Radiometric in vitro susceptibility testing of Mycobacterium tuberculosis

ANNLDO 3(8) 55-62, 1986

ISSN 0738-1751

VOLUME 3, NUMBER 8, AUGUST 1986

EDITORIAL BOARD

Editor

Associate Editors

DANIEL AMSTERDAM, PhD,

RONALD N. JONES, MD,

State University of N e w York at Buffalo a n d Erie C o u n t y Laboratory

Kaiser-Permanente Medical Care Program

CLYDETHORNSBERRY, PhD, Center for Infectious Diseases, Centers for Disease Control

HAROLD C. NEU, MD,

LOWELLS. YOUNG, MD,

College of Physicians and Surgeons, Columbia University

UCLA School of Medicine

RADIOMETRIC IN VITRO SUSCEPTIBILITY TESTING OF MYCOBA C ~ I ~ U M TUBERCULOSIS EDITOR'S NOTE

55

D. AMSTERDAM

Radiometric In Vitro Susceptibility Testing of

Mycobacterium tuberculosis 55 C. B. [NDERLIED L. S. YOUNG

What's In a N a m e ? M o r e on M e t h i c i l l i n (or is it Oxacillin?)- Resistant Staphylococci 60 C. THORNSBERRY L. K. McDOUGAL

EDITOR'S NOTE

In this month's issue of Tile AMN, Drs. lnderlied and Young review the status of susceptibility testing of Mycobacterium tuberculosis using radiometric methodology. It is interestingto note that although this approach has achieved some degree of acceptance, it has not been applied in a similar routine for aerobic (or anaerobic) microorganisms. The advantages of the instrumental technique are obvious in that one can determine susceptibility more rapidly and additionalIv monitor for the presence of drug resistance. As pointed out by the authors, there are as yet no guidelines for even the more conventional proportional method for testing the susceptibility of M. tuberculosis. The National Committee

ELSEVIER

C L A R K B. INDERLIED A N D L O W E L L S. YOUNG

The clinical relevance of the laboratory diagnosis of mycobacteria, including detection, isolation,

identification and susceptibility testing, often is diminished by the length of time necessary to obtain results. Using conventional methods, the isolation of mycobacteria from clinical specimens commonly takes 4 to 8 weeks and susceptibility test results another 3 to 4 weeks. In the mid to late 1970s radiometric methods for detecting the growth of mycobacteria were developed and an instrument became commercially available that

on Clinical Laboratory Standards (NCCLS) has, under the Chairmanship of Dr. Herbert M. Sommers, formed the Antimycobacterial Susceptibility Testing Subcommittee and it is anticipated that guidelines will be forthcoming. In a previous issue of Tile A M N (Vol. 2, No. 12, December 1985), Wallace, Swenson, and Silcox reported on the susceptibility testing of the rapidly growing mycobacteria. The authors presented tentative zone sizes and minimal inhibitory concentration (MIC) standards for M. fortut'tum and M. chehmac. In another section of this month's issue of The AMN, Clyde Thornsberry and Linda K. McDougal deal in part with the underlying nonscientific problems related to methicillin-resistant staphylococci. An issue they dis-

cuss has been referred to as the noun/adjective dilemma, wherein scientific writers and scientists attempt to limit the length of text by using a string of nouns as modifiers for another noun. Frequently we furthur aggravate the problem when we attempt to abbreviate or form acronyms for the chain of descriptors. Tile authors have attempted to sort out the polyglot of terms that frequently confronts the reader. In this corner, we heartily endorse the suggestions and recommendations and hope that in time they will become accepted and utilized. More importantly, the newly suggested terminology more accurately conveys the dynamics and interactions among microorganisms and drugs and attempts to describe the mechanistic nature of the events that are observed.

Department of Pathology, School of Medicine, University of Southern California, Children's Hospital Of Los Angeles; and Kuzell Institute for Arthritis and Infectious Diseases, Pacific Presbyterian Medical Center, San Francisco, Califoruia

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permitted the automated detection of 14CO2(Bactec, Johnston Laboratories, Towson, Maryland)J '2 This radiometric approach was based on the fact that virtually all mycobacteria, including Mycobacterium tuberculosis (MTB), catabolize fatty acid substrates to carbon dioxide. If these fatty acids are radioactively labeled with 14C, the carbon dioxide end product can be easily detected and quantitated. The result is a rapid and sensitive method for detecting the growth of mycobacteria. The adaptation of this method for in vitro susceptibility testing of MTB was a straightforward extension of the growth-detecting capability. B'4 Over the intervening decade several studies have confirmed the clinical usefulness of the Bactec radiometric method. For example, three recent studies showed that the average time to detection of MTB in clinical specimens by Bactec was 8-12 days earlier than by conventional methods, s- 7 Furthermore, the recovery rates for treated respiratory specimens, untreated tissues and fluids, and smear positive and negative specimens were comparable for the two methods. THE CONVENTIONAL PROPORTION METHOD

The traditional method for susceptibility testing of MTB is by the proportion method, used in either a direct or indirect manner. 8"~The principle of the proportion method is important to understand since the same principle is applied in the radiometric method. By proportion testing, an isolate of MTB is considered susceptible if less than 1% of the population of mycobacteria tested survive exposure to a particular drug at a concentration known to correlate with clini-

THE ANT1MICROBIC NEWSLETTER, V O L U M E 3, N U M B E R 8, A U G U S T 1986

cal effectiveness. The criterion for 1% survival is growth on 7H10 or 7 H l l agar-containing drug that is more than the growth of the inoculum diluted 1:100 and plated on the same medium without drug. The reasoning is that populations of MTB may consist of both resistant and susceptible strains; however, clinical correlation studies have shown that a drug is effective if 99% of the entire population can be inhibited by the drug. In practice, multiple drugs are used in the treatment of M. tuberculosis disease to assure that resistant mutants do not emerge during the course of therapy. Since the frequency of mutation of MTB to resistance for a single drug such as isoniazid (INH) may be 10-5; for two drugs the frequency would be 10-lO or lower, which is an unlikely event given the usual concentration of organisms in clinical specimens. Thus the use of multiple drugs for the treatment of tuberculosis was never based on a presumption that the drugs act synergistically, but rather to prevent the emergence of resistance that can readily occur with single drug therapy. There are two methods commonly used in performing the proportion susceptibility test. The method most frequently used in the United States was developed at the Center for Disease Control (CDC), 10.11or is a modification of this procedure. The method advocated by the World Health Organization (WHO) 12'13 is used in many other parts of the world. There are significant differences between these methods in terms of drug concentrations, media, and preparation of drug-containing media. Although the proportion method is considered the standard method for in vitro susceptibility testing of M. tuberculosis, there is a

lack of uniformity in how the method, either CDC or WHO, is utilized. BACTEC RADIOMETRIC METHOD

The Bactec method utilizes a modified Middlebrook 7H12 TB medium, which is Middlebrook 7H9 broth supplemented with bovine serum albumin (fraction V), catalase, casein hydrolysate and 4 ~Ci of t4C-palmitic acid. 9 The medium (4 mL) is contained in a small glass vial sealed with a septum. The growth of mycobacteria is detected by measuring the amount of 14CCO2 released during 48-72-hour periods over 3-4 weeks and then weekly for an additional 3-4 weeks. The Bactec 460 instrument automatically evacuates the head space above the culture medium, replaces the displaced gas with 510% CO2 in air, and measures the amount of 14CO2 in the evacuated gas in terms of a Growth Index (GI); the GI values are arbitrary units of radioactivity. The amount of 14CO2 released is a measure of growth and if cumulative GI values are plotted as a function of time, the resulting curve approximates a standard growth curve. Therefore, susceptibility to drugs can be defined in terms of decreased GI values compared with a control. Either a direct or indirect method of susceptiblity testing also can be used with the Bactec procedure. The direct method uses a positive Bactec vial, originating from a clinical specimen, as the source of inoculum for susceptibility tests. The positive vial is incubated until the daily GI value is ~ 300-500 and then incubated one additional day. For the indirect method, a small quantity of growth of an isolated organism on solid media is homogenized with glass beads in an

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albumin-polysorbate 80 diluent, then the suspension is adjusted to a McFarland No. 0.5-1 standard with diluent. An acid-fast smear should be made of the inoculum with either the direct or indirect method, to check for purity and consistency (medium or specimen carryover can contribute to turbidity). The primary antimycobacterial drugs are tested at the following concentrations: streptomycin (2.0 and 6.0 I~g/mL), isoniazid (0.2 and 1.0 ~g/mL), rifampin (2.0 I~g/mL), and ethambutol (2.5 and 7.5 p,g/ mL). Note that some concentrations are different from the concentrations described in early package inserts for 7H12A medium provided by Johnston Laboratories. A TB product and procedure manual that describes these changes is available free of charge from the manufacturer. Vials containing drug are inoculated with 0.1 mL of the suspension prepared by either the direct or indirect method and a control vial (without drug) is inoculated with 0.1 mL of a 1:100 dilution of the same suspension. For susceptibility tests the vials are read daily and when the daily GI value of the control vial is 30 or more, the results are interpreted as follows: & GI (Control) > & GI (Drug)--+ SUSCEPTIBLE A GI (Control) ~< & GI (Drug)--* RESISTANT A GI (Control) = k GI (Drug)--+ BORDERLINE, where the A GI is the difference between the GI of the day of interpretation and the GI of the previous day. However, if the GI of the 1:100 control is >30 before day 4, testing should be continued until day 4. Presumably this assists in distinguishing an effect on lag phase from a true inhibition of growth. COMPARISON OF METHODS

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sensitivity is defined as the ability of a test to detect resistance and specificity as the ability of a test to detect susceptibility. From the sensitivity and specificity of a method one can calculate predictive values; however, as with any such measure, the predictive value also is dependent on incidence which in this case is the incidence of resistance with a population of clinical isolates of MTB. Two large collaborative studies that compared the rapid Bactec radiometric method with the conventional proportion method of susceptibility testing of MTB were published in 1981 (Table 1). Siddigi et al4 tested 106 strains of MTB against streptomycin (SM), isoniazid (INH), ethambutol (EMB), and rifampin (RIF) by both methods. Overall there was 98(/,, agreement between the two methods. Of the eight discrepant results, six were resistant only by the proportion method and two were resistant only by the radiometric method. The sensitivity for INH, EMB, and RIF ranged from 83 (RIF) to 100% (EMB), but was only 77% for SM. The specificity for all drugs ranged from 99 to 100%. They pointed out that many of the SMresistant strains had a low proportion of resistant organisms. In this study susceptibility results were available for 99% of the tests in six days using the radiometric method. Snider et al z4 tested nearly 300 strains of MTB against SM, INH, EMB, RIF, and para-aminosalicylic acid (PAS) by both methods, although the method for interpreting the radiometric data varied between the two groups that participated in this study. The radiometric results were compared with results obtained by the CDC using the proportion method. The overall agreement varied from 9198% and 87-97% for the two groups. As with the previously mentioned study, most of the discrepancies were with strains called resistant by the proportion method

and susceptible by the Bactec method. The sensitivity was 80 (EMB) to 100% (RIF) for one group, and 43% (INH at 5 ~g) to 96% (RIF) for the other group. Radiometric testing of SM at low concentrations was a problem for both groups. The specificity was 9298% and 88-99%, respectively. This study also concluded that the degree of resistance was an important factor in the ability of the radiometric method to detect resistance, ie, as the degree of resistance within a population increased, the ability to detect resistance by the radiometric method also increased. Also, there appeard to be a direct correlation between sensitivity and drug concentration. Three more recent studies with large numbers of M. tubercuh~sis clinical isolates reached similar conclusions (Table 1). Roberts et al ~ tested 126 MTB strains using a proportion method and 170 MTB strains using the Bactec radiometric method. Again, all strains were tested against SM, INH, RIF, and EMB. The overall agreement was 97% with a sensitivity of 67 (EMB) to 99% (INH) and a specificity of 98 to 100%. The low sensitivity value was attributed to discrepancies with ethambutol; the radiometric method called four strains susceptible. Overall there were 20 discrepancies between the two methods. The Bactec method called nine strains susceptible that were resistant by the proportion method, and 11 strains that were resistant by the radiometric method were susceptible by the conventional method. The overall time to recovery and susceptibility results by the radiometric method was 18 days as opposed to nearly 39 days by the conventional method. In the second study, Laszlo et al ~ tested 216 strains of MTB of which 134, or 55%, were resistant to one drug. For the same primary antimycobacterial drugs the sensitivity ranged from 18 (EMB) to 100% (RIF), and specificity from 98 to 100%. Again, the majority of

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THE ANTIMICROBIC NEWSLETTER, VOLUME 3, NUMBER 8, AUGUST 1986

TABLE 1. Sensitivity and Specificity of Radiometric and Proportion Susceptibility Testing of M. tuberculosis"

Drug

Concentration (p~g/mL)

SM SM INH INH INH PAS RIF EMB EMB

2.0 10.0 0.2 1.0 5.0 4.0 1.0 5.0 10.0

Study 1 SE SP 0.83

0.95

0.90

0.98

0.88 1.00 0.80

0.92 0.98 0.97

Study 2 SE SP

Study 3 SE SP

Study 4~' SE SP

Study 5' SE SP

0.67 0.78 0.71 0.77 0.43

0.95 0.99 0.96 0.95 0.96

0.77

1.00

0.91

0.98

0.96

1.00

0.88

0.99

0.99

0.99

0.91

0.98

0.96 0.77

0.97 0.88

0.83

1.00

1.00

0.99

0.98 0.67

1.00 1.00

1.00

1.00

0.18

0.99

Study 6 SE SP 0.95

0.93

0.99

0.89

0.93 0.54

0.99 0.99

Study 7" SE SP

Study 8' SE SP

0.97

0.96

0.51

1.00

0.98

0.99

0.97

0.94

1.00 0.88 0.54

1.00 0.99 0.98

0.98 0.88

0.98 0.91

SE = sensitivity; SP = specificity; SM = streptomycin; INH = isoniazid; PAS = para-aminosalicyclic acid; RIF - rifampin; and EMB = ethambutol. " Data were taken from the following references: Study ],,4 Study 2,14 Study 3, ~4 Study 4, ~ Study 5, ~5 Study 6, ~ Study 7, '7 and Study 8. '7 b C o m b i n e d data irrespective of drug concentration. " In this study SM at 4 and 4, RIF at 2 and 40, and EMB at 10 and 2 #g/mL by radiometric (Bactec) and proportion testing, respectively; all other drugs at the indicated concentrations. In this study SM at 4 and 10, RIF at 2 and 1, and EMB at 2.5 and 5 p~g/mL by radiometric (Bactec) and proportion testing, respectively; all other drugs at the indicated concentrations. In this study SM at 4 and 4, RIF at 2 and 40, and EMB at 2.5 and 2 ~g/mL by radiometric (Bactec) and proportion testing respectively; all other drugs at the indicated concentrations.

discrepancies, 29 of 35, were radiometric susceptible and proportion resistant. This s t u d y concluded that the radiometric m e t h o d can be a d o p t e d w i t h o u t reservation if the incidence of resistant strains is or exceeds 5%. The s t u d y by Steadh a m et a116 e x a m i n e d p r o p o r t i o n and radiometric results for 199 strains of MTB. The overall agreem e n t for INH, EMB, and RIF was 96 and 94%. For a larger panel of ten drugs including SM, PAS, ethionamide, kanamycin, amikacin, capreomycin, and cycloserine, the sensitivity was 25 (ethionamide) to 100%, or 88 (PAS) to 100% if ethionamide and ethambutol were excluded. The specificity was 28-100% or 88-100% if cycloserine was excluded. In this case the overall time to susceptibility results by the radiometric m e t h o d was 4.8 days. The problem of detecting resistance was recently analyzed by Siddigi et al ~7 in a report on the interlaboratory susceptibility testing of M. tuberculosis by radiometric and two (CDC and W H O ) proportion methods. They tested 224 clinical isolates that were multiply resistant to antimycobacterial drugs. Overall there was good correlation

b e t w e e n the m e t h o d s using a population of organisms that included a high frequency of resistance, eg, approximately 50% of isolates were resistant to 0.2 ~g/mL INH, about 25% to 2 p,g/mL RIF, about 20% to 4 ~g/mL SM, and about 25% to 2.5 p,g/mL EMB. As in other studies, SM and EMB were most problematic with overall sensitivities of 51% for SM at 4 ~g/mL and 54% for EMB at 10 ~g/mL w h e n radiometric results were c o m p a r e d with p r o p o r t i o n results. These authors concluded that alterations in the concentration of SM and EMB used in the radiometric m e t h o d would improve the sensitivity in comparison with the proportion method. Indeed, changes in SM and EMB concentrations have been

incorporated in the new (March 1986) Johnston Laboratories procedure for MTB susceptibility testing (Table 2). H o w e v e r , these authors also suggested that the concentrations of EMB used in the proportion m e t h o d be reevaluated. Hawkins ~8 recently reiterated these concerns and e m p h a s i z e d the importance of accuracy in testing INH, the primary antituberculotic medication, and s h o w e d that the radiometric m e t h o d m a y not detect as m a n y as 20% of isolates resistant to INH if d r a w n from a patient population with a high freq u e n c y of INH resistance. O t h e r recent studies, 7'19 with smaller n u m b e r s of MTB strains but including secondary drugs, ~4 confirm the overall a g r e e m e n t be-

TABLE 2. Equivalent Drug Concentrations Used in Proportion and Radiometric (Bactec) In Vitro Susceptibility Tests with M. tuberculosis

Drug Isoniazid Rifampin Ethambutol Streptomycin

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Proportion Concentration

Radiometric (p,g/mL)"

0.2

0.2

1.0

1.0

1.0 5.0 10.0 2.0 10.0

2.0 2.5 7.5 2.0 6.0

"Final concentration in 7H10 agar (proportion) or 7H12 broth (radiometric).

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THE ANTIMICROBIC NEWSLETTER, VOLUME 3, NUMBER 8, AUGUST 1986

tween the radiometric and proportion m e t h o d and emphasize the comparatively short time necessary to obtain results by the radiometric method. In addition, Hawkins 1~'2<21 reported on a schedule for radiometric susceptibility testing that obviates the problem of w e e k e n d staff scheduling. CONCLUSIONS The radiometric m e t h o d for testing the susceptibility of MTB to antituberculotic drugs is rapid and convenient and appears to be reliable. In addition, this m e t h o d facilitates testing combinations of drugs and screening new drugs for potential antimycobacterial activity. For example, Heifets et a122 reported on the susceptibility of 65 MTB strains to a variety of cephalosporins using radiometric and conventional methods. They found there was good correlation between the m e t h o d s and by testing a range of drug concentrations they could report minimal inhibitory concentration (MIC) values using the radiometric method. In addition, this m e t h o d has been used to test other mycobacteria, including the M. avium complex, and the m e t h o d shows promise with these organisms as well. 23'24 The clearest advantage of the radiometric m e t h o d is the decreased time necessary to obtain results. The implication for the treatment of patients is obvious. The sooner resistance can be detected, either in primary isolates or in isolates from patients with reactivated or unresponsive disease, the sooner therapy can be altered to exclude ineffective drugs or include more effective drugs. Based on the various studies reported here, the overall correlation between the radiometric and proportion m e t h o d s is good. However, the ability of the radiometric m e t h o d to detect resistance seen with the proportion m e t h o d remains a b o t h e r s o m e concern. The recent report by Siddigi et al, w which indicates that alteration of the concentrations of 0738-1731/86/$0.00

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SM and EMB improves the radiometric-proportion correlation, is encouraging. Although there is evidence that ethambutol, the most problematic drug in the comparative studies reviewed here, may be more of a problem with the proportion m e t h o d than with the radiometric method. 2s Kubica et al 2~ e m p h a s i z e d the importance of experience in performing clinical mycobacteriology and suggested that susceptibility testing be offered only in laboratories that perform at least ten susceptibility tests per week. ~ In our view the facility of the Bactec radiometric m e t h o d is not a sufficient reason at this time to a b a n d o n these guidelines. Until the m e t h o d s of susceptibility testing of mycobacteria are as rigorously standardized, verified, and controlled as are the in vitro susceptibility testing m e t h o d s used with rapidly growing aerobic and facultative anaerobic bacteria, extensive experience is the prevailing factor in assuring accuracy and precision in the susceptibility testing of MTB. The National Committee on Clinical Laboratory Standards (NCCLS) has formed a subcommittee with the objective of developing a standard m e t h o d for the susceptibility testing of mycobacteria. This committee met for the first time at the most recent American Society for Microbiology meeting in Washington, D.C. and expects to issue guidelines for the proportion testing of M. tuberculosis in approximately 12 months. Following the establishment of these guidelines, the committee will consider radiometric susceptibility testing as well as m e t h o d s for testing atypical and rapidly growing mycobacteria. REFERENCES

1. Cummings DM, Rostraph D: Radiometric detection of metabolic activity of Mycobacterium tuberculosis. J Iqucl Med 16:1189-1191, 1975. 2. Middlebrook G, Reggiardo Z, Tigertt WD: Automatable radiometric detection of growth of Mycobacte

3.

4.

5.

6.

7.

8.

rium tuberculosis in selective media. Am Rev Respir Dis 115:1066-1069, 1977. Kertcher JA, Chen MF, Charache P, Hwangbo CC, Camargo EE, Mclntyre PA, Wagner HN: Rapid radiometric susceptibility testing of Mycobacterium tuberculosis. Am Rev Respir Dis 117:631-637, 1978. Siddigi SH, Libonati JP, Middlebrook G: Evaluation of a rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 13:908-912, 1981. Roberts GD, Goodman NL, Heifets L, et al: Evaluation of the Bactec radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear-positive specimens. J Clin Microbiol 18:689-696, 1983. Fadda G, Roe SL: Recovery and susceptibility testing of Mycobacterium tuberculosis from extrapulmonary specimens by the Bactec radiometric method. J Clin Microbiol 19:720-721, 1984. Kirihara JM, Hillier SL, Coyle MB: Improved detection times for Mycobacterium avium complex and Mycobacterium tuberculosis with the Bactec radiometric system. J Clin Microbiol 22:841-845, 1985. Hawkins JE: Drug Susceptibility Testing. In GP Kubica, LG Wayne (ed), The Mycobacteria: A Sourcebook, Part A. New York, Marcel Dekker, Inc., 1984, pp 177-193.

9. McClatchy JK: Antimycobacteria drugs: Mechanisms of action, drug resistance, susceptibility testing, and assays of activity in biological fluids. In V Lorian (ed.), Antibiotics in Laboratory Medicine, 2nd Edition. Baltimore, Williams and Wilkins, 1986, pp 181-222. 10. Vestal AL: Procedures for the isolation and identification of mycobacteria. U.S. Department of Health, Education and Welfare, publication no. (CDC) 76-82301, Atlanta, Center for Disease Control, 1975, pp 97-110. 11. Strong BE, Kubica GP: Isolation and identification of Mycobacterium tuberculosis. A guide for level II laboratories. U.S. Department of Health and Human Services publication no. (CDC) 81-8390. Atlanta, Center for Disease Control, 1981, pp 115 125. 12. Canetti G, Fox W, Khomenko A, et al: Advances in techniques of testing mycobacterial drug sensi-

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13.

14.

15.

16.

17.

THE ANTIMICROBIC NEWSLETTER, VOLUME 3, NUMBER 8, AUGUST 1986

tivity and the use of sensitivity test in tuberculosis control programrues. Bull WHO 41:21~t3, 1969. Canetti G, Froman S, Grosset J, et al: Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull WHO 29:565-578, 1963. Snider DE, Good RC, Kilburn JO, Laskowski LF Jr, Lusk RH, Marr JJ, Reggiardo A, Middlebrook G: Rapid drug-susceptibility testing of Mycobacterium tuberculosis. Am Rev Respir Dis 123:402-406, 1981. Laszlo A, Gill P, Hondzel V, et al: Conventional and radiometric drug susceptibility testing of Mycobacterium tuberculosis complex. J Clin Microbiol 18:1335-1339, 1983. Steadham JE, Stall SK, Simmank JL: Use of the Bactec system for drug susceptibility testing of Mycobacterium tuberculosis, M. kansasii, and M. avium complex. Diagn Microbiol Infect Dis 3:33-40, 1985. Siddiqi SH, Hawkins JE, Laszlo A: Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods. J Clin Microbiol 22:919-923, 1985.

WHAT'S IN A NAME? More on Methicillin (or is it Oxacillin?)-Resistant Staphylococci CLYDE THORNSBERRY AND LINDA K. McDOUGAL Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia In the 1950s, more and more staphylococci became resistant to penicillin, the d r u g of choice, because they were acquiring plasmids containing genes for beta-lactamase production. It was not surprising that the introduction of methicillin--a penicillinase-resistant penicillin ( P R P ) - - w a s a heralded e v e n t in the history of microbiology and infectious disease. It also was not surprising that the almost immediate isolation of a methicillin-resistant Staphylococcus aureus strain was disappointing. This methicillin-resistant staphylo-

18. Hawkins JE: Rapid rnycobacterial susceptibility tests. Clin Microbiol Newsltr 8:101-104, 1986. 19. Laszlo A, Helbecque DM, Tostowaryk W: Proficiency testing of radiometric drug susceptibility tests of M. tuberculosis. Washington DC, Abstracts of the Annual Meeting of the American Society of Microbiology, (abstract #V-41), 1986, p 126. 20. Gross WM, Hawkins JE: Radiometric susceptibility testing of Mycobacterium tuberculosis with secondary drugs. Washington DC, Abstracts of the Annual Meeting of the American Society of Microbiology, (Abstract #C-378), 1986, p 391. 21. Hawkins JE: Nonweekend schedule for Bactec drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 23:934-937, 1986. 22. Heifets LB, Iseman MD, Cook JL, et al: Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporines by radiometric and conventional methods. Antimicrob Agents Chemother 27:11-15, 1985.

coccus p r o v e d to be a very unusual microorganism, which has fascinated and frustrated microbiologists and clinicians for the two and one-half decades since its discovery. The m e c h a n i s m s of the resistance to methicillin were not readily detected and for years there was not even a g r e e m e n t as to w h e t h e r it was chromosomally or plasmid mediated. It was, h o w e v e r , the biologic nature of the organism that created the most interest a m o n g microbiologists and also caused t h e m the most problems in susceptibility testing. It was evident early on that a culture of this organism contained two subpopulations, and e v e n t h o u g h the subpopulations were v e r y different, they could not be separated. Because of this characteristic the organisms were called heteroresistant. Thus the stage was set for a parade of other n a m e s that w o u l d be attached to

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23. Inderlied CB, Bruckner DA, Young LS: Susceptibility of Mycobacterium avium-intracellulare (MAI) to amikacin, BMY-28142 and ciprofloxacin using a radiometric method (Abstract #395) In Program and abstracts of the 24th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington DC, American Society of Microbiology, 1984. 24. Inderlied CB, Yamada JK, Young LS: Comparison of T100, Bactec and proportion in vitro susceptibility test of Mycobacterium avium complex (MAC) (Abstract #629). In Program and Abstracts of the 25th Interscience Conference on Antimicrobial Agents and Chemotherapy. Minneapolis, MN, American Society of Microbiology, 1985. 25. Gangadharam PR, Gonzales ER: Influence of the medium on the in vitro susceptibility of Mycobacterium tuberculosis to ethambutol. Am Rev Respir Dis 102:653-655, 1970. 26. Kubica GP, Gross WM, Hawkins JE, et al: Laboratory services of mycobacterial diseases. Am Rev Respir Dis 112:773-787, 1975.

these organisms. Most of these terms are described in Table 1. It is obvious that we have too m a n y terms and excessive confusion in the descriptions we use for these organisms. At the risk of a d d i n g to the p r o b l e m , we will make some r e c o m m e n d a t i o n s . Oxacillin should be the reference antibiotic since it is the test agent that should be used in most institutions. We should use terms that will indicate the m e c h a n i s m of resistance, ie, w h e t h e r it is resistant d u e to altered or low affinity PBPs or due to beta-lactamase. Since the coagulase-negative staphylococci are an increasingly i m p o r t a n t pathogen, and since a large percentage of t h e m are resistant to PRPs, there is little reason to separate t h e m from S. aureus in this terminology. We suggest the following two major classifications, definitions, and a c r o n y m s (if desired) of terms: 0738-1751/86/$0.00 + 2.20