The primary pharmacology of ceftazidime/avibactam: resistance in vitro.

This article reviews resistance to ceftazidime/avibactam as an aspect of its primary pharmacology, linked thematically with recent reviews of the basic in vitro and in vivo translational biology of the combination (J Antimicrob Chemother 2022; 77: 2321-40 and 2341-52). In Enterobacterales or Pseudomonas aeruginosa, single-step exposures to 8×  MIC of ceftazidime/avibactam yielded frequencies of resistance from <∼0.5 × 10-9 to 2-8 × 10-9, depending on the host strain and the ß-lactamase harboured. ß-Lactamase structural gene mutations mostly affected the avibactam binding site through changes in the Ω-loop: e.g. Asp179Tyr (D179Y) in KPC-2. Other mutations included ones proposed to reduce the permeability to ceftazidime and/or avibactam through changes in outer membrane structure, up-regulated efflux, or both. The existence, or otherwise, of cross-resistance between ceftazidime/avibactam and other antibacterial agents was also reviewed as a key element of the preclinical primary pharmacology of the new agent. Cross-resistance between ceftazidime/avibactam and other ß-lactam-based antibacterial agents was caused by MBLs. Mechanism-based cross-resistance was not observed between ceftazidime/avibactam and fluoroquinolones, aminoglycosides or colistin. A low level of general co-resistance to ceftazidime/avibactam was observed in MDR Enterobacterales and P. aeruginosa. For example, among 2821 MDR Klebsiella spp., 3.4% were resistant to ceftazidime/avibactam, in contrast to 0.07% of 8177 non-MDR isolates. Much of this was caused by possession of MBLs. Among 1151 MDR, XDR and pandrug-resistant isolates of P. aeruginosa from the USA, 11.1% were resistant to ceftazidime/avibactam, in contrast to 3.0% of 7452 unselected isolates. In this case, the decreased proportion susceptible was not due to MBLs.

The primary pharmacology of ceftazidime/avibactam: in vitro translational biology.

Previous reviews of ceftazidime/avibactam have focused on in vitro molecular enzymology and microbiology or the clinically associated properties of the combination. Here we take a different approach. We initiate a series of linked reviews that analyse research on the combination that built the primary pharmacology data required to support the clinical and business risk decisions to perform randomized controlled Phase 3 clinical trials, and the additional microbiological research that was added to the above, and the safety and chemical manufacturing and controls data, that constituted successful regulatory licensing applications for ceftazidime/avibactam in multiple countries, including the USA and the EU. The aim of the series is to provide both a source of reference for clinicians and microbiologists to be able to use ceftazidime/avibactam to its best advantage for patients, but also a case study of bringing a novel ß-lactamase inhibitor (in combination with an established ß-lactam) through the microbiological aspects of clinical development and regulatory applications, updated finally with a review of resistance occurring in patients under treatment. This first article reviews the biochemistry, structural biology and basic microbiology of the combination, showing that avibactam inhibits the great majority of serine-dependent ß-lactamases in Enterobacterales and Pseudomonas aeruginosa to restore the in vitro antibacterial activity of ceftazidime. Translation to efficacy against infections in vivo is reviewed in the second co-published article, Nichols et al. (J Antimicrob Chemother 2022; 77: 2341-52).

Discovery of an Orally Available Diazabicyclooctane Inhibitor (ETX0282) of Class A, C, and D Serine ß-Lactamases.

Multidrug resistant Gram-negative bacterial infections are an increasing public health threat due to rapidly rising resistance toward ß-lactam antibiotics. The hydrolytic enzymes called ß-lactamases are responsible for a large proportion of the resistance phenotype. ß-Lactamase inhibitors (BLIs) can be administered in combination with ß-lactam antibiotics to negate the action of the ß-lactamases, thereby restoring activity of the ß-lactam. Newly developed BLIs offer some advantage over older BLIs in terms of enzymatic spectrum but are limited to the intravenous route of administration. Reported here is a novel, orally bioavailable diazabicyclooctane (DBO) ß-lactamase inhibitor. This new DBO, ETX1317, contains an endocyclic carbon-carbon double bond and a fluoroacetate activating group and exhibits broad spectrum activity against class A, C, and D serine ß-lactamases. The ester prodrug of ETX1317, ETX0282, is orally bioavailable and, in combination with cefpodoxime proxetil, is currently in development as an oral therapy for multidrug resistant and carbapenem-resistant Enterobacterales infections.

Narrow-Spectrum Antibacterial Agents-Benefits and Challenges.

