Detection of Methicillin-Resistant Staphylococcus aureus

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p Vol. 32, No /94/$ Copyright (C 1994, American Society for Microbiology Comparison of Phenotypic Methods and DNA Hybridization for Detection of Methicillin-Resistant Staphylococcus aureus PASCALE RICHARD,'* MICHEL MEYRAN,2 ESTELLE CARPENTIER,' ANDRE THABAUT,2 AND HENRI B. DRUGEON1 Laboratoire de Bacteriologie B, Hopital G.R. Laennec, Nantes Cedex,' and Laboratoire de Biologie Medicale, Hopital d'instruction des Arm&es Begin, Saint-Mande2 France Received 22 September 1993/Returned for modification 12 November 1993/Accepted 24 November 1993 One hundred thirty-eight Staphylococcus aureus isolates from patients with severe staphylococcal infections were collected in 15 French hospitals. Detection of the mec gene was performed by dot blot hybridization with a specific DNA probe. Dot blot results were used to characterize the isolates as methicillin susceptible (77 isolates) or resistant (61 isolates). The isolates were screened for methicillin resistance by an agar spread method on Mueller-Hinton plates containing oxacillin (2 and 10,ug/ml) and were incubated at 37 C, with 108 CFU as the inoculum. MICs of oxacillin and methicillin were determined by the agar dilution method on Mueller-Hinton plates without NaCl, by using 105 CFU per spot, after 24 and 48 h of incubation at 30 or 37 C. Moderately elevated MICs were found for 20 isolates (14.5%). The mec gene was detected in six (30%) of the isolates expressing a low level of resistance to methicillin and/or oxacillin. As determined by comparison with probe hybridization results, the spread plate method with oxacillin at 2,ug/ml was more sensitive (sensitivity, 100%) and specific (specificity, 100%o) than agar dilution with either methicillin or oxacillin in identifying methicillin resistance or susceptibility. Determinations of methicillin and oxacillin MICs by the agar dilution method had a specificity of 99 to 100% depending on the conditions of incubation, but the sensitivity was below 85% whatever the duration or temperature of incubation. The presence of a low-affinity penicillin-binding protein (PBP 2a) encoded by the mec gene is the main factor responsible for methicillin resistance in staphylococci (2, 14). The mec gene and its product (PBP 2a) have never been found in methicillin-susceptible Staphylococcus aureus, while they have been detected in almost all methicillin-resistant S. aureus isolates (MRSA) examined so far (1, 2, 7, 11, 14, 18, 21, 24, 25). Despite the invariable presence of these resistance determinants in MRSA, phenotypic expression of methicillin resistance is strain specific and is often heterogeneous (4, 12, 13). Thus, routine susceptibility tests may fail to identify MRSA, particularly strains expressing a very low level of resistance. Although incubation at temperatures below 37 C, prolonged incubation, use of media containing NaCl, and use of large inocula have been proposed for improving the detection of MRSA, there is no universal agreement on the choice of either an optimal procedure or MIC breakpoints for the identification of methicillin resistance (4, 5, 8, 12, 17, 19, 23). Detection of the mec gene by DNA hybridization or PCR has been proved useful for identification of MRSA (1, 7, 11, 14, 18, 21, 24, 25). Although these methods require specialized staff and equipment and may not be applied in every clinical microbiology laboratory, they provide a useful tool for the evaluation of procedures based on phenotypic expression of resistance. S. aureus isolates with a borderline level of resistance to methicillin or oxacillin have been reported from time to time (7, 15, 16, 22). Three mechanisms of resistance have been associated with this phenotype: mec-encoded resistance (expression class 1), overproduction of penicillinase, and modifications of normal PBPs (7, 15, 16, 22). Since there is no evidence that the efficacy of methicillin or oxacillin is impaired * Corresponding author. Mailing address: Laboratoire de Bacteriologie A, Institut de Biologie des H6pitaux de Nantes, 9 Quai Moncousu, Nantes Cedex, France. Phone: Fax: for isolates with overproduction of penicillinase, it seems important to differentiate these isolates from those with