Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a ß-lactam/ß-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane ß-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine ß-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of ß-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

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: microbiology from clinical studies, and development of resistance during treatment.

As one of a series of thematically linked reviews of the primary pharmacology of the ß-lactam/ß-lactamase inhibitor combination, ceftazidime/avibactam, this article reviews the microbiological findings in drug-exposed patients. Earlier articles in the series focused on basic in vitro and in vivo translational biology (J Antimicrob Chemother 2022; 77: 2321-40 and 2341-52) and the development and mechanisms of resistance in vitro (J Antimicrob Chemother 2023: Epub ahead of print. doi: 10.1093/jac/dkac449). In clinical trials of ceftazidime/avibactam, combined favourable microbiological responses for evaluable patients infected at baseline by susceptible Enterobacterales or Pseudomonas aeruginosa were 86.1% (851/988). The corresponding percent favourable among patients infected by ceftazidime/avibactam-resistant pathogens was 58.8% (10/17), noting that the majority (15/17) of the resistant examples were P. aeruginosa. Microbiological response rates to comparator treatments in the same clinical trials ranged between 64% and 95%, depending on the type of infection and the analysis population. Uncontrolled case studies over a wide range of patients infected by antibiotic multiresistant Gram-negative bacteria have demonstrated that ceftazidime/avibactam can elicit microbiological clearance of ceftazidime/avibactam-susceptible strains. In case studies where a matched cohort of patients had been treated with antibacterial agents other than ceftazidime/avibactam, microbiological outcomes were comparable between treatments, mostly being observationally more favourable for ceftazidime/avibactam (recognizing that numbers were too small for definitive superiority assessments). Development of resistance to ceftazidime/avibactam during therapy is reviewed. The phenomenon has been reported multiple times, mostly in difficult-to-treat patients infected by KPC-producing Enterobacterales. Molecular mechanisms, when determined, have frequently been observed previously in vitro, such as the 'Ω-loop' D179Y (Asp179Tyr) substitution found in KPC variant enzymes. In human volunteers exposed to therapeutic levels of ceftazidime/avibactam, faecal numbers of Escherichia coli, other enterobacteria, lactobacilli, bifidobacteria, clostridia and Bacteroides spp. decreased. Clostridioides difficile was detected in the faeces, but this was of uncertain significance, because no unexposed controls were studied.

Efficacy and safety of ceftazidime/avibactam in patients with infections caused by ß-lactamase-producing Gram-negative pathogens: a pooled analysis from the Phase 3 clinical trial programme.

OBJECTIVES: This post hoc pooled analysis evaluated clinical and microbiological outcomes and safety in patients with infections caused by ß-lactamase-producing Gram-negative pathogens across five Phase 3, randomized, controlled, multicentre trials of ceftazidime/avibactam in adults with complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI)/pyelonephritis and nosocomial pneumonia (NP), including ventilator-associated pneumonia (VAP). METHODS: In each trial, RECLAIM/RECLAIM 3 (cIAI), REPRISE (cIAI/cUTI), RECAPTURE (cUTI) and REPROVE (NP, including VAP) patients were randomized 1:1 to IV ceftazidime/avibactam (plus metronidazole for patients with cIAI) or comparators (carbapenems in >97% patients) for 5-21 days. Clinical and microbiological responses at the test-of-cure visit were assessed for patients with ESBLs, and/or plasmidic and/or overexpression of chromosomal AmpC, and/or serine carbapenemases without MBLs identified in baseline Gram-negative isolates by phenotypic screening and molecular characterization in the pooled microbiological modified ITT (mMITT) population. RESULTS: In total, 813 patients (ceftazidime/avibactam, n = 389; comparator, n = 424) had ≥1 ß-lactamase-producing baseline pathogen identified, amongst whom 792 patients (ceftazidime/avibactam, n = 379; comparator, n = 413) had no MBLs. The most frequent ß-lactamase-producing pathogens across treatment groups were Escherichia coli (n = 381), Klebsiella pneumoniae (n = 261) and Pseudomonas aeruginosa (n = 53). Clinical cure rates in the pooled non-MBL ß-lactamase-producing mMITT population were 88.1% (334/379) for ceftazidime/avibactam and 88.1% (364/413) for comparators; favourable microbiological response rates were 76.5% (290/379) and 68.8% (284/413), respectively. The safety profile of ceftazidime/avibactam was consistent with previous observations. CONCLUSIONS: This analysis provides supportive evidence of the efficacy and safety of ceftazidime/avibactam in patients with infections caused by ESBLs, AmpC and serine carbapenemase-producing Gram-negative pathogens. TRIAL REGISTRATION: NCT01499290; NCT01726023; NCT01644643; NCT01595438/NCT01599806; NCT01808092.