The number of antibacterial agents in clinical and preclinical development possessing activity against a narrow spectrum of bacterial pathogens is increasing, with many of them being nontraditional products. The key value proposition hinges on sparing antibiotic use and curtailing the emergence of resistance, as well as preventing the destruction of a beneficial microbiome, versus the immediate need for effective treatment of an active infection with a high risk of mortality. The clinical use of a targeted spectrum agent, most likely in combination with a rapid and robust diagnostic test, is a commendable goal with significant healthcare benefits if executed correctly. However, the path to achieving this will come with several challenges, and many scientific and clinical development disciplines will need to align their efforts to successfully change the treatment paradigm.

Ceftaroline efficacy against high-MIC clinical Staphylococcus aureus isolates in an in vitro hollow-fibre infection model.

Objectives: The current CLSI and EUCAST clinical susceptible breakpoint for 600 mg q12h dosing of ceftaroline (active metabolite of ceftaroline fosamil) for Staphylococcus aureus is ≤1 mg/L. Efficacy data for S. aureus infections with ceftaroline MIC ≥2 mg/L are limited. This study was designed to generate in-depth pharmacokinetic/pharmacodynamics (PK/PD) understanding of S. aureus isolates inhibited by ≥ 2 mg/L ceftaroline using an in vitro hollow-fibre infection model (HFIM). Methods: The PK/PD target of ceftaroline was investigated against 12 diverse characterized clinical MRSA isolates with ceftaroline MICs of 2 or 4 mg/L using q8h dosing for 24 h. These isolates carried substitutions in the penicillin-binding domain (PBD) and/or the non-PBD. Additionally, PD responses of mutants with ceftaroline MICs ranging from 2 to 32 mg/L were evaluated against the mean 600 mg q8h human-simulated dose over 72 h. Results: The mean stasis, 1 log10-kill and 2 log10-kill PK/PD targets were 29%, 32% and 35% f T>MIC, respectively. In addition, these data suggest that the PK/PD target for MRSA is not impacted by the presence of substitutions in the non-PBD commonly found in isolates with ceftaroline MIC values of ≤ 2 mg/L. HFIM studies with 600 mg q8h dosing demonstrated a sustained long-term bacterial suppression for isolates with ceftaroline MICs of 2 and 4 mg/L. Conclusions: Overall, efficacy was demonstrated against a diverse collection of clinical isolates using HFIM indicating the utility of 600 mg ceftaroline fosamil for S. aureus isolates with MIC ≤4 mg/L using q8h dosing.

Antibacterial FabH Inhibitors with Mode of Action Validated in Haemophilus influenzae by in Vitro Resistance Mutation Mapping.

Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (ß-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery.

Identification of non-PBP2a resistance mechanisms in Staphylococcus aureus after serial passage with ceftaroline: involvement of other PBPs.

OBJECTIVES: Ceftaroline (the active metabolite of ceftaroline fosamil) is a cephalosporin that possesses activity against MRSA due to its differentiating high affinity for PBP2a. It is known that PBP2a sequence variations, including some outside of the transpeptidase-binding pocket, impact ceftaroline susceptibility and recent evidence suggests involvement of non-PBP2a mechanisms in ceftaroline resistance. This study evaluated the potential of ceftaroline to select for resistant Staphylococcus aureus clones during serial passage. METHODS: Selection experiments were performed by up to 20 daily passages of three S. aureus isolates (two MRSA and one MSSA) in broth with increasing selective pressure. Mutants that emerged were tested for changes in ceftaroline susceptibility and genetically characterized. RESULTS: The MSSA isolate developed mutations in PBP2 and PBP3 that increased the ceftaroline MIC by 16-fold and increased the MICs of other ß-lactams. A Glu447Lys substitution in the PBP2a transpeptidase pocket in one MRSA isolate elevated the ceftaroline MIC to 8 mg/L. Selective pressure in a ceftaroline-resistant MRSA isolate generated mutations in LytD, as well as changes in the pbp4 promoter previously shown to result in PBP4 overexpression, the one PBP not inhibited by ceftaroline. Elevated ceftaroline MIC was reversed when tested in combination with extremely low levels of methicillin or meropenem that could inhibit the function of PBP4. CONCLUSIONS: These studies demonstrate that resistance to ceftaroline can be manifested through numerous mechanisms. Further, they support a hypothesis where PBP4 can functionally provide the essential transpeptidase activity required for MRSA cell wall biogenesis when PBP2a is inhibited.

Structural and sequence analysis of class A ß-lactamases with respect to avibactam inhibition: impact of Ω-loop variations.