mecrelated resistance (3). However, the frequency of the borderline resistance phenotype and the relative frequency of the corresponding resistance determinants have not been studied extensively. The purposes of this study were to compare phenotypic methods for identification of MRSA with detection of the mec gene by DNA hybridization and to evaluate the frequency of mec-encoded borderline resistance among French S. aureus isolates. (Part of this work was presented at the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., 1992.) MATERIALS AND METHODS Isolates. In 1990, the microbiologists from 15 French hospitals (in Bordeaux, Brest, Clermont-Ferrand, Dijon, Grenoble, Lille, Lyon, Montpellier, Nantes, Nice, Paris-Begin, Paris-Saint Antoine, Poitiers, and Strasbourg) were requested to send all isolates of S. aureus collected from patients with systemic or deep-seated infections to the Groupe d'etude des Infections S6veres a Staphylocoques. A total of 138 isolates were collected: 118 from blood, 8 from skin or wound abscesses, 7 from intravenous catheters, and 5 from other sources. Spread plate method. All isolates were screened for oxacillin resistance by using a modification of the spread plate test described by Archer and Pennell (1). A 100-,ul portion of an overnight Mueller-Hinton broth (Difco Laboratories) culture (approximately 108 CFU) was spread on Mueller-Hinton plates (Difco Laboratories) containing 5% NaCl and oxacillin at concentrations of 2 and 10 p,g/ml. Any growth after 2 days of incubation at 37 C was considered indicative of resistance. MIC determinations. MICs of oxacillin and methicillin were determined by agar dilution on Mueller-Hinton plates (Difco

2 614 RICHARD ET AL. J. CLIN. MICROBIOL. TABLE 1. Detection of methicillin resistance by DNA hybridization and routine microbiologic susceptibility tests in 138 S. aureus isolatesa No. of No. of isolates with indicated MIC (jig/ml) under incubation conditions shown Hybridization result isolates with spread plate Oxacillin Methicillin OSb result ORc 300C 24 h 30 C 48 h 370C 24 h 37 C 48 h 300C 24 h 300C 48 h 37 C 24 h 37 C 48 h c2.4 c2.4 c2.4 s2.4 c8.16 '8.16 c8.16 s8.16 mec positive (n =61) mec negative (n =77) Total a Detection of the mec gene was performed by dot-blot hybridization with a specific DNA probe. The isolates were screened for oxacillin resistance by an agar spread plate method on oxacillin (2 and 10,ug/ml)-containing Mueller-Hinton plates incubated at 370C, with 108 CFU as the inoculum. MICs of oxacillin and methicillin were determined by the agar dilution method on Mueller-Hinton plates without NaCl, using 105 CFU per spot, after 24 and 48 h of incubation at 30 or 37 C. b OS, oxacillin susceptible, i.e., isolates not growing on the plates with 2 p.g of oxacillin per ml. c OR, oxacillin resistant, i.e., isolates growing on the plates with 2,ug of oxacillin per ml. Laboratories) without NaCl, after 24 and 48 h of incubation at 30 or 37 C, by using 105 CFU per spot. Susceptibility and resistance breakpoints were c2 and.4,ug/ml for oxacillin and.8 and.16,ug/ml for methicillin (19, 20). MICs of oxacillin in the presence of clavulanic acid were also determined by the agar dilution method after incubation at 30 C, by using a fixed concentration of clavulanate (4 jig/mi).,-lactamase assays were performed with nitrocefin disks (Cefinase; biomerieux SA). Dot blot hybridization. The mec-specific DNA probe was obtained from John Kornblum, Public Health Research Institute, New York, N.Y. The origin of this probe has been described previously (22). For hybridization experiments, the probe was radiolabeled with 32P by using a random-priming kit (Amersham France SA, Les Ullis, France). Whole-cell lysates were prepared from 1 ml of an overnight broth culture. Bacterial cells were pelleted by centrifugation and then resuspended and incubated at 37 C for 30 min in the following lysis buffer: 25 mm Tris hydrochloride (ph 8.0), 10 mm EDTA, 16 jig of lysostaphin per ml (Sigma Chemical Co., St. Louis, Mo.) and 8 jig of muramidase per ml (Sigma). The resulting protoplasts were lysed with sodium dodecyl sulfate (0.1%) and 20,ug of proteinase K per ml (Sigma) for 1 h at 37 C. Samples (25 RI each) of lysates were loaded into the wells of a dot blot apparatus (Schleicher & Schuell, Inc., Keene, N.H.) and transferred to a nitrocellulose membrane. Hybridization and washings were carried out by standard methods (13). Autoradiographs were exposed for 4 h at - 70 C. Interlaboratory agreement. Susceptibility testing and detection of the mec gene were performed at two separate laboratories, and results were reported to an independent investigator for comparison. For strains which showed discrepancies between hybridization results and susceptibility testing results and for all strains with a low level of resistance, the hybridization results were verified. A transportation medium (bacterial strains storage medium; Sanofi Diagnostics Pasteur, Marnes- La-Coquette, France) was inoculated from the culture used for DNA preparation and sent to the second laboratory for MIC determinations in duplicate. The MIC determination results were validated if the differences between all determinations were within one log 2 dilution. For six isolates, the discrepancies between the hybridization and MIC results were attributed to a contamination of the storage culture used for the first determination. For the other 26 isolates that were retested, the hybridization and susceptibility testing results were reproducible. RESULTS Comparison of the probe and spread plate results (Table 1). The plates were read after 24 and 48 h of incubation because the first reading was difficult for some isolates (13 isolates) giving pinpoint colonies. The reading was easier after 48 h of incubation, but the interpretation remained unchanged. Among the 138 staphylococcal isolates examined, 77 isolates (55.8%) did not grow on the plates containing the lowest concentration of oxacillin (2 p.g/ml) and were classified as oxacillin susceptible, while 59 isolates (42.7%) grew on the plates containing the highest concentration of oxacillin (10,ig/ml) and were considered oxacillin resistant. Two isolates (1.5%) grew only on the plates containing oxacillin at 2,ug/ml. Of the 61 isolates growing on the plates containing oxacillin at 2,ug/ml, 100% gave a positive signal with the mec probe, while 100% of the 77 oxacillin-susceptible isolates showed no detectable hybridization. Thus, compared with the hybridization procedure, the spread plate test with oxacillin at 2,ug/ml had a sensitivity and a specificity of 100%; with oxacillin at 10,ug/ml, the sensitivity and the specificity of the spread plate test were 96.7 and 97.4%, respectively. Comparison of the probe and MIC results (Table 1). The MICs of methicillin were.8 jig/ml for all mec-negative isolates, and the MICs of oxacillin were.2,ug/ml for 76 of the 77 mec-negative isolates. Under all test conditions, oxacillin MICs for 7 (11.5%) of the 61 mec-positive isolates were.2 jig/ml and methicillin MICs for 9 (14.75%) were.8 jig/ml. However, MICs of both oxacillin and methicillin were either inferior or equal to the National Committee for Clinical Laboratory Standards (NCCLS) breakpoints for susceptibility for only four mec-positive isolates (6.5%). With the NCCLS breakpoints, MIC determinations were highly specific (specificity, 99 to 100% under all conditions) but lacked sensitivity. After 24 h of incubation, the agar dilution had sensitivities of 57% (30 C) and 73% (37 C) for oxacillin and sensitivities of 83% (30 C) and 80% (37 C) for methicillin. When the incubation lasted 48 h, the results obtained with methicillin did not change but the sensitivities for oxacillin increased to 62% (30 C) and 77% (37 C). Changing the MIC breakpoints did not result in sensitivity and specificity both greater than 90%. Low-level resistance. Twenty isolates (14.5%) expressed a low or borderline level of resistance to methicillin or oxacillin (MICs of methicillin, 4 to 16,ig/ml; MICs of oxacillin, 1 to 4,ug/ml) under any of the test conditions. Six of the 61 mecpositive isolates (9.8%) fell in this category. Four of them (6.5%) would have been considered susceptible to both methi-

