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).

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.

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.

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.

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.

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.

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.

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.

Characterization of the novel DNA gyrase inhibitor AZD0914: low resistance potential and lack of cross-resistance in Neisseria gonorrhoeae.

The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.

Characterization of Escherichia coli NDM isolates with decreased susceptibility to aztreonam/avibactam: role of a novel insertion in PBP3.

OBJECTIVES: The spread of NDM-1 amongst Enterobacteriaceae has highlighted a significant threat to the clinical management of serious infections. The combination of aztreonam and avibactam, a non-ß-lactam ß-lactamase inhibitor, may provide a much-needed therapeutic alternative. This combination was potent against most NDM-containing Enterobacteriaceae, although activity was diminished against many Escherichia coli isolates. These E. coli isolates were characterized to elucidate the mechanism of decreased susceptibility to aztreonam/avibactam. METHODS: MIC determinations were performed using broth microdilution, and whole-genome sequencing was performed to enable sequence-based analyses. RESULTS: The decreased susceptibility was not due to avibactam being unable to inhibit the serine ß-lactamases found in the E. coli isolates. Rather, it was manifested by a four-amino-acid insertion in PBP3. This same insertion was also found in non-NDM-containing E. coli that had reduced susceptibility to aztreonam/avibactam. Construction of an isogenic mutant confirmed that this insertion resulted in decreased susceptibility to aztreonam and several cephalosporins, but had no impact on carbapenem potency. Structural analysis suggests that this insertion will impact the accessibility of the ß-lactam drugs to the transpeptidase pocket of PBP3. CONCLUSIONS: The acquisition of ß-lactamases is the predominant mechanism of ß-lactam resistance in Enterobacteriaceae. We have demonstrated that small PBP3 changes will affect the susceptibility to a broad range of ß-lactams. These changes were identified in multiple MLST lineages of E. coli, and were enriched in NDM-containing isolates. However, they were not present in other key species of Enterobacteriaceae despite significant conservation among the PBP3 proteins.

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.

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.

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.

Kinetics of avibactam inhibition against Class A, C, and D ß-lactamases.

Avibactam is a non-ß-lactam ß-lactamase inhibitor with a spectrum of activity that includes ß-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.

Activity of avibactam against Enterobacter cloacae producing an extended-spectrum class C ß-lactamase enzyme.

BACKGROUND: Extended-spectrum AmpC (ESAC) ß-lactamase enzymes, which are either chromosomally encoded or plasmid encoded, have minor structural changes that broaden their substrate hydrolysis profile. The derepressed AmpC enzyme found once in Enterobacter cloacae CHE was shown to contain a six residue deletion in the H-10 helix in close proximity to the active site. Avibactam is a non-ß-lactam inhibitor of Ambler class A, class C and some class D ß-lactamases that is in clinical development with several ß-lactam agents. It has been shown to inhibit AmpC enzymes, but its microbiological activity against isolates carrying different ESAC enzymes is less well understood. METHODS: MICs were determined using the broth microdilution technique. RT-PCR analyses were performed to measure the level of ampC expression and whole genome sequencing was performed to enable sequence-based analyses. RESULTS: Structural analyses of avibactam bound to a representative AmpC ß-lactamase suggested that the H-10 helix deletion would impact the potency of the inhibitor. Under standard conditions, the ceftazidime/avibactam and ceftaroline/avibactam MIC values for E. cloacae CHE were 64 and 4 mg/L, respectively, representing a significant decrease in susceptibility over control E. cloacae isolates. However, use of higher avibactam concentrations restored the susceptibility of E. cloacae CHE in a dose-dependent manner. Comparison with other E. cloacae isolates carrying derepressed AmpC enzymes suggested that this difference in inhibition by avibactam was unrelated to the level of AmpC being produced. CONCLUSIONS: The E. cloacae CHE ESAC enzyme is inhibited less efficiently by avibactam than other E. cloacae AmpC proteins due to a subtle rearrangement of the binding site. Although the variants are not commonly observed, the different ESAC enzymes may be inhibited to varied extents by avibactam.

Analysis of Staphylococcus aureus clinical isolates with reduced susceptibility to ceftaroline: an epidemiological and structural perspective.

OBJECTIVES: Ceftaroline, approved in Europe in 2012, has activity against methicillin-resistant Staphylococcus aureus (MRSA), with MIC90 values of 1-2 mg/L depending on geographical location. During a global 2010 surveillance programme, conducted prior to the European launch, 4 S. aureus isolates, out of 8037 tested, possessing ceftaroline MIC values of >2 mg/L were identified. The objective of this study was to characterize these four isolates to elucidate the mechanism of ceftaroline resistance. METHODS: MIC determinations were performed using broth microdilution and whole genome sequencing was performed to enable sequence-based analyses. RESULTS: The only changes in proteins known to be required for full expression of methicillin resistance that correlated with the ceftaroline MIC were in penicillin-binding protein 2a (PBP2a). Isolates with a ceftaroline MIC of 2 mg/L had a Glu239Lys mutation in the non-penicillin-binding domain whereas the four isolates with ceftaroline MIC values of 8 mg/L carried an additional Glu447Lys mutation in the penicillin-binding domain. The impact of these mutations was analysed using the known X-ray structure of S. aureus PBP2a and a model for ceftaroline resistance proposed. Analysis of the core genomes showed that the isolates with reduced susceptibility to ceftaroline were epidemiologically related. CONCLUSIONS: Mutations in PBP2a can affect the activity of ceftaroline against MRSA. Although a rare event, based on surveillance studies, it appears a first-step change in the non-penicillin-binding domain together with a second-step in the penicillin-binding domain may result in elevation of the ceftaroline MIC to >2 mg/L.

Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.

Structural insight into potent broad-spectrum inhibition with reversible recyclization mechanism: avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC ß-lactamases.

Although ß-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of ß-lactamase enzymes that are not affected by currently marketed ß-lactam/ß-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-ß-lactam ß-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D ß-lactamases. Here we describe the structures of two clinically important ß-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum ß-lactamase and the class C Pseudomonas aeruginosa AmpC ß-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.