BACKGROUND: There exists a significant diversity among class A ß-lactamases and the proliferation of these enzymes is a significant medical concern due to the ability of some members to efficiently hydrolyse both extended-spectrum cephalosporins and carbapenems. Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that, in combination with ceftazidime, has recently obtained regulatory approval in the USA. Although avibactam is known to efficiently inhibit key class A enzymes, the diversity of this enzyme family warranted a more complete investigation to understand the breadth of the potential spectrum of inhibition. METHODS: Using the known residues critical for avibactam binding, a thorough structural and sequence-based conservation analysis was performed across >650 class A enzymes. Several variations that had the potential to impact avibactam inhibition were observed and representative enzymes were cloned and expressed isogenically to evaluate the impact of these variations. RESULTS: The majority of the key residues involved in avibactam binding were well conserved across the different sub-families of class A ß-lactamases, although some differences were observed. The differences in the Ω-loop of PER enzymes were found to impact the ability of avibactam to effectively protect ß-lactams against hydrolysis. However, substitutions in a key hydrogen-bonding residue (N170) in some of the GES variants were found to not have a significant impact on avibactam inhibition. CONCLUSIONS: Overall, the computational and experimental analyses suggest that the vast majority of class A ß-lactamases should be well inhibited by avibactam, although a very small number of outliers exist.

Multi-center and multi-method evaluation of in vitro activities of ceftaroline against S. aureus.

This five-site study was performed to assess the reproducibility of ceftaroline MIC and disk results for Staphylococcus aureus. Three commercial broth microdilution, three gradient diffusion and ceftaroline 5µg disk diffusion methods were compared to a reference broth microdilution method against challenge isolates (n = 41) and isolates collected at four European sites (n = 30/site). For four MIC methods (Sensititre and three gradient diffusion methods), 99.0% of consolidated MIC results were within +/- 1 dilution of the reference MIC. Categorical agreement rates based on EUCAST breakpoints for the challenge isolates were 75.6-100% and for disk testing were 78.0-92.7%. There was no clear distinction between isolates with MIC results of 1 and 2mg/L with regard to variation in MIC or molecular genotyping results. The addition of an intermediate category for isolates with MIC results of 2mg/L would help to identify these isolates as borderline susceptible/non-susceptible isolates.

Identification of Novel VEB ß-Lactamase Enzymes and Their Impact on Avibactam Inhibition.

Ceftazidime-avibactam has activity against Pseudomonas aeruginosa and Enterobacteriaceae expressing numerous class A and class C ß-lactamases, although the ability to inhibit many minor enzyme variants has not been established. Novel VEB class A ß-lactamases were identified during characterization of surveillance isolates. The cloned novel VEB ß-lactamases possessed an extended-spectrum ß-lactamase phenotype and were inhibited by avibactam in a concentration-dependent manner. The residues that comprised the avibactam binding pocket were either identical or functionally conserved. These data demonstrate that avibactam can inhibit VEB ß-lactamases.

Potential of Staphylococcus aureus isolates carrying different PBP2a alleles to develop resistance to ceftaroline.

OBJECTIVES: Infections caused by MRSA continue to cause significant morbidity worldwide. Ceftaroline (the active metabolite of the prodrug ceftaroline fosamil) is a cephalosporin that possesses activity against MRSA due to its having high affinity for PBP2a while maintaining activity against the other essential PBPs. PBP2a sequence variations, including some outside of the transpeptidase binding pocket, impact ceftaroline susceptibility. This study evaluated the potential of ceftaroline to select for resistant Staphylococcus aureus clones in isolates containing a variety of PBP2a alleles and with a range of ceftaroline MIC values from different MLST lineages. METHODS: Direct resistance selection experiments were performed by plating 20 S. aureus isolates (18 MRSA and 2 MSSA) on agar plates containing increasing concentrations of ceftaroline. Colonies that emerged were tested by standard broth microdilution for changes in ceftaroline susceptibility and genetically characterized. RESULTS: The frequency of spontaneous resistance to ceftaroline was low for all isolates and, although resistant variants were not obtained on plates containing ≥4-fold the MIC of ceftaroline, six MRSA isolates had a small number of colonies emerge on plates containing 2-fold the MIC of ceftaroline and had a 2- to 8-fold elevation of the ceftaroline MIC, while also impacting the MIC of methicillin compared with the parental isolate. Additional PBP2a mutations located in the ceftaroline-binding pocket, Y446N or A601S, were observed in several of the resistant isolates. CONCLUSIONS: These studies demonstrate that there is a low risk of generating ceftaroline-resistant MRSA isolates, which appears independent of any pre-existing variation in the PBP2a protein sequence or initial ceftaroline MIC.

