Durlobactam, a Broad-Spectrum Serine ß-lactamase Inhibitor, Restores Sulbactam Activity Against Acinetobacter Species.

Sulbactam-durlobactam is a pathogen-targeted ß-lactam/ß-lactamase inhibitor combination in late-stage development for the treatment of Acinetobacter infections, including those caused by multidrug-resistant strains. Durlobactam is a member of the diazabicyclooctane class of ß-lactamase inhibitors with broad-spectrum serine ß-lactamase activity. Sulbactam is a first-generation, narrow-spectrum ß-lactamase inhibitor that also has intrinsic antibacterial activity against Acinetobacter spp. due to its ability to inhibit penicillin-binding proteins 1 and 3. The clinical utility of sulbactam for the treatment of contemporary Acinetobacter infections has been eroded over the last decades due to its susceptibility to cleavage by numerous ß-lactamases present in this species. However, when combined with durlobactam, the activity of sulbactam is restored against this problematic pathogen. The following summary describes what is known about the molecular drivers of activity and resistance as well as results from surveillance and in vivo efficacy studies for this novel combination.

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.

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

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

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

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

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.

Structural and Biochemical Characterization of the Novel CTX-M-151 Extended-Spectrum ß-Lactamase and Its Inhibition by Avibactam.

The diazabicyclooctane (DBO) inhibitor avibactam (AVI) reversibly inactivates most serine ß-lactamases, including the CTX-M ß-lactamases. Currently, more than 230 unique CTX-M members distributed in five clusters with less than 5% amino acid sequence divergence within each group have been described. Recently, a variant named CTX-M-151 was isolated from a Salmonella enterica subsp. enterica serovar Choleraesuis strain in Japan. This variant possesses a low degree of amino acid identity with the other CTX-Ms (63.2% to 69.7% with respect to the mature proteins), and thus it may represent a new subgroup within the family. CTX-M-151 hydrolyzes ceftriaxone better than ceftazidime (kcat/Km values 6,000-fold higher), as observed with CTX-Ms. CTX-M-151 is well inhibited by mechanism-based inhibitors like clavulanic acid (inactivation rate [kinact]/inhibition constant [Ki ] = 0.15 µM-1 · s-1). For AVI, the apparent inhibition constant (Kiapp), 0.4 µM, was comparable to that of KPC-2; the acylation rate (k2/K) (37,000 M-1 · s-1) was lower than that for CTX-M-15, while the deacylation rate (koff) (0.0015 s-1) was 2- to 14-fold higher than those of other class A ß-lactamases. The structure of the CTX-M-151/AVI complex (1.32 Å) reveals that AVI adopts a chair conformation with hydrogen bonds between the AVI carbamate and Ser70 and Ser237 at the oxyanion hole. Upon acylation, the side chain of Lys73 points toward Ser130, which is associated with the protonation of Glu166, supporting the role of Lys73 in the proton relay pathway and Glu166 as the general base in deacylation. To our knowledge, this is the first chromosomally encoded CTX-M in Salmonella Choleraesuis that shows similar hydrolytic preference toward cefotaxime (CTX) and ceftriaxone (CRO) to that toward ceftazidime (CAZ).

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.

Interplay between Meropenem and Human Serum Albumin on Expression of Carbapenem Resistance Genes and Natural Competence in Acinetobacter baumannii.

Acinetobacter baumannii A118, a carbapenem-susceptible strain, and AB5075, carbapenem resistant, were cultured in lysogeny broth (LB) or LB with different supplements, such as 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5% HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation in A. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus-associated genes. The combination meropenem plus 3.5% HSA produced a synergistic enhancement in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumannii AMA16 blaNDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may increase the stress response, the expression of genes involved in natural competence is increased in A. baumannii. This process may favor the acquisition of foreign DNA and accelerate evolution.

Staphylococcus aureus Potentiates the Hemolytic Activity of Burkholderia cepacia Complex (Bcc) Bacteria.