3 VOL. 32, 1994 COMPARISON OF METHODS FOR DETECTION OF MRSA 615 TABLE 2. Characteristics of borderline MRSA Oxacillin spread MIC(s) (,ug/ml) under indicated incubation conditions Strain" Presence of plate results" Oxacillin Methicillin penlicillinase 2 plg/ml 10,ug/ml 30(C, 24 h 30 C, 48 h' 37 C, 24 h 37 C, 48 h 30 C, 24 h 30 C, 48 h 37 C, 24 h 37 C, 48 h mec negative , , , , , , , , , , , , , I 1, mec positive , , , , , , "iiec negative and itiec positive, no hybridization and hybridization with the mec-specific probe, h respectively. After 48 h of incubation at 37"C. +, growth; -, absence of growth. First and second values were obtained without and with clavulanic acid (4,ug/ml), respectively. cillin and oxacillin according to their MICs. With the exception of two isolates, all mec-positive borderline resistant isolates grew on plates containing oxacillin at the highest concentration (10,ug/ml) in the spread plate test (Table 2). Of the 77 mec-negative isolates, 14 (18.2%) had a borderline resistance to oxacillin (9 of 77, i.e., 11.6%) and/or methicillin (6 of 77, i.e., 7.8%), but none grew on the plates containing 2 p.g of oxacillin per ml (Table 2). Clavulanic acid (4,ug/ml) did not significantly reduce the MICs of oxacillin for these borderline-resistant isolates, and two of them were penicillinase negative (Table 2). The MICs of penicillin G for these two penicillinase-negative strains were 0.05.g/ml. DISCUSSION Because the choice of antimicrobial therapy and isolation procedures for patients with staphylococcal infections depends on the susceptibility of the isolates to methicillin, there is a need for reliable methods for detection of methicillin resistance. However, because of the heterogeneous expression of resistance, some methicillin-resistant isolates may be misidentified as methicillin susceptible by the conventional methods of in vitro testing. Various procedures have been proposed for improving the detection of methicillin resistance, but there is no standardized procedure for the detection of methicillin resistance that has been accepted worldwide (4, 6, 12, 19, 20). For agar dilution and broth microdilution testing, the NCCLS recommends the addition of 2% NaCl, incubation at 35 C for 24 h and the use of either methicillin or oxacillin, although oxacillin is preferred because of its stability and the reproducibility of its test results (19, 20). In addition, for confirmation of resistance, the NCCLS recommends an agar screen method using 6 plg of oxacillin per ml in Mueller-Hinton plates containing 4% NaCl and incubated for 24 h at 35 C. In France, the Comite Francais de l'antibiogramme suggests using oxacillin instead of methicillin and either adding 5% NaCl to the medium and incubating the plates at 37 C for 18 h or using Mueller-Hinton medium and incubating the plates at 30 C for 18 h (5). In addition, there is no universal agreement on the MIC breakpoints for defining methicillin resistance. Isolates for which the MICs of oxacillin are higher than 2,ug/ml are usually classified as MRSA, while isolates for which the MICs of methicillin are higher than 2, 4, or 8,ug/ml are considered resistant (5, 8, 17, 19, 20). These problems about the choice of an optimal method for the detection of methicillin resistance are due to the absence of a reference procedure. Because of the mec gene's invariable presence in MRSA, its detection provides an accurate method for identification of MRSA, independent of environmental conditions that may affect the phenotypic expression of resistance (1, 7, 11, 18, 21, 24, 25). As determined by comparison with DNA hybridization, the spread plate method and agar dilution were accurate in identifying methicillin-susceptible S. aureus. The agar dilution test had a poor sensitivity for the detection of MRSA despite the use of an increased inoculum (105 CFU) compared with the NCCLS recommendations (104 CFU). This is probably related to the absence of NaCl in the test medium, since other authors obtained similar results using Mueller-Hinton agar without NaCl and noted a substantial improvement of sensitivity in the presence of NaCl (10, 11). According to Huang et al., the best agreement between the NCCLS reference broth test and agar dilution was achieved when 2% NaCl was added to the medium, although this cannot be achieved without sacrificing specificity (10). For oxacillin, more mec-positive isolates were detected after incubation at 37 than at 30 C; this is unlikely to be a temperature effect since, as previously reported, incubation at 30 instead of 37 C improved the sensitivity of the agar dilution test for methicillin (11, 12).

