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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 -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 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 contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were -negative staphylococci with 24-h oxacillin MICs of <2 FIg/ml. Ninety-five isolates were mec gene negative, including seven strains of 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 . 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 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 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 for 24 h. Nitrocefin (500 ,ug/ml) was then dropped onto the freezer. colonies on the , and the test results were read For testing, isolates were removed from storage, streaked after 15 min. onto a 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. Agar dilution plates were incubated at 35'C for dilution, E test, and disk diffusion assays at each salt 24 h. concentration tested. Finally, the MIC results of broth and Broth microdilution test. Broth microdilution MIC tests agar dilution, E test, and disk diffusion assays, at each salt were performed as described in NCCLS standard M7-A2 concentration tested, were compared by using the Wilcoxon (21) with cation-adjusted Mueller-Hinton broth. Oxacillin signed-rank test and also by the percentage of isolates concentrations ranged from 0.016 to 256.0 ,ug/ml. Parallel yielding identical results, within the accuracy limits of +±1 sets of microdilution plates containing oxacillin in the con- log2 dilution, as previously described (2, 15). centrations cited above and 0, 2, 4, or 5% NaCl were prepared by using a Quickspense (Sandy Springs, Md.). RESULTS Plates were incubated at 35'C for 24 h. The MIC tests of some coagulase-negative strains of staphylococci were incu- Accuracy of the broth dilution reference method. The bated for an additional 24 h if discrepancies were observed oxacillin MICs for 99 strains of S. aureus and 124 strains of between the MIC result and the results of the mec DNA other Staphylococcus species were determined by broth probe test. microdilution testing with 2% NaCl, which is the current E test. E test strips were tested on Mueller-Hinton II agar NCCLS reference method. All isolates also were tested for containing 0, 2, 4, or 5% NaCl. Agar plates (150-mm diam- the presence of the mec gene by using a DNA probe. The eter) were inoculated by swabbing the surfaces with the results of the tests are shown in Table 1. All of the strains of inoculum in three directions. After the surfaces of the S. aureus containing the mec gene were resistant to oxacillin inoculated plates had dried, E test strips were placed on the (MIC, .4 ,ug/ml). However, three strains of S. epidermidis surfaces. Care was taken not to move a strip once it had and three of S. simulans, all of which had oxacillin MICs touched the agar. The plates were incubated at 35'C in ranging from 0.5 to 2.0 Rg/ml (data not shown), were ambient air for 24 h. consistently mec gene probe positive. One isolate of S. Disk diffusion test. Disk diffusion tests were performed as haemolyticus that was mec probe positive produced discrep- described in NCCLS standard M2-A4 (22). A cotton-tipped ant MIC results when tested in duplicate, giving MICs of 2 swab was dipped into the standardized suspension, the and 4 ,ug/ml. When the MIC tests for these seven organisms excess liquid was expressed, and Mueller-Hinton II agar were incubated for an additional 24 h, the oxacillin MICs for plates containing 0, 2, 4, or 5% NaCl were streaked in three the S. epidermidis and S. haemolyticus isolates increased to directions until the entire surface of the plate was covered 4 to >16 ,ug/ml, which would be interpreted as resistant, VOL. 31, 1993 OXACILLIN TESTING METHODS 2685