Polymicrobial lung infections in individuals with Cystic Fibrosis (CF) contribute to the complexity of this disease and are a major cause of morbidity and mortality in the CF community. The microorganisms most commonly associated with severe airway infections in individuals with CF are the opportunistic pathogens S. aureus, P. aeruginosa and bacteria from the Burkholderia cepacia complex (Bcc), particularly B. cenocepacia and B. multivorans. Three Bcc strains, two S. aureus wild-type strains, and two derivative mutants were used to investigate the interplay between S. aureus and Bcc with a focus on the hemolytic activity of Bcc. Our results revealed that extracellular products from S. aureus potentiated the hemolysis of Bcc strains. Moreover, this effect was influenced by the composition of the medium in which S. aureus is grown. These findings contribute towards the understanding of the impact of interactions between S. aureus and Bcc and their possible implications in the context of co-infections by these pathogens in individuals with CF.

Effect of Serum Albumin, a Component of Human Pleural Fluid, on Transcriptional and Phenotypic Changes on Acinetobacter baumannii A118.

Acinetobacter baumannii is a multidrug-resistant pathogen that causes numerous infections associated with high mortality rates. Exposure to human body fluids, such as human pleural fluid (HPF) and human serum, modulates gene expression in A. baumannii, leading to changes in its pathogenic behavior. Diverse degrees of effects at the transcriptional level were observed in susceptible and carbapenem-resistant strains. The transcriptional analysis of AB5075, a hyper-virulent and extensively drug-resistant strain showed changes in genes associated with quorum sensing, quorum quenching, fatty acids metabolism, and high-efficient iron uptake systems. In addition, the distinctive role of human serum albumin (HSA) as a critical component of HPF was evidenced. In the present work, we used model strain to analyze more deeply into the contribution of HSA in triggering A. baumannii's response. By qRT-PCR analysis, changes in the expression level of genes associated with quorum sensing, biofilm formation, and phenylacetic acid pathway were observed. Phenotypic approaches confirmed the transcriptional response. HSA, a predominant component of HPF, can modulate the expression and behavior of genes not only in a hyper-virulent and extensively drug-resistant A. baumannii model, but also in other strains with a different degree of susceptibility and pathogenicity.

Structural Insights into Ceftobiprole Inhibition of Pseudomonas aeruginosa Penicillin-Binding Protein 3.

Ceftobiprole is an advanced-generation broad-spectrum cephalosporin antibiotic with potent and rapid bactericidal activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, as well as susceptible Gram-negative pathogens, including Pseudomonas sp. pathogens. In the case of Pseudomonas aeruginosa, ceftobiprole acts by inhibiting P. aeruginosa penicillin-binding protein 3 (PBP3). Structural studies were pursued to elucidate the molecular details of this PBP inhibition. The crystal structure of the His-tagged PBP3-ceftobiprole complex revealed a covalent bond between the ligand and the catalytic residue S294. Ceftobiprole binding leads to large active site changes near binding sites for the pyrrolidinone and pyrrolidine rings. The S528 to L536 region adopts a conformation previously not observed in PBP3, including partial unwinding of the α11 helix. These molecular insights can lead to a deeper understanding of ß-lactam-PBP interactions that result in major changes in protein structure, as well as suggesting how to fine-tune current inhibitors and to develop novel inhibitors of this PBP.

A Standard Numbering Scheme for Class C ß-Lactamases.

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

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.

Structural Insights into the Inhibition of the Extended-Spectrum ß-Lactamase PER-2 by Avibactam.

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-ß-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D ß-lactamases (i.e., k2/K of ≈103 M-1 s-1) but lower than other class A ß-lactamases (i.e., k2/K = 104 to 105 M-1 s-1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum ß-lactamases (ESBLs). Comparatively, relebactam combined with a ß-lactam is more potent against Escherichia coli producing PER-2 variants than ß-lactam-AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

Beyond Piperacillin-Tazobactam: Cefepime and AAI101 as a Potent ß-Lactam-ß-Lactamase Inhibitor Combination.

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 ß-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 ß-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 ß-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.

Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC.

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) ß-lactamase inhibitor that inactivates class A and C ß-lactamases and exhibits intrinsic antibiotic and ß-lactam "enhancer" activity against Enterobacteriaceae In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-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 Ki [Ki app] = 31 ± 3 µM) more efficiently than the K234R variant (Ki app = 270 ± 27 µM) and displayed a faster acylation rate (k2/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 ß-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

"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.

Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative ß-Lactamases.

Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly ß-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative ß-lactamase-encoding genes. In general, these genes have more than 93% identity with ß-lactamase genes found in other Bcc species. Two ß-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22-44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum ß-lactamase (ESBL) phenotype. These results provide insight into the presence of ß-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.