Susceptibility Tests

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1982, p. 213-217 Vol. 16, No. 2 0095-1137/82/080213-05$02.00/0 In Vitro Studies with Cefotaxime: Disk Diffusion Susceptibility Tests SMITH SHADOMY* AND EDWARD L. CHAN Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Received 22 February 1982/Accepted 29 April 1982 Until recently two sets of conflicting interpretive criteria existed for use with the standardized 30-,ug cefotaxime diffusion disk [National Committee for Clinical Laboratory Standards (NCCLS) M2-A2S, supplement 1; product information package insert for Claforan (cefotaxime sodium), edition 10/81.] The latter criteria recently were superseded by a third set which differs radically from the first two in both minimal inhibitory concentration (MIC) and zone size breakpoints. The first two sets of criteria differed mainly in the zone of inhibition diameters used to predict resistant and intermediate organisms. The accuracies of these two sets of criteria were evaluated by paired agar dilution (World Health Organization- International Collaborative Study) and disk diffusion tests (Food and Drug Administration) with 347 clinical isolates of aerobic bacteria. A total of 274 isolates (79%) were clinically susceptible by agar dilution as defined by NCCLS (MIC.8,ug/ml) and 61, including 48 Pseudomonas spp., were of intermediate susceptibility (MIC of 16 or 32 p,g/ml). Twelve were resistant (MIC.64,ug/ml). The original product information package insert criteria proved to be most unreliable in identification of the intermediate organisms (zone of inhibition diameters of 18 to 22 mm); only 5 were correctly predicted as being intermediate, whereas 54 were predicted as being resistant, and 2 were predicted as being susceptible. In contrast, the NCCLS criteria (zone of inhibition diameter of 15 to 22 mm) predicted 41 as being intermediate and 18 as being resistant; again, 2 were susceptible. The 12 isolates resistant to cefotaxime by agar dilution were correctly predicted to be resistant by either set of breakpoints (zone of inhibition diameter of.14 or.17 mm). These results suggest that the package insert criteria previously recommended for use with the 30-,ug cefotaxime disk were overly conservative and required revision to bring them into agreement with NCCLS performance standards. Unfortunately however, the recent revision of the package insert criteria has further confused this issue [product information package insert for Claforan (cefotaxime sodium), edition 2/82]. Cefotaxime (HR 756; CTX) is a broad-spectrum cephalosporin antibiotic possessing a high degree of resistance to,-lactamases (4, 7). It is intended for parenteral use in treatment of serious infections caused by susceptible organisms, both gram-negative and gram-positive. The in vitro spectrum of CTX includes many organisms resistant to other cephalosporins and penicillins (9). Because of this extended spectrum, a separate 30-,ug CTX disk has been developed for use in disk diffusion susceptibility tests. Until recently, two sets of interpretive "breakpoint" criteria had been promulgated for use with the 30-,ug CTX disk. One set comprises those included in the standards recently proposed by the National Committee for Clinical Laboratory Standards (NCCLS; 8). These standards agree with and appear to be derived from those proposed by Fuchs et al. (3). The second set of 213 criteria comprises those previously contained in the product information package insert for Claforan (CTX sodium; 5). These two sets of criteria differed primarily in the zone of inhibition diameters used to define intermediate and resistant organisms (Table 1). In terms of corresponding minimal inhibitory concentration (MIC) values, both sets of criteria regarded 8,ug/ml as the upper limit of probable clinical susceptibility. In terms of clinical resistance, NCCLS defines this as an MIC.64 p.g/ml, whereas the package insert used an MIC of >16 jig/ml. Left undefined by these criteria is the intermediate, or indeterminate, category as suggested by Fuchs et al. (3), which, by default, appears to be 16 or 32,ug/ ml. The study reported here had two specific goals: evaluation of the performance characteristics of the 30-,ig CTX disk and evaluation of the reliability of each of the two sets of criteria

