Evaluation of a Rapid Bauer-Kirby Antibiotic Susceptibility

ANTIMICROBIAL AGENTS AND CHEMoTHERAPY, Mar. 1975. p. 250-255 Copyright 0 1975 American Society for Microbiology Vol. 7, No. 3 Printed in USA. Evaluation of a Rapid Bauer-Kirby Antibiotic Susceptibility Determination DANIEL F. LIBERMAN* AND RICHARD G. ROBERTSON Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 23 October 1974 To reduce the incubation time requirement in the Bauer-Kirby antibiotic susceptibility test, comparisons were made of the test results at 18 to 20 h (standard) and 7 to 8 h (rapid) utilizing 100 recent clinical isolates. The zone diameters for 664 disks were monitored by using the standard classification: resistant, intermediate, or susceptible. The susceptibility determination was unchanged in 558 out of 664 instances (84.0%). An analysis of the remaining 106 sets revealed that an initial interpretation of intermediate in zone size, subsequently determined resistant or susceptible, accounted for 49 of the observed differences. The reverse changes, initial resistant or susceptible subsequently classified as intermediate, accounted for 20 of the changes. In five instances the interpretation changed from susceptible to resistant; in two cases the interpretation changed from resistant to susceptible. The remaining 30 determinations were classified as indeterminant due to (i) insufficient growth at the early (7 to 8 h) determination, and to (ii) zones which were so large that they could not be measured accurately. The data indicate that zone sizes when measured to the nearest 0.1 mm can be interpreted with reasonable accuracy and the results can be available 10 to 14 h sooner. Recently, the Food and Drug Administration has recommended the Bauer-Kirby technique as the standardized procedure for the determination of antimicrobial disk susceptibility (6). The test is a single, high-potency disk method which appears to correlate quite closely with broth dilution techniques (2, 3) and appears to be gaining in widespread use throughout this country and the world (3). General acceptance of the in vitro disk susceptibility method has been aided by its simplicity. However, the prolonged incubation interval required (18 to 24 h) between the initiation and completion of the test has remained a distinct disadvantage. Boyle and co-workers (3) have recently described a modified Bauer-Kirby test which utilized a tetrazolium dye, 2-(p-iodophenyl)-3- p-nitrophenyl-5-phenyl tetrazolium chloride, to enhance the distinction between areas of bacterial growth and zones of inhibition produced by antimicrobial agents. This dye-reduction test was rapid (6 to 7 h) and correlated quite well with the standard Bauer-Kirby method. Barry et al. have also observed that zones of inhibition were evident after a reduced incubation period (5 to 6 h) and that reliable data were obtainable for a majority of drugs and bac- 250 terial genera without using the dye-reduction technique (1). Recently, we described a disk bioassay for clindamycin that utilized the dye-reduction procedure (8). During the course of this study, we frequently noticed that the growth on the susceptibility plate was extensive: zones were readily discernible after 6 or 7 h of incubation without using the tetrazolium dye. In view of the report of Barry et al. (1), we thought it necessary to re-investigate the reliability of determining antibiotic susceptibilities at an earlier time period (as compared to the more conventional incubation time, i.e., 18 to 20 h) under routine conditions in the clinical laboratory without using the dye-reduction technique. In this communication we shall describe our experience with this comparison. MATERIALS AND METHODS Organisms. The gram-negative organisms utilized in this study are listed in Table 1 and were all recent isolates selected at random and subsequently identified in the bacteriology laboratory of Strong Memorial Hospital by standard techniques. Routine susceptibility test. Antibiotic susceptibility was determined by the Bauer-Kirby technique (2, 6). Antibiotic disks were obtained from BioQuest