Consensus on ß-Lactamase Nomenclature.

Assigning names to ß-lactamase variants has been inconsistent and has led to confusion in the published literature. The common availability of whole genome sequencing has resulted in an exponential growth in the number of new ß-lactamase genes. In November 2021 an international group of ß-lactamase experts met virtually to develop a consensus for the way naturally-occurring ß-lactamase genes should be named. This document formalizes the process for naming novel ß-lactamases, followed by their subsequent publication.

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).

The primary pharmacology of ceftazidime/avibactam: in vivo translational biology and pharmacokinetics/pharmacodynamics (PK/PD).

This review describes the translational in vivo and non-clinical pharmacokinetics/pharmacodynamics (PK/PD) research that supported clinical trialling and subsequently licensing approval of ceftazidime/avibactam, a new ß-lactam/ß-lactamase inhibitor combination aimed at the treatment of infections by Enterobacterales and Pseudomonas aeruginosa. The review thematically follows on from the co-published article, Nichols et al. (J Antimicrob Chemother 2022; 77: 2321-40). Avibactam protected ceftazidime in animal models of infection with ceftazidime-resistant, ß-lactamase-producing bacteria. For example, a single subcutaneous dose of ceftazidime at 1024 mg/kg yielded little effect on the growth of ceftazidime-resistant, blaKPC-2-carrying Klebsiella pneumoniae in the thighs of neutropenic mice (final counts of 4 × 108 to 8 × 108 cfu/thigh). In contrast, co-administration of avibactam in a 4:1 ratio (ceftazidime:avibactam) was bactericidal in the same model (final counts of 2 × 104 to 3 × 104 cfu/thigh). In a rat abdominal abscess model, therapy with ceftazidime or ceftazidime/avibactam (4:1 w/w) against blaKPC-2-positive K. pneumoniae resulted in 9.3 versus 3.3 log cfu/abscess, respectively, after 52 h. With respect to PK/PD, in Monte Carlo simulations, attainment of unbound drug exposure targets (ceftazidime fT>8 mg/L and avibactam fT>1 mg/L, each for 50% of the dosing interval) for the labelled dose of ceftazidime/avibactam (2 and 0.5 g, respectively, q8h by 2 h IV infusion), including dose adjustments for patients with impaired renal function, ranged between 94.8% and 99.6% of patients, depending on the infection modelled.

Epidemiology of ß-Lactamase-Producing Pathogens.