214 SHADOMY AND CHAN TABLE 1. Interpretive criteria for use in disk diffusion susceptibility tests with the 30-Rg CTX disk Category Zone diam (mm) breakpoints Claforan package insert criteria (10/81 NCCLS interpretive standards edition) Susceptible.23 (.8)" 23 (s8) Intermediate 18-22 15-22 Resistant.17 (>16) <14 (.64) a Corresponding MIC (pg/ml). previously promulgated for use in interpretation of results obtained with this disk as well as those now contained in the revised Claforan product information package insert (6). MATERIALS AND METHODS Organisms. A total of 347 isolates of gram-negative and gram-positive aerobic bacteria were tested. These included 78 organisms collected from several different medical centers in this country, as well as a diverse population of gram-negative organisms of Asiatic origin. Identifications of all isolates were confirmed by either accepted laboratory criteria (2, 7) or the API 20E system (Analytab Products) as required. Stock cultures were maintained in cystine Trypticase (BBL Microbiology Systems) agar medium (CTA medium). Susceptibility tests. Paired agar dilution and disk diffusion tests were performed with all 347 isolates. The agar dilution tests employed the World Health Organization-International Collaborative Study procedure as described by Washington and Sutter (10); final test concentrations of CTX (HR 756, Hoechst-Roussel Pharmaceuticals, Inc.; lot 17011) ranged from 0.13 to 64,ug/ml. The medium employed in the agar dilution tests was a commercial dehydrated Mueller-Hinton agar (MH; BBL). The disk diffusion tests were performed by the standardized Food and Drug Administration procedure as described by Barry and Thornsberry (1). A standardized 30-p.g CTX disk (BBL; lot 104026, assayed content of 30 pg) and commercially prepared 150-mm-diameter MH plates (MH-11; BBL) were used in the disk diffusion tests. Inocula for the disk diffusion tests were prepared from overnight broth cultures and then adjusted to match the turbidity of a 0.5 MacFarland standard. Inocula for the agar dilution tests were prepared to contain approximately 2 x 106 colony-forming units per ml or 5 x 103 colony-forming units per spot. Both agar dilution and disk diffusion tests then were performed. Inoculated plates were incubated overnight at 35 C. The MIC was defined as being the lowest concentration of drug yielding no more than one or two colonies; barely discernable or hazy responses were ignored. Zones of inhibition in the disk diffusion tests were measured in triplicate to the nearest 0.1 mm and averaged. Data analyses. Original data were evaluated in terms of numbers of organisms inhibited at each given concentration of CTX and their corresponding zones of inhibition. All measurable zones of inhibition for each given concentration were then averaged. Results of J. CLIN. MICROBIOL. the paired tests then were analyzed in terms of each tested drug concentration (log2 value) and the corresponding average zone of inhibition using standard regression analysis (least-squares method). Statistics from this analysis were then used to derive disk diffusion zone size correlates for MIC values of 8 (susceptible) and 64 (resistant) p.g/ml. Finally, individual paired zone size and MIC responses were analyzed in terms of agreement between actual MIC values and susceptibility categories as predicted by the corresponding zone size responses. Three sets of criteria were used in this analysis: the two previously published sets of zone size criteria described above (5, 8) and the zone size criteria now contained in the current edition of the Claforan product information package insert (6). RESULTS In vitro susceptibility data. A total of 274 isolates had CTX MIC values of 8 jig/ml or less. Thus, by NCCLS MIC standards, these were susceptible organisms (Table 2). A total of 61 isolates had MIC values of 16 or 32 plg/ml and thus were of intermediate or indeterminant susceptibility. Ten isolates were clinically resistant, with MIC values of 64 plg/ml, and 2 more were totally resistant (MIC >64 ptg/ml). Isolates of Pseudomonas spp. accounted for 68 of the 73 organisms in the latter two categories. Regression analysis. The average zones of inhibition for all isolates inhibited at each given drug concentration were calculated (Table 2). Data for several isolates were rejected from these calculations because of either apparent noncongruency, two values, or nonmeasurable values (no measurable zone of inhibition or MIC values of >64,ug/ml, seven values). Regression analysis was then performed, matching the log2 value of each concentration against the average zone TABLE 2. In vitro susceptibility to CTX as determined by paired agar dilution and disk diffusion tests Drug No. Avg zone of Excluded (pg/mi) inhibited inhibition data" 0.13 204 31.10 2 0.25 19 30.05 0 0.5 21 28.83 0 1 6 28.52 0 2 6 26.35 0 4 7 26.55 0 8 11 20.49 1 16 45 16.00 1 32 16 14.61 1 64 10 10.96 2 >64 2 6.9 2 a Data rejected from further regression analysis: contamination, two values; no measurable zones or MICs >64,g/ml, seven values.

