Comparison of Agar Diffusion Methodologies for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Bacterial strains.Two hundred fifty isolates were randomly selected from the collection of clinical isolates at the CF Center at the Children's Hospital and Regional Medical Center and the University of Washington in Seattle. An additional 250 isolates were selected from strains sent to the CF Referral Center for Susceptibility and Synergy Testing of Multiply Resistant Organisms at Columbia University in New York, N.Y. Strains were stored frozen at −80°C and checked for purity and mucoid phenotype by plating on MacConkey agar, followed by streaking onto Mueller-Hinton agar to confirm purity and detect pigment production. The following strain definition of P. aeruginosawas used: oxidase positive, catalase positive, growth at 42°C, and pigment production (5). Of the 500 isolates tested, 147 did not meet these phenotypic criteria for P. aeruginosa and thus were further tested by colony hybridization and probing with the exotoxin A gene from P. aeruginosa (13). Seven were found to be exotoxin A negative and were further identified using the API 20 NE strip (bioMerieux Vitek, Inc.) in accordance with the manufacturer's instructions. Two of these seven were not identified asP. aeruginosa and were excluded from further analysis. The 1.4% (7 of 498) proportion of exotoxin A-negative CF isolates ofP. aeruginosa is comparable to that reported in the literature (17).

The entire collection of 498 isolates was available at each center and numbered sequentially. Three hundred forty-eight isolates were tested in the laboratory at the Children's Hospital and Regional Medical Center (all odd-numbered isolates and 99 even-numbered isolates), and 249 were tested in the laboratory at Columbia University (even-numbered isolates). An overlap of 99 isolates was tested at both centers to assess the interlaboratory reproducibility identified in an earlier phase of this study using 98 different strains (12).

Antimicrobials tested.The following drugs were obtained from Sigma (St. Louis, Mo.) or from their respective U.S. manufacturers: amikacin, gentamicin, tobramycin, ticarcillin, ticarcillin-clavulanic acid (2-μg/ml fixed concentration), piperacillin, piperacillin-tazobactam (4-μg/ml fixed concentration), ceftazidime, aztreonam, ciprofloxacin, imipenem, and meropenem. The ranges of concentrations tested by the reference broth microdilution method were 0.06 to 8 μg/ml for ciprofloxacin; 0.25 to 16 μg/ml for meropenem and imipenem; 0.5 to 64 μg/ml for gentamicin, tobramycin, aztreonam, and ceftazidime; and 2 to 256 μg/ml for all of the other drugs. For disk diffusion, concentrations were in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. For Etest, the concentration ranges were 0.002 to 32 μg/ml for ciprofloxacin, meropenem, and imipenem and 0.016 to 256 μg/ml for all of the other agents tested.

Susceptibility test methods.The broth microdilution method (6) was selected as the reference method for this study (12). Drug- and cation-adjusted Mueller-Hinton broth medium-containing trays were custom manufactured by PML Microbiologicals (Wilsonville, Oreg.). Trays were stored at −80°C and allowed to come to room temperature prior to use. Testing was performed in accordance with the recommendations of NCCLS document M7-A4 (6). However, inoculated trays were incubated for 48 h and readings were taken between 18 and 24 h, based on growth in the control well, in accordance with the manufacturer's recommendations, and additionally at 48 h. The MIC endpoint was the lowest concentration of an antimicrobial that completely visibly inhibited the growth in a well.

Disk diffusion susceptibility testing was performed in accordance with the recommendations of NCCLS document M2-A6 (7). Cartridges of antimicrobial-containing disks were obtained from BBL (Becton Dickinson Microbiology Systems, Cockeysville, Md.), stored between 4 and −20°C, and allowed to come to room temperature prior to use. Mueller-Hinton plates (PML Microbiologicals) were incubated for a total of 48 h after inoculation with organisms and placement of disks. Zones of inhibition were measured at both 18 to 21 h and again at 48 h.

Etest strips were purchased from AB BIODISK, with the exception of meropenem strips, which were kindly provided by Zeneca Pharmaceuticals (Wilmington, Del.), and strips were used in accordance with the manufacturer's instructions. They were stored at −20°C and brought to room temperature prior to use. Mueller-Hinton agar plates were inoculated as for the disk diffusion method (7); however, in accordance with the verbal recommendation of the manufacturer, the inocula of mucoid isolates were equivalent to 1.0 and were 0.5 McFarland standards for nonmucoid isolates. Six strips were applied radially to the surface of each Mueller-Hinton plate, and plates were incubated for 48 h with readings taken both between 18 and 21 h, in accordance with the recommendations of the manufacturer, and at 48 h.

Interpretive criteria for susceptibility for all of the test methods were in accordance with NCCLS document M100-S9 (8). Results from all of the methods were validated using American Type Culture Collection quality control strains P. aeruginosa ATCC 27853 and ATCC 35218 and Escherichia coli ATCC 25922.

Retesting of isolates.In the cases in which serious categorical interpretation errors were identified, retesting of the isolates was performed to determine reproducible error. Interpretation errors were classified based on their impact on clinical management. Serious errors included very major errors, defined as susceptible by the test method and resistant by the reference test, and major errors, defined as resistant by the test method and susceptible by the reference method. Minor errors were defined as an intermediate result for either the test or the reference method. For the penicillins, where there is no intermediate category, all errors were either very major or major. All strains with single- or multiple-drug serious errors at the 18- to 21-h incubation were retested in triplicate using the test methodology and the reference broth microdilution test at a third reference laboratory (University of Iowa College of Medicine) by a single experienced technologist. Only retest results are shown in the tables.

Statistical analysis.MICs were converted to a log₂ + 9 values to permit appropriate comparison with each other and with disk diffusion results (millimeters) and entered into the Statistical Analysis System statistical package (SAS Institute, Inc., Cary, N.C.). Pearson correlation coefficients (r values), regression slope equations, and categorical error rates were generated for each antimicrobial agent indexed by susceptibility test method and by mucoid phenotype. A perfect statistical correlation result is 1.00 for the correlation coefficient and/or the slope, and ≥0.90 (r value) is considered desirable and ≥0.80 (r value) is considered an acceptable result.

Limitations of the statistical analysis included exclusion of strains due to insufficient growth for certain test methods (broth microdilution, 3 strains; Etest, 21 strains; disk diffusion, 20 strains), an increased correlation with increased sample size, and inclusion of differing off-scale results (≤ or > values for MIC tests and 6-mm results for the disk diffusion test) from certain antimicrobials in the analysis, the latter usually resulting in a slightly stronger correlation. Off-scale Etest results occurred at the upper end of the tested ranges, while off-scale broth microdilution results occurred at the lower and upper ends of the tested ranges.