Research Article
25 May 2018

Assessment of 30/20-Microgram Disk Content versus MIC Results for Ceftazidime-Avibactam Tested against Enterobacteriaceae and Pseudomonas aeruginosa

ABSTRACT

We evaluated the correlation between MIC and disk diffusion inhibition zones when testing ceftazidime-avibactam, using the 30/20-μg disk and the disk diffusion and MIC breakpoints established by the U.S. FDA and the Clinical and Laboratory Standards Institute (CLSI). Organisms used included 2 groups of Enterobacteriaceae isolates and 2 groups of Pseudomonas aeruginosa isolates; 1 group of each consisted of randomly selected isolates and the second group consisted of a challenge group from thousands of surveillance isolates with an increased proportion of organisms displaying ceftazidime-avibactam MIC values close to the breakpoints. Broth microdilution, disk diffusion tests, and data analysis were performed according to reference standardized methods. Ceftazidime-avibactam breakpoints of ≤8/4 (susceptible) and ≥16/4 μg/ml (resistant) for MIC and ≥21/≤20 mm for disk diffusion, as established by the U.S. FDA and the CLSI, were applied for Enterobacteriaceae and P. aeruginosa. Ceftazidime-avibactam MIC and disk zone (30/20-μg disk) correlation were acceptable when testing Enterobacteriaceae (overall, very major [VM] and major [Ma] error rates of 0.4% and 0.0%, respectively) and nearly so when testing P. aeruginosa (2.3% VM and 2.9% Ma errors). In summary, disk diffusion and broth microdilution testing results demonstrated good categorical agreement for ceftazidime-avibactam against Enterobacteriaceae and P. aeruginosa, using 30/20-μg disks.

INTRODUCTION

In antimicrobial susceptibility testing, categorical breakpoints are initially defined for reference broth microdilution or agar dilution methods by correlation of microbiologic/clinical outcome with MIC data for the infecting organisms (1). Once MIC breakpoints have been determined, disk diffusion (zone diameter) breakpoints are developed by plotting a scattergram of zone diameters versus MIC values for isolates tested by both methods (2, 3). Because of the inherent variation of these susceptibility testing methods, the correlation between MIC and zone diameter is not completely linear. Thus, a statistical method, denominated the error rate-bounded method, was developed for setting zone diameter interpretative criteria; this method involves selecting zone diameters that provide the lowest intermethod error rates (1, 3, 4).
Ceftazidime-avibactam is a combination antimicrobial consisting of the β-lactamase inhibitor avibactam and the broad-spectrum cephalosporin ceftazidime (5). Ceftazidime-avibactam was approved by the United States Food and Drug Administration (U.S. FDA) in 2015 and by the European Medicine Agency in 2016 to treat complicated intraabdominal infections in combination with metronidazole, as well as to treat complicated urinary tract infections, including pyelonephritis (6, 7). Ceftazidime-avibactam is also approved to treat nosocomial pneumonia in Europe and has been studied in pediatric patients (ClinicalTrials.gov registration no. NCT01893346) (8).
Ceftazidime-avibactam MIC breakpoints of ≤8/4 μg/ml for susceptible and ≥16/4 μg/ml for resistant isolates, and zone diameter breakpoints of ≥21 mm for susceptible and ≤20 mm for resistant isolates, when using a 30/20 μg ceftazidime-avibactam disk reagent, were approved by the U.S. FDA (6) and later by the Clinical and Laboratory Standards Institute (CLSI) (9) for Enterobacteriaceae and Pseudomonas aeruginosa; whereas European Committee on Antimicrobial Susceptibility Testing established MIC breakpoints of ≤8/4 and >8/4 μg/ml (for susceptible and resistant isolates, respectively) for Enterobacteriaceae and Pseudomonas aeruginosa and disk diffusion breakpoints for a 10/4 μg disk reagent of ≥13/<13 mm (susceptible/resistant) for Enterobacteriaceae and ≥17/<17 mm for P. aeruginosa (10). In the present study, we evaluated the correlation between MIC values and disk diffusion inhibition zones, using the 30/20 μg disk reagent and the disk diffusion and MIC breakpoints established by the U.S. FDA.