ß-Lactam antibiotics have been widely used as therapeutic agents for the past 70 years, resulting in emergence of an abundance of ß-lactam-inactivating ß-lactamases. Although penicillinases in Staphylococcus aureus challenged the initial uses of penicillin, ß-lactamases are most important in Gram-negative bacteria, particularly in enteric and nonfermentative pathogens, where collectively they confer resistance to all ß-lactam-containing antibiotics. Critical ß-lactamases are those enzymes whose genes are encoded on mobile elements that are transferable among species. Major ß-lactamase families include plasmid-mediated extended-spectrum ß-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases now appearing globally, with geographic preferences for specific variants. CTX-M enzymes include the most common ESBLs that are prevalent in all areas of the world. In contrast, KPC serine carbapenemases are present more frequently in the Americas, the Mediterranean countries, and China, whereas NDM metallo-ß-lactamases are more prevalent in the Indian subcontinent and Eastern Europe. As selective pressure from ß-lactam use continues, multiple ß-lactamases per organism are increasingly common, including pathogens carrying three different carbapenemase genes. These organisms may be spread throughout health care facilities as well as in the community, warranting close attention to increased infection control measures and stewardship of the ß-lactam-containing drugs in an effort to control selection of even more deleterious pathogens.

In Vitro Activity of Ceftazidime-Avibactam against Isolates from Respiratory and Blood Specimens from Patients with Nosocomial Pneumonia, Including Ventilator-Associated Pneumonia, in a Phase 3 Clinical Trial.

Nosocomial pneumonia (NP), including ventilator-associated pneumonia (VAP), is increasingly associated with multidrug-resistant Gram-negative pathogens. This study describes the in vitro activity of ceftazidime-avibactam, ceftazidime, and relevant comparator agents against bacterial pathogens isolated from patients with NP, including VAP, enrolled in a ceftazidime-avibactam phase 3 trial. Gram-positive pathogens were included if coisolated with a Gram-negative pathogen. In vitro susceptibility was determined at a central laboratory using Clinical and Laboratory Standards Institute broth microdilution methods. Of 817 randomized patients, 457 (55.9%) had ≥1 Gram-negative bacterial pathogen(s) isolated at baseline, and 149 (18.2%) had ≥1 Gram-positive pathogen(s) coisolated. The most common isolated pathogens were Klebsiella pneumoniae (18.8%), Pseudomonas aeruginosa (15.8%), and Staphylococcus aureus (11.5%). Ceftazidime-avibactam was highly active in vitro against 370 isolates of Enterobacteriaceae, with 98.6% susceptible (MIC90, 0.5 µg/ml) compared with 73.2% susceptible for ceftazidime (MIC90, >64 µg/ml). The percent susceptibility values for ceftazidime-avibactam and ceftazidime against 129 P. aeruginosa isolates were 88.4% and 72.9% (MIC90 values of 16 µg/ml and 64 µg/ml), respectively. Among ceftazidime-nonsusceptible Gram-negative isolates, ceftazidime-avibactam percent susceptibility values were 94.9% for 99 Enterobacteriaceae and 60.0% for 35 P. aeruginosa MIC90 values for linezolid and vancomycin (permitted per protocol for Gram-positive coverage) were within their respective MIC susceptibility breakpoints against the Gram-positive pathogens isolated. This analysis demonstrates that ceftazidime-avibactam was active in vitro against the majority of Enterobacteriaceae and P. aeruginosa isolates from patients with NP, including VAP, in a phase 3 trial. (This study has been registered at ClinicalTrials.gov under identifier NCT01808092.).

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.

In vitro activity of sulbactam/durlobactam against clinical isolates of Acinetobacter baumannii collected in China.

