this antibody (i.e., detected <500 pg of purified PenA, all 37 PenA variants in B. multivorans, and Pen-like ß-lactamases from other species within the Burkholderia cepacia complex). The significant sequence heterogeneity found in PenA may have occurred due to selective pressure (e.g., exposure to antimicrobial therapy) within the host. The contribution of these changes warrants further investigation.

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients.

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-ß-lactam ß-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major ß-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of (2 ± 1) × 106 µM-1 s-1 and a slow koff of (2 ± 1) × 10-3 s-1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA-avibactam revealed several interactions that stabilized the acyl-enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime-avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime-avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime-avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.