In Vitro Activity of Ceftaroline against Staphylococcus aureus Isolated in 2012 from Asia-Pacific Countries as Part of the AWARE Surveillance Program.

Ceftaroline, the active metabolite of the prodrug ceftaroline-fosamil, is an advanced-generation cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). This investigation provides in vitro susceptibility data for ceftaroline against 1,971 S. aureus isolates collected in 2012 from seven countries (26 centers) in the Asia-Pacific region as part of the Assessing Worldwide Antimicrobial Resistance and Evaluation (AWARE) program. Broth microdilution as recommended by the CLSI was used to determine susceptibility. In all, 62% of the isolates studied were MRSA, and the ceftaroline MIC90 for all S. aureus isolates was 2 µg/ml (interpretive criteria: susceptible, ≤1 µg/ml). The overall ceftaroline susceptibility rate for S. aureus was 86.9%, with 100% of methicillin-sensitive S. aureus isolates and 78.8% of MRSA isolates susceptible to this agent. The highest percentages of ceftaroline-nonsusceptible MRSA isolates came from China (47.6%), all of which showed intermediate susceptibility, and Thailand (37.1%), where over half (52.8%) of isolates were resistant to ceftaroline (MIC, 4 µg/ml). Thirty-eight ceftaroline-nonsusceptible isolates (MIC values of 2 to 4 µg/ml) were selected for molecular characterization. Among the isolates analyzed, sequence type 5 (ST-5) was the most common sequence type encountered; however, all isolates analyzed from Thailand were ST-228. Penicillin-binding protein 2a (PBP2a) substitution patterns varied by country, but all isolates from Thailand had the Glu239Lys substitution, and 12 of these also carried an additional Glu447Lys substitution. Ceftaroline-fosamil is a useful addition to the antimicrobial agents that can be used to treat S. aureus infections. However, with the capability of this species to develop resistance to new agents, it is important to recognize and monitor regional differences in trends as they emerge.

In Vitro Activity of Ceftaroline against Staphylococcus aureus Isolates Collected in 2012 from Latin American Countries as Part of the AWARE Surveillance Program.

The in vitro activities of ceftaroline and comparators, using broth microdilution, were determined against 1,066 Staphylococcus aureus isolates from hospitalized patients. Seventeen medical centers from Latin American countries contributed isolates. Methicillin-resistant S. aureus (MRSA) percentages ranged from 46% (Brazil) to 62% (Argentina). All methicillin-susceptible S. aureus (MSSA) isolates were susceptible to ceftaroline. Ceftaroline activity against MRSA varied with MIC90s of 0.5 (Venezuela) to 2 (Brazil, Chile, and Colombia) µg/ml, which was the highest MIC value. ST-5 was the most common sequence type.

Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases.

With the diminishing effectiveness of current antibacterial therapies, it is critically important to discover agents that operate by a mechanism that circumvents existing resistance. ETX0914, the first of a new class of antibacterial agent targeted for the treatment of gonorrhea, operates by a novel mode-of-inhibition against bacterial type II topoisomerases. Incorporating an oxazolidinone on the scaffold mitigated toxicological issues often seen with topoisomerase inhibitors. Organisms resistant to other topoisomerase inhibitors were not cross-resistant with ETX0914 nor were spontaneous resistant mutants to ETX0914 cross-resistant with other topoisomerase inhibitor classes, including the widely used fluoroquinolone class. Preclinical evaluation of ETX0914 pharmacokinetics and pharmacodynamics showed distribution into vascular tissues and efficacy in a murine Staphylococcus aureus infection model that served as a surrogate for predicting efficacious exposures for the treatment of Neisseria gonorrhoeae infections. A wide safety margin to the efficacious exposure in toxicological evaluations supported progression to Phase 1. Dosing ETX0914 in human volunteers showed sufficient exposure and minimal adverse effects to expect a highly efficacious anti-gonorrhea therapy.

Insights into the mechanism of inhibition of novel bacterial topoisomerase inhibitors from characterization of resistant mutants of Staphylococcus aureus.

