JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1993, p. 2683-2688 Vol. 31, No. 10 0095-1137/93/102683-06$02.00/0 Copyright X 1993, American Society for Microbiology Two Percent Sodium Chloride Is Required for Susceptibility Testing of Staphylococci with Oxacillin when Using Agar-Based Dilution Methods MING BO HUANG,' T. ELAINE GAY,1 CAROLYN N. BAKER,1 SHAILEN N. BANERJEE,2 AND FRED C. TENOVERl* Nosocomial Pathogens Laboratory Branch' and Statistics and Information Systems Branch, 2 Hospital Infections Program, National Centerfor Infectious Disease, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Received 19 May 1993/Returned for modification 7 July 1993/Accepted 20 July 1993 The need to add NaCl to agar media to ensure accuracy of results when testing staphylococci with oxacillin was investigated. The results of four antimicrobial susceptibility testing methods (agar and broth dilution, E test, and disk diffusion) in which the growth medium contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were coagulase-negative staphylococci with 24-h oxacillin MICs of <2 FIg/ml. Ninety-five isolates were mec gene negative, including seven strains of Staphylococcus aureus with oxacillin MICs of 24 tag/ml. The oxacillin MICs for mec gene-positive, oxacillin-resistant strains of staphylococci increased two- to fourfold with the addition of NaCl to the test medium, while the MICs for mec gene-negative strains did not change in the presence ofadded salt. Very major error rates for the agar dilution and E test methods in the absence of salt ranged from 18.2 to 20.2%. Major error rates for mec gene-negative S. aureus isolates were >17% for all test methods when 4 or 5% NaCl was added to the test medium. The addition of 2% NaCl to Mueller-Hinton agar for testing of oxacillin resulted in very major error rates of < 1% for the agar dilution and E test methods although the major error rates for the two methods with added NaCl were 8.5 and 6.91%, respectively. The disk diffusion test did not perform well in this study, showing essential error rates of >18.3%. We recommend the addition of 2% NaCl to Mueller- Hinton agar when testing staphylococci with oxacillin by either the agar dilution or E test method. NaCl should not be added for the disk diffusion test. Over the last decade, a number of modifications to tradi- accurately detected oxacillin resistance in staphylococci tional antimicrobial susceptibility testing techniques have without the addition of NaCl to the agar (2); however, a been suggested to improve the accuracy of detecting oxacil- second study using a larger collection of strains suggested lin resistance in all staphylococcal species, including Staph- that 2% NaCl should be added (15). Ngui-Yen et al., how- ylococcus aureus. These include prolonged time of incuba- ever, have recommended the addition of 4% NaCl to the tion, incubation at 30 or 35°C, and the addition of NaCl to the medium for testing semisynthetic penicillins by the E test test medium (5, 7, 17, 18, 21, 22, 25, 26). More recently, method (23). DNA probes (1, 6, 12, 29) and the polymerase chain reaction This study examined a large, genetically diverse collection (19, 27) have been used to detect the mec gene in staphylo- of staphylococci to determine whether (i) adding salt to agar cocci as an indication of resistance. The DNA-based meth- improves the accuracy of agar dilution and E test MIC ods are probably the most accurate means of identifying results when testing staphylococci with oxacillin compared resistance to the semisynthetic penicillins in staphylococci with the broth microdilution method and (ii) whether salt even though these techniques will not detect those rare should be added to oxacillin disk diffusion tests. strains of staphylococci that have low-level resistance be- cause they possess penicillin-binding proteins with reduced affinity for beta-lactam antimicrobial agents compared with MATERIALS AND METHODS wild-type strains (28). Strains that produce large quantities of 1-lactamase can also produce borderline-resistant MICs, Antimicrobial agents, E test strips, and disks. Oxacillin and the interpretation of such strains as oxacillin susceptible powder was purchased from Sigma Chemical Company (St. or resistant has often proven to be problematic (4, 8, 10, 14, Louis, Mo.) for broth microdilution and agar dilution testing. 