category
Regression analyses of individual zone diameters and the sum of zone diameters of various combinations of the oxacillin, methicillin, and penicillin disks against mean reference MICs were performed. These scatterplots are depicted in Fig.
1 and
2, and essential agreement rates, correlation coefficients, and error rates are shown in Table
2. The individual disk correlation coefficients were 0.84, 0.92, and 0.93 for oxacillin, methicillin, and penicillin, respectively. Essential agreement scores of MICs calculated from individual oxacillin and methicillin disks were 79% and 87%, respectively. The penicillin disk, however, had the best essential agreement of the single disks (93%), although the MIC ratios for only 54% of the strains were 1. The oxacillin and methicillin disks did not differentiate Pen
i from Pen
r strains, and penicillin disks showed considerable overlap between categories.
| FIG. 1Regression lines of penicillin, methicillin, and oxacillin disk zones plotted against mean reference MICs. Diagonal dotted lines on either side of regression lines indicate 1 doubling-dilution variation. Zone diameter breakpoints corresponding to penicillin (more ...) |
| FIG. 2Regression lines of combination disk zones plotted against mean reference MICs. Diagonal dotted lines on either side of regression lines indicate 1 doubling-dilution variation. Zone diameter breakpoints corresponding to penicillin MIC breakpoints are (more ...) |
| TABLE 2Comparison of MIC ratios of MICs, calculated from zone diameter measurements and mean reference MICs, and categorical errorratesa |
The sum of the oxacillin, methicillin, and penicillin disks and the sum of the methicillin and penicillin disks produced better agreement with mean reference MICs than did the single disks. The correlation coefficient was 0.97 for both disk combinations, and essential agreement between MICs calculated by the three-disk procedure (oxacillin, methicillin, and penicillin) with the mean reference MICs was 98.4%, and that between MICs calculated by the two-disk procedure (methicillin and penicillin) was 98.9%. In addition, MIC ratios of 1 were obtained for 76% of strains by the three-disk method and for 81% by the two-disk method.
Analysis of discrepancies in Pens, Peni, and Penr categories between calculated MICs and mean reference MICs showed all discrepancies to be minor, with most being within 1 doubling dilution of a breakpoint. The fewest discrepancies occurred with the three-disk, two-disk, and methicillin disk procedures, with values similar to those obtained by the various MIC methods. Oxacillin discrepancies were substantially higher (27.9%), even when discrepancies within 1 dilution of breakpoints were excluded (11.5%).
Further analysis of oxacillin disk and individual MIC method data with the NCCLS oxacillin zone interpretations of ≥20 mm as Pens and ≤19 mm as Peni or Penr showed agreement for 181 of the 183 strains (98.9%) with E-test categories; the MICs for the discrepant strains were 0.06 and 0.12 μg/ml. Oxacillin zones were in agreement with agar dilution categories for 173 strains (94.5%); the MICs for the discrepant strains were 0.06 μg/ml (9 strains) and 0.12 μg/ml (1 strain). Agreement with microdilution category occurred for 164 strains (89.6%); the MICs for the discrepant strains were 0.03 μg/ml (1 strain), 0.06 μg/ml (17 strains), and 0.12 μg/ml (1 strain).
The MICs that can be calculated from the two- and three-disk combination procedures include a wide range (from 0.004 to 16 μg/ml), assuring that all currently encountered susceptible, intermediate, and resistant strains can be differentiated (Fig.
2). This is not the case with the single-disk oxacillin procedure, which is unable to differentiate strains for which MICs are 1.1 μg/ml from strains for which MICs are higher (Fig.
1). Similarly, the highest MIC predicted by the methicillin disk is 2.5 μg/ml. A wide range of MIC results can be calculated with the penicillin disk (0.012 to 48 μg/ml); however, the intermediate range (from 19 to 35 mm) has a 5% minor error rate, with both susceptible and resistant organisms in this range.
Validation of the data generated by this study with 60 recently isolated strains (20 Pen
s, 20 Pen
i, and 20 Pen
r) showed essential agreement rates of 95.0% for the three-disk procedure and 96.7% for the two-disk procedure (Table
3). Calculated penicillin MICs were correctly categorized for all Pen
s and Pen
r strains. Of the 20 Pen
i strains, 18 were correctly categorized by the three-disk method and 19 were correctly categorized by the two-disk method; calculated MICs for the misclassified strains were 1.1 to 1.3 μg/ml, while reference MICs for these two strains were 1.0 μg/ml.
| TABLE 3Comparison of MIC ratios of MICs calculated from sums of zone diameters with microdilution MICs for 60strains |
Resistance of S. pneumoniae to agents other than β-lactams is usually high level, and strains therefore have bimodal distributions to these agents, with few (if any) intermediate strains ( 16, 18). β-Lactam resistance, on the other hand, results from multiple changes to one or more penicillin-binding proteins in strains for which penicillin MICs are 0.06 μg/ml to as high as 64 μg/ml, depending on the number of changes and the affinities of the penicillin-binding proteins ( 9). Disk diffusion has been shown to be an accurate method for obtaining categorical susceptibility results with S. pneumoniae for many non-β-lactams, including erythromycin, clindamycin, trimethoprim-sulfamethoxazole, tetracycline, and chloramphenicol ( 7, 11, 17, 22). Prior to this study, the use of disk diffusion for β-lactams has been limited to screening for penicillin susceptibility with 1-μg oxacillin disks ( 14, 21, 24). In addition, the use of the oxacillin disk is limited by the fact that strains for which penicillin MICs are 0.06 μg/ml, the upper limit of the susceptible category, usually have oxacillin zones of ≤19 mm ( 14, 16– 18). This occurs more frequently when MICs are determined by microdilution, which was the case in this study, in which 17 such discrepancies occurred with microdilution MICs, compared to 9 with agar dilution, and 1 with the E-test. Although this limits the value of the oxacillin disk as a screening method, in the current study most of the discrepant Pen s strains had oxacillin zones of 11 to 19 mm, while most Pen i strains had zones of ≤10 mm. This study has shown that the standard NCCLS disk diffusion method for testing S. pneumoniae can be adapted to provide accurate MICs and qualitative category (Pen s, Pen i, and Pen r) results for penicillin that are comparable to E-test, agar dilution, and microdilution MIC methods. By summing zone diameter measurements from either three (penicillin, oxacillin, and methicillin) or two (penicillin and methicillin) disks, accurate MIC results can be determined from regression plots at a considerably lower cost than that for any MIC method that is currently available. The best results were obtained with the sum of the three zone diameters from standard penicillin, methicillin, and oxacillin disks. The sum of two disks also provided excellent results, with only minimally more minor category discrepancies than with the three disks. In fact, the essential agreement rates of 98.4% for MICs determined by the three-disk procedure and 98.9% for MICs determined by the two-disk procedure were higher than the essential agreement rate of 95.1% for MICs obtained by microdilution versus agar dilution and microdilution versus E-test. MICs can be determined manually from regression plots or generated electronically with the BIOMIC system programmed to perform these conversions ( 3, 19). The validation of the two- and three-disk procedures performed as part of this study confirmed the accuracy of these methods for determining penicillin MICs. Essential agreement rates for penicillin (95.0 and 96.7%) were similar to those found with several commercially available systems (91.3 to 100%) which have been approved for use in diagnostic laboratories ( 25). The few categorical errors found in the validation study were minor, and the frequency of these minor errors was similar to or lower than recently published values ( 25). In summary, this study has demonstrated the value of disk diffusion in accurately and economically determining the penicillin susceptibility of S. pneumoniae at both a categorical level and a calculated MIC. This approach may have additional applications with other β-lactams. |
ACKNOWLEDGMENTS We acknowledge the interest of Ronald N. Jones in this work, the support of David Gibbs for the data analysis, and the editorial assistance of Laura Koeth in manuscript preparation. |
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