TABLE 1. Comparison of broth microdilution oxacillin MIC aureus had similar penicillin and oxacillin MICs, the oxacil- results with mec gene probe results for 223 strains lin MICs were not changed by clavulanic acid, and each of staphylococcia strain was mec gene probe negative. Thus, all seven isolates No. of strains (%) with result were classified as MOD strains. A statistical comparison of Species and no. . . the categorical interpretations of the two tests gave a Mc- of strains mec gene positive mec gene negative Nemar x2 value of 0.03 and a kappa value of 0.87, indicating S R S R a high degree of agreement between the results of the two tests. When taken together, these data confirm the overall S. aureus (99) 0 37 (37.4) 55 (55.6) 7 (7.0) accuracy of the broth reference method. S. epidermnidis (57) 3 (5.3) 50 (87.7) 4 (7.0) 0 The effect of adding NaCl to the susceptibility test S. haemolyticus (22) 1 (4.5)b 21 (95.5) 0 0 medium. S. saprophyticus (13) 0 1 (7.6) 12 (92.3) 0 The oxacillin MICs for the 223 strains of staphylococci were S. hominis (12) 0 8 (66.7) 4 (33.3) 0 determined by agar and broth microdilution and E test with S. simulans (7) 3 (42.8) 3 (42.8) 1 (14.0) 0 media containing 0, 2, 4, or 5% NaCl. The results of the MIC S. lugdunensis (6) 0 0 6 (100) 0 tests compared with the results of broth dilution in media S. warneri (4) 0 1 (25.0) 3 (75.0) 0 containing 2% NaCl are shown in Table 2. Broth MIC results S. xylosus (2) 0 0 2 (100) 0 without added NaCl were significantly lower than the results S. capitis (1) 0 0 1 (100) 0 with 2% NaCl (P < 0.001; Wilcoxon signed-rank test), while a S, susceptible (<2 ,ug/ml); R, resistant (24 Ag/ml in broth microdilution broth tests using 4 and 5% salt showed percent agreement test [with 2% NaCl]). values of >90% (P = 0.159 and 0.172, respectively) and did b The results of duplicate testing with this strain were discrepant. One result not show a similar shift. The agar, broth, and E test results was susceptible (MIC = 2 1ig/ml), and the other was resistant (MIC = 4 determined without added NaCl also showed significantly Ag/ml). lower MICs when compared with results from broth with 2% NaCl (P < 0.001; Wilcoxon signed-rank test), and those with any added NaCl showed MICs significantly higher than although the MIC for all of the S. simulans strains was 2 those in broth with 2% NaCl (P < 0.001). The best agree- ,ug/ml at 48 h. Seven isolates of S. aureus demonstrated ment (kappa value, >0.92) between the reference broth test resistance to oxacillin (MIC, 24 ,ug/ml) but were consis- and E test, and the reference broth test and agar dilution, tently negative with the mec probe. Two of these strains was achieved in both cases when 2% NaCl was added to the were previously shown to contain penicillin-binding proteins medium for testing. The need for the addition of 2% NaCl for with reduced affinity for beta-lactam antimicrobial agents but agar and E test is also reflected in the categorical agreement did not contain the novel PBP 2a encoded by the mec data for these tests as shown in Table 3. Thus, the most determinant (28). These strains, referred to as MOD strains favorable essential agreement (fewest number of very major (for modified penicillin-binding proteins), do not produce and major errors) was achieved when 2% NaCl was added to large quantities of 1-lactamase, as judged by their low the test medium for both the agar dilution (0.8% very major penicillin MICs (8 to 32 ,ug/ml), and have oxacillin MICs (4 to and 8.5% major errors) and E test methods (0.4% very major 8 ,ug/ml) that are not lowered by more than a sihgle dilution and 6.9% major errors), although the major error rates were by clavulanic acid (data not shown). Thus, these strains were still high (Table 3). A comparison of the agar dilution and the deemed to be correctly classified as mec gene probe negative E test results, when supplemented with additional 0, 2, 4, but oxacillin resistant. The remaining five strains of S. and 5% NaCl, with the results of the mec gene probe also

