APPLIED MICROBIOLOGY, Aug. 1972, p. 240-247Copyright 0 1972 American Society for Microbiology

Vol. 24, No. 2Printed in U.S.A.

Reliability of the Kirby-Bauer Disc DiffusionMethod for Detecting Methicillin-Resistant

Strains of Staphylococcus aureusW. LAWRENCE DREW, A. L. BARRY, RICHARD O'TOOLE, AND JOHN C. SHERRIS

Department of Pathology and Laboratory Medicine, Mount Zion Hospital and Medical Center, SanFrancisco, California 94120; Hospital Infection Research Laboratory, Departments of Medical Microbiologyand of Medicine, College of Medicine, University of California, Irvine, California 92650; and Departments ofMedicine and Microbiology, University of Washington and Harborview Medical Center, Seattle, Washington

98104

Received for publication 13 September 1971

The resistance of Staphylococcus aureus to methicillin and related drugs canbe reliably determined by using the Kirby-Bauer method of susceptibilitytesting if the incubation temperature is 35 C or below, but resistance may bemissed at 37 C. The 1-aug discs of oxacillin and nafcillin or the 5-gg discs ofmethicillin may be used for this purpose but not the 1-,gg discs of cloxacillin.The latter fail to discriminate between sensitive and resistant staphylococciby zone measurement; some resistant strains of staphylococci may show largerzones of inhibition than sensitive strains. Stability of these antibiotic-containingdiscs was studied under conditions of temperature and humidity variation thatmight be encountered in a clinical laboratory refrigerator. Oxacillin discs were

the most stable and are to be preferred for susceptibility testing. Nafcillin discswere less stable, and methicillin discs lose their potency rapidly unless carefullystored in a refrigerator with a desiccant.

Strains of Staphylococcus aureus which areresistant to methicillin and other penicillinase-stable penicillins have been isolated regularlyfrom infected patients in the hospitals of Eu-rope and the United Kingdom. In the UnitedStates, there have been only a few reportedoutbreaks of infections due to these organisms(4, 8, 15).

Disc diffusion methods for susceptibilitytesting may fail to detect all methicillin-re-sistant strains of S. aureus, largely becausesuch strains are remarkably heterogenous intheir resistance (3, 9, 10, 12, 16, 20, 21). That isto say, the majority of cells within a clone maybe fully susceptible, and only a small propor-tion may be capable of growing in the presenceof an increased concentration of methicillin.On ordinary media, the resistant portion of thepopulation may grow rather slowly and tend toform small colonies (17). An incubation periodof 48 hr has been recommended for detectingthe resistant portion of the population, butthis can be decreased by using a hypertonicmedium (2, 10) or by incubating the tests at a

lower temperature (1, 12, 13). The presentstudy was undertaken to determine whetherthe disc diffusion method of Bauer et al. (16),without modification, would suffice for de-tecting methicillin resistance among isolates ofS. aureus or whether an additional type ofscreening test must be utilized. Because cloxa-cillin, nafcillin, and oxacillin are structurallyand pharmacologically related to methicillin,detection of resistance to these agents was alsostudied.

MATERIALS AND METHODSTests were carried out with 52 isolates of methi-

cillin-resistant S. aureus obtained from severalsources and 25 isolates of methicillin-susceptible S.aureus obtained from the clinical laboratories of theUniversity Hospital, Seattle, Wash.

Strains of methicillin-resistant S. aureuswere kindly supplied by E. J. Benner, Univer-sity of California, Davis (24 strains); R. J. Bul-ger, University of Washington, Seattle (6strains); S. Cohen, University of California,San Francisco (3 strains); and R. Sutherland,Beecham Research Laboratories, Brockham