16, 18, 29). E test strips were purchased from AB Biodisk (Culver City, Although it is recommended that 2% NaCl be added to Calif.). The 1-,ug oxacillin disks used for disk diffusion tests broth when testing staphylococci with semisynthetic penicil- were purchased from Becton Dickinson Microbiology Sys- method it is not clear tems (Cockeysville, Md.). lins by the broth microdilution (21, 26), Bacterial strains and growth conditions. Two hundred and whether a similar concentration of NaCl should be added to twenty-three organisms from the strain collection of the agar when testing staphylococci by either the agar dilution or were used in this initial that the E test Centers for Disease Control and Prevention E test method. Our study suggested study. Many of these isolates were sent to the Centers for Disease Control and Prevention for antimicrobial suscepti- bility testing because they proved to be difficult to classify as * Corresponding author. susceptible or resistant to oxacillin by automated methods. 2683 2684 HUANG ET AL. J. CLIN. MICROBIOL. The isolates included 99 strains of S. aureus, which repre- with the suspension. Oxacillin-containing disks were placed sented a broad array of bacteriophage and ,-lactamase on the plates, and the plates were incubated in ambient air at subtypes; 57 strains of S. epidermidis; 22 strains of S. 35'C. Zone diameters were measured after 24 h of incuba- haemolyticus; 13 strains of S. saprophyticus; 12 strains of S. tion. hominis; 7 strains of S. simulans; 6 strains of S. lugdunensis; 1-Lactamase. P-Lactamase production was determined 4 strains of S. wameni; 2 strains of S. xylosus; and 1 strain of after induction with subinhibitory concentrations of oxacil- S. capitis. Many of the isolates had oxacillin MICs of 2 to 8 lin. Organisms were grown on Mueller-Hinton agar plates ,ug/ml (i.e., borderline resistance to oxacillin). Isolates were containing 2% NaCl and 0.25 jig of oxacillin per ml at 35'C stored in rabbit blood at c - 120'C in a liquid nitrogen for 24 h. Nitrocefin (500 ,ug/ml) was then dropped onto the freezer. colonies on the agar plate, and the test results were read For testing, isolates were removed from storage, streaked after 15 min. onto a Trypticase soy agar plate supplemented with 5% DNA probe preparation and hybridization conditions. sheep blood (Becton Dickinson), and incubated under aero- Staphylococci were lysed as described by Goering and Ruff bic conditions at 35°C for 24 h. An isolated colony was (13), and the lysates were applied in duplicate to positively picked from each plate, streaked onto a new Trypticase soy charged nylon membranes (Zeta probe; Bio-Rad Laborato- agar plate, and incubated for 18 to 24 h. All inocula were ries, Richmond, Calif.) by using a slot blot apparatus (Bio- prepared from this subculture. The control strains used in Rad). The mec gene probe was a 400-bp intragenic ClaI- this study included S. aureus ATCC 43387, ATCC 43300, HincII restriction fragment of plasmid pG0164 (a 1.1-kb PBP ATCC 25923, and ATCC 29213. 2a gene fragment cloned into pUC18 [1]) kindly provided by Antimicrobial susceptibility testing. Five colonies were Gordon Archer, University of Virginia, Charlottesville. The transferred into a tube containing 5 ml of Mueller-Hinton probe was labeled with digoxigenin by using the Genius broth (Difco Laboratories, Detroit, Mich.) to prepare a labeling kit, and the filters were hybridized, washed, and suspension equivalent in density to that of a 0.5 McFarland developed in accordance with the manufacturer's (Boehr- barium sulfate standard. All four susceptibility tests were inger Mannheim Corporation, Indianapolis, Ind.) instruc- performed with the same adjusted inoculum. Quantitative tions. Positive controls included S. aureus ATCC 43300, and subcultures demonstrated that the final inocula contained negative controls included S. aureus ATCC 25923 and approximately 108 CFU/ml for E test and disk diffusion, 5 x ATCC 29213. 10 CFU/ml for broth microdilution, and 104 CFU per spot Statistics. The validity of using the broth microdilution test for agar dilution. All testing was performed in duplicate. containing 2% NaCl as the reference method was verified by Agar dilution tests. Agar dilution MIC testing was per- comparing the broth microdilution MIC results with those of formed as described in National Committee for Clinical a mec gene probe assay in which a positive result in the latter Laboratory Standards (NCCLS) standard M7-A2 (21) by test indicated resistance. The association between the two using an inoculum-replicating device to inoculate 0.001 ml of methods was determined by using McNemar's x2 test for the inoculum suspension of each isolate onto the surfaces of matched pairs (11); kappa, a measurement of interrater Becton Dickinson Mueller-Hinton II agar plates containing agreement, was also calculated to assess agreement, correct- oxacillin in concentrations ranging from 0.016 to 256.0 ing for chance (11). The x2 and kappa values were also used pLg/ml. Organisms were tested in parallel on Mueller-Hinton to estimate the agreement between the interpretive results media containing similar oxacillin concentrations and 0, 2, 4, (R, I, and S) of broth microdilution and the results of agar or 5% NaCl.