Reassessment of the "Class" Concept of Disk Susceptibility Testing

Reassessment of the "Class" Concept of Disk Susceptibility Testing Disks versus Minimal Inhibitory Concentrations with Eleven Cephalosporins ARTHUR L. BARRY, PH.D., CLYDE THORNSBERRY, PH.D., RONALD N. JONES, M.D., PETER C. FUCHS, M.D., THOMAS L. GAVAN, M.D. AND E. HUGH GERLACH, PH.D. Barry, Arthur L., Thornsberry, Clyde, Jones, Ronald N., Fuchs, Peter C, Gavan, Thomas L., and Gerlach, E. Hugh: Reassessment of the "class" concept of disk susceptibility testing. disks versus minimal inhibitory concentrations with eleven cephalosporins. Am J Clin Pathol : 99-9, 9. Studies were carried out to determine whether susceptibility or resistance to cephalosporins could be predicted reliably from the results of tests with a single cephalothin disk. The cephalosporins were tested with a microdilution technic and with a standardized disk test. Strains susceptible to a cephalothin disk were predictably susceptible to all other cephalosporins. However, -% of the strains were resistant to cephalothin disks but were susceptible to the more active parenteral drugs cefoxitin, cephamandole, cefuroxime, and BL-S6. Because of differences in antimicrobial activities, the cephalosporins could be divided into three subgroups for purposes of susceptibility testing: one subgroup includes the majority of cephalosporins and may be represented by tests with cephalothin, the second subgroup includes three active parenteral drugs (cephamandole, cefuroxime, and BL-S6) and may be represented by tests with cefuroxime, and the third subgroup consists of cefoxitin, a cephamycin with a unique broad spectrum of activity. Until the drugs in the second and third subgroups are released for general therapeutic use, the practice of testing only one cephalosporin disk appears to be a reasonably reliable procedure. (Key words: Antibiotic disk susceptibility tests; Class concept of disk testing; Cephalosporins; disk susceptibility tests.) Received June, 9; received revised manuscript August, 9; accepted for publication August, 9. Arthur L. Barry: Clinical Microbiology Laboratories, University of California (Davis) Sacramento Medical Center, Sacramento, California 9. Clyde Thornsberry: Antimicrobic Testing Laboratory, Center for Disease Control, Atlanta, Georgia. Ronald N. Jones: Department of Pathology, Kaiser Foundation Hospital Laboratories, Portland, Oregon 9. Peter C. Fuchs: Department of Pathology, St. Vincent Hospital and Medical Center, Portland, Oregon 9. Thomas L. Gavan: Department of Microbiology, Cleveland Clinic Foundation, Cleveland, Ohio 446. E. Hugh Gerlach: Microbiology Laboratory, St. Francis Hospital, Wichita, Kansas 64. Address reprint requests to Dr. Barry: Clinical Microbiology Laboratories, University of California (Davis) Sacramento Medical Center, Sacramento, California 9. Clinical Microbiology Laboratories University of California (Davis) Sacramento Medical Center, Sacramento, California THE ROUTINE ANTIMICROBIC susceptibility test cannot include all of the antimicrobial agents that are currently available for use. Normally it is necessary to test only one representative from each class of related drugs with similar specta of activity. This so-called class concept of disk testing has been endorsed by the Food and Drug Administration 4 and by the National Committee for Clinical Laboratory Standards (NCCLS). 6 Specifically, cephalothin has been designated the preferred representative of the cephalosporin group. As part of recent studies to evaluate some newly developed cephalosporins, we were able to reassess the class concept of disk testing. Eleven cephalosporins were tested by a microdilution technic, and standardized Bauer-Kirby disk tests were performed with disks containing either cephalothin or one of seven other cephalosporins. Materials and Methods The present report accumulates data collected from the two separate studies. The first study included tests with cephalothin and four orally administered cephalosporins (cefaclor, cephalexin, cephradine, and cefatrizine [BL-S64]). Microdilution tests were performed with isolates, but disk tests were performed with only isolates, including Escherichia coli, Enterobacter spp., Klebsiella spp., Proteus mirabilis, indole-positive Proteus spp., Staphylococcus aureus, and Streptococcus faecalis. The second study included tests with cephalothin, cefuroxime, cephamandole, cefazolin, cefoxitin, cephaloridine, cephradine, and BL-S6. Both microdilution and disk diffusion tests were used to study 9 isolates, includ- -9///99 $. American Society of Clinical Pathologists 99 Downloaded from https://academic.oup.com/ajcp/article-abstract//6/99/ on 6 April

9 BARRY ET AL. A.J.C.P. December 9 > 6 id r. =. RAT. UJ <J z,. >-. m. i. Z = o.oe. \, v!v '! BL-S6 %*- I PFFAMANnm F - M. M. NSW fcl LCrHnHHUULC «^%r. ' CEFOITIN * + * + < i*^ * ' rffiinnyinr, ^%.**. CEPHALOTHIN >!," * N, * ^S-W. -. ^» ^ k* V. *. ^N. x ^ ^.N '^ ' ' ^fa* ^ *. ^ o - ^ I ^. f. "^ ^ "» v. * v >» **» ^."v *<, ' >*> ^fw v. "' * ^ * ^ ' \ v ^. *. ZONE DIAMETER (MM) - MCG DISCS FIG.. Regression analyses of the data correlating zone sizes with MIC values. ing S. aureus, Staphylococcus epidermidis, S. faecalis, Streptococcus bovis, Streptococcus durans, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae (including ten ampicillin-resistant strains), 6Aeromonas hydrophilia, \OPseudomonas aeruginosa, "hpseudomonas cepacia, Pseudomonas maltophilia, 4 Enterobacter spp. (E. cloacae, E. aerogenes, and 4E. agglomerans), Serratia spp., Klebsiella pheumoniae, E. coli, P. mirabilis, Proteus vulgaris, Proteus morganii, and Proteus rettgeri. Minimal inhibitory concentrations were determined for each cephalosporin, using the microdilution technic previously described. The microdilution trays were prepared with doubling dilutions of each cephalosporin, ranging from to. fig/m\. The trays were stored frozen during the course of the study (as long as six weeks). Tests with control organisms demonstrated no significant inactivation of the drugs during the storage period. The trays were inoculated so as to achieve a final inoculum density of about viable cells per ml, and minimal inhibitory concentrations (MICs) were determined after 6- hours at C. Disk susceptibility tests were performed with the use of the Bauer-Kirby method, as outlined by the NCCLS. 6 In addition to fig cephalothin disks, cefuroxime, cephamandole, cefoxitin, BL-S6, cefatrizine, cephaloridine, and cefaclor disks were tested. The disks were either supplied by the appropriate manufacturer or prepared in the testing laboratory: all disks contained fig of drug. Results Sufficient data were accumulated with five cephalosporins to permit regression analyses of the data correlating zone sizes with minimal inhibitory concentration values (Fig. ). The regression formulas are described in Table. Correlation coefficients are excellent, and the intercepts of the regression lines are very similar to those published for other cephalosporins. -9 Although the slopes of the regression lines are somewhat different for individual drugs, the lines tend to converge at the 4- -mm zone size break points. With cephalothin disks, a zone of mm or more is interpreted as indicating susceptibility (MIC ^ /xg/ml). Using the regression formulas listed in Table, the MIC values that correlated with a zone of mm were calculated for the drugs; they ranged from. to 9. pig/ml. For that reason, the interpretive zone standards used for cephalothin disks might also apply to tests with the other cephalosporin disks. Disk susceptibility tests were interpreted according to established zone standards (resistant S 4, intermediate -, susceptible, S mm). The interpretation of the cephalothin disk test was then compared with the MIC category (resistant g, intermediate 6, susceptible ^ fig/ml) for each of the cephalosporins. Discrepancies were considered minor when one test was intermediate and the other susceptible or resistant. Discrepancies were considered major when the organisms were resistant with the disk test but the MICs indicated susceptibility (MIC ^ fig/ml). Very major Downloaded from https://academic.oup.com/ajcp/article-abstract//6/99/ on 6 April

Vol.. No. 6 CEPHALOTHIN DISK TESTS 9 J J Table I. Regression Formulas Describing MIC: Zone Size Correlations with Five Different Cephalosporins Antimicrobial Agent No. of Strains Tested* Correlation Coefficient Slope Regression line Interceptt MIC Correlate to Zone of mmt BL-S6.9.96.9.9.96 -. -.9 -. -.6 -..6.4...4 6.6 /ug/ml. /xg/ml. /xg/ml 9. fig/ml.4 /xg/ml * Strains with no zones or MICs outside of the range tested were excluded from the calculations. t MIC expressed as the loga. discrepancies were recorded when resistant organisms (MIC ^ /xg/ml) were reported to be susceptible with the disk test. The relative accuracy of cephalothin disks for predicting susceptibility or resistance to other cephalosporins is assessed by the data in Table. Complete agreement between dilution test categories and the interpretation of tests with cephalothin disks occurred with -9% of the strains tested, and -% of the strains showed only minor discrepancies. A small proportion of strains (-6%) would have been reported susceptible with cephalothin disks but proved to be resistant to three cephalosporins (cephalexin, cefaclor, and cefatrizine). With the parenteral cephalosporins, such very major discrepancies occurred with ^% of the strains. However, -% of the strains were considered resistant when tested with cephalothin disks but were susceptible to other cephalosporins; i.e., there were major discrepancies. Seven cephalosporin disks, other than