BACKGROUND: Durlobactam is a broad-spectrum inhibitor of class A, C and D ß-lactamases. Sulbactam is a generic ß-lactam most commonly used as a ß-lactamase inhibitor in combination with ampicillin; however, it has a unique property in that it has selective intrinsic activity against Acinetobacter baumannii. Currently, there is widespread resistance caused by multiple ß-lactamases including class A carbapenemases and class C and class D enzymes. The addition of durlobactam to sulbactam restores in vitro activity against MDR A. baumannii that possess multiple ß-lactamases. OBJECTIVES: Previously, susceptibility data for sulbactam/durlobactam were limited to isolates from patients in Western countries. This study was undertaken to determine the activity of sulbactam/durlobactam against A. baumannii isolated from patients in mainland China. METHODS: Nine hundred and eighty-two recent A. baumannii clinical isolates were collected from 22 sites across mainland China during 2016-18. The isolates were collected from lower respiratory tract, intra-abdominal, urinary tract and skin and skin structure infections. The in vitro activities of sulbactam/durlobactam and comparators were determined by broth microdilution. RESULTS: The addition of durlobactam restored the activity of sulbactam against the majority of the strains tested. The MIC90 of sulbactam/durlobactam was 2 mg/L for all A. baumannii, compared with 64 mg/L for sulbactam alone. The MIC90 of sulbactam/durlobactam of 2 mg/L remained unchanged for 831 carbapenem-resistant isolates. Colistin was the only comparator with comparable activity (MIC90 = 1 mg/L). CONCLUSIONS: This study demonstrated the potential utility of sulbactam/durlobactam for the treatment of infections caused by A. baumannii in China.

In vitro activity of omadacycline against pathogens isolated from Mainland China during 2017-2018.

Antibiotic resistance of bacterial pathogens isolated in China is a major concern. Omadacycline is a novel tetracycline derivative that has been approved for use in skin infections and community-acquired pneumonia. This study was conducted to determine the in vitro activity of omadacycline against a large collection of patient isolate medical centers across Mainland China. A total of 1041 recent clinical isolates are obtained from patients hospitalized in 29 provinces and municipalities across China. The in vitro activity of omadacycline and comparator agents was assessed using the microbroth dilution methodology. Omadacycline was active against methicillin-susceptible and -resistant Staphylococcus aureus with MIC90 values of 0.25 and 1 mg/L, respectively. All isolates of Enterococcus faecalis and Enterococcus faecium, including vancomycin-resistant isolates, were inhibited by ≤ 0.25 mg/L of omadacycline. It was active against Streptococcus pneumoniae irrespective of susceptibility to penicillin or macrolides (MIC90 =0.12 mg/L). The minimum inhibitory concentration (MIC) distribution of omadacycline was nearly identical against (extended-spectrum beta-lactamases) ESBL-positive, ESBL-negative, and carbapenemase-producing Escherichia coli (MIC90 = 4 mg/L). Omadacycline also showed good activity against Acinetobacter baumannii, inhibiting all isolates at ≤ 8 mg/L. Against Hemophilus influenzae and Moraxella catarrhalis, the MICs of omadacycline were low and not influenced by the presence of ß-lactamase. Overall, the activity of omadacycline was very good against isolates commonly associated with skin infections and pneumonia, and the susceptibility of Chinese isolates was similar to that reported for these pathogens from large surveillance studies outside China. This suggests that omadacycline could be an option for treatment of these infections in Chinese patients.

Antimicrobial Activity of Exebacase (Lysin CF-301) against the Most Common Causes of Infective Endocarditis.

Exebacase, a recombinantly produced lysin (cell wall hydrolase), and comparator antibiotics were tested by the broth microdilution method against strain sets of Staphylococcus and Streptococcus spp., which are the most common causes of infective endocarditis in humans. Exebacase was active against all Staphylococcus spp. tested, including S. aureus and coagulase-negative staphylococci (MIC50/90, 0.5/1 µg/ml). Activity against Streptococcus spp. was variable, with S. pyogenes, S. agalactiae, and S. dysgalactiae (MIC50/90, 1/2 µg/ml) among the most susceptible.

Characterization of ß-Lactamase Content of Ceftazidime-Resistant Pathogens Recovered during the Pathogen-Directed Phase 3 REPRISE Trial for Ceftazidime-Avibactam: Correlation of Efficacy against ß-Lactamase Producers.