MATERIALS AND METHODS

Organism collection.

The organism collection included 2 groups of Enterobacteriaceae isolates and 2 groups of P. aeruginosa isolates. One group of each consisted of randomly selected (nonchallenge) isolates with ceftazidime-avibactam and ceftazidime MIC distributions similar to those found in the International Network for Optimal Resistance Monitoring (INFORM) Program (11, 12). The second group consisted of challenge organisms, as described below.

Enterobacteriaceae.

The randomly selected group consisted of 200 isolates (108 Escherichia coli, 47 Klebsiella spp., and 45 isolates from other species) collected from U.S. medical centers in 2013, and the challenge group included 50 molecularly characterized β-lactamase-producing strains from the ceftazidime-avibactam phase 2 complicated urinary tract infection and complicated intraabdominal infection clinical trials. This challenge collection included CTX-M-producing strains and ST131 clonal representatives, isolates containing multiple β-lactamase enzymes, and metallo-β-lactamase-producing strains (13). None of the isolates carried a blaKPC gene, and all 7 Enterobacteriaceae isolates with a ceftazidime-avibactam MIC of >32 μg/ml that were included in the study had a metallo-β-lactamase (MBL) gene, including blaNDM-1 (4 isolates), blaVIM-1 (2 isolates), and blaIMP-1 (1 isolate).

P. aeruginosa.

The randomly selected group consisted of 53 isolates collected from 42 U.S. medical centers in 2013, and the challenge group included 81 isolates from 37 U.S. medical centers (27 states) collected in 2014, which were selected based on previous ceftazidime-avibactam MIC results. An increased number of isolates with ceftazidime-avibactam MIC values close to the breakpoint (4/4 to 32/4 μg/ml) were included in this group to provide a better evaluation of the categorical agreement between results from disk diffusion and broth microdilution methods. This challenge collection of P. aeruginosa included 23 (28.4%) isolates with ceftazidime-avibactam MIC values of 8/4 or 16/4 μg/ml (±1 dilution of the breakpoint) and 39 (48.1%) isolates with ceftazidime-avibactam MIC values of 4/4 to 32/4 μg/ml (±2 dilutions of the breakpoint); whereas data from the U.S. INFORM Program indicated that only 7.3% and 21.9% of P. aeruginosa isolates had ceftazidime-avibactam MIC values of 8/4 to 16/4 μg/ml and 4/4 to 32/4 μg/ml, respectively (11).

Susceptibility testing.

The disk diffusion tests followed the CLSI standardized method (2, 14) and used Mueller-Hinton agar and quality control (QC) recommendations for all tested strains (15). Mueller-Hinton agar medium for disk diffusion testing was obtained commercially (Becton, Dickinson and Company, Sparks, MD; Remel, Lenexa, KS) and prepared under regulated good manufacturing practice conditions. Ceftazidime disks (Becton, Dickinson and Company) were tested as a control. Disk diffusion tests were performed in duplicate (using a different disk lot from the same manufacturer [Becton, Dickinson and Company] and bacterium inocula for each test for all Enterobacteriaceae isolates and for the P. aeruginosa randomly selected group against ceftazidime-avibactam and ceftazidime (2 disk diffusion results for each MIC result) (2). For the P. aeruginosa challenge group, 3 disks of ceftazidime-avibactam (different disk lots [from 1 manufacturer [Mast Group Ltd.] and bacterial inocula for each test) and 1 disk lot of ceftazidime (Becton, Dickinson and Company) was tested against each isolate.
The broth microdilution tests followed the CLSI standardized method (16) and used cation-adjusted Mueller-Hinton broth and QC recommendations (15). Susceptibility test panels were produced from freshly prepared antimicrobial stocks of ceftazidime and avibactam, and the panel lot was stored frozen at −70°C or lower until used.
QC organisms were tested concurrently, and inoculum density was monitored by colony counts. The QC reference organisms tested were Escherichia coli ATCC 25922 and ATCC 35218, Klebsiella pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853.

Results analysis.