cephalothin, were tested, and the interpretations of such tests compared with MIC categories (Table ). The errors encountered by testing only cephalothin disks were reduced but not eliminated by testing the specific cephalosporin disk. One could easily fill an entire test plate with disks containing different cephalosporins, but in most cases, the added information that would be gained could not justify the time and expense involved. The major problem involves the magnitude of the differences in the antimicrobial activities of the cephalosporins. Although the actual MICs differ, most isolates are categorized as being susceptible (MIC S /u.g/ml) or resistant (MIC g /xg/ml) to all cephalosporins. With the staphylococci, streptococci, and Haemophilus spp., our isolates were either susceptible or resistant to all cephalosporins tested; but some of the gram-negative bacilli showed significant differences in their susceptibilities to different cephalosporins. Tables and 4 summarize a comparison of MIC determinations made in two separate series of tests with different drugs and two different collections of test strains. The number of strains that were clearly susceptible to one drug and t MIC value that correlated with a zone of mm, as calculated from the regression formulas. resistant to another was tabulated, ignoring those strains with intermediate MICs (6 /xg/ml). The cephalothin-susceptible strains were rarely resistant to any other cephalosporin. However, a significant proportion of the cephalothin-resistant strains were susceptible to cephamandole, cefuroxime, BL-S6, and cefoxitin. With the seven other drugs, susceptibility or resistance can be predicted reasonably well from the results of tests with cephalothin or with any one of the Table. Relative Accuracies of Disk Tests for Determining Susceptibility or Resistance to Cephalosporins* Antimicrobic vs. -/ug Disks MICs Cefazolin MICs Cephaloridine MICs vs. cephaloridine disks MICs vs. cefuroxime disks Cefamandole MICs vs. cephamandole disks BL-S6 MICs vs. BL-S6 Disks MICs vs. cefoxitin disks Cefaclor MICs vs. cefaclor disks Cefatrizine MICs vs. cefatrizine disks MICs Cephalexin MICs % Full Agreement 9 9 9 9 9 9 6 9 96 9 Minor % Discrepancies 6 9 4 6 6 Major Very Major 4 6 6 9 6 4 * Based on disk tests and MIC determinations with 9 isolates (see text); tests with cefaclor, cefatrizine, and cephalexin limited to isolates. Downloaded from https://academic.oup.com/ajcp/article-abstract//6/99/ on 6 April

9 BARRY ET AL. A.J.C.P.. December 9 Table. Cross-resistance among Eight Parenteral Cephalosporins Tested against Gram-negative Bacilli* Percentage of Strains Resistant (MIC > 6 ng/ml) Percentage of Strains Susceptible (MIC =s jug/ml) BL-S6 Cephaloridine Cefazolin Cephaloridine Cefazolin BL-S6 I I I 4 I I x!' 6 4 ^ 9 4 r x ~i * Includes tests with E. coli, K. pneumoniae, P. mirabilis, indole-positive Proteus spp., E. cloacae, E. aerogenes, 4 E. agglomerans, Serratia spp., P. aeruginosa, P. cepacia, P. maltophilia, and 6 A. hydrophilia. seven cephalosporins. At the most, a % very major error and -6% major error would occur as a result of assuming complete cross-resistance between cephalothin and the seven drugs. The major errors were principally due to increased cefazolin activity compared with the other members of this subgroup of cephalosporins., cefuroxime, and BL-S6 showed similar spectra of activity: they were the most active cephalosporins tested., a cephamycin drug, had a spectrum unlike that of any other cephalosporin tested. Discussion The routine susceptibility test should include a limited number of antimicrobial agents, selected to represent different families of drugs. The so-called class concept of disk testing presumes that when an organism is susceptible to the class (family) representative, it should also be susceptible to other, related, agents. It also follows that an organism that is resistant to the class representative should be resistant to other Table 4. Cross-resistances of Four Orally Administered Cephalosporins and Tested against 6 Gram-negative Bacilli* Percentage of Strains Resistant (MIC>6^g/ml) Cephalexin Cefaclor Cefatrizine Percentage of Strains Susceptible (MIC = /ng/ml) Cepha- Cepha- Ceph- Cefa- Cefalothin lexin radine clor trizine * Includes tests with E. coli, 9 K. pneumoniae, E. agglomerans, 9 E. aerogenes, E. cloacae, Serratia spp., P. mirabilis, 6 indole-positive Proteus spp., E. tarda, Shigella spp., 6 Salmonella spp., and P. aeruginosa. agents within that group of related drugs. By using the class concept of disk testing, one would occasionally rule out the use of an effective drug, i.e., when the organism was resistant to the family representative but susceptible to one or more other drugs in the family. Such a major error is considered less dangerous than the reverse situation, when