REPRISE was a pathogen-directed (ceftazidime-resistant) phase 3 prospective, open-label, randomized, multicenter trial that evaluated the efficacy, safety, and tolerability of ceftazidime-avibactam (CAZ-AVI) and best available therapy (BAT) in the treatment of hospitalized adults with complicated intra-abdominal infections (cIAI) and complicated urinary tract infections (cUTI). This study characterized the ß-lactamase content of ceftazidime-resistant Enterobacteriaceae and Pseudomonas aeruginosa recovered during the baseline visits of patients enrolled in REPRISE. Ceftazidime had MIC90 results of >64 µg/ml against baseline Enterobacteriaceae and P. aeruginosablaCTX-M variants were the most common ß-lactamases found in Escherichia coli (detected in 94.3% of all E. coli isolates) and Klebsiella pneumoniae (91.2%), whereas Proteus mirabilis often carried plasmid AmpC (pAmpC) (66.7%). blaKPC (6 isolates), blaNDM-1 (3), blaOXA-48 (3), and blaVIM (2) were detected in 4.9% (14/284) of Enterobacteriaceae Overall, clinical cure rates against the Enterobacteriaceae were 91.2% and 90.8% for the CAZ-AVI and BAT groups, respectively, or 92.5% and 92.9% in the subset of patients infected with isolates harboring blaCTX-M Patients with baseline isolates carrying AmpC genes (pAmpC and/or overexpression of intrinsic AmpC) showed clinical cure rates of 80.0% and 89.5% for CAZ-AVI and BAT arms, respectively. Favorable microbiological responses were generally lower than clinical cure rates in both arms, but CAZ-AVI (80.0 to 85.0%) showed microbiological response rates consistently higher than those for BAT (57.9 to 64.3%) among patients with non-carbapenemase-producing Enterobacteriaceae Lower microbiological response rates (50.0%) were found in patients with carbapenemase producers from both arms. This study expands on efficacy data analysis of CAZ-AVI among patients infected with ceftazidime-resistant pathogens, especially blaCTX-M-carrying isolates, and although clinical cure rates for CAZ-AVI and BAT were similar, eradication rates for CAZ-AVI were higher than those for BAT. (This study has been registered at ClinicalTrials.gov under identifier NCT01644643.).

Determination of MIC Quality Control Ranges for the Novel Gyrase Inhibitor Zoliflodacin.

This report describes the results of two different, multilaboratory quality control (QC) studies that were used to establish QC ranges for the novel gyrase inhibitor zoliflodacin against the ATCC strains recommended by the Clinical and Laboratory Standards Institute (CLSI). Following the completion of an eight-laboratory, CLSI document M23-defined tier 2 study, the agar dilution MIC QC range for zoliflodacin against the Neisseria gonorrhoeae QC strain ATCC 49226 was defined as 0.06 to 0.5 µg/ml and was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. This QC range will be used for in vitro susceptibility testing of zoliflodacin during phase 3 human clinical trials and surveillance studies, and eventually it will be implemented in clinical labs. In a separate study, broth microdilution MIC quality control ranges for zoliflodacin against additional QC strains were determined to be 0.12 to 0.5 µg/ml for Staphylococcus aureus ATCC 29213, 0.25 to 2 µg/ml for Enterococcus faecalis ATCC 29212, 1 to 4 µg/ml for Escherichia coli ATCC 25922, 0.12 to 0.5 µg/ml for Streptococcus pneumoniae ATCC 49619, and 0.12 to 1 µg/ml for Haemophilus influenzae ATCC 49247. These MIC QC ranges were also approved by CLSI for use in future in vitro susceptibility testing studies against organisms other than N. gonorrhoeae.

In Vitro Activity of Ceftazidime-Avibactam against Isolates from Patients in a Phase 3 Clinical Trial for Treatment of Complicated Intra-abdominal Infections.