Ceftazidime-avibactam breakpoints of ≤8/4 (susceptible) and ≥16/4 μg/ml (resistant) for MIC and ≥21/≤20 mm (susceptible/resistant) for disk diffusion were applied for Enterobacteriaceae and P. aeruginosa, as established by the U.S. FDA (6). For ceftazidime, breakpoints of ≤4/8/≥16 μg/ml (susceptible/intermediate/resistant) for MIC and ≥21/18 to 20/≤17 mm (susceptible/intermediate/resistant) for disk diffusion were applied for Enterobacteriaceae, and breakpoints of ≤8/≥16 μg/ml (susceptible/resistant) for MIC and ≥18/≤17 mm (susceptible/resistant) for disk diffusion were applied for P. aeruginosa, as established by the U.S. FDA (17). Of note, CLSI has established different ceftazidime breakpoints for P. aeruginosa, i.e., ≤8/16/≥32 μg/ml (susceptible/intermediate/resistant) for MIC and ≥18/15 to 17/≤14 mm (susceptible/intermediate/resistant) for disk diffusion, but these breakpoints were not applied in this study (15).
Discrepancy rates between MIC values and zone diameter test results were calculated according to the CLSI M23-A4 document (2). Discrepancies involving false susceptible disk results and a resistant ceftazidime-avibactam MIC value were defined as very major (VM) errors, whereas false resistant disk diffusion results and a susceptible ceftazidime-avibactam MIC were defined as major (Ma) errors. Discrepancies involving the intermediate category were defined as minor (Mi) errors (2).

RESULTS

Ceftazidime-avibactam and Enterobacteriaceae.

No error was observed for the group of randomly selected isolates, and error rates were low for the challenge group (2.0% [2/100] very major [VM] and no major [Ma] errors). The VM error of 2.0% was due to a single Providencia stuartii strain (duplicate tests) with a MIC value of 16/4 μg/ml and an inhibitory zone diameter of 21 mm (at breakpoint). This is an isolate from the complicated intraabdominal infection (cIAI) clinical trial, and the ceftazidime-avibactam MIC was 8/4 μg/ml when it was tested during the trial. When isolates of both collections were combined, the VM error rate was only 0.4% (Table 1 and Fig. 1).
TABLE 1
TABLE 1 Summary of error rates
Antimicrobial drug or organism groupNo. of testsaRates of error typesb:
VMMaMi
Ceftazidime-avibactam    
    Enterobacteriaceae    
        Randomly selectedc4000.00.0NA
        Challenge groupd1002.00.0NA
        All isolates5000.40.0NA
    P. aeruginosa    
        Randomly selectedc1061.90.9NA
        Challenge groupd2432.53.7NA
        All isolatese3492.32.9NA
Ceftazidime    
    Enterobacteriaceae    
        Randomly selectedc4000.00.02.0
        Challenge groupd1000.00.013.0
        All isolates5000.00.04.2
    P. aeruginosa    
        Randomly selectede1067.60.0NA
        Challenge groupf813.70.0NA
        All isolates1875.90.0NA
a
Number of tests indicates number of MIC/disk diffusion test pairs performed.
b
VM, very major error, i.e., false susceptible by disk diffusion; Ma, major error, i.e., false resistant by disk diffusion; Mi, minor error, i.e., errors involving the intermediate category; NA, not applicable due to the lack of intermediate category.
c
Includes randomly selected isolates; tested in duplicate.
d
Includes β-lactamase-producing strains selected from complicated urinary tract infection and complicated intraabdominal infection ceftazidime-avibactam clinical trials; tested in duplicate.
e
Includes a challenge set of 81 isolates with increased number of isolates with ceftazidime-avibactam MIC results at the breakpoints (28.4% at 8 or 16 μg/ml). The isolates were tested against 3 lots of ceftazidime-avibactam disks and 1 lot of ceftazidime disks.
f
Includes 53 randomly selected isolates tested in duplicate and 81 challenge isolates tested in triplicate by disk diffusion.
FIG 1
FIG 1 Scattergram comparing the results of ceftazidime-avibactam broth microdilution MIC values (μg/ml) and disk diffusion zone diameters (mm) of a 30/20-μg disk when testing 250 Enterobacteriaceae isolates (250 isolates tested in duplicate) (n = 500). Horizontal and vertical broken lines indicate ceftazidime-avibactam breakpoints (U.S. FDA and CLSI). The table at the bottom describes the number of isolates tested (n) and very major (VM) and major (Ma) error rates for each category (13). NA, not applicable.

Ceftazidime-avibactam and P. aeruginosa.

Error rates were low for the randomly selected group (1.9% [2/106] VM and 0.9% [1/106] Ma errors), and slightly elevated for the challenge group (2.5% [6/243] VM and 3.7% [9/243] Ma errors) and for the combined collection (2.3% [8/349] VM and 2.9% [10/349] Ma errors, with susceptible [S] and resistant [R] at 10.4% for VM and 13.0% for Ma errors) of P. aeruginosa isolates (Table 1 and Fig. 2).
FIG 2
FIG 2 Scattergram comparing the results of ceftazidime-avibactam broth microdilution MIC values (μg/ml) and disk diffusion zone diameters (mm) for a 30/20-μg disk when testing Pseudomonas aeruginosa isolates (349 results for 134 isolates, including 53 randomly selected isolates tested in duplicate and 81 challenge isolates tested in triplicate). Horizontal and vertical broken lines indicate ceftazidime-avibactam breakpoints (U.S. FDA and CLSI). The table at the bottom describes the number of isolates tested (n) and very major (VM) and major (Ma) error rates for each category (13). NA, not applicable.

Ceftazidime and Enterobacteriaceae.

Error rates were low and acceptable for the group of randomly selected isolates (no VM or Ma errors and only 2.0% [8/400] minor [Mi] errors); however, the Mi rate was elevated (13.0% [13/100]; acceptable at ≤10.0%) for the challenge (clinical trial) organism group, due to this collection containing only β-lactamase-producing strains. The minor error rate would improve (from 13.0% to 4.0%) if the disk breakpoint was moved 1 mm upward (from ≤17/≥21 mm to ≤18/≥22 mm) or if both enteric organism populations were combined (4.2% [21/500]; Table 1 and Fig. 3).
FIG 3
FIG 3 Scattergram comparing the results of ceftazidime broth microdilution MIC values (μg/ml) and disk diffusion zone diameters (mm) for a 30-μg disk when testing 250 Enterobacteriaceae isolates (250 isolates tested in duplicate) (n = 500). Horizontal and vertical broken lines indicate ceftazidime breakpoints (U.S. FDA and CLSI). The table at the bottom describes the number of isolates tested (n) and very major (VM), major (Ma), and minor (Mi) error rates for each category (13). NA, not applicable.

Ceftazidime and P. aeruginosa.

The VM error rates were elevated, at 7.5% (8/106; no Ma errors) for the randomly selected group of isolates, 3.7% [3/81] for the challenge group, and 5.9% [11/187] for all isolates combined, and no Ma errors were observed when U.S. FDA breakpoints were applied (susceptible/resistant at ≤8/≥16 μg/ml for MIC and ≥18/≤17 mm for disk diffusion; Table 1 and Fig. 4). If CLSI breakpoints were applied (susceptible/intermediate/resistant at ≤8/16/≥32 μg/ml for MIC and ≥18/15 to 17/≤14 mm for disk diffusion), the overall minor error rate would be elevated at 11.2%, with 0.5% VM and no Ma errors (data not shown).
FIG 4
FIG 4 Scattergram comparing the results of ceftazidime broth microdilution MIC values (μg/ml) and disk diffusion zone diameters (mm) for a 30-μg disk when testing Pseudomonas aeruginosa isolates (187 results for 134 isolates). Horizontal and vertical broken lines indicate ceftazidime breakpoints (U.S. FDA). The table at the bottom describes the number of isolates tested (n) and very major (VM) and major (Ma) error rates for each category (13). NA, not applicable.

DISCUSSION

Discrepancy rates, i.e., VM, Ma, and Mi error rates, are directly proportional to the percentage of isolates with MIC values in the range of ±1 doubling dilution of the breakpoints (2). Thus, when the vast majority of isolates are highly susceptible, as is common with newer agents, it is difficult to define disk breakpoints using a “typical” or randomly selected organism collection. Consequently, it is difficult to evaluate the accuracy of defined disk breakpoints when nonsusceptibility is uncommon. Thus, because ceftazidime-avibactam is active against >99.0% of Enterobacteriaceae and around 97.0% of P. aeruginosa isolates in U.S. hospitals (11, 12), it is necessary to “artificially” increase the percentage of isolates with MIC values close to the breakpoints (within ±1 doubling dilution) to be able to properly evaluate the disk diffusion susceptibility breakpoints. It is also important to note that large surveys of susceptibility do not necessarily reflect the typical isolates that will be tested against these antimicrobial agents in a clinical laboratory.
The results of this investigation indicate that the ceftazidime-avibactam 30/20-μg disk performed very well when testing Enterobacteriaceae isolates, with only 0.4% VM and no Ma errors overall (all isolates combined). An important factor that contributed to the optimal performance of the ceftazidime-avibactam disk was the low occurrence of isolates with ceftazidime-avibactam MIC values within ±1 doubling dilution of the breakpoint, even in the challenge group (only 1 isolate). It is also important to note that disproportionately large zones of inhibition compared to the MIC values have been observed for other cephalosporins when testing indole-positive Proteeae (15). This low occurrence of isolates with elevated ceftazidime-avibactam MIC values reflects the contemporary population of Enterobacteriaceae isolates causing infections in U.S. hospitals. A recent investigation that evaluated more than 36,000 Enterobacteriaceae isolates collected from 94 U.S. medical centers in the 2013 to 2016 period revealed that 99.9% of isolates were ceftazidime-avibactam-susceptible, and <0.1% of isolates (28/36,380) displayed ceftazidime-avibactam MIC values of 8/4 or 16/4 μg/ml (12). It is important to note, however, that a recent study reported elevated major errors (false-resistant) when testing ceftazidime-avibactam by disk diffusion (30/20-μg disk) against carbapenem-resistant Enterobacteriaceae (CRE) isolates. Eighteen of 74 isolates (24.3%) were categorized as resistant by disk diffusion and as susceptible (MIC values of 0.5 to 8 μg/ml) by reference broth microdilution method (BMD) (18).
When testing P. aeruginosa against ceftazidime-avibactam, discrepancy rates were nearly acceptable for the unselected collection, but slightly higher than the CLSI acceptable limits, which are <1.5% for VM errors and <3% for Ma errors (2). The elevated VM (2.5%) and Ma (3.7%) rates observed with the challenge group reflects the increased percentage of isolates with MIC values within the range of ±1 doubling dilution of the breakpoint (28.4%) compared to that of the “typical” unselected population (approximately 7% [11, 12]). Furthermore, our results indicated that the disk diffusion breakpoints of ≥21/≤20 mm (S/R) established by the U.S. FDA provide the minimum combination of VM and Ma errors and appear to be the most appropriate zone diameter breakpoints. If the disk breakpoints move 1 mm upward (to ≥22/≤21 mm), overall VM errors would decrease slightly (from 2.3% to 1.7%) and the Ma error rate would increase significantly (from 2.9% to 8.0%), whereas if the breakpoints move 1 mm downward (to ≥20/≤19 mm) VM errors would increase from 2.3% to 4.3%.
The ceftazidime disk performance was acceptable for Enterobacteriaceae (no VM or Ma errors and 4.2% Mi errors), but results can be improved by moving the disk breakpoints 1 mm (from ≥21/≤17 mm to ≥22/≤18 mm). The overall Mi errors would decrease from 4.2% to 2.6%. However, the ceftazidime disk performed poorly when testing P. aeruginosa, resulting in elevated VM errors (5.9% overall). The main factors that contributed to the poor performance of the ceftazidime disk were the lack of intermediate MIC and disk breakpoints and the elevated percentage of P. aeruginosa isolates with ceftazidime MIC values within ±1 doubling dilution of the breakpoint (8 to 16 μg/ml), which was slightly higher (15.5%) compared to that observed in the INFORM Program (11.6%; JMI Laboratories, data on file). If CLSI breakpoint criteria (9), which include an intermediate category for MIC and disk breakpoints, were applied, the majority of VM errors would become Mi errors; thus, the VM error rate would be acceptable (0.5%; data not shown), but the Mi errors would be above the acceptable range (11.2%; data not shown).
Accurate and timely performance of antimicrobial susceptibility testing is crucial for treatment of life-threatening infections; however, the ability of laboratories to effectively perform susceptibility testing is challenged by several factors, including the extensive delays between approval of new antimicrobials and marketing of commercially available tests for these new compounds (19). The results of this investigation indicate that the disk diffusion test represents a valuable alternative option for accurately testing ceftazidime-avibactam against Enterobacteriaceae and P. aeruginosa using the 30/20-μg disk. Continued monitoring of the performance of this disk is recommended since the occurrence of clinical isolates with ceftazidime-avibactam MIC values at the breakpoint (8 to 16 μg/ml) may increase and new mechanisms of resistance to ceftazidime-avibactam might emerge.

ACKNOWLEDGMENTS

This study was supported by Cerexa, Inc., and Forest Research Institute, Inc. (an affiliate of Actavis, Inc.). Both companies were involved in the design and decision to present these results. H.S.S., P.R.R., M.D.H., R.K.F., and R.N.J. are employees of JMI Laboratories, and were paid consultants to Pfizer in connection with the development of the manuscript. G.G.S. was an employee of AZ at the time of this study and is currently an employee of Pfizer Inc. I.A.C. is an employee of Allergan and was an employee of either Cerexa, Inc., or Forest Research Institute, Inc. (an affiliate of Actavis, Inc.) during the study period. AstraZeneca's rights to ceftazidime-avibactam were acquired by Pfizer in December 2016.
JMI Laboratories contracted to perform services in 2016 for Achaogen, Actelion, Allecra Therapeutics, Allergan, AmpliPhi Biosciences, API, Astellas Pharma, AstraZeneca, Basilea Pharmaceutica, Bayer AG, BD, Biomodels, Cardeas Pharma Corp., CEM-102 Pharma, Cempra, Cidara Therapeutics, Inc., CorMedix, CSA Biotech, Cutanea Life Sciences, Inc., Debiopharm Group, Dipexium Pharmaceuticals, Inc., Duke, Entasis Therapeutics, Inc., Fortress Biotech, Fox Chase Chemical Diversity Center, Inc., Geom Therapeutics, Inc., GSK, Laboratory Specialists, Inc., Medpace, Melinta Therapeutics, Inc., Merck & Co., Inc., Micromyx, MicuRx Pharmaceuticals, Inc., Motif Bio, N8 Medical, Inc., Nabriva Therapeutics, Inc., Nexcida Therapeutics, Inc., Novartis, Paratek Pharmaceuticals, Inc., Pfizer, Polyphor, Rempex, Scynexis, Shionogi, Spero Therapeutics, Symbal Therapeutics, Synlogic, TenNor Therapeutics, TGV Therapeutics, The Medicines Company, Theravance Biopharma, ThermoFisher Scientific, VenatoRx Pharmaceuticals, Inc., Wockhardt, and Zavante Therapeutics, Inc. We have no speakers' bureaus or stock options to declare.

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Information & Contributors

Information

Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 56Number 6June 2018
eLocator: 10.1128/jcm.01960-17
Editor: Sandra S. Richter, Cleveland Clinic

History

Received: 13 December 2017
Returned for modification: 24 January 2018
Accepted: 11 March 2018
Published online: 25 May 2018

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Keywords

  1. error rate-bounded method
  2. broth microdilution
  3. disk diffusion

Contributors

Authors

Helio S. Sader
JMI Laboratories, North Liberty, Iowa, USA
Paul R. Rhomberg
JMI Laboratories, North Liberty, Iowa, USA
Michael D. Huband
JMI Laboratories, North Liberty, Iowa, USA
Ian A. Critchley
Allergan plc, Irvine, California, USA
Gregory G. Stone
Pfizer Inc., New York, New York, USA
Robert K. Flamm
JMI Laboratories, North Liberty, Iowa, USA
Ronald N. Jones
JMI Laboratories, North Liberty, Iowa, USA

Editor

Sandra S. Richter
Editor
Cleveland Clinic

Notes

Address correspondence to Helio S. Sader, [email protected].

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