VOL. 16, 1982 CEFOTAXIME DISK DIFFUSION TESTS 215 of inhibition for that concentration. Results of this analysis indicated excellent correlation (r = 0.9551, P < 0.001; Fig. 1). Regression statistics a(26.78) and r(-2.2872) then were used to calculate disk diffusion zone size correlates for MIC values of 8 (Y, = 19.92 mm) and 64 (YI. = 13.06 mm) p.g/ml. These values were rounded to 20 and 14 mm, respectively, leaving an intermediate region of 15 to 19 mm. Analysis of disk diffusion test results. Paired MIC and disk diffusion test data were analyzed for all 347 isolates. According to MIC data, using NCCLS standards, there were three groups of organisms: resistant (MIC.64,ug/ml), 12; intermediate (MIC of 16 or 32,ug/ml), 61; susceptible (MIC s8,ug/ml), 274. Application of the zone size breakpoints generated by these same data correctly predicted the probable category of susceptibility for 315 isolates (91%). Incorrect predictions involved 22 isolates that were intermediate by MIC and 10 isolates that were susceptible by MIC. Of the 22 intermediate isolates, 4 were predicted to be susceptible, and 18 were predicted to be resistant. Of the 10 isolates susceptible by MIC, 3 were predicted to be resistant, and 7 were predicted to be intermediate. Results similar to those above were obtained when the NCCLS interpretive zone size standards were applied (Table 3). Both sets of data were in agreement for 309 (89%) isolates. There were 38 instances of disagreement between mea- 27-6 - 11-5 - <1I O O ( -I_ -2-3i I sured MIC values and interpretation of zone size responses. The isolates for which data were inconsistent involved 20 intermediate by MIC and 18 susceptible by MIC. Of the intermediate organisms, two were predicted by the disk test as being susceptible, and 18 were predicted as being resistant. The latter were the same organisms predicted as being resistant with the regression statistics correlates. Two isolates each of Pseudomonas spp. and Escherichia coli and one isolate each of Enterobacter spp. and Proteus mirabilis, susceptible by MIC and predicted as being susceptible by the regression statistic correlates, now were placed in the intermediate category. Application of the original package insert criteria to the results of the disk diffusion tests resulted in 74 (21%) instances of disagreement between paired MIC and disk diffusion test data. Three isolates of Pseudomonas susceptible by MIC and predicted as being intermediate by the NCCLS standards now were predicted to be resistant. More importantly, 38 isolates intermediate by MIC now were placed in the resistant category. Only 5 (8%) of the 61 isolates intermediate by MIC were predicted as being intermediate by these criteria. Recently and subsequent to the performance of these studies, the package insert criteria were revised (6). The revision suggests use of MIC respectively, to define clinical susceptibility and resistance. Or- values of -16 and.64 jig/ml, 41 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 3 2 2 4 4-2 2 1 3 3 12 3 12 2 2 E 2 3(8)- 1 12 1 1 1 0 2 2-1\ 1 2 I1 2 3 1 1 5 1 1 3 2 1 4 I1 1 2 I I 1 2 1 2 1 6 13 17 27 3634K 5 1 5 I I I I I I I I I 1 I I I I I 1 I I I I I I slo 15 20 25 30 35 >38 ZONE OF INHIBITION (AVG. Z.D.,ea MIC), mm, 30-pg CTX DISC FIG. 1. Correlation between CTX agar dilution MIC values and zones of inhibition obtained with a 30-,ug CTX diffusion disk. Numbers reflect distribution of individual zone size responses (rounded to nearest millimeter). Z.D., Zone diameter.

