Detection of Intrinsically Resistant - Journal of Clinical Microbiology

Vol. 25, No. 2

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1987, p. 412-415 0095-1137/87/020412-04$02.00/0 Copyright C 1987, American Society for Microbiology

Detection of Intrinsically Resistant (Heteroresistant) Staphylococcus aureus with the Sceptor and AutoMicrobic Systems SHARON L. HANSEN12* AND THOMAS J. WALSH2 Laboratory Service' and Infectious Diseases,' Veterans Administration Medical Center, Baltimore, Maryland 21218 Received 22 July 1986/Accepted 21 October 1986

Modified procedures for the Sceptor Gram-Positive MIC Panel and the Vitek AutoMicrobic System GPS-M Card were evaluated for their ability to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. A total of 398 clinical isolates (including 222 methicillin-resistant S. aureus) obtained from 10 hospitals were tested. Both systems had 2% NaCl in the oxacillin wells. Sceptor MIC panels were inoculated with an organism suspension prepared from an 18- to 24-h blood agar plate and were inoculated for a full 24 h at 35C before MICs were read. All methicillin-resistant S. aureus isolates were detected as resistant to oxacillin at .8 ,ug/ml by the Sceptor method and at >2 ,ug/ml by the Vitek method. All 176 oxacillin-susceptible, methicillinsusceptible S. aureus isolates were correctly distinguished from methicillin-resistant S. aureus isolates by Sceptor. However, with the Vitek system 29 methicillin-susceptible S. aureus isolates tested as falsely resistant to oxacillin and four isolates tested as falsely resistant to vancomycin. The modified testing procedure with the Sceptor system can be used reliably for accurate susceptibility testing of methicillin-resistant and methicillinsusceptible S. aureus. The Vitek GPS-M card does not accurately discriminate between methicillin-resistant and methicillin-susceptible S. aureus with an oxacillin breakpoint of >2 ,ug/ml. standard M2-A3, except that Mueller-Hinton plates were incubated for a full 24 h at 35°C before measurement of zone diameters (9). Zones of inhibition with oxacillin were examined carefully for small-colony variants or a haze of growth within the zone margins. (ii) AMS. The AutoMicrobic System (AMS) GramPositive Susceptibility Cards (GPS-M; Vitek Systems, Inc., Hazelwood, Mo.) with 2% NaCl incorporated into the oxacillin well were used in conjunction with the AMS microprocessor program, AMSP 14 POC. Computer mode 3 was used to obtain an MIC and a category interpretation of susceptible, moderately susceptible, or resistant. (iii) Sceptor procedure. Sceptor MIC panels containing doubling dilutions of the following selected antibiotics were custom prepared at our request at Johnston Laboratories, Towson, Md., by Johnston manufacturing procedures: oxacillin (0.5 to 32 ptg/ml); oxacillin-2% NaCl (0.5 to 32 ,ug/ml); gentamicin (0.25 to 16 ptg/ml); clindamycin (0.12 to 8 ,ug/ml); erythromycin (0.12 to 8 ,ug/ml); penicillin (0.12 to 16 ,ug/ml); vancomycin (0.25 to 16 p.g/ml); cephalothin (0.5 to 32 ,ug/ml); rifampin (0.12 to 8 Fxg/ml); novobiocin (0.12 to 8 kg/ml); and trimethoprim-sulfamethoxazole (0.12 and 2.4 to 8 and 152 ,ug/ml). MIC panels were tested according to the instructions of the manufacturer with the following modifications. Inoculum was prepared from stationary-phase (18 to 24 h) colonies grown on sheep blood agar with Prompt (3M, St. Paul, Minn.) so that the final concentration was approximately 5 x 105 CFU/ml (5 x 104 CFU per well). Incubation was completed at 35°C for a full 24 h before MIC endpoints were read. The number of strains that gave endpoints of c1, 2, 4, and .8 ,ug of oxacillin per ml with and without 2% NaCI was determined to assess the ability of the Sceptor system to discriminate between susceptible and heteroresistant strains. (iv) Sceptor screening tests. Included in the Sceptor panels were four single wells containing nafcillin (6 ug/Iml); oxacillin (6 pg/ml); methicillin (10 pug/rnl); and methicillin-oxacillin

The difficulty in accurately detecting intrinsically methicillin-resistant Staphylococcus aureus strains has been the topic of numerous studies. Automated, commercial MIC, and reference methods have not reliably detected resistance to the cephalosporins and penicillinase-resistant penicillins in all strains of S. aureus (1-3, 5). New recommendations for in vitro testing of staphylococci have been included in the work of the National Committee for Clinical Laboratory Standards (NCCLS) (9, 10). Additional recommendations suggested by McDougal and Thornsberry include (i) using breakpoints of 2

pLg/ml]

[-2

p.g/ml]

0 (0) 147 (83.5)

(10 and 6 ,ug/ml, respectively). Each well contained 2% NaCl. RESULTS Analysis of the 398 strains of S. aureus in the Vitek AMS with the GPS-M card was completed, and results were available in an average time of 5.0 h. Sensitivity of the Vitek AMS GPS-M card for detecting methicillin-resistant strains was 100% and specificity was 83%. Of the methicillinresistant strains, 100% were accurately detected as resistant to oxacillin plus 2% NaCI in the AMS (Table 1). Moreover, the multiresistance characteristics of these strains as determined by disk diffusion were accurately detected in the GPS-M card. Of the 176 methicillin-susceptible strains, 29 (16.5%) were reported as resistant and 147 (83.5%) were reported as susceptible to oxacillin in the GPS-M card. In addition to these discrepancies, four methicillin-susceptible S. aureus strains were also reported as resistant to vancomycin. None of these 29 strains gave multiresistance patterns with other antibiotics either in the AMS or by disk diffusion testing. Repeat testing of these 29 strains in the GPS-M card with careful inoculum standardization resulted in correct determination of susceptibility to oxacillin in 5 of 29 and vancomycin in 4 of 4. Incorporation of 2% NaCi into the oxacillin wells, preparation of the inoculum from stationary-phase cultures, incubation for a full 24 h at 35°C before reading the MIC trays, and use of a resistance breakpoint of -8 ,ug/ml provided successful discrimination between methicillin-resistant and -susceptible S. aureus for all 398 tested strains with the Sceptor system (Table 2). MICs for all methicillin-resistant strains were -8 ,ug/ml when tested with oxacillin plus 2% NaCi. Without the addition of 2% NaCI to the oxacillin wells, 15 strains tested as borderline (2 ,Lg/ml) or intermediate (4 ,ug/ml) in susceptibility to oxacillin. On the other hand, 29 methicillinsusceptible strains gave the same results when tested with the addition of NaCI. However, none of the methicillinsusceptible strains had oxacillin M1Cs of -8 ,ug/ml. None of the 176 methicillin-susceptible strains grew in the four methicillin resistance screening wells included in the Sceptor panels. Two of the methicillin-resistant strains did not grow in either the oxacillin (6 ,ug/ml) or the combination methicillin (10 ,ug/ml)-oxacillin (6 ,ug/ml) well. Oxacillin MICs for each of these two strains were 8 ,ug/ml. Sceptor oxacillin plus 2% NaCl MIC results with the 29 methicillin-susceptible strains which tested as falsely resistant by the Vitek AMS GPS-M card were s1 (17 strains), 2 (7 strains), and 4 ,ug/ml (5 strains). Endpoints for all antimicrobial agents tested in the Sceptor system were easily read, with the exception of those of trimethoprim-sulfamethoxazole, as diffuse slight turbidity was present frequently throughout the microdilution wells.

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For five of the methicillin-susceptible strains, MICs of 4 ,ug of oxacillin plus 2% NaCI per ml were recorded, which would be categorized as resistant by NCCLS MIC interpretive criteria (.4 ,ug/ml). Repeat disk diffusion testing of these five strains with amoxicillin-clavulanic acid (Augmentin) confirmed that beta-lactamase production affected the oxacillin MIC result, as these five strains were susceptible to amoxicillin-clavulanic acid (.20-mm zone size). Interpretive results for all other antibiotics, except rifampin, in the Sceptor panels agreed with disk diffusion results when available for all 398 strains tested. Novobiocin MICs for all strains were