VOL. 7, 1975 (Cockeysville, Md.). All disks were stored as recommended by the manufacturer. Modified test. The only modification of the standard technique was the extent of incubation at 37 C. Duplicate sets of 150-mm Mueller-Hinton plates (Bio- Quest) were streaked evenly in three planes with a cotton swab, excess suspension having been removed from the swab by rotating it against the side of the tube containing the standardized bacterial suspension (3, 6). The swab was reloaded before the second plate was inoculated. After the inoculum had dried (15 min), disks were put in place on the agar surface and the plates were incubated disk down at 37 C. After 7 h of incubation, one plate from each set was removed and the zone sizes were measured with a Fisher-Lilly antibiotic zone reader to the nearest 0.1 mm. After an additional 11 to 13 h of incubation, the second plate was removed from the incubator and the zone sizes were measured on the zone reader. RESULTS During the course of this study, occasional differences in zone sizes were observed when the rapid (7 h) result was compared to the regular TABLE 1. Organisms in rapid versus regular test study and source material Organisma No. Source No. Acinetobacter calcoaceticus Acinetobacter Iwoffi Escherichia coli Enterobacter sp. Klebsiella sp. Hafnia sp. Proteus mirabilis Proteus morganii Proteus rettgeri Pseudomonas sp. 2 1 33 10 21 1 14 1 4 13 Wounds and exudates Urine Throat a Organisms used in this study were acquired from three work stations in the bacteriology laboratory of Strong Memorial Hospital. The organisms were selected at random from gram-negative isolates. TABLE 2. BAUER-KIRBY ANTIBIOTIC SUSCEPTIBILITY TEST 251 (18 to 20 h) susceptibility determination. There were 664 pairs of zone diameters measured in this study. In 313 sets (47%), zone diameters were greater with the additional 11 to 13 h of incubation. Of the remaining 351 pairs, 160 (24%) were smaller, 161 (or 24%) remained unchanged, and the remaining 4.5% were classified into the indeterminant group. A summary of the changes in zone size (increase or decrease) according to the extent of the change is presented in Table 2. Some (24.2%) of the sets showed no change, whereas 4.5% of the sets of disks could not be adequately evaluated. One hundred ninety-eight (29%) showed a 1.0-mm or less change in zone size and 124 (18.6%) were greater than 1.0 but less than or equal to 2.0 mm. Sixty-five of the sets were between 2 and 3 mm (or 10%), 37 were between 3 and 4, and 49 were greater than 4 mm in difference. In essence then 483 of 664 zones showed no change or less than a 2-mm change between the two time points utilizing two separate plates. Since it was evident that in certain instances there were differences between the early and late readings, it was extremely important to evaluate whether or not these changes would influence the susceptibility report, i.e., susceptible, intermediate, or resistant. We evaluated the effect of these zone size changes on the interpretation of antibiotic susceptibility. The zone diameters recommended by the Food and Drug Administration (6) had to be modified since the Fisher-Lilly antibiotic zone reader was used to evaluate the diameter size. The zone reader is accurate to the nearest 0.2 mm but estimates to 0.1 mm can be readily made. The modification consisted of redefining the cutoff points for the interpretation, using polymyxin B as an example: 8, 9 to 11, and 12 (6) became 8.9, 9.0 to 11.9, and 12.0 for resistant, intermediate, and susceptible, respectively. Susceptibility de- Summary of zone determinations No. of U Zone diameter changeb Antibiotic (11 g) a mina- deter- changed Un- deter- mined 01- -1 1.1-166 2 3.1-2 31-> 4- tions mnd 0.5 061 1.5 162 2.5 26 3 36-5.. Streptomycin (10) 64 21 1 12 7 6 7 4 1 2 1 2 Chloramphenicol (30) 100 4 8 11 14 13 15 8 9 3 7 8 Tetracycline (20) 100 22 10 14 10 10 6 9 6 2 4 7 Kanamycin (30) 100 18 2 17 13 13 14 3 5 1 6 8 Cephalothin (30) 100 25 4 11 10 12 7 5 5 4 2 15 Ampicillin (10) 100 51 2 5 6 6 8 1 7 2 3 9 Polymyxin b (300) 100 20 3 46 22 6 1 1 1 a Potency of commercial disk. b Both increases and decreases in zone size.