The increasing prevalence of multidrug-resistant Gram-negative pathogens has generated a requirement for new treatment options. Avibactam, a novel non-ß-lactam-ß-lactamase inhibitor, restores the activity of ceftazidime against Ambler class A, C, and some class D ß-lactamase-producing strains of Enterobacteriaceae and Pseudomonas aeruginosa The in vitro activities of ceftazidime-avibactam versus comparators were evaluated against 1,440 clinical isolates obtained in a phase 3 clinical trial in patients with complicated intra-abdominal infections (cIAI; ClinicalTrials.gov identifier NCT01499290). Overall, in vitro activities were determined for 803 Enterobacteriaceae, 70 P. aeruginosa, 304 Gram-positive aerobic, and 255 anaerobic isolates obtained from 1,066 randomized patients at baseline. Susceptibility was determined by broth microdilution. The most commonly isolated Gram-negative, Gram-positive, and anaerobic pathogens were Escherichia coli (n = 549), Streptococcus anginosus (n = 130), and Bacteroides fragilis (n = 96), respectively. Ceftazidime-avibactam was highly active against isolates of Enterobacteriaceae, with an overall MIC90 of 0.25 mg/liter. In contrast, the MIC90 for ceftazidime alone was 32 mg/liter. The MIC90 value for ceftazidime-avibactam (4 mg/liter) was one dilution lower than that of ceftazidime alone (8 mg/liter) against isolates of Pseudomonas aeruginosa The ceftazidime-avibactam MIC90 for 109 ceftazidime-nonsusceptible Enterobacteriaceae isolates was 2 mg/liter, and the MIC range for 6 ceftazidime-nonsusceptible P. aeruginosa isolates was 8 to 32 mg/liter. The MIC90 values were within the range of susceptibility for the study drugs permitted per the protocol in the phase 3 study to provide coverage for aerobic Gram-positive and anaerobic pathogens. These findings demonstrate the in vitro activity of ceftazidime-avibactam against bacterial pathogens commonly observed in cIAI patients, including ceftazidime-nonsusceptible Enterobacteriaceae (This study has been registered at ClinicalTrials.gov under identifier NCT01499290.).

A Systematic Approach to the Selection of the Appropriate Avibactam Concentration for Use with Ceftazidime in Broth Microdilution Susceptibility Testing.

Selection of the avibactam concentration to combine with ceftazidime in susceptibility testing was determined using Gram-negative isolates with characterized ß-lactamases predefined as susceptible or resistant, based on the known inhibition spectrum of avibactam. MIC values were determined by broth microdilution of ceftazidime with fixed concentrations and ratios of avibactam. A constant concentration of 4 µg/ml of avibactam was selected for susceptibility testing with ceftazidime because of its ability to correctly categorize susceptible and resistant isolates while minimizing categorical errors.

In Vitro Activity of Ceftazidime-Avibactam and Aztreonam-Avibactam against OXA-48-Carrying Enterobacteriaceae Isolated as Part of the International Network for Optimal Resistance Monitoring (INFORM) Global Surveillance Program from 2012 to 2015.

Enterobacteriaceae producing the Ambler class D OXA-48 carbapenemase, combined with additional resistance mechanisms, such as permeability defects or cocarriage of class A, B, or C ß-lactamases, can become highly resistant to most ß-lactams currently in use, including carbapenems. A total of 45,872 Enterobacteriaceae clinical isolates collected in 39 countries as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance study in 2012 to 2015 were tested for susceptibility to ß-lactams and comparator agents using the Clinical and Laboratory Standards Institute broth microdilution methodology and screened for the presence of ß-lactamases. The blaOXA-48 and blaOXA-48-like genes were detected in 333 isolates across 14 species of Enterobacteriaceae collected in 20 countries across the globe. Few agents tested were effective in vitro against the overall collection of OXA-48-producers (n = 265), with tigecycline (MIC90, 2 µg/ml; 92.5% susceptible), ceftazidime-avibactam (MIC90, 4 µg/ml; 92.5% susceptible), and aztreonam-avibactam (MIC90, 0.5 µg/ml; 99.6% of isolates with MIC ≤8 µg/ml) demonstrating the greatest activity. Similarly, colistin (MIC90, 1 µg/ml; 94.2% susceptible), tigecycline (MIC90, 2 µg/ml; 92.6% susceptible), ceftazidime-avibactam (MIC90, >128 µg/ml; 89.7% susceptible), and aztreonam-avibactam (MIC90, 4 µg/ml; 100% of isolates with MIC ≤8 µg/ml) were most active against OXA-48-like-positive isolates (n = 68). The in vitro activity of ceftazidime-avibactam was improved against the subset of metallo-ß-lactamase (MBL)-negative, OXA-48- and OXA-48-like-positive isolates (99.2% and 100% susceptible, respectively). The data reported here support the continued investigation of ceftazidime-avibactam and aztreonam-avibactam for the treatment of infections caused by carbapenem-resistant Enterobacteriaceae carrying OXA-48 and OXA-48-like ß-lactamases in combination with serine- or metallo-ß-lactamases.