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determined by storing cartridges in open containersand in a closed storage rack with a desiccant whichwas replaced when needed. Both types of containerswere stored at room temperature (12 to 28 C, 10 to50% relative humidity) and in a household refriger-ator located in the center of a clinical microbiologylaboratory. During the working day, the door to thisrefrigerator was frequently opened and closed and,consequently, the temperature varied from 3 to 18 Cand the relative humidity from 56 to 80%. Once aweek, for 12 weeks, the discs were removed fromstorage and tested in triplicate against a fully sus-ceptible clinical isolate of S. aureus by using theagar overlay modification of the Kirby-Bauermethod; (5) the diameters of the inhibitory zoneswere recorded after overnight incubation. Becausechloramphenicol is very stable under a variety ofconditions of temperature and humidity, discs con-taining this drug were also included as a method-ology control. The zone diameters around chloram-phenicol discs varied no more than 1 mm during the12-week study, regardless of storage conditions, thusconfirming the reproducibility of the test method.

RESULTSThe inactivation of antibiotic-containing

discs during 12 weeks of storage under four dif-ferent conditions is depicted in Fig. 1. Whenexposed to the temperature and humidity ofthe laboratory, methicillin discs lost all ac-

Park, Batchwork, Surrey, England (17 strains).All filter-paper discs were obtained from DifcoLaboratories. For agar dilution studies methi-cillin, cloxacillin, and oxacillin were obtainedfrom Bristol Laboratories, Syracuse, N.Y., andnafcillin was obtained from Wyeth Laborato-ries, Philadelphia, Pa.

Susceptibility tests were performed by the discdiffusion method of Bauer et al. (6) with Mueller-Hinton agar (Difco). Zones of inhibition were meas-ured after 18 hr and again after 48 hr of incubationat 35 C. Studies were also carried out by incubatingthe plates at 35 and 37 C. Simultaneous agar dilu-tion susceptibility tests were performed with thesame suspension of bacteria as prepared for the disctest but diluted so that a Steers-Foltz (19) inoculumreplicator would deposit approximately 105 viablecells of each strain onto each plate. Doubling dilu-tions of antibiotic were incorporated into Mueller-Hinton agar (Difco) for these tests. The minimal in-hibitory concentration (MIC) was recorded as thelowest concentration of drug permitting the growthof no more than one colony after 48 hr of incubationat 35 C. Population analyses were carried out in asimilar manner but by inoculating the plates withvarying dilutions of the bacterial suspension. Thenumber of colony-forming units capable of growingin the presence of each concentration of drug wasthen determined.The comparative stability of discs containing

methicillin, oxacillin, cloxacillin, and nafdillin was

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FIG. 1. Stability of methicillin, nafcillin, cloxacillin, and oxacillin discs at refrigerator or room tempera-ture, with and without desiccant.

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METHICILLIN NAFCILLIN CLOXACILLIN OXACILLIN5 AG I ,G I G I G

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DREW ET AL.

tivity after 6 weeks, and nafcillin was com-pletely inactivated after 12 weeks. Inactivationof the drugs was not as pronounced when thediscs were refrigerated with a desiccant. Evenunder these conditions, methicillin lost a sig-nificant amount of activity during the 12-weekstudy period, but the other three drugs werenot markedly affected. Of the four drugstested, oxacillin and cloxacillin were somewhatmore stable than nafcillin, and all three weremuch more stable than methicillin.These four drugs were then tested for their

ability to separate methicillin-resistant S. au-reus from susceptible strains by the disc diffu-sion technique of Bauer et al. (6) The relation-ship between the sizes of inhibitory zones readafter 18 hr and the 48-hr MIC is described inFig. 2 and 3. Oxacillin and nafcillin discs (1gug) both readily detected resistant strains(MIC > 1.25 Ag/ml) all of which gave no zoneof inhibition, whereas susceptible strains(MIC < 0.62 gg/ml) gave zones of 11 mm orgreater. Methicillin discs (5 ,ug) were also ca-pable of separating resistant strains from sus-ceptible strains; with one exception, resistantstrains (MIC > 5.0 Ag/ml) grew up to the edgeof the disc, and all the susceptible strains(MIC < 2.5 Ag/ml) gave zones 15 mm in di-ameter or greater.Some resistant strains showed reduced den-

sity of growth in the immediate vicinity of thediscs; occasionally an "outer" zone of inhibi-tion could actually be measured. In these in-stances bacterial growth in the "inner" zonecould be seen with careful inspection. Thisappearance was more frequent around nafcillin

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discs than around oxacillin or methicillindiscs.With 1-,ug cloxacillin discs, zone measure-

ments provided no clear separation of resistantand sensitive staphylococci. Resistant strains,with MIC ranging from 1.5 to 100,ug/ml, gaveinhibitory zones 13 to 19 mm in diameter,whereas susceptible strains (MIC < 1.25ug/ml) had zones measuring 14 to 23 mm.Plates seeded with resistant organisms oftenrevealed tiny colonies within the zone of inhibi-tion, but these colonies were not always vis-ible to the naked eye and did not always ap-pear even after 48 hr of incubation. Figure 4demonstrates the type of zones observed withan isolate of S. aureus which is actually re-sistant to all four drugs by agar dilution tests.Limited experience with dicloxacillin discsrevealed results similar to those observed withcloxacillin.Ten isolates were retested by the disc diffu-

sion technique and incubated at two differenttemperatures (Table 1). The ten resistant iso-lates gave very small zones, if any, aroundoxacillin, nafcillin, and methicillin discs at 35C, but at 37 C there was a marked increase inthe zone sizes. With cloxacillin discs the sizesof the zones of inhibition were not affected bythe temperature of incubation.Population survival studies were carried out

to determine whether the percentage of cellsresistant to high concentrations of cloxacillinis smaller than that with comparable anti-biotics and accounts for the apparent suscep-tibility of these bacteria in disc diffusion sus-ceptibility tests. Figure 5 represents the re-

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0* O6le

4 8 12 16 20 24

ZONE DIAMETER (MM.)

FIG. 2. Relationship of zone sizes to minimal inhibitory concentration (MIC).

4 8 12 16 20

ZONE DIAMETER (MM.)

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METHICILLIN5A G DISC

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ZONE DIAMETER (MM.) ZONE DIAMETER (MM.)

FiM. 3. Relationship of zone sizes to minimal inhibitory concentrations (MIC).

FIG. 4. Appearances of inhibitory zones withmethicillin-resistant S. aureus and the followingdiscs: oxacillin (OX, upper right); cloxacillin (CX,upper left); nafeillin (NF, lower right); and methicil-lin (DP, lower left).

sults of such an experiment in which the num-ber of viable cells capable of growing in thepresence of increasing concentrations of clox-acillin was compared with the same capabilityin nafcillin. The resistant isolate tested in thisexperiment was capable of growing on agarplates containing 100 Mug of either drug per ml,but cloxacillin was much more active in thatthe percentage of resistant cells surviving in-termediate concentrations was much lower.Indeed, 99.99% of the viable cells in this cul-ture was inhibited by 1.6 Mg of cloxacillin/ml

whereas 50 ,ug of nafcillin/ml was required forcomparable results.A similar population survival study was car-

ried out with two other isolates of S. aureus,both of which had a cloxacillin MIC of 50sg/ml. One isolate showed confluent growthright up to the edge of a i-gg cloxacillin disc,whereas the other had the more typical patternof a 16-mm zone with several inner colonies.The latter organism was more susceptible tointermediate concentrations of cloxacillin thanthe former (Fig. 6). In this instance, 50 Mg

of cloxacillin/ml was required to inhibit 99.99%of the cells in the more resistant culture,whereas only 1.6 Mg/ml was required for com-

parable inhibition of the culture which gavelarge zones of inhibition with the disc method.

DISCUSSIONThe data reported here indicate that the disc

diffusion technique of Bauer et al. (6) is reli-able for the detection of S. aureus resistant tomethicillin and related to antimicrobials whenincubated at 35 C. However, several precau-tions must be taken to avoid erroneous results.In practice, many "methicillin-resistant S.aureus" are actually coagulase-negative S. epi-dermidis which have been misidentified forone reason or another. Methicillin resistance isnot uncommon among strains of S. epidermi-dis, and mixtures of resistant S. epidermidisand susceptible S. aureus are often found inclinical specimens. The identification of resis-tant colonies within a zone of inhibition shouldbe confirmed before being reported.

Inactivation of the susceptibility test discs is

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TABLE 1. Effect of temperature of incubation onzone-size measurements

Zone diameters (mm)'Strain Temp Oxa- Naf- Methi- Cloxa-

cillin cillin cillin cillin

CCAU 35 6b 6b 6 1737 13 16 14 17

N37 35 6 6 6 1637 6 (11) 14 11.5 16

387 35 6 6b 6 1737 14 17 15 18

UC2 35 6 (11) 6 (13) 6b 16.537 13 16 10 17

68/8585 35 6 6 6 1637 6 6b 6b 15

1484 35 6 6 6 1637 12 17 15 16

7913 35 6 6 6 1637 6 (11) 14 12 16

996 35 6 6 6 1937 13 14 12 17

968 35 6 (14) 6 (15) 6 (10) 1937 15 18 6 (13) 19

E164 35 6 (12) 12 6 (11) 1637 13 17 14 16

a A diameter of 6 mm equals no inhibition becausethis is the diameter of the disc. Figures in paren-theses equal the "outer" zone in those tests wherethere was marked reduction of the density of growthin the immediate vicinity of the disc.

' These tests showed only a suggestion of an outerzone.

another significant source of error in the clin-ical laboratory. Griffith and Mullins (11)studied a number of storage conditions anddocumented inactivation of penicillinase-stable penicillins under conditions of highhumidity and temperature. They recom-mended that the stock supply of discs shouldbe stored in a vacuum desiccator at -20 C toprovide maximum stability. The desiccatorshould be removed from the freezer at least 1hr before the discs are used to prevent conden-sation on the discs with subsequent inactiva-tion of the drug. The present study documentsthe extremes of temperature and humidity towhich working supplies of discs may be ex-posed within one laboratory. In other parts ofthe country, greater or lesser extremes of tem-

perature and humidity certainly could be en-countered. The conditions of storage duringtransit from the manufacturer to the labora-tory is always an unknown factor. Once in thelaboratory, the discs can be brought in and outof the refrigerator several times before theworking supply is consumed. For this reason itis difficult to determine how long the discs canbe used before the drug is inactivated. Dailyquality control tests should be carried out tocheck the performance of the discs being usedwith each batch of tests. Obviously, fewertechnical problems will be encountered if onlythe more stable penicillinase-resistant penicil-lins are used for routine testing. Our studiesindicate that oxacillin is satisfactorily stableunder the storage conditions tested. Nafcillinwould be the next best choice on the basis ofstability alone, with methicillin the most likelyto become inactivated during storage. Cloxa-cillin discs failed to discriminate betweenstrains which were sensitive and resistant bythe dilution test; thus, although stable, theyare not suitable for use.

Because oxacillin discs are the most stableand provide clearcut separation of susceptibleand resistant populations when tested with thedisc method of Bauer et al. (6), they are pre-ferred for routine testing purposes. Nafcillindiscs also discriminate between susceptibleand resistant S. aureus but are somewhat lessstable than those of oxacillin. Also, some re-sistant strains may show only a fine film ofbacterial growth around these discs suggestinga zone of inhibition. Methicillin discs are suit-ably discriminatory, but their instability canprovide a serious potential source of technicalerror unless they are stored under optimalconditions (18).

Zone size criteria for these discs are shownin Table 2. Interpretive zone standards forcloxacillin could not be established becausesome resistant strains gave large, clear zones ofinhibition which were as large or larger thanthose seen with susceptible strains.

Other reports have indicated that methi-cillin resistance can be detected with filter-paper discs only when the agar medium is sta-bilized with 5% NaCl (2, 10, 12) or by incu-bating the test plates at 30 C rather than 35 to37 C (1, 12, 13). However, most of these studieswere done with lighter inocula and more po-tent discs which could cause resistant staphy-lococci to show large zones of inhibition underthe usual conditions of temperature and to-nicity. With the Kirby-Bauer disc diffusiontechnique, using the recommended disc con-tents and an incubation temperature of 35 C,

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MCG. NAFCILLIN OR CLOXACILLIN/ML.

FIG. 5. Comparison of the survival of one strain of methicillin-resistant S. aureus when exposed to in-creasing concentrations of nafcillin and cloxacillin.

1070U1 STRAIN 967I.. 0 (6MM CLOXACILLIN ZONE)Z

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FIG. 6. Comparison of the survival of two strains of methicillin-resistant staphylococci when exposed toincreasing concentrations of cloxacillin. Strain 967 had shown a 6-mm zone of inhibition around the 1-ugcloxacillin disc when tested by the Kirby-Bauer method; strain 896 had shown a 16-mm zone of inhibition.

these additional procedures appear to be un-necessary.The success of the Kirby-Bauer method in

detecting methicillin-resistant staphylococcidepends on close control of incubator tempera-ture. Increasing the temperature of incubationfrom 35 to 37 C may cause resistant strains toappear sensitive. Another laboratory has hadsimilar findings (Thornsberry et al., personalcommunication). Regulation of incubationtemperature in a clinical laboratory can be

quite difficult due to frequent opening andclosing of the incubator doors. For accuratesusceptibility testing of staphylococci, incuba-tion temperature must not exceed 35 Cthroughout incubation. To ensure this, incu-bator temperature should be checked afterovernight use, before the doors are opened inthe morning.The failure of cloxacillin discs to detect

strains which show high cloxacillin MIC andare resistant to other penicillinase-stable peni-

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TABLE 2. Disc zone sizes and standards

Inhibitory zone MICDisc diameter (mm) (;&g/ml)

Drug potency Resist- Inter-(JAg) ant medi- Suscep- Suscep-

(mm) ate tible tible

Methicillin 5 9 or 10-13 14 or 2.5 orless more less

Nafcillin 1 10 or 11-12 13 or 0.6 orless more less

Oxacillin 1 10 or 11-12 13 or 0.6 orless more less

cillins has been reported previously. Kayser(14) found discrepancies between MIC andzone sizes. Hewitt et al. (13) also documentedthat cloxacillin discs failed to discriminatebetween sensitive and resistant staphylococci,even when a hypertonic medium was used.The inability of cloxacillin discs to detect all

resistant staphylococci is due, in part at least,to the low proportion of resistant cells withinthe population. Intermediate concentrations ofcloxacillin inhibit a greater proportion of theindividual cells than do other related drugs.Therefore, few, if any, colonies are able to growon the agar in the immediate vicinity of thecloxacillin disc where the drug concentration isrelatively high. With methicillin, oxacillin, ornafcillin growth around the discs may showreduced density but, nonetheless, there isusually confluent growth right up to the edgeof the disc. We have not yet found a strain ofS. aureus with a sufficiently low proportion ofresistant cells to yield a "false-sensitive" disctest with these agents under the specifiedtesting conditions.Whether the extreme heterogeneity of staph-

ylococci resistant to cloxacillin provides anadvantage in the clinical use of the antibioticis uncertain, but any potential advantage ofcloxacillin is probably offset by its high degreeof protein binding. Clinical studies adequatelyanswering this question are not available. Itmay be that more quantitative approaches tosusceptibility testing, such as population sur-vival studies, are necessary for determining therelative efficacy of these agents.The current data confirm that isolates of S.

aureus can be separated into two distinctgroups (resistant and susceptible to methicillinand related antimicrobials) and indicate thatthe Kirby-Bauer disc test will distinguish be-tween these groups provided the incubatortemperature is 35 C or below. Cloxacillin and

dicloxacillin discs should not be used. Oxa-cillin discs are preferred; if methicillin discsare used, careful attention must be paid to theconditions of storage. Use of nafcillin discsrequires more careful inspection of the "inner"zone of inhibition to detect the growth of re-sistant staphylococci.

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service training grants 5-T01 AI 00169-08 and AI 00146-09from the National Institute of Allergy and Infectious Dis-ease.We thank Jacques J. Roy and Robert Hoo for expert tech-

nical assistance.