252 LIBERMAN AND ROBERTSON terminations were altered in 76 of the 664 instances when the rapid test was compared to the regular test. Changes were classified (Table 3) into six groups: intermediate to susceptible; intermediate to resistant; resistant to intermediate; susceptible to intermediate; susceptible to resistant; and resistant to susceptible. An initial reading (7 to 8 h) of intermediate, which was subsequently (18 to 20 h) read as ANTIMICROB. AGEMS CHEMOTHER. susceptible or resistant, accounted for 64.5% (49 out of 76) of the changes. Five of the changes which occurred fell into the susceptible-changing-to-resistant category when the rapid and the regular tests were compared, whereas in two instances the reverse transition was observed. Tetracycline accounted for two of the susceptible-to-resistant changes and one resistant to susceptible; chloramphenicol was associated TABLE 3. Susceptibility changes, organisms, and antibiotics Type of change, Antibiotic I - R I - S R-I S_I SR R_coS Changes c 0 o zc [ o zc zc zcs Streptomycin Chloramphenicol Tetracycline Kanamycin Ampicillin Cephalothin Polymyxin B Percentage 1.6 5.7 8.0 8.5 9.3 9.7 10.9 4.9 5.0 2.0 4.5 1.5 1.7 0.8 2.7 4.3 9.0 27.6 Ac. Hf. 1.0. 2.5 2.7 4.2 2.2 3.4 3.8 0.5 4.0 4.2 1.2 1.4 1.5 2.6 2.7 3.0 5.0 5.4 5.6 6.8 4.6 6.2 8.7 10.1 0.8 1.4 36.8 Hf. Ac. 1.3 Hf. 0.3 1.9 0.1 2.5 2.8 4.2 4.9 2.2 2.8 3.0 3.0 5.7 1.3 1.9 3.2 0.2 0.8 1.1 2.8 25.0 Al. Kl. Pm. Pm. 15.6 5.7 13.7 Kl. Kl. 17.8 Pm. 5.6 Pm. 13.2 10.0 Pm.I 19.7 13.1 2.6 1 17.1 a I, Intermediate; R, resistant; S, susceptible; ZC, zone difference in millimeters between early and late readings; 0, organism; Ac, Acintobacter calcoaceticus; Ps, Pseudomonas sp.; Kl, Klebsiella sp.; Ec, Escherichia coli; Pm, Proteus mirabilis; Hf, Hafnia sp.; En, Enterobacter sp; Al, Acinetobacter Iwoffi. 17.1 17.1 9.2

VOL. 7, 1975 with one of each transition, whereas ampicillin and cephalothin accounted for the two remaining susceptible-to-resistant changes. The organisms encountered in these susceptibility changes are also presented in Table 3. Klebsiella sp., Escherichia coli, and Pseudomonas sp. were the organisms most frequently encountered. Pseudomonas sp. (see Table 4) accounted for 23.7% (18 out of 76) of the changes. A change from intermediate to resistant was noted in 11 of 18 instances. A change from susceptible to intermediate was noted in five instances. Klebsiella and E. coli accounted for 28.9 and 26.3% of the changes, respectively. In both cases, a change from intermediate to susceptible was the most frequently observed change, 11 of 22 and 13 of 20, respectively. The actual zone size change is also presented in Table 3 for each of the 76 differences in susceptibility results. Some (30%) of the transitions were due to a change of less than 2 mm. Eight of these changes fell into the intermediate-to-resistant group; seven into the intermediate-to-susceptible group; one resistant to inter- TABLE 4. Bacterial organisms involved in susceptibility changes Organism Totala Percent" Enterobacter sp. 5 6.58 Klebsiella sp. 22 28.94 Pseudomonas sp. 18 23.69 Acinetobacter lwoffi 1 1.31 Escherichia coli 20 26.31 Hafnia 3 3.93 Acinetobacter calcoaceticus 2 2.62 