The type II topoisomerases DNA gyrase and topoisomerase IV are clinically validated bacterial targets that catalyze the modulation of DNA topology that is vital to DNA replication, repair, and decatenation. Increasing resistance to fluoroquinolones, which trap the topoisomerase-DNA complex, has led to significant efforts in the discovery of novel inhibitors of these targets. AZ6142 is a member of the class of novel bacterial topoisomerase inhibitors (NBTIs) that utilizes a distinct mechanism to trap the protein-DNA complex. AZ6142 has very potent activity against Gram-positive organisms, including Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. In this study, we determined the frequencies of resistance to AZ6142 and other representative NBTI compounds in S. aureus and S. pneumoniae. The frequencies of selection of resistant mutants at 4× the MIC were 1.7 × 10(-8) for S. aureus and <5.5 × 10(-10) for S. pneumoniae. To improve our understanding of the NBTI mechanism of inhibition, the resistant S. aureus mutants were characterized and 20 unique substitutions in the topoisomerase subunits were identified. Many of these substitutions were located outside the NBTI binding pocket and impact the susceptibility of AZ6142, resulting in a 4- to 32-fold elevation in the MIC over the wild-type parent strain. Data on cross-resistance with other NBTIs and fluoroquinolones enabled the differentiation of scaffold-specific changes from compound-specific variations. Our results suggest that AZ6142 inhibits both type II topoisomerases in S. aureus but that DNA gyrase is the primary target. Further, the genotype of the resistant mutants suggests that domain conformations and DNA interactions may uniquely impact NBTIs compared to fluoroquinolones.

Molecular characterization of MRSA isolates bracketing the current EUCAST ceftaroline-susceptible breakpoint for Staphylococcus aureus: the role of PBP2a in the activity of ceftaroline.

OBJECTIVES: The objectives of this study were to characterize contemporary MRSA isolates and understand the prevalence and impact of sequence variability in PBP2a on ceftaroline susceptibility. METHODS: A total of 184 MRSA isolates collected from 28 countries were collected and characterized. RESULTS: WT PBP2a proteins were found in MRSA distributed evenly over the ceftaroline MIC range of 0.5-2 mg/L (n=56). PBP2a variations found in 124 isolates fell into two categories: (i) 12 isolates contained a substitution in the transpeptidase pocket located in the penicillin-binding domain and exhibited significantly decreased ceftaroline susceptibility (typically 8 mg/L); and (ii) isolates with substitutions in the non-penicillin-binding domain (nPBD) in a region proposed to be functionally important for cell wall biogenesis. The majority (71%) of isolates containing only nPBD variations were inhibited by 2 mg/L ceftaroline, 23% by ≤1 mg/L and 6% by 4 mg/L. These data suggest that the WT MRSA distribution extends beyond the current EUCAST and CLSI susceptible breakpoints and includes isolates inhibited by 2 mg/L ceftaroline. SCCmec type IV was the predominant type in the ceftaroline-susceptible population (68%), whereas it only represented 6% of the non-susceptible population. The variations of MLST lineages were fewer among the non-susceptible group. CONCLUSIONS: This study suggests that MRSA populations with a WT PBP2a and those with nPBD variations overlap significantly and that PBP2a sequence-independent factors contribute to ceftaroline susceptibility. Whereas characterization of isolates with a ceftaroline MIC of 2 mg/L enriched for isolates with nPBD variations, it was not a discrete population. In contrast, the rare isolates containing a substitution in the transpeptidase-binding pocket were readily differentiated.

Novel broad-spectrum inhibitors of bacterial methionine aminopeptidase.

With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections.

Selection and molecular characterization of ceftazidime/avibactam-resistant mutants in Pseudomonas aeruginosa strains containing derepressed AmpC.

OBJECTIVES: Pseudomonas aeruginosa is an important nosocomial pathogen that can cause a wide range of infections resulting in significant morbidity and mortality. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, is being developed in combination with ceftazidime and has the potential to be a valuable addition to the treatment options for the infectious diseases practitioner. We compared the frequency of resistance development to ceftazidime/avibactam in three P. aeruginosa strains that carried derepressed ampC alleles. METHODS: The strains were incubated in the presence of increasing concentrations of ceftazidime with a fixed concentration (4 mg/L) of avibactam to calculate the frequency of spontaneous resistance. The mutants were characterized by WGS to identify the underlying mechanism of resistance. A representative mutant protein was characterized biochemically. RESULTS: The resistance frequency was very low in all strains. The resistant variants isolated exhibited ceftazidime/avibactam MIC values that ranged from 64 to 256 mg/L. All of the mutants exhibited changes in the chromosomal ampC gene, the majority of which were deletions of various sizes in the Ω-loop region of AmpC. The mutant enzyme that carried the smallest Ω-loop deletion, which formed a part of the avibactam-binding pocket, was characterized biochemically and found to be less effectively inhibited by avibactam as well as exhibiting increased hydrolysis of ceftazidime. CONCLUSIONS: The development of high-level resistance to ceftazidime/avibactam appears to occur at low frequency, but structural modifications in AmpC can occur that impact the ability of avibactam to inhibit the enzyme and thereby protect ceftazidime from hydrolysis.