216 SHADOMY AND CHAN J. CLIN. MICROBIOL. TABLE 3. Analysis of disk diffusion test results-correlation of MIC responses with results of disk diffusion tests using three different sets of interpretive criteria No. in category by:a CTX MIC (p.g/ NCCLS zone size standards with Original package insert criteria, Revised package insert criteria, ml) (no. zone size diam (mm) of: 10/81 edition, with zone size diam 2/82 edition, with zone size diam inhibited) Res. Intermed. Suscept. Res. Intermed. Suscept. Res. Intermed. Suscept..14 15-22 -23.17 18-22 -23.14 15-19 >20 264 (12) 12 0 0 12 0 0 12 0 0 32(16) 7 9 0 16 0 0 7 9 0 16 (45) 11 32 2 38 5 2 11 30 4 8 (11) 1 7 3 3 5 3 1 5 5 c4(263) 4 8 253 3 7 253 2 2 259 a Res, Resistant; Intermed., intermediate; suscept., susceptible. ganisms having MICs between 16 and 64,ug/ml are defined as being "... susceptible if high dosage is used or if the infection is confined to tissues... in which high antibiotic levels are attained (6)." The corresponding zone size correlates are as follows: susceptible,.20 mm; intermediate, 15 to 19 mm; resistant, <14 mm. These new zone size and MIC criteria were applied to the previously described data with the following results (Table 3). Of 274 organisms having MIC values of 8 jig/ml or less, the new zone size correlates predicted 264 (96%) to be susceptible and 10 to be resistant or intermediate. However, of 45 organisms having MIC values of 16,ug/ml and now considered to be susceptible by MIC, only 4 were predicted to be susceptible by the disk test, whereas 30 were predicted to be intermediate, and 11 were predicted to be resistant. Of the latter 41 organisms, 39 were isolates of Pseudomonas. Most instances of disagreement between measured MIC values and categories of susceptibility predicted by the 30-,ug CTX disk diffusion test involved isolates of Pseudomonas spp. (Table 4). Paired data for 78 such isolates were analyzed. Ten isolates were resistant by MIC; all 10 were correctly predicted as being resistant by the NCCLS standards and both sets of package insert criteria. Ten were susceptible to 8,ug/ ml or less; only two were correctly predicted as being susceptible by either the NCCLS criteria or the original package insert criteria. The original package insert criteria predicted resistant for two and intermediate for six of the remaining eight, whereas the NCCLS standards predicted intermediate for all eight. A total of 58 Pseudomonas isolates had intermediate CTX MIC values as defined by NCCLS, 15 at 32 p.g/ml and 43 at 16,ug/ml. Application of the NCCLS standards to the disk diffusion test data for these 58 isolates correctly predicted 40 (70%) as being intermediate; two were predicted to be susceptible, and 16 were predicted to be resistant. In contrast, when the original package insert criteria were applied, 52 (90%) were predicted to be resistant, with only 4 as being intermediate and two as being susceptible. Only confusion resulted when the revised package insert criteria were applied to the data for Pseudomonas spp. According to the revised MIC criteria, 53 isolates could be considered to be susceptible. The disk test data predicted the following for these 53 isolates: susceptible, 8; intermediate, 34; resistant, 11. Of 15 isolates now intermediate by MIC, the disk test data predicted 10 to be intermediate and 5 to be resistant. TABLE 4. Performance of the 30-p.g CTX disk in disk diffusion tests with 78 isolates of Pseudomonas spp. No. in category by:a CTX MIC (,ug/ NCCLS zone size standards with Original package insert criteria, Revised package insert criteria, C mlc(n.g/ NCCS zone size stan s w 10/81 edition, with zone size diam 2/82 edition, with zone size diam m') (no. zone size diam (mm) of: (mm) of: '(mm) of: inhibited) Res. Intermed. Suscept. Res. Intermed. Suscept. Res. Intermed. Suscept..14 15-22.23.17 18-22.23.14 15-19 >20 264 (10) 10 0 0 10 0 0 10 0 0 32(15) 5 10 0 15 0 0 5 10 0 16 (43) 11 30 2 37 4 2 11 28 4 8 (8) 0 7 1 2 5 1 0 5 3 '4(2) 0 1 1 0 1 1 0 1 1 a See Table 3, footnote a for abbreviations.