4 616 RICHARD ET AL. The spread plate method was the most sensitive phenotypic method for the detection of MRSA, probably because it uses both a high salt content and a large inoculum. Gerberding et al. have previously noted that agar screening with oxacillin at 6,ug/ml lacked specificity when a larger inoculum (107 CFU) was used, especially when NaCl was added to the medium (9). In our study, the specificity of the spread plate test was better, despite the use of lower concentrations of oxacillin (2 ju.g/ml) and of a greater inoculum (108 CFU), probably because we did not select for troublesome isolates, while most strains tested by Gerberding et al. expressed borderline resistance (9). We found a borderline resistance to methicillin and/or oxacillin in 20 of 138 French S. aureus isolates (14.5%) from patients with severe infections. mec-encoded resistance accounted for this phenotype in 30% (6 of 20) of the isolates. Of 77 mec-negative isolates, 14 (18%) had a borderline resistance to oxacillin and/or methicillin, a quite high frequency that might be due in part to the large inoculum (105 CFU) used for agar dilution. However, using an inoculum of 103 CFU, de Lencastre et al. found MICs of methicillin of -4 pug/ml for 5% (11 of 215) of recent methicillin-susceptible S. aureus isolates, compared with 8% (6 of 77) (P = 0.4) in our study (7). The spread plate test with oxacillin at 2 p.g/ml was useful in identifying the strains with mec-encoded resistance among isolates with ambiguous MICs. Four mec-negative isolates yielding oxacillin MICs of -2 [Lg/ml with an inoculum of 105 CFU did not grow on the agar screen plates with 2 pug of oxacillin per ml despite the use of a larger inoculum; this was possibly related to temperature, since the MICs of oxacillin at 37 C were.0.25,ug/ml for all these isolates. Two of the 14 mec-negative isolates with a borderline level of resistance had no detectable r-lactamase activity. Although we did not look for the biochemical basis of resistance, it is likely that the borderline resistance phenotype was also independent of penicillinase production in the 12 other mec-negative isolates, since the addition of clavulanic acid did not significantly reduce the MICs of oxacillin. These findings are consistent with those of de Lencastre et al., who have suggested that, in addition to the presence of a penicillinase, many contemporary isolates of S. aureus have a low-level intrinsic resistance to methicillin that is possibly related to altered PBPs (7, 22). Since the spread plate method used in this study identified MRSA with a sensitivity and a specificity of 100%, this method should be used routinely for detection of methicillin resistance. Determinations of methicillin and oxacillin MICs by agar dilution at 30 and 37 C, after 24 and 48 h of incubation on Mueller-Hinton plates without NaCl, had a poor sensitivity and gave ambiguous results for 14.5% of the isolates. Compared with MIC determinations, the spread plate method with oxacillin at 2,Lg/ml was much more efficient for identification of mec-encoded resistance in isolates expressing a low level of resistance. Detection of the mec gene should be used as the procedure of reference when routine susceptibility testing gives ambiguous results. ACKNOWLEDGMENTS We are grateful to John Kornblum for providing the DNA probe used in the present study, to our colleagues from the Groupe d'etude des Infections Severes a Staphylocoques for supplying the isolates included in the study, and to Herve Richet for suggestions and helpful discussion. REFERENCES 1. Archer, G. L., and E. Pennell Detection of methicillin resistance in staphylococci by using a DNA probe. Antimicrob. Agents Chemother. 34: J. CLIN. MIC1ROBIOL. 2. Beck, W. D., B. Berger-Bachi, and F. H. Kayser Additional DNA in methicillin-resistant Staplhylococcus aureus and molecular cloning of nwec-specific DNA. J. Bacteriol. 165: Chambers, H. F., G. Archer, and M. Matsuhashi Low-level methicillin resistance in strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 33: Chambers, H. F., and C. J. Hackbarth Effect of NaCl and nafcillin on penicillin-binding protein 2a and heterogeneous expression of methicillin resistance in Staphylococcus alreus. Antimicrob. Agents Chemother. 