the family representative indicates susceptibility but the test organism is resistant to related drugs (very major errors). In general, the family concept may be considered valid when the number of major errors is held to a minimum and very major errors are extremely rare. The number of major errors that can be tolerated within the family concept cannot be defined easily: the clinical relevance of the discrepancies must be taken into consideration. Many of the cephalosporins included in the present study have not yet been subjected to enough clinical use to judge their place as first-line drugs that must be considered in the routine susceptibility test. If the more active cephalosporins find a place as second-line drugs, they may be reserved for in vitro testing only when necessitated by the clinical situation, i.e., when firstline drugs cannot be used for one reason or another. The class concept of disk testing with the cephalosporins can be justified by the data in the present report. Essentially all of the cephalothin-susceptible strains were also susceptible to all other cephalosporins. However, a small proportion of cephalothin-resistant strains were found to be susceptible to the more active parenteral cephalosporins, i.e., cefuroxime, cephamandole, BL-S6, and cefoxitin. If these drugs become first-line drugs of choice, the errors that occur from testing only cephalothin disks may be significant enough to justify the use of more than one cephalosporin., cephamandole, or BL- S6 should be tested, because of the differences in their spectra of activity, but all three drugs are not needed, because of their similarities. In addition, tests Downloaded from https://academic.oup.com/ajcp/article-abstract//6/99/ on 6 April

Vol. No. 6 CEPHALOTHIN DISK TESTS 9 with cefoxitin can be justified on the grounds of its broader and unique spectrum of activity. would be a reasonable representative of the former subgroup, thus minimizing the number of major and very major errors. For the other cephalosporins, cephalothin appears to remain a reasonably reliable representative. The correlation between MIC categories and interpretation of disk tests was reasonably good when disks containing cephalothin were compared with MICs for different cephalosporins. The correlation was improved by using the disks that contained the same cephalosporin as in the MIC determinations. However, major and very major discrepancies still occurred with -% of the strains even when the MIC and disk tests were both performed with the same drug. When cephalothin disks were used, major and very major discrepancies were seen with -% of the strains. The question then arises as to whether these discrepancies are important enough to justify the routine use of more than one cephalosporin disk. At this time, it is probably not justified from a practical point of view. One should continue testing only cephalothin disks, and if the test organism is susceptible, it will be susceptible to other cephalosporins. If, on the other hand, the organism is resistant or equivocal with a cephalothin disk, additional tests with other cephalosporins (when available) may be justified, providing that the clinical situation warrants therapy with a more active cephalosporin or cephamycin, i.e., when alternative drugs are not available for safe use. In such unusual clinical situations, dilution tests are the most appropriate methods for in-vitro testing, because the disk is only 9-96% accurate. References. Barry AL: The Antimicrobic Susceptibility Test: Principles and Practices. Philadelphia, Lea and Febiger, 96. Bauer AW, Kirby WMM, Sherris JC, et al: Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 4:49-494, 966. Ericsson HM, Sherris JC: Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol Microbiol Scand [B] (Suppl), 9 4. Federal Register, Rules and regulations; antibiotic susceptibility discs. Fed Regist :-9, 9. Kirby WMM: Selection of antibiotics for disc susceptibility testing, The Clinical Laboratory as an Aid in Chemotherapy of Infectious Diseases. Edited by Bondi A, Bartola JT, Prier JE. Baltimore, Md., University Park Press, 9, pp. - 6. NCCLS Subcommittee for Antimicrobial Susceptibility Tests, Performance standards for Antimicrobial Disc Susceptibility Tests. National Committee for Clinical Laboratory Standards, E. Lancaster Ave., Villanova, Pa., 9, 9. Sherris JC.Rashad AL, LighthartGA: Laboratory determination of antibiotic susceptibility to ampicillin and cephalothin. Ann NY Acad Sci 4:4-6, 96. Washington, JA, Yu, PK: Regression curve analysis of cephalosporin activity. Appl Microbiol 9:9-9, 9 9. Wick WE: Delineation of the differences of various bacterial susceptibility tests with cephalexin. Antimicrob Agents Chemother 96, 4-44, 969 Downloaded from https://academic.oup.com/ajcp/article-abstract//6/99/ on 6 April