Proteus mirabilis 5 6.58 a Total for all antibiotics. Percent of total changes, i.e., Enterobacter sp. were involved with 6.58% of the susceptibility transitions observed. Druga TABLE 5. BAUER-KIRBY ANTIBIOTIC SUSCEPTIBILITY TEST 253 mediate; and the remaining eight into the susceptible-to-intermediate group. The susceptibility plates that were used in this study were obtained from several work stations in the clinical laboratory. At each location, the technologist would dilute the inoculum suspension to approximate a standard suspension. Since these dilutions were performed manually and were dependent upon varying degrees of visual acuity, variations in inoculum size were anticipated. To test for inoculum size effects, a recently isolated strain of E. coli was tested against nine antibiotics. The test organism (which was susceptible to all the antibiotics) was diluted to several different optical densities (as determined by a Spectronic 20 at 650 nm). The densities were selected so as to cover the visual range from which the technologist was most likely to select as an end point. This range included the optical density that corresponded to the McFarland standard recommended for the disk test, 0.1 optical density unit (3). Duplicate plates were set up as described previously and incubated at 37 C. One plate was monitored with the zone reader at 7 to 8 h and the second plate at 18 to 20 h. The results of this experiment are presented in Table 5. In both rapid and regular groups, the trend of decreasing zone size diameter with increasing inoculum size was evident. DISCUSSION The data presented here demonstrate the feasibility of utilizing a rapid procedure for the determination of susceptibilities of bacteria to antibiotic disks. In this study, only gram-negative organisms that grew on Mueller-Hinton agar without added sheep blood were utilized. Gram-positive organisms were excluded from this study, because in our laboratory Mueller- Hinton plates with 5% sheep blood are often Effect of inoculum size and time on zone size 0.01k 0.05 0.1 0.4 7to8hc 18to24h 7to8h 18to24h 7to8h 18to24h 7to8h 18to24h Streptomycin... 15.2 (0.21) 16.3 (0.39) 14.9 (0.22) 16.0 (0.29) 14.8 (0.22) 15.6 (0.44) 14.8 (0.24) 15.3 (0.40) Chloramphenicol... 2 (0.29) 22.6 (0.32) 21.3 (0.41) 21.3 (0.22) 20.1 (0.41) 20.9 (0.25) 20.0 (0.70) 20.6 (1.25) Tetracycline...... 21.7 (0.48) 22.5 (0.42) 21.0 (0.78) 21.9 (0.28) 21.0 (0.53) 21.5 (0.41) 20.7 (0.42) 20.2 (0.57) Kanamycin...:.19.4 (0.56) 20.9 (0.42) 19.0 (0.63) 21.1 (0.20) 18.8 (0.35) 20.3 (0.76) 18.4 (0.50) 19.3 (0.29) Cephalothin... 21.5 (0.77) 23.6 (0.39) 20.9 (0.73) 22.8 (0.19) 21.1 (0.85) 22.9 (0.81) 20.0 (0.62) 21.4 (0.33) Ampicillin... 18.8 (0.26) 19.9 (0.20) 18.4 (0.22) 19.5 (0.22) 18.2 (0.50) 19.3 (0.85) 17.3 (0.90) 18.7 (1.05) Poly.myxin B. 15.1 (0.27) 14.6 (0.23) 15.0 (0.22) 14.5 (0.29) 15.0 (0.41) 14.2 (0.26) 13.9 (0.75) 13.5 (0.25) a Commercial disks. " Optical density measured on Spectronic 20 colorimeter at 650 nm. 0.1 corresponds to the McFarland turbidity standard recommended for Bauer-Kirby procedure. c Results show mean zone size in millimeters after hours of incubation. Parentheses show standard error for five determinations.