TABLE 2. Effect of sodium chloride on oxacillin MIC results for 223 staphylococci tested in duplicatea Method No. of isolates (% of total) with the foilowing difference in MIC" compared and % with results of broth dilution plus 2% NaCl % Agree- pd NaClNaCl<-2 -2 -1 0 +1 +2 >+2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ment' Agar 0 39 (8.6) 41 (9.2) 103 (23.1) 158 (35.4) 86 (19.3) 17 (3.8) 2 (0.4) 77.8 ± 2.0 <0.001 2 2 (0.4) 2 (0.4) 24 (5.4) 208 (46.6) 139 (31.2) 51 (11.4) 20 (4.4) 83.2 ± 1.8 <0.001 4 6 (1.3) 11 (2.5) 48 (10.8) 125 (28.0) 145 (32.5) 74 (16.6) 37 (8.0) 71.3 ± 2.1 <0.001 5 4 (0.8) 7 (1.6) 58 (13.0) 118 (26.5) 146 (32.7) 69 (15.5) 44(9.8) 72.2 ± 2.1 <0.001 Broth 0 81 (18.0) 76 (17.0) 122 (27.4) 137 (30.7) 28 (6.3) 2 (0.4) 0 (0.0) 64.4 ± 2.3 <0.001 4 0 (0.0) 10 (2.2) 77 (17.3) 250 (56.1) 94 (21.1) 15 (3.4) 0 (0.0) 94.5 ± 1.1 0.159 5 3 (0.7) 21 (4.7) 105 (23.5) 201 (45.1) 98 (22.0) 18 (4.0) 0 (0.0) 90.6 ± 1.4 0.172 E test 0 51 (11.4) 45 (10.1) 91 (20.4) 134 (30.0) 91 (20.4) 16 (3.6) 18 (4.0) 70.9 ± 2.2 <0.001 2 1 (0.2) 4 (0.8) 47 (10.5) 175 (39.2) 124 (27.8) 51 (11.4) 44 (9.6) 77.6 ± 2.0 <0.001 4 3 (0.6) 2 (0.4) 39 (8.7) 133 (29.8) 136 (30.5) 66 (14.8) 67 (14.9) 69.1 ± 2.2 <0.001 5 3 (0.6) 5 (1.1) 35 (7.8) 131 (29.4) 130 (29.1) 71 (15.9) 71 (15.9) 66.4 ± 2.2 <0.001 a All method concentrations were compared with cation-adjusted Mueller-Hinton broth plus 2% NaCl (NCCLS reference method). Each result of replicate testing is included. b Indicates number of strains for the given MIC differences. c Percentage of strains within acceptable test limits (±1 log2 dilution) + standard error. d P value obtained from Wilcoxon signed-rank test. 2686 HUANG ET AL. J. CLIN. MICROBIOL.

TABLE 3. Categorical agreement of susceptibility testing methods compared with broth dilution plus 2% NaCl for 223 isolates of staphylococcia No. of isolates (% of total) with result Method and % Agreement Disagreement NaCl Susceptible Resistant Minor Major Very major errors errors errors Agar 0 187 (98.9) 210 (82.0) NAb 2 (1.1) 47 (18.2) 2 173 (91.5) 255 (99.2) NA 16 (8.5) 2 (0.8) 4 140 (74.1) 253 (98.4) NA 49 (26.0) 4 (1.6) 5 137 (72.5) 253 (98.4) NA 52 (27.5) 4 (1.6) Broth 0 189 (100.0) 200 (77.8) NA 0 57 (22.1) 4 156 (82.5) 253 (98.4) NA 33 (17.5) 4 (1.6) 5 154 (81.5) 253 (98.4) NA 35 (18.5) 4 (1.6) E test 0 188 (99.5) 205 (79.8) NA 1 (0.5) 52 (20.2) 2 176 (93.1) 256 (99.6) NA 13 (6.9) 1 (0.4) 4 146 (77.2) 254 (98.8) NA 43 (22.8) 3 (1.2) 5 140 (74.1) 253 (98.4) NA 49 (26.0) 4 (1.6) Disk diffusion 0 165 (87.3) 219 (85.2) 19 (4.3) 11 (5.8) 32 (12.4) 2 138 (73.0) 256 (99.6) 14 (3.1) 38 (20.1) 0 (0.0) 4 127 (67.2) 255 (99.2) 12 (2.7) 52 (27.5) 0 (0.0) 5 125 (66.1) 254 (98.8) 10 (2.2) 55 (29.1) 2 (0.8) a Perfonned in cation-adjusted Mueller-Hinton broth; each result of replicate testing is included. b NA, not applicable. indicated the strongest agreement with additional 2% NaCl use of Mueller-Hinton broth with 2% NaCl for testing for both agar dilution and the E test (kappa values = 0.84 and oxacillin against staphylococci was favored by Thornsberry 0.87, respectively; data not shown). and McDougal and subsequently recommended by the NC- Disk diffusion tests for oxacillin resistance. When NaCl was CLS in 1985. However, no recommendations were made for added to the medium for disk diffusion testing, none of the testing this same organism-drug combination by agar dilution salt concentrations appeared to give satisfactory results (20). (Table 3). The best essential agreement with the broth In an attempt to resolve the issue of the need for supple- reference test was achieved with no added NaCl, although mentation of the agar growth media with NaCl, we compared this resulted in a large number of very major errors (12.4%). the results of agar dilution, broth microdilution, E test, and No very major errors were noted when 2% NaCl was added disk diffusion with added 0, 2, 4, or 5% NaCl with the results to the medium; however, the high level of major errors of a hybridization assay using a mec gene probe. The mec (20.1%) and the presumed effect of NaCl on the results of probe was used as the "gold standard," as advocated in other antimicrobial agents that would be tested on the same several studies (1, 12, 29). The isolates used in this study plate preclude us from recommending the addition of NaCl showed a wide range of oxacillin MICs and included both to agar for disk diffusion testing. hetero- and homogeneously resistant organisms as well as organisms that proved difficult to test by automated meth- DISCUSSION ods. Many of the latter organisms had oxacillin MICs around the resistance breakpoint and tested oxacillin susceptible on Although used routinely by relatively few laboratories, one day (MIC, 1 to 2 ,ug/ml) but oxacillin resistant (MIC, 4 to agar dilution susceptibility testing continues to be an impor- 8 ,ug/ml) on subsequent days. tant reference method for evaluating new types of suscepti- In the first part of this study, we compared the interpretive bility testing methods, such as the E test (2). Thus, it is category results obtained by using broth microdilution with important that the accuracy of agar dilution be verified for all added 2% NaCl (NCCLS reference method) with the results organism-drug combinations. of the mec gene probe. Strains that were positive with the The idea of adding NaCl to the growth medium to enhance probe were designated as resistant. Agreement between the the detection of methicillin-resistant staphylococci was sug- test results was high; there were only 14 discrepant results gested by both Barber (3) and Churcher (9) in the 1960s. among the 223 strains tested. Seven of the discrepant iso- Barry and Badal then recommended the addition of 5% NaCl lates were S. aureus isolates that demonstrated the MOD to Mueller-Hinton broth for susceptibility tests with oxacillin phenotype (28), and would be considered to be correctly (4). In 1983, Thornsberry and McDougal reported no quali- classified as oxacillin resistant even though they were mec tative differences in oxacillin MIC results when either 2 or probe negative. The other seven isolates were coagulase- 5% NaCl was added to Mueller-Hinton broth (26). However, negative staphylococci that were probe positive but had while 5% NaCl enhanced growth, it also made some border- borderline-susceptible oxacillin MICs. While the S. epider- line-susceptible strains appear to be resistant. Therefore, the midis and S. haemolyticus strains produced MICs in the VOL. 31, 1993 OXACILLIN TESTING METHODS 2687 resistant range after an additional 24 h of incubation, this ACKNOWLEDGMENTS was not true of the S. simulans strains, which remained We thank Linda McDougal and Jana Swenson for helpful discus- oxacillin susceptible with MICs of 2 ,ug/ml. We have no data sions and suggestions and Leticia Dy for help in preparing the as to whether the mec gene is functional in these isolates. manuscript. Coagulase-negative staphylococci that contain functional mec have been noted previously (19, 24, 30), and some REFERENCES 1. Archer, G. L., and E. Pennell. 1990. Detection of methicillin of these strains produced borderline-susceptible MIC results resistance in staphylococci by using a DNA probe. Antimicrob. (24, 30). Thus, the effectiveness of the broth microdilution Agents Chemother. 34:1720-1724. test with added 2% NaCl when compared with the mec gene 2. Baker, C. N., S. A. Stocker, D. H. Culver, and C. Thornsberry. probe was confirmed. While detection of the mec gene as an 1991. Comparison of the E test to agar dilution, broth microdi- indicator of oxacillin resistance is probably a more accurate lution, and agar diffusion susceptibility testing techniques by test, the absence of a commercially manufactured kit makes using a special challenge set of . J. Clin. Microbiol. this technology unavailable to most laboratories and pre- 29:533-538. cludes us from proposing the probe test as a reference 3. Barber, M. 1964. Naturally-occurring methicillin-resistant method. While polymerase chain reaction may facilitate staphylococci. J. Gen. Microbiol. 35:183-190. 4. Barry, A. L., and R. E. Badal. 1977. 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Rahal, and concentration of NaCl is too high and will generate false A. Tomasz. 1991. Multiple mechanisms of methicillin resistance the agar and improved methods in clinical isolates of Staphylococcus resistance in some strains. We found that incubating aureus. Antimicrob. Agents Chemother. 35:632-639. dilution MIC tests for coagulase-negative staphylococci an 11. Fleiss, J. L. 1981. Statistical methods for rates and proportions. additional 24 h was helpful in resolving discrepancies be- John Wiley & Sons, Inc., New York. tween the MIC and probe data. However, we do not have 12. Gerberding, J. L., C. Miick, H. H. Liu, and H. F. Chambers. enough data to ensure that routine incubation of susceptibil- 1991. Comparison of conventional susceptibility tests with di- ity tests for 48 h for this group of organisms will not lead to rect detection of penicillin-binding 2a in borderline an unacceptable rate of false-positive results. 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Accuracy of the E test for determining antimicrobial mance of the disk diffusion test when compared with the susceptibilities of staphylococci, enterococci, DNA probe results was also noted by de Lencastre and jejuni, and gram-negative bacteria resistant to antimicrobial agents. J. Clin. Microbiol. 30:3243-3248. coworkers in their study of the various classes of methicillin 16. Liu, H., G. Bulscher, N. Lewis, and D. Jungkind. 1990. Detec- resistance in staphylococci (10). We have no data as to tion of borderline oxacillin resistant Staphylococcus aureus and whether changes in either disk potencies or breakpoints differentiation from methicillin resistant strains. Eur. J. Clin. would enhance the accuracy of this test. Microbiol. Infect. Dis. 9:717-724. In conclusion, 2% NaCl is necessary for increasing the 17. Maple, P. A. C., J. M. T. Hamilton, and M. W. Brumfitt. 1989. accuracy of results if agar dilution or E test strips are used World-wide resistance in methicillin-resistant Staph- for testing staphylococci with oxacillin. However, salt ylococcus aureus. Lancet ii:537-540. should not be added to disk diffusion tests because of the 18. McDougal, L. K., and C. Thornsberry. 1986. The role of on other antimicrobial agents (9) and the high 3-lactamase in staphylococcal resistance to penicillin-resistant adverse effects penicillin and cephalosporins. J. Clin. Microbiol. 23:832-839. number of major errors produced. Our results also suggest 19. Murakami, K., W. Minamide, K. Wada, E. Nakamura, H. that the accuracy of the oxacillin screen test, which incor- Teraoka, and S. Watanabe. 1991. Identification of methicillin- porates 4% NaCl into agar containing 6 pLg of oxacillin, may resistant strains of staphylococci by polymerase chain reaction. need to be reevaluated, particularly in light of the results of J. Clin. Microbiol. 29:2240-2244. Gerberding et al. (12), which showed that no additional salt 20. National Committee for Clinical Laboratory Standards. 1985. was superior to 4% NaCl for the recognition of oxacillin- Methods for dilution antimicrobial susceptibility tests for bac- resistant staphylococci. teria that grow aerobically, vol. 8. Tentative standard M7-A. 2688 HUANG ET AL. J. CLIN. MICROBIOL.

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