VOL. 16, 1982 DISCUSSION Two conclusions can be drawn from the results of this study. First, the zone size criteria previously recommended by the Claforan product information package insert (5) for use with the 30-,ug CTX disk diffusion test were overly conservative. This applied primarily to organisms of intermediate or indeterminate susceptibility (MICs of 16 or 32 jig/ml). Many such organisms will be predicted by disk diffusion testing to be resistant when these older criteria are applied. Revision of the package insert zone size criteria, bringing them in line with current NCCLS standards for the 30-,ug CTX disk, would have been desirable. Unfortunately, the recent revision of these criteria now differs from NCCLS standards in both MIC and zone size criteria. This only adds confusion to the issue. Second, disk diffusion susceptibility tests with the 30-,Ig CTX disk and isolates of Pseudomonas spp. appear to be a most unreliable way of predicting probable clinical susceptibility or resistance regardless of which criteria are used. More importantly, and as already cautioned by others (3), sufficient clinical experience does not yet exist to permit definition of absolute in vitro breakpoints for CTX and Pseudomonas spp. For these reasons it is recommended that results of disk diffusion tests with the 30-,ug CTX disk not be used in making therapeutic decisions regarding Pseudomonas spp. and CTX. LITERATURE CITED 1. Barry, A. L., and C. Thornsberry. 1980. Susceptibility testing: diffusion test procedures, p. 463-474. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant CEFOTAXIME DISK DIFFUSION TESTS 217 (eds.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, 2. R. R. Facklam. 1980. Streptococci and aerococci, p. 88-110. In E. H. Lennette, A. Balows, W. J. Hausler, Jr.. and J. P. Truant (eds.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology. Washington, 3. Fuchs, P. C., A. L. Barry, C. Thornsberry, R. N. Jones, T. L. Gavan, E. H. Gerlach, and H. M. Sommers. 1980. Cefotaxime: in vitro activity and tentative interpretive standards for disk susceptibility testing. Antimicrob. Agents Chemother. 18:88-93. 4. Heymes, R., A. Lutz, and E. Schrinner. 1978. Experimental evaluation of HR 756, a new cephalosporin derivative, p. 823-824. In W. Siegenthaler and R. Luthy (ed.), Current chemotherapy. American Society for Microbiology, Washington, 5. Hoechst-Roussel Pharmaceuticals, Inc. 1981. Claforan (cefotaxime sodium) Sterile. Product information package insert, ed. 10/81. Hoechst-Roussel Pharmaceuticals, Inc.. Somerville, N.J. 6. Hoechst-Roussel Pharmaceuticals, Inc. 1982. Claforan (cefotaxime sodium) Sterile. Product Information package insert, ed. 2/82. Hoechst-Roussel Pharmaceuticals, Inc.. Somerville, N.J. 7. Kloos, W. E., and P. B. Smith. 1980. Staphylococci, p. 83-87. In E. H. Lennette, A. Balows, W. J. Hausler. Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, 8. National Committee for Clinical Laboratory Standards Subcommittee on Antimicrobial Disc Susceptibility Testing. 1981. Performance standards for antimicrobic disc susceptibility tests, 2nd ed., first supplement (NCCLS standard M2-A2). National Committee for Clinical Laboratory Standards Publication 1:141-156. 9. Peters, G., and G. Pulverer. 1980. Comparative in vitro activity of cefotaxime (HR 756). Chemotherapy 27:177-183. 10. Washington, J. A., and V. L. Sutter. 1980. Dilution susceptibility test: agar and macro-broth dilution procedures. p. 453-458. In E. H. Lennette, A. Balows, W. J. Hausler. Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington,