Impact of Prolonged Incubation on Disk Diffusion Susceptibility Test ...

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paste from the tops of four or five isolated colonies was inoculated into 5.0 ml oftryptic soy broth (Calscott, Carson,. Calif.) and incubated for 2 to 4 h at 35°C. The ...
Vol. 25, No. 5

JOURNAL OF CLINICAL MICROBIOLOGY, May 1987, p. 840-844

0095-1137/87/050840-05$02.00/0 Copyright © 1987, American Society for Microbiology

Impact of Prolonged Incubation on Disk Diffusion Susceptibility Test Results for Staphylococcus aureus MAURY E. MULLIGAN,12 3* DIANE M. CITRON,' RICHARD Y. Y. KWOK,2 J. PATRICK WHEELOCK,' F. KIAIE FARROHI,2 JANET A. HINDLER,4 AND LYNN JOHNSTON' Medical' and Research2 Services, West Los Angeles Veterans Administration Medical Center, Los Angeles, California 90073, and Laboratory Service, University of California, Los Angeles, Center for the Health Sciences,4 and University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 Received 2 September 1986/Accepted 3 December 1986

Because strains of Staphylococcus aureus that are resistant to penicillinase-resistant penicillins may be difficult to detect in the clinical laboratory, a variety of changes in methodology have been suggested to increase their detection. In 1984, the West Los Angeles Veterans Administration Medical Center experienced an increase in clinically significant strains of oxacillin-resistant S. aureus. To insure that such strains would not be missed by the disk diffusion test methods employed for routine testing, changes in methodology were insituted. These included interpreting zone diameters around oxacillin disks at 48 h of incubation. We collected 139 isolates from patients thought to have oxacillin-resistant S. aureus based on these test results and later retested the isolates using microdilution MIC testing. Only 85 isolates (61%) had microdilution oxacillin MICs of .8.0 ,ig/ml, whereas 54 (39%) had oxacillin MICs of -2.0 ,ug/ml. A review of medical records revealed that in 1 year there were 98 patients with isolates appearing resistant by disk diffusion but not confirmed by microdilution MICs; many patients were placed in isolation and treated with specific antimicrobial agents. We conclude that incubation of oxacillin disk diffusion tests for longer than 24 h in conjunction with disregard for resistance to other classes of antimicrobial agents may result in an unacceptably high degree of false resistance results. Because the resistance of S. aureus has important therapeutic and infection control implications, it is necessary to recognize problems that may result in ambiguous or inaccurate susceptibility results.

The penicillinase-resistant penicillins, oxacillin, methicillin, and nafcillin, are antimicrobial agents of choice for treating Staphylococcus aureus infections. However, clinical isolates that are resistant to these agents are becoming more common. Generally, oxacillin is the agent that is used for in vitro susceptibility tests in the clinical laboratory to detect resistance to this group of drugs. Some isolates of oxacillin-resistant S. aureus can be detected readily by using standard test conditions. With these isolates, virtually all cells in the test population express resistance and are often described as demonstrating homogeneous resistance (11). The detection of oxacillin resistance in other S. aureus isolates can present a problem in the clinical laboratory. Under standard test conditions, only a small number of cells express resistance, whereas the majority appear to be susceptible. This phenomenon has been referred to by many designations, including heteroresistance and occult resistance (4, 12). It is believed that this varied expression of resistance is simply a phenotypic trait and that all cells in the population have the genetic predisposition for intrinsic resistance and will demonstrate this resistance when tested under appropriate conditions. Variations of in vitro test methods that have been shown to enhance detection include increasing osmolality of test media, increasing inoculum size, using inocula prepared from 24-h colonies, decreasing incubation temperature below 37°C, and increasing the length of incubation. Generally, isolates with such intrinsic resistance demonstrate oxacillin MICs of .8.0 ,ug/ml when tested by microdilution in broth supplemented with 2% NaCi, which is the medium commonly employed at this time in clinical laboratories (13). *

More recently, another type of oxacillin resistance has been described. This more subtle type of resistance has been shown to be due to the lack of complete stability of oxacillin to staphylococcal beta-lactamase and production of excessive amounts of beta-lactamase by some strains of S. aureus. These isolates, which have oxacillin MICs at or very near the breakpoint for susceptibility (2.0 to 4.0 ,ug/ml), have been referred to as "borderline" isolates (7). Their betalactamase-mediated resistance appears to be distinct from the classic intrinsic resistance that is not dependent upon beta-lactamase production. Although the clinical significance of this type of resistance is unknown, recognition of these strains has further complicated the problem of differentiating oxacillin-resistant from susceptible S. aureus strains. As clinically significant isolates of oxacillin-resistant S. aureus have been encountered with increasing frequency, changes in methodology designed to insure detection of the difficult-to-detect heteroresistant strains have been suggested. At the West Los Angeles Veterans Administration Medical Center, most strains of S. aureus (those not recovered from normally sterile sites) are tested for susceptibility by the disk diffusion method. An increase in frequency of oxacillin-resistant isolates noted in April 1984 prompted a change in methodology in keeping with then-current opinions: standard Kirby-Bauer procedures were employed, with the exception that susceptibility plates for isolates of S. aureus were reincubated for an additional day at 30°C before the final reading of the zone diameter around the oxacillin disk. It became apparent that this change in methodology caused a much larger number of isolates to be identified as resistant to oxacillin if any haze or growth of isolated colonies within the zone of inhibition was considered significant. With the goal of identifying optimal methods for our

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routine disk diffusion susceptibility testing, we subsequently reevaluated the susceptibilities of selected isolates by using supplemented microdilution broth MIC testing. We also attempted to assess the impact on patient care resulting from the change in method for detecting oxacillin resistance in isolates of S. aureus. MATERIALS AND METHODS Bacteria. All isolates of S. aureus were obtained from specimens submitted to the clinical microbiology laboratory at West Los Angeles Veterans Administration Medical Center for routine culture and susceptibility testing. The identity ofthese isolates was established by a positive coagulase test. Coagulase tests were performed either by the slide method or tube method or both with citrated rabbit plasma (Difco Laboratories, Detroit, Mich.). Isolates that were reported to be oxacillin resistant according to disk diffusion results obtained during initial routine testing were collected and saved for further study. Initially, 139 isolates, each from a different patient, were examined. These isolates were stored at -40°C in 10% skim milk and then subcultured to 5% sheep blood agar plates two times before testing. Quality control of disk diffusion and MIC tests included testing of S. aureus ATCC 25923 and two additional highly resistant S. aureus isolates obtained in house. Initial disk diffusion testing of clinical isolates. The disk diffusion test (1) described in the National Committee for Clinical Laboratory Standards (NCCLS) protocol M2-A2 (8) (the protocol still in use when the study was initiated) was followed for routine susceptibility testing of clinical isolates of S. aureus with modifications as indicated below. Cell paste from the tops of four or five isolated colonies was inoculated into 5.0 ml oftryptic soy broth (Calscott, Carson, Calif.) and incubated for 2 to 4 h at 35°C. The turbidity of this log-phase suspension was adjusted to that of a McFarland 0.5 standard before inoculation of commercially prepared Mueller-Hinton agar plates (BBL Microbiology Systems, Cockeysville, Md.). The following disks (Difco) were tested: chloramphenicol (30 ,ug), clindamycin (2 ,ug), erythromycin (15 ,ug), gentamicin (10 ktg), oxacillin (1 ,ug), penicillin (10 U), tetracycline (30 ,ug), trimethoprim-sulfamethoxazole (1.25 ,ug-23.75 ,ug), and vancomycin (30 ,ug). After 16 to 18 h of incubation at 35°C in a non-CO2-supplemented environment, zones of inhibition around all disks except oxacillin were measured by using reflected light and interpreted according to NCCLS recommendations. Any zone around an oxacillin disk was examined by using transmitted light and evaluated as follows: (i) isolates with l10-mm zones at 18 h were called oxacillin resistant; (ii) isolates with >10-mm zones at 16 to 18 h were incubated for an additional 24 h at 30°C and reexamined. The appearance of any colonies or haze within the 10-mm zone after the 2 days of incubation was considered significant. Follow-up disk diffusion testing. The disk diffusion procedure described in NCCLS protocol M2-A3 (10), which became the approved standard in December 1984 and included specific recommendations for testing for methicillinresistant staphylococci, was used to study the previously collected isolates with modifications as indicated. Cell paste from the tops of four or five colonies no greater then 24 h old was used to prepare a suspension equivalent to a 0.5 McFarland standard and inoculated onto Mueller-Hinton Il agar plates freshly prepared (within 24 h) in house by

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following the instructions of the manufacturer (BBL). In some cases, this suspension was also inoculated onto commercially prepared Mueller-Hinton agar plates. Disks were applied and plates were incubated, examined, and evaluated as described above. In-house-prepared oxacillin disks (4 ,ug)

were also evaluated. Microdilution MIC testing. Microdilution MIC trays were manufactured in house by using the MIC-2000 System (Dynatech Laboratories, Inc., Alexandria, Va.) by the procedure described in NCCLS protocol M7-T (9). Log2 dilutions of oxacillin at concentrations of 0.125 to 128.0 pg/ml were prepared in Mueller-Hinton broth supplemented with calcium and magnesium. In addition, 2% NaCl was added (13). Cell paste from the tops of four or five colonies no greater than 24 h old was used to prepare a suspension equivalent to a McFarland 0.5 standard (the same bacterial suspension used for follow-up disk diffusion testing) and was then further diluted 1:10 in water. MIC trays were inoculated with disposable inoculators (Dynatech) that delivered 0.01 ml to obtain a final concentration of approximately 1.5 x 106 CFU/ml (1.5 x 105 CFU per well). Trays were sealed and incubated for 24 h at 35°C in a non-CO2-supplemented environment. The MIC was defined as the lowest concentration that inhibited growth as determined visually. Ail tests were reevaluated after an additional 24 h of incubation at

300C. Evaluation of clinical impact of changes in methodology of susceptibility testing. Reflecting the increased frequency of highly resistant S. aureus (oxacillin MIC, -8.0 ,ug/ml), our hospital policy was to treat patients with suspected or confirmed serious infections due to S. aureus with vancomycin until the clinical laboratory could confirm the absence of oxacillin-resistant S. aureus. In addition, any patient with an isolate thought to be oxacillin-resistant S. aureus was placed in isolation for infection control purposes. Because commercial systems for microdilution MIC testing to determine susceptibility of staphylococci to methicillin or oxacillin were reported to be unreliable at the time (3), clinical and infection control judgements were made on the basis of disk diffusion susceptibility results, with the exception that selected isolates from patients with serious infections were occasionally referred to a reference laboratory for determination of oxacillin MICs. Because some of the isolates that appeared resistant by disk diffusion testing only after 48 h of incubation were found to have oxacillin MICs of 2 or 4 ,ug/ml (a result that would now be called borderline), we felt that it would be most prudent to consider resistant all isolates suspected of resistance on the basis of disk diffusion results that incorporated a final evaluation at 48 h. At the time, it seemed likely that most of the isolates would have oxacillin MICs of not less than 2.0 ,ug/ml when subsequently tested. All patients believed to have oxacillin-resistant S. aureus were monitored throughout their hospitalization for infection control purposes. These records as well as patients' medical charts were reviewed to determine any antimicrobial therapy given and any infection control measures instituted as a result of having an isolate recovered that was thought to be oxacillin-resistant S. aureus. The exact number of patients with cultures revealing S. aureus during the study period is not known because microbiology laboratory results had not yet been computerized. Therefore, to estimate the frequency of recovery of all S. aureus from clinical specimens, microbiology reports for a single typical month were retrieved for review to determine the disk diffusion susceptibility profile of isolates considered oxacillin resistant by the initial disk diffusion testing as well

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TABLE 1. Correlation of disk diffusion susceptibility results using commercially prepared media with microdilution MICs

Length (h) rsist diffusion, By *isk :incubationb

of

P + OX P + OX + ER P + OX + ER + TE P + OX + GM P + OX + ER + CC

P + OX + ER + CC + GM P + OX + ER + CC + GM + TE P + OX + ER + CC + GM + SXT P + OX + ER + CC + GM + TE + CHL

24 48 24 48 48 48 24 48 24 48 24 24 24

No. of isolates

6 32 1 8 1

2 41 2 24 1 14 5 2

0.25

No. (%) of isolates with oxacillin MICs 0.5 2 1

2 (33)

5 (16) 0

2(25) 1 (100) 1 (50) 0 0 0 1 (100) 0 0 0

0 4 (12) 0 5 (63) 0 1 (50) 0 1 (50) 0 0 0 0 0

3 (50) 19 (59) 0 1 (13) 0 0 0 1 (50) 0 0 0 0 0

(uWg/ml) of:

1 (17) 4 (13) 1 (100) 0 0 0 0 0 1 (4)

0 0 0 0

16

0 0 0 0 0 0 41 (100) 0 23 (96) 0 14 (100) 5 (100) 2 (100)

a Abbreviations for drugs: P, penicillin; OX, oxacillin; ER, erythromycin; TE, tetracycline; GM, gentamicin; CC, clindamycin; SXT, trimethoprimsulfamethoxazole; CHL, chloramphenicol. b Hours of incubation required for resistance to be detected by initial disk diffusion testing.

the time of incubation at which oxacillin resistance was reported. RESULTS Comparison of susceptibility test results. The initial collection and storage of isolates of S. aureus considered oxacillin resistant by disk diffusion occurred from April 1984 through August 1985. Comparison with MIC determinations was completed in December 1985. The results of these tests correlated with the disk diffusion results are given in Table 1. Because cephalosporin susceptibility results were considered unreliable and sometimes were inconsistent for oxacillin-resistant S. aureus, these results are not presented. Of the 139 isolates tested, 54 had oxacillin MICs of s2.0 ,ug/ml at 24 h. Of these, 38 isolates (approximately 27% of ail isolates tested) did not demonstrate resistance to any of the non-beta-lactam antimicrobial agents tested. Only six of these isolates were read as oxacillin resistant by disk diffusion within 24 h; five of these strains had oxacillin MICs of '1.0 ,ug/ml, and one had an oxacillin MIC of 2.0 ,ug/ml. Of the 32 isolates that were resistant to only beta-lactam agents and were read as oxacillin resistant by disk diffusion only at the 48-h reading, 28 isolates had oxacillin MICs of 1.0 ,uglml, whereas the other 4 isolates had oxacillin MICs of 2.0 ,ug/ml. After the initial phase of the study, 116 additional isolates that appeared resistant by disk diffusion because of haze or isolated colonies within the zone of inhibition at 24 or 48 h but were susceptible to ail non-beta-lactam drugs were tested; none was intrinsically resistant S. aureus. Evaluation of microtiter tests at 48 h showed that no change from 24-h results occurred for 71 isolates (51%). A one-step increase was noted for 52 isolates (37%), and 16 isolates (12%) had increased oxacillin MICs greater than one step. as

Isolates with disk diffusion resistance to the beta-lactams plus erythromycin, to the beta-lactams plus erythromycin and tetracycline, or to the beta-lactams plus gentamicin had in all cases oxacillin MICs of s2.0 ,ug/ml. Of the 86 isolates demonstrating disk diffusion resistance to oxacillin at 24 h plus resistance to clindamycin and any other antibiotic, 85 (98%) had oxacillin MICs of -8.0 ,ug/ml. The three strains with multiple resistance of this type that were read as oxacillin resistant by disk diffusion only after 48 h were found to have oxacillin MICs of l20 1 2 1 3 1 1 1 1 1 5 3 5 6 1 1i 1 0.5 1 2 1 2 1 2 1 1 0.25 2 3 4 3 2 1 3 0.125

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NCCLS protocol M2-A3 with zones of inhibition around the 1-,ug oxacillin disk determined at 24 h. Prolonged incubation was not performed for any isolate with a zone diameter of .13 mm unless resistance to other classes of drugs was noted. When there was resistance to multiple classes of drugs or any question of oxacillin resistance based on 24-h disk diffusion results, isolates were further evaluated by the now more reliable commerical microdilution MIC testing. In addition, because disk diffusion could not be relied upon to detect isolates with borderline susceptibility, we decided that microdilution MICs would be determined for all isolates of S. aureus recovered from normally sterile sites. Clinical impact of altered methodology. During the period from April 1984 to December 1985, when extended incubation was being employed, isolates from 190 patients were considered oxacillin resistant by initial disk diffusion. Original laboratory records were not available for five isolates. Using microdilution MIC results, we determined that of the 185 isolates for which sufficient original laboratory data were available, 121 (65%) did not have oxacillin MICs of .8.0 ,ug/ml. Thus, although borderline resistance to oxacillin occurred within this group, we considered that isolates of 121 patients had been incorrectly labeled oxacillin resistant if that term is understood to indicate the classic intrinsic resistance. Of the 121 patient isolates, 98 were identified in 1985. We reviewed the patient records to determine the impact of their susceptibility test results. During 1985, approximately 948 patients had an isolate of S. aureus recovered from a clinical specimen, and only 37 patients had isolates that would be considered oxacillin resistant by MIC testing (MIC, -8.0 ,ug/ml), whereas 98 patients had borderline or susceptible isolates. Of these 98 patients, 61 were inpatients in 1985. Of these, four died within 5 days of recovery of S. aureus and eight were discharged within 5 days. The remaining 49 patients were subjected to isolation procedures for infection control purposes. The duration of isolation ranged from 4 to 70 days with an average of 23 days. Complete records of antimicrobial therapy could be obtained for 55 of the 61 patients. Of these, 40 received antimicrobial therapy directed against oxacillin-resistant S. aureus. Parenteral vancomycin was given to 34 patients (although two of these who were on maintenance hemodialysis would probably have received this drug regardless of culture results). One of the vancomycin-treated patients also received a course of oral rifampin plus trimethoprim-sulfamethoxazole. Three other patients who had no parenteral therapy were given that oral regimen, and two received trimethoprim-sulfamethoxazole alone. Thus, 38 of the 55 patients (69%) whose complete records could be located received antimicrobial therapy directed against oxacillin-resistant S. aureus. DISCUSSION first Although recognized in the 1960s, strains of S. aureus that are resistant to semisynthetic penicillins such as methicillin and oxacillin have become common in the United States only in recent years (2). Their increased prevalence has led to increased attention to methods for their detection (6, 13), an appropriate attention because detection can be difficult for heteroresistant strains that have only a small portion of cells displaying resistance in standard laboratory conditions. However, it is important to emphasize that relatively minor changes in methodology can lead to ambiguous or inaccurate results. We were unfortunate in that our hospital experienced a marked increase in recovery of oxacillin-resistant S. aureus

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(with a variety of susceptibility patterns rather than with one distinctive epidemic antibiogram) at a time when there were changes occurring in the recommendations for testing susceptibility of S. aureus. Because commercial methods for microdilution MIC testing for detection of heteroresistant S. aureus had not yet been perfected, we had to rely on disk diffusion results or resort to expensive and delayed testing by sending isolates to a reference laboratory for MIC determinations. An additional misfortune is that we encountered isolates with oxacillin MICs of 2 to 4 ,ug/ml among our first problem isolates. These isolates with borderline resistance have only recently been described and clearly differentiated from the classic isolates with intrinsic resistance (7). Because we had anecdotal evidence to suggest that the nowtermed borderline isolates might display clinically significant resistance, we felt that laboratory methods that would detect these strains as well as those with intrinsic resistance would be prudent. Until the clinical significance of borderline resistance is determined, laboratories must decide how carefully to look for these organisms and how to report them to clinicians, and institutions must determine what therapy and isolation precautions should be instituted for patients with these isolates. It was most disconcerting to us that it was not until we determined microdilution MICs for representative strains that we realized the frequency with which even the clearly susceptible strains (with oxacillin MICs of .1.0 ,ug/ml) can be considered resistant if oxacillin disk diffusion results at 48 h are used as the only criterion. In our initial study, 47 of 139 isolates (34%) thought to be oxacillin resistant by disk testing had oxacillin MICs of '1.0 ,ug/ml. We feel that is very important to point out the impact that can result when a relatively minor change in methodology has very major clinical consequences. Although current recommendations do not advocate routine incubation of all oxacillin disk diffusion tests for S. aureus for 48 h (10), the potential problems resulting from this practice have not been widely publicized. Because the methodology has evolved so rapidly, current standard texts still suggest that this practice may be considered (5). Our findings confirm the wisdom of current recommendations that advise against routine evaluation of disk diffusion susceptibility results at 48 h of incubation, since this practice results in a high degree of ambiguous or inaccurate results. However, even with incubation limited to 24 h, a small number of isolates may appear resistant by disk diffusion but may be found to be susceptible by microdilution. Although it has been reported that different commercial media give different results in susceptibility testing of true oxacillinresistant S. aureus (4), the problem that we observed, that of truly susceptible isolates appearing to be resistant, has not been reported. The use of freshly prepared media for ail disk diffusion testing of S. aureus is not a practical procedure for most laboratories, but the fact that variations in media may influence results is noteworthy and suggests the need for further study and standardization. Since we studied media from only one manufacturer, we cannot comment on the degree to which there may be variation with other products. We found that considering disk diffusion susceptibilities to non-beta-lactam agents was a very useful adjunct for differentiating isolates that would prove to be oxacillin susceptible from those oxacillin resistant by broth microdilution. All of the isolates with oxacillin MICs of .8.0 ,ug/ml that we identified in this study were resistant to clindamycin as well as to beta-lactam drugs. Erythromycin resistance alone was much less useful as a predictor of intrinsic oxacillin resistance. Because there is good evidence that different geo-

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graphic areas encounter different strains of S. aureus (14), the utility of clindamycin resistance seen in our study cannot be generalized to other areas. Indeed, since performing this study, we have encountered an increase in clindamycin resistance among strains of oxacillin-susceptible S. aureus at our institution. Continued surveillance at our institution has indicated that 48 h of incubation has been required to detect oxacillin resistance by disk diffusion for a very small number of strains. All of these have been resistant to multiple other classes of agents, however. It is of note that the recently described borderline resistance of S. aureus is not detectable with current standard disk diffusion methods. Realization of this has led to our present practice of determining the MIC for any isolate of S. aureus recovered from a normally sterile site. Although the clinical significance of such resistance has not been determined, the information may be of value when a serious infection due to S. aureus is present. Studies are needed to determine the appropriate therapy and infection control measures for these organisms. The practical problems with laboratory identification of oxacillin-resistant S. aureus may have very important consequences. At our institution, it is the policy to treat serious confirmed or suspected infections due to S. aureus with vancomycin until susceptibility to beta-lactam agents is demonstrated by the laboratory. Patients thought to be infected or colonized are also placed in isolation. Considerable hardship for patients and health care workers as well as increased cost and possible increased toxicity may result when susceptible strains are incorrectly considered to be resistant. In addition, placement in other facilities may be denied to patients who are labeled as carriers of oxacillinresistant S. aureus. Because our facility provides care for patients with acute and chronic illnesses, we could not determine the degree to which the inaccurate label of oxacillin-resistant S. aureus prolonged hospitalization. Nevertheless, isolation measures imposed during that time are themselves costly. As improved methodology increases our ability to detect bacterial resistance, it may also lead to confusion as previously unrecognized types of resistance are identified (such as borderline resistance of S. aureus). In addition, as changes in methodology are instituted, there can be initial unexpected consequences such as ambiguous or inaccurate results. ACKNOWLEDGMENTS We thank the medical technologists of the Clinical Microbiology Laboratory and the Infection Control Practitioners, Veterans Administration West Los Angeles Medical Center, and Kimi Ishii for manuscript preparation.

LITERATURE CITED 1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antimicrobial susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. 2. Haley, R. W., A. W. Hightower, R. F. Khabbaz, C. Thornsberry, W. J. Martone, J. R. Allen, and J. M. Hughes. 1982. The emergence of methicillin-resistant Staphylococcus aureus infections in the United States. Ann. Intern. Med. 97:297-308. 3. Hansen, S. L., and P. K. Freedy. 1984. Variation in the abilities of automated, commerical, and reference methods to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. J. Clin. Microbiol. 20:494-499. 4. Hindler, J. A., and C. B. Inderlied. 1985. Effect of the source of Mueller-Hinton agar and resistance frequency on the detection of methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 21:205-210. 5. Kloos, W. E., and J. H. Jorgensen. 1985. Staphylococci, p. 151. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed), Manual of clinical microbiology. American Society for Microbiology, Washington, D.C. 6. McDougal, L. K., and C. Thornsberry. 1984. New recommendations for disk diffusion antimicrobial susceptibility testing for methicillin-resistant (heteroresistant) staphylococci. J. Clin. Microbiol. 19:482-488. 7. McDougal, L. K., and C. Thornsberry. 1986. The role of p-lactamase in staphylococcal resistance to penicillinaseresistant penicillins and cephalosporins. J. Clin. Microbiol. 23:832-839. 8. National Committee for Clinical Laboratory Standards. 1979. Approved standard M2-A2. Performance standards for antimicrobic disk susceptibility tests, 2nd ed. National Committe for Clinical Laboratory Standards, Villanova, Pa. 9. National Committee for Clinical Laboratory Standards. 1983. Tentative standard M7-T. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Laboratory Standards, Villanova, Pa. 10. National Committee for Clinical Laboratory Standards. 1984. Performance standards for antimicrobial disk susceptibility tests, 3rd ed. Approved standard M2-A3. National Committee for Clinical Laboratory Standards, Villanova, Pa. 11. Peacock, J. E. Jr., D. R. Moorman, R. P. Wenzel, and G. L. Mandell. 1981. Methicillin-resistant Staphylococcus aureus: microbiologic characteristics, antimicrobial susceptibilities, and assessment of virulence of an epidemic strain. J. Infect. Dis. 144:575-582. 12. Sabath, L. D. 1982. Mechanisms of resistance to beta-lactam antibiotics in strains of Staphylococcus aureus. Ann. Intern. Med. 97:339-344. 13. Thornsberry, C., and L. K. McDougal. 1983. Successful use of broth microdilution in susceptibility tests for methicillinresistant (heteroresistant) staphylococci. J. Clin. Microbiol.

18:1084-1091. 14. Welch, W. D., and P. M. Southern. 1984. Unusual susceptibility of methicillin-resistant Staphylococcus aureus to erythromycin, clindamycin, gentamicin, and tetracycline at 30 but not 35°C. J. Clin. Microbiol. 19:831-833.