31: Comite de l'antibiogramme de la Societe Francaise de Microbiologie Communique 1992 du Comite de l'antibiogramme de la Societ Francaise de Microbiologie. Pathol. Biol. 40: Coudron, P. E., D. L. Jones, H. P. Dalton, and G. L. Archer Evaluation of laboratory tests for detection of methicillin-resistant Stalphylococcus aurcus and Staphylococcus epidermidis. J. Clin. Microbiol. 24: de Lencastre, H., A. M. Sa Figueiredo, C. Urban, J. Rahal, and A. Tomasz Multiple mechanisms of methicillin resistance and improved methods for detection in clinical isolates of Staphylococcus aureus. Antimicrob. Agents Chemother. 35: French, G. L., J. Ling, Y. W. Hui, and H. K. T. Oo Determination of methicillin-resistance in Staphylococcus aureus by agar dilution and disc diffusion methods. J. Antimicrob. Chemother. 20: Gerberding, J. L., C. Miick, H. H. Liu, and H. F. Chambers Comparison of conventional susceptibility tests with direct detection of penicillin-binding proteini 2a in borderline oxacillin-resistant strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 35: Huang, M. B., T. E. Gay, C. N. Baker, S. N. Banerjee, and F. C. Tenover Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods. J. Clin. Microbiol. 31: Ligozzi, M., G. M. Rossolini, E. A. Tonin, and R. Fontana Nonradioactive DNA probe for detection of gene for methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 35: Madiraju, M. V. V. S., D. P. Brunner, and B. J. Wilkinson Effects of temperature, NaCl, and methicillin on penicillin-binding proteins, growth, peptidoglycan synthesis, and autolysis in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 31: Maniatis, T. E., F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Matsuhashi, M., M. D. Song, F. Ishino, M. Wachi, M. Doi, M. Inoue, K. Ubukata, N. Yamashita, and M. Konno Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to,b-lactam antibiotics in Staphylococcus aureus. J. Bacteriol. 167: McDougal, L. K., and C. Thornsberry The role of 3-lactamase in staphylococcal resistance to penicillinase-resistant penicillinis and cephalosporins. J. Clin. Microbiol. 23: Montanari, M. P., E. Tonin, F. Biavasco, and P. E. Varaldo Further characterization of borderline methicillin-resistant Staphylococcus aureus and analysis of penicillin-binding proteins. Antimicrob. Agents Chemother. 34: Mouton, R. P., S. L. T. Mulders, J. De Knijff, and J. Hermans Comparison of test systems for recognition of methicillin resistance in Staphyloco-ccus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 8: Murakami, K., W. Minamide, K. Wada, E. Nakamura, H. Teraoka, and S. Watanabe Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J. Clin. Microbiol. 29: National Committee for Clinical Laboratory Standards Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 2nd ed. Approved standard M7-A2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 20. National Committee for Clinical Laboratory Standards Performance standards for antimicrobial susceptibility testing.

5 VOL. 32, 1994 COMPARISON OF METHODS FOR DETECTION OF MRSA 617 M1(0-S4. National Committee for Clinical Laboratory Standards, Villanova, Pa. 21. Tokue, Y., S. Shoji, K. Satoh, A. Watanabe, and M. Motomiya Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 36: Tomasz, A., H. B. Drugeon, H. M. de Lencastre, D. Jabes, L. McDougall, and J. Bille New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob. Agents Chemother. 33: Tomasz, A., S. Nachman, and H. Leaf Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci. Antimicrob. Agents Chemother. 35: Ubukata, K., S. Nakagami, A. Nitta, A. Yamane, S. Kawakami, M. Sugiura, and M. Konno Rapid detection of the meca gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products. J. Clin. Microbiol. 30: Unal, S., J. Hoskins, J. E. Flokowitsch, C. Y. E. Wu, D. A. Preston, and P. L. Skatrud Detection of methicillin-resistant staphylococci by using the polymerase chain reaction. J. Clin. Microbiol. 30: