Development of EUCAST zone diameter breakpoints and quality control criteria for ceftazidime-avibactam 10-4 µg.

Ceftazidime-avibactam disk studies were performed for disk mass selection and for establishing EUCAST quality control ranges and zone diameter breakpoints. The disk mass study included disk diffusion testing with ceftazidime-avibactam 10-4 and 10-6 µg disks and broth microdilution MIC testing for challenge set of 94 Enterobacteriaceae and 45 Pseudomonas aeruginosa. EUCAST SOP 9.0-based QC and MIC-disk correlations studies were followed for development of ceftazidime-avibactam 10-4 µg ranges for Escherichia coli ATCC 25922, P. aeruginosa ATCC 27583, and Klebsiella pneumoniae ATCC 700603 and for zone diameter breakpoint determination. The ceftazidime-avibactam 10-4 and 10-6 µg disks performed similar in comparison to broth microdilution, with zones ≤ 14 mm for all resistant strains. The 10-4 µg disk was selected and used in QC and breakpoint studies. There was minimal variation of ceftazidime-avibactam 10-4 µg QC study results between disks, media, and sites. The QC ranges were within 7 mm for all strains. The zone diameter breakpoint study demonstrated good correlation of MIC and disk results. The established zone diameter breakpoints resulted in false susceptible rates of 1.6 and 4.0% for Enterobacteriaceae and P. aeruginosa. EUCAST selected the ceftazidime-avibactam 10-4 µg disk and established QC ranges for E. coli 25922 of 24-30 mm, P. aeruginosa ATCC 27853 of 21-27 mm, and K. pneumoniae ATCC 700603 of 18-24 mm. The zone diameter breakpoints that correlated best with the MIC breakpoints of susceptible ≤ 8 mg/L and resistant > 8 mg/L were Enterobacteriaceae (S ≥ 13, R < 13 mm) and P. aeruginosa (S ≥ 17, R < 17 mm).

Analysis of MIC and Disk Diffusion Testing Variables for Gepotidacin and Comparator Agents against Select Bacterial Pathogens.

This study was conducted to determine the effect of testing parameters on the in vitro activity of gepotidacin, a new triazaacenaphthylene antibacterial agent for the treatment of conventional and biothreat pathogens. CLSI methods, and variations of those methods, were used to test 10 Staphylococcus aureus, 10 Streptococcus pneumoniae, 10 Haemophilus influenzae, and 5 Escherichia coli isolates by MIC and 30 S. aureus, 15 S. pneumoniae, and 15 S. pyogenes isolates by disk diffusion (DD) methods. Levofloxacin and linezolid were tested as comparator agents for MIC and DD methods, respectively. Broth microdilution (BMD), macrodilution (MD), and agar dilution (AD) methods were compared. Variations in media, temperature, incubation time, CO2 level, and inoculum concentration were tested by all methods, and variations in pH, calcium, magnesium, zinc, potassium, thymidine, and polysorbate 80 levels were tested by BMD and DD. The addition of albumin, serum, and lung surfactant was studied by BMD. The variables that impacted the results the most were high inoculum and pH 5.5 (no growth of H. influenzae and S. pneumoniae by BMD). Gepotidacin AD MIC levels were increased and disk zone diameters were decreased for all species in 10% CO2 incubation. The following variables had a minimal effect on gepotidacin results: pH, agar method, atmospheric condition, temperature, and addition of serum and albumin for broth methods. There were also some slight differences in gepotidacin disk results between disk manufacturers and some agar types and also with potassium and thymidine for S. pneumoniae For all other variations, gepotidacin MIC and disk results were considered comparable to reference results.

Development of EUCAST zone diameter breakpoints and quality control range for Staphylococcus aureus with ceftaroline 5-µg disk.

This ceftaroline MIC/disk comparison study for Staphylococcus aureus was performed for the purpose of establishing EUCAST zone diameter breakpoints. Ceftaroline susceptibility for a challenge set of 70 methicillin resistant- and 30 methicillin susceptible-S. aureus was determined by 5-µg disk diffusion and broth microdilution methods. Seventeen isolates were retested by disk and MIC, and the remaining 83 isolates were retested by MIC. Molecular testing was performed on 19 isolates with borderline susceptible ceftaroline MIC results to assess any differences in mecA and epidemiological correlation. An additional set of 101 consecutive clinical S. aureus isolates were tested using the 5-µg disk. S. aureus ATCC 29213 was tested by multiple sites and media for QC range determination. Replicate MIC results were within ±1 doubling dilution, with tendency for slightly lower repeat MICs, and there was minimal variation in replicate zone results. Based on susceptible breakpoints for MIC of ≤1 mcg/mL and for disk of >20 mm, there was 100 % categorical agreement for 30 MSSA and 92 % categorical agreement for 70 MRSA. There were no common MLST or PBP changes for strains with MICs of 1 and 2 mcg/mL. All ceftaroline disk results for the consecutively collected isolates were >20 mm. EUCAST selected the ceftaroline 5-µg disk breakpoint of Susceptible ≥20, Resistant <20 mm because it correlated best with the MIC breakpoint of Susceptible ≤1, Resistant >1 mg/L. A ceftaroline 5-µg disk QC range for S. aureus ATCC 29213 of 24-30 mm was also established by EUCAST.

Comparison of daptomycin Etest MICs on Mueller Hinton, IsoSensitest and brain heart infusion agars from Europe against 20 Staphylococcus aureus isolates.

The Etest manufacturer (bioMerieux) recommends Mueller-Hinton agar (MHA) with calcium concentration of 25-40 mg/L for daptomycin testing. A two phase study was performed to evaluate the effect of European agars on daptomycin Etest MICs. Broth microdilution (BMD) testing was performed and compared to Etest MICs for a challenge set of 20 Staphylococcus aureus with daptomycin MICs near the susceptible breakpoint and S. aureus 29213. In the first phase, Etest MICs were determined using agar plates prepared from MHA, IsoSensitest Agar (ISA) and Brain-Heart Infusion Agar (BHIA) and supplemented with various levels of calcium. In the second phase, Etest MICs were determined using commercially prepared MHA from Mast (MST), BD, Oxoid (OX), BioRad (BR), bioMerieux (BM) and E&O (EO) and ISA plates from OX, EO and MST. The calcium concentration of each agar was determined using ion selective electrode methodology. The best correlation of Etest and BMD MICs was obtained in phase 1 using MHA with 42.8 mg/L calcium and in phase 2 with BD MHA. The phase 2 MHA calcium concentration ranged from 19.2 to 63.6 mg/L. 87.5% of strains with BMD MICs of 1 mg/L, had Etest MICs of 1.5 or 2 mg/L using MHA with recommended calcium levels. Etest MICs using BHIA and ISA were higher than the BMD MICs and therefore are not recommended for use with the daptomycin Etest. The high percentage of major errors using Etest in this study, even with use of the most optimal medium, suggests that Etest MICs of 1.5 or 2 mg/L using MHA should be retested by BMD.

Antimicrobial resistance of Streptococcus pneumoniae and Haemophilus influenzae in Sao Paulo, Brazil from 1996 to 2000.

This study was undertaken to assess the in vitro activity of several antimicrobial agents against Brazilian isolates of Streptococcus pneumoniae and Haemophilus influenzae from 1996 to 2000. The antibiotics used were penicillin, amoxicillin/clavulanic acid (A/C), ampicillin, amoxicillin, cefaclor, cefdinir, cefixime, cefprozil, ceftriaxone, cefuroxime, azithromycin, clarithromycin, erythromycin, ciprofloxacin, levofloxacin, ofloxacin, chloramphenicol, clindamycin, doxycycline and trimethoprim/sulphamethoxazole (T/S). MICs were determined by the National Committee for Clinical Laboratory Standards (NCCLS) method and interpreted using NCCLS and PK/PD breakpoints. For S. pneumoniae 80.0% were penicillin susceptible, 18.3% intermediate, 1.7% resistant; most active agents were amoxicillin, A/C, ceftriaxone and levofloxacin; T/S was the least active agent. Beta-lactamase was produced by 13.7% of H. influenzae. All were susceptible to A/C, cefdinir, cefixime, ceftriaxone and quinolones. The least active agents were T/S and macrolides.

Comparison of cation-adjusted Mueller-Hinton broth with Iso-Sensitest broth for the NCCLS broth microdilution method.

Comparison of MIC results obtained in different parts of the world is currently difficult because of variations in methods. In this study, cation-adjusted Mueller-Hinton broth, the NCCLS-recommended medium, was compared with Iso-Sensitest broth, which is widely used in Europe. Microbroth dilution testing, using the NCCLS procedure, was performed on 124 Gram-positive (staphylococci and enterococci) and Gram-negative (Enterobacteriaceae and Pseudomonas aeruginosa) isolates from the CDC reference set, with the only variable being the medium used. Twelve antimicrobial agents were tested: amoxycillin-clavulanic acid, ampicillin, ciprofloxacin, erythromycin, gentamicin, imipenem, levofloxacin, oxacillin, gemifloxacin, trimethoprim- sulphamethoxazole, tetracycline and vancomycin. Vancomycin, erythromycin and oxacillin were only evaluated for the Gram-positive organisms. Trimethoprim-sulphamethoxazole was only evaluated for a subset of Gram-negative organisms because of off-scale results. The 124 isolates were tested in one American and one UK laboratory with two batches of cation-adjusted Mueller-Hinton broth and two of Iso-Sensitest broth. A statistical evaluation of the data used a 24 fully specified factorial analysis to determine if there were significant differences in results owing to Gram reaction, site of testing and type and/or batch of broth. In addition, the cumulative results for each antimicrobial agent in each broth were plotted against the range of MIC dilutions tested. MICs of ciprofloxacin, levofloxacin, gemifloxacin, gentamicin and tetracycline were slightly higher (half a doubling dilution) with Iso-Sensitest broth than with Mueller-Hinton broth. MIC results for the other antimicrobial agents were equivalent. Essential and category agreement rates were comparable for all agents (88.4-100% and 88.2-99.0%, respectively).