254 LIBERMAN AND ROBERTSON used for susceptibility testing and zone sizes cannot be evaluated with the antibiotic zone reader. Our data confirm the observations of Barry et al. (1). They observed differences in 95 of 960 tests (9.8%) as compared to our 76 out of 664 or 11.44%. They divided their differences into two groups, i.e., major and minor. The major discrepancy group consisted of those that were resistant at one time and susceptible at the other (these correspond to the resistant-to-susceptible and susceptible-to-resistant transition groups in the present study). They observed 33 out of 960 (3.4%) such instances whereas we observed 7 out of 664 (1%). The larger number of major discrepancies in the Barry et al. study may be due to the fact that they evaluated the zone size at 5 to 6 h and we measured them at 7 to 8 h. In our experience, the additional incubation time improved the accuracy since the cutoff for the inhibition zone is more visible. The minor discrepancy group consisted of changes involving an intermediate at one time point and susceptible or resistant at the other (this corresponds to the remaining four groups in this present study). Sixty-two (6.5%) of their comparisons fell into this group whereas we found 69 out of 664 (10.4% incidence) for corresponding changes. The higher percentage incidence reported in the present study may reflect our utilization of the antibiotic zone reader. Borderline zones (between intermediate and resistant and intermediate and susceptible) may be classified as resistant or susceptible when measured with a caliper or ruler by eye, but are actually intermediate in susceptibility when measured under the 4 times magnification of the zone reader. Boyle et al. (3) found that there was close agreement when antibiotic susceptibilities were determined at 6 to 7 h as compared to 18 to 20 h when utilizing the tetrazolium-dye-reduction technique. We have found similar agreement without the use of the dye when an additional hour of incubation is permitted. It is important to note that there is nothing sacred about the 18- to 20-h incubation time. It is more of a convenience than a requirement for the Bauer-Kirby test. Susceptibility plates can be set up on 1 day and read the next without any major alterations in the work-day of the technologist. The Food and Drug Administration regulations indicate that susceptibility plates can be read 6 to 8 h after incubation if there is sufficient growth. However, they do recommend that the results be confirmed by rereading after overnight incubation (6). ANTIMICROB. AGENTS CHEMOTHER. This brings up an interesting question. What constitutes the optimum time period for incubation? It appears from Barry et al. (1), Boyle et al. (3), and the data presented here that there is a difference of approximately 10% between an early and standard reading of susceptibility plates. It is generally assumed that the standard incubation (18 to 20 h) procedure is the more correct of the two. In 1952, Humphrey and Leightbown gave a detailed description for the diffusion of antibiotics into agar media (7). Cooper and Gillespie (4) amplified this information by presenting the concept of critical time which states that after a certain time interval a microorganism will be able to grow in the presence of many times the minimal inhibitory concentration of antibiotic. In other words, the antibacterial effect of antibiotics is confined to a relatively short period, the "critical time." Thus, bacteria which are reached by a minimal inhibitory concentration of drug during this critical period will be inhibited. On the other hand, after bacterial density has reached a certain point, the antimicrobial will no longer affect growth of the organism. In Cooper's studies (4, 5), the critical time appeared to be four times the microbial generation time plus the lag phase, or between 2.5 to 4.0 h. The zone sizes, therefore, become established during this critical time period before macroscopic growth can be observed. Cooper's studies (4, 5) show clearly that disk susceptibility tests depend not only on the organism and its susceptibility but also on the diffusion, i.e., the concentration gradient, of the antibiotic contained in the disk. It would appear then that it may be more proper to consider the early reading as the more correct. It is hoped that further experimentation will clarify this point. The data presented here and the studies of Boyle et al. (3) and Barry et al. (1) indicate that Bauer-Kirby susceptibility plates can be read earlier than is conventionally recommended with reasonably high accuracy. Approximately 4.7% of the tests in the present study (30 out of 664) could not be interpreted. Some (76) of the 664 tests demonstrated a change in the susceptibility evaluation (11.5%). Approximately 90% of these changes could be classified into the Barry et al. (1) minor group. The remaining 10% fell into the major group. The latter group represents 1% of all the sets of disks monitored and, as indicated before, some of these major discrepancies may be due to diffusion kinetics. The data presented in Tables 3 and 4 indicate that the various transitions that were observed were spread throughout the organisms studied in this

VOL. 7, 1975 BAUER-KIRBY ANTIBIOTIC SUSCEPTIBILITY TEST 255 report with several notable exceptions. E. coli and Klebsiella sp. accounted for 24 of the 28 intermediate-to-susceptible transitions. More importantly though is the observation that Pseudomonas accounted for over half of the intermediate-to-resistant transitions we observed (11 out of 21). In six of these 11 intermediate-to-resistant transitions, the early observation indicated that a zone was evident and that when the later observations were recorded no zones were observed. Pseudomonas sp., generally extremely resistant to most drugs, are a constant source of concern in hospital burn units and with the compromised host population. If a drug has some efficacy against the pathogen, i.e., it is intermediate in susceptibility, then the clinician may be able to use this drug. However, if the test indicates that the organism is resistant, then the drug is often disregarded. Since it is paramount to insure the most accurate information possible, it is imperative then to determine whether or not growth or overgrowth phenomena are responsible, in part, for the intermediate-to-resistant changes we have observed for this group of organisms. Recently, Barry et al. (1) stated that the early reading may be more accurate for certain groups of organisms. They were primarily concerned with swarming observed with Proteus mirabilis. It should be emphasized that, as was previously mentioned, only gram-negative bacilli were utilized in this present study. In our clinical laboratory all gram-positive coccisusceptibility testing is performed on Mueller- Hinton supplemented with sheep blood. This precluded the inclusion of Staphylococci (both aureus and epidermidis). Streptococci, in general, are slower growing organisms, hence they may not lend themselves to this procedure and we would recommend the more conventional incubation time. If we assume that a change of 1.0 mm or less is actually no change, then 161 unchanged zones plus 198 0.1- to 1.0-mm zones equals 359. This would mean that 54% (359 out of 664) of the zones represented no change. We would then reduce the number of transitions we report from 76 to 69 (Table 3) or 10.4%. The seven major changes which occurred would then represent about 10% of the transitions noted in this study. This means that only 4.6% of the changes would fall into this category or that 95.4% would not. This numerology assumes that all changes greater than 1.0 mm in diameter result in a change in the susceptibility determination. Clearly, from the data presented here, only 76 of the 473 zone sizes which changed resulted in a transition in susceptibility. (This would correspond to 69 in 289 if the 1.0-mm difference is considered unchanged, i.e., 24.5%.) This places the error of a major discrepancy at the 2.5% level. Frequency estimates for the other transitions can likewise be calculated from the data. If there is any question about a certain determination, the plate can be reincubated. If the uncertainty is still present, one can always run a tube dilution test and determine the minimal inhibitory concentration. ACKNOWLEDGMENTS This investigation was supported in part by Public Health Service training grant no. A1-00427-03 from the National Institute of Allergy and Infectious Diseases. We wish to express our gratitude to the technicians in The Bacteriology Laboratory of Strong Memorial Hospital for their cooperation in this study. LITERATURE CITED 1. Barry, A. L., L. J. Joyce, A. P. Adams, and E. J. Benner. 1973. Rapid determination of antimicrobial susceptibility for urgent clinical situations. Am. J. Clin. Pathol. 59:639-699. 2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:439-496. 3. Boyle, V. J., M. E. Fancher, and R. W. Ross, Jr. 1973. Rapid modified Bauer-Kirby susceptibility test with single high-concentration antimicrobial disk. Antimicrob. Agents Chemother. 3:418-424. 4. Cooper, K. E., and W. A. Gillespie. 1952. The influence of temperature on streptomycin inhibition zones in agar cultures. J. Gen. Microbiol. 7:1-7. 5. Cooper, K. E., and A. H. Linton. 1952. The importance of the temperature during the early hours of incubation of agar plates in assays. J. Gen. Microbiol. 7:8-17. 6. Food and Drug Administration. 1972. Antibiotics intended for use in the laboratory diagnosis of disease. Fed. Regist. 37:20525-20529. 7. Humphrey, J. H., and J. W. Leightbown. 1952. A general theory for plate assays of antibiotics with some practical applications. J. Gen. Microbiol. 7:129-143. 8. Liberman, D. F., J. Fitzgerald, and R. G. Robertson. 1974. A rapid disk test for determining clindamycin serum levels. Antimicrob. Agents Chemother. 5:458-461. 9. Masten, J. M., M. J. H. Koepcke, and P. G. Quie. 1970. Evaluation of the Bauer-Kirby-Sherris-Turck single disc diffusion method of antibiotic susceptibility testing, p. 445-453. Antimicrob. Agents Chemother. 1969.