LITERATURE CITED1. Annear, D. 1968. The effect of temperature on the resist-

ance of Staphylococcus aureus to methicillin andsome other antibiotics. Med. J. Aust. 1:444-446.

2. Barber, M. 1964. Naturally occurring methicillin-re-sistant staphylococci. J. Gen. Microbiol. 35:183-190.

3. Barber, M., and P. M. Waterworth. 1962. Antibacterialactivity of the penicillins. Brit. Med. J. 1:1159-1164.

4. Barrett, F. F., R. F. McGehee, and M. Finland. 1968.Methicillin-resistant Staphylococcus aureus at BostonCity Hospital. N. Engl. J. Med. 279:441-448.

5. Barry, A. L., F. Garcia, and L. D. Thrupp. 1970. Animproved single disc method for tksting the antibioticsusceptibility of rapidly-growing pathogens. Amer. J.Clin. Pathol. 53:149-158.

6. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M.Turck. 1966. Antibiotic susceptibility testing by astandardized single disk method. Amer. J. Clin. Pa-thol. 45:493:496 (4).

7. Benner, E. J., and V. Morthland. 1967. Methicillin-re-sistant Staphylococcus aureus. N. Engl. J. Med. 277:678-680.

8. Bulger, R. J. 1967. A methicillin-resistant strain ofStaphylococcus aureus. Clinical and laboratory expe-rience. Ann. Intern. Med. 67:81-89.

9. Chabbert, Y. A., J. G. Baudens, J. F. Acar, and G. R.Gerbaud. 1965. La resistance naturelle des staphylo-coques a la methicilline et l'oxacilline. Revue Fr.Etud. Clin. Biol. 10:495-506.

10. Churcher, G. M. 1968. A screening test for the detectionof methicillin-resistant staphylococci. J. Clin. Pathol.21:213-217.

11. Griffith, L. J., and C. G. Mullins. 1968. Drug resistanceas influenced by inactivated sensitivity discs. Appl.Microbiol. 16:656-658.

12. Hallander, H. O., G. Laurell, and K. Dornbusch. 1969.Determination of methicillin-resistance of Staphylo-coccus aureus: heterogeneity and its influence on thedisc diffusion method. Scand. J. Infect. Dis. 1:169-174.

13. Hewitt, J. H., A. W. Coe, and M. T. Parker, 1969. Thedetection of methicillin-resistance in Staphylococcusaureus. J. Med. Microbiol. 2:443-456.

14. Kayser F. H. 1967. Zur Resistenzprufung der Staphylo-kokeen gengen penicillinase-feste penicilline und ce-phalosporin. Pathol. Microbiol. 30:381-391.

15. O'Toole, R., W. L. Drew, B. J. Dahlgren, and H. N.Beaty. 1970. An outbreak of methicillin-resistantStaphylococcus aureus infection. Observations in hos-pital and nursing home. J. Amer. Med. Ass. 213:257-263.

16. Parker, W. T., and M. P. Jevons. 1964. A survey of

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methicillin-resistance in Staphylococcus aureus. Post-grad. Med. 40 (Suppl):170-281.

17. Seligman, S. J. 1966. Methicillin-resistant staphylo-cocci. Genetics of a minority population. J. Gen. Mi-crobiol. 42:315-322.

18. Sherris, J. C., A. L. Rashad, and G. A. Lighthart. 1967.The laboratory determination of antibiotic suscepti-bility to ampicillin and cephalothin. Ann. N. Y. Acad.Sci. 145:248-267.

LND S. AUREUS STRAINS 247

19. Steers, E., E. Foltz, B. S. Graves, and J. Riden. 1959.An inocula replicating apparatus for routine testing ofbacterial susceptibility to antibiotics. Antibiot. Chem-other. 9:307-311.

20. Sutherland, R. 1964. Discussion of paper by Parker andJevons. Postgrad. Med. 40 (Suppl):170-281.

21. Sutherland, R., and G. N. Rolinson. 1964. Characteris-tics of methicillin-resistant staphylococci. J. Bacte-riol. 87:887-899.

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