Genomic characterization of IMP and VIM carbapenemase-encoding transferable plasmids of Enterobacteriaceae.

Objectives: IMP and VIM carbapenemases, in association with class 1 integrons, have spread globally among multiple Enterobacteriaceae species. We characterized IMP- and VIM-encoding transferable plasmids of clinical Enterobacteriaceae collected from two global surveillance programmes. Methods: We performed conjugation and transformation experiments for 38 IMP and 89 VIM producers. Plasmids, obtained from transconjugants or transformants, were sequenced with Illumina next-generation sequencing and analysed for replicon types and antimicrobial resistance genes. Results: A total of 41 transconjugants (blaIMP, n = 22; blaVIM, n = 19) and 10 transformants (blaIMP, n = 1; blaVIM, n = 9) were obtained. Broad-host-range IncL/M, IncC and IncN plasmids were associated with blaIMP and blaVIM, contained various integrons and showed inter-species and international distribution. Narrow-host-range IncFII(K) plasmids were limited to Klebsiella pneumoniae with blaIMP-26 from the Philippines, while IncR and IncHI1B-IncFIB(Mar) plasmids were restricted to K. pneumoniae with blaVIM from Greece and Spain. IncA-like hybrid plasmids were detected in Enterobacter xiangfangensis from Italy and K. pneumoniae from Spain. Transferable plasmids were major contributors of antimicrobial resistance genes among Enterobacteriaceae with blaIMP and blaVIM. Conclusions: This is the first report, to the best of our knowledge, describing blaIMP on IncFII(K) plasmids and blaVIM on IncL/M, IncN2, IncHI1B-IncFIB(Mar) and IncX3-IncC-like plasmids and showed that broad-host-range and narrow-host-range plasmids have contributed to the global spread of blaIMP and blaVIM among different species. This study highlights the importance of molecular analysis of plasmids in providing insight into horizontal spread of these carbapenemases.

In Vitro Activity of Ceftazidime-Avibactam against Isolates in a Phase 3 Open-Label Clinical Trial for Complicated Intra-Abdominal and Urinary Tract Infections Caused by Ceftazidime-Nonsusceptible Gram-Negative Pathogens.

The in vitro activity of ceftazidime-avibactam was evaluated against 341 Gram-negative isolates from 333 patients in a randomized, phase 3 clinical trial of patients with complicated urinary tract or intra-abdominal infections caused by ceftazidime-nonsusceptible pathogens (NCT01644643). Ceftazidime-avibactam MIC90 values against Enterobacteriaceae and Pseudomonas aeruginosa (including several class B or D enzyme producers that avibactam does not inhibit) were 1 and 64 µg/ml, respectively. Overall, the ceftazidime-avibactam activity against ceftazidime-nonsusceptible isolates was comparable to the activity of ceftazidime-avibactam previously reported against ceftazidime-susceptible isolates. (This study has been registered at ClinicalTrials.gov under identifier NCT01644643.).