Vol. 30, No. 8
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 1985-1988
0095-1137/92/081985-04$02.00/0 Copyright © 1992, American Society for Microbiology
Evaluation of Commercial and Standard Methodology for Determination of Oxacillin Susceptibility in Staphylococcus aureus MARTIN SKULNICK,l* ANDREW E. SIMOR,1'2 DANIEL GREGSON,3'4 MOHAN GLEN W. SMALL,1 BARRY KREISWIRTH,5 DEBORAH HATHOWAY,3 AND DONALD E. LOW1'2
PATEL,'
Department of Microbiology, Mount Sinai Hospital,1 and University of Toronto, 2 Toronto, Ontario MSG JX5, and Department ofMicrobiology, St. Joseph's Hospital, 3 and Department ofMedicine, University of Western Ontario, 4 London, Ontario N6A 4LB, Canada, and The Public Health Research Institute, New York; New York 10O165 Received 23 January 1992/Accepted 15 May 1992
Agar dilution with and without 4% NaCI, broth microdilution with 2% NaCI, the dried MicroScan Rapid Positive MIC 1 panel (Baxter Health Care Corp., West Sacramento, Calif.), the Vitek GPS-SA card (Vitek Systems, Hazelwood, Mo.), and the oxacillin agar screen plate were compared with a DNA probe encoding the mec gene for their abilities to detect oxacillin resistance in 506 clinical isolates of Staphylococcus aureus. The results of testing for the mec gene showed that there were 254 oxacillin-resistant and 252 oxacillin-susceptible isolates of S. aureus. There were 14.2% very major errors with Vitek (a resistant isolate was interpreted as susceptible) and 6.7% very major errors with MicroScan. Fewer major errors were seen: 0.8% with MicroScan (a susceptible isolate was interpreted as resistant) and 0.4% with Vitek. No very major errors but 2.4% major errors occurred by agar dilution with 4% NaCl supplementation, whereas there were 0.8% very major and 0.4% major errors without 4% NaCl supplementation. By broth microdilution there were 2.01% very major and 0.8% major errors. The results of the oxacillin agar screen plate method were 100l% concordant with those of the mec gene probe method. cooled on ice, and 50 ,ul of 1 N HCl and 0.2 ml of Soak II (0.5 M Tris, 3 M NaCl [pH 7.2]) were added. The sample was then applied to the manifold and spotted onto a nitrocellulose membrane (Schleicher & Schuell) under vacuum. The DNA was fixed to the nitrocellulose membrane by baking in a vacuum oven for 1.5 h at 80°C. Strains RN6354 (10) and RN6390 (8) were spotted as positive and negative controls, respectively. Prehybridization and hybridization were performed in the presence of 5x SSC (0.75 M NaCl plus 0.075 M sodium citrate) at 65°C, and the membranes were washed at the same temperature in the presence of lx SSC (9, 11). The filters were dried and exposed to X-ray film (Fuji RX) for 16 h at room temperature, and the film was developed on the following day. A 1.1-kb BglII-XbaI fragment unique to the methicillin resistance gene was cloned into an Escherichia coli pUC plasmid, forming pGO164 (1). This mec-specific plasmid construct was nick translated with [a-32P]dATP and was used as the hybridization probe to determine the presence or absence of the resistance determinant (17). Probing with the pUC plasmid alone did not allow hybridization of pUC to S. aureus DNA. Broth microdilution. Broth microdilution testing was performed according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (14). Dynatech MIC-2000 panels (Dynatech Laboratories Inc., Alexandria, Va.) containing oxacillin (range, 0.5 to 1,024 ,ug/ml) in cation-supplemented Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) and 2% NaCl were used. The panels were inoculated with 10 pul of the test suspension to give a final inoculum of 5 x 104 CFU per well. All panels were incubated at 35°C for 24 h. MicroScan. Testing was performed with the dried Micro-
Oxacillin-resistant Staphylococcus aureus has become important nosocomial pathogens (12, 21). The accurate and rapid detection of these organisms is important for the early implementation of appropriate infection control precautions and for the institution of effective antimicrobial therapy. The detection of oxacillin resistance, however, is complicated by the fact that resistance in most strains is heterogeneous (5, 18, 19). This has resulted in the development of several laboratory techniques that are used to enhance the expression of this resistance in vitro (2, 14, 15). In the study described here, we examined the abilities of several standard and commercial testing methodologies compared with that of a probe encoding for the mec gene to accurately detect oxacillin resistance in S. aureus. MATERIALS AND METHODS Strains. A total of 506 clinical isolates of S. aureus collected from 14 hospitals across Canada and the Public Health Research Institute, New York, N.Y., were tested. The isolates were stored frozen at -70°C in skim milk before testing. At the time of testing, each isolate was subcultured twice onto Columbia sheep blood agar to ensure viability and purity. All of the isolates were identified as S. aureus by tube coagulase and DNase testing (7). DNA probe. S. aureus DNA was extracted by the method of Projan et al. (16). The DNA was denatured and then spotted onto a 96-well manifold (Schleicher & Schuell, Keene, N.H.) (4). A 10-,ul aliquot of DNA was added to 0.2 ml of Soak I (0.5 M NaOH, 1.5 M NaCl), and the solution was incubated at 95°C for 10 min. The tubes were then *
Corresponding author. 1985
1986
SKULNICK ET AL.
Scan Rapid Positive MIC 1 panel (Baxter Health Care Corp., West Sacramento, Calif.) according to the manufacturer's instructions. The panels were then processed by using the MicroScan Walkaway system. Vitek. Susceptibility testing with the Vitek GPS-SA card (Vitek Systems, Hazelwood, Mo.) was performed according to the manufacturer's instructions. A Vitek 120 readerincubator was used to process the cards. Agar dilution. The MIC for each isolate was determined by agar dilution according to guideline M7-A2 of NCCLS (14) by using BBL Mueller-Hinton II agar (Becton Dickinson, Cockeysville, Md.). Additional susceptibility tests with Mueller-Hinton II agar supplemented with 4% NaCl and Mueller-Hinton II agar with 4 p,g of clavulanic acid (SmithKline Beecham Pharmaceuticals, Oakville, Ontario, Canada) per ml were performed. For each series of tests, serial twofold dilutions of oxacillin (Sigma Chemical Co., St. Louis, Mo.) ranging from 0.5 to 1,024 p,g/ml were tested, but for plates containing clavulanic acid, a range of 0.5 to 16 ,ug of oxacillin per ml was used. The inoculum was prepared directly from overnight subcultures of each isolate by touching several colonies into 5 ml of normal saline to achieve a 0.5 McFarland turbidity standard. These inocula were then further diluted 1:10 in saline and were loaded into a multipoint inoculator. This dilution was then inoculated onto the plates to give a final inoculum of 104 CFU. The plates were incubated at 35°C for 24 h before they were examined. The following strains of S. aureus served as controls: ATCC 29213 (oxacillin susceptible), ATCC 43387 (borderline resistant), American MicroScan (Baxter Health Care Corp.) 241 and 242 (both of which are oxacillin resistant). Susceptibility and resistance breakpoints were .2 and 24 ,ug/ml, respectively (14). Oxacillin agar screen plate. Agar screen plates were prepared as directed in NCCLS guidelines (14). Mueller-Hinton II agar was supplemented with 6 ,ug of oxacillin per ml and 4% NaCl. The isolates were spot inoculated onto the agar by using a cotton swab dipped into a 0.5 McFarland suspension of each test isolate, and the agar plates were incubated for 24 h at 35°C. In addition to the screen plate, a growth control plate was used. If growth was evident on the agar screen plate, the isolate was considered oxacillin resistant. RESULTS Of the 506 isolates tested, 254 were oxacillin resistant and 252 were oxacillin susceptible, as determined by the presence of the mec gene. When the strains were tested by using the oxacillin agar screen plate, there was total agreement with the gene probe results. Compared with the mec gene probe, MicroScan and Vitek failed to detect resistance in 17 and 36 oxacillin-resistant isolates, respectively (Table 1), resulting in 14.2% very major errors for Vitek and 6.7% very major errors for MicroScan. However, 20 of the 36 resistant isolates not detected by Vitek were "flagged" by Vitek as being possibly oxacillin resistant because of multiple resistance to other antibiotics, and so the results could be classified as partially correct. There were few major errors, with Vitek and MicroScan having 0.4 and 0.8% major errors, respectively. Both systems incorrectly identified the resistances of nine isolates. The mean times required to generate a final report for MicroScan with oxacillin-resistant and oxacillin-susceptible strains were 5.8 and 5.1 h, respectively, and for Vitek the times were 6.9 and 6.6 h, respectively.
J. CLIN. MICROBIOL.
TABLE 1. Distribution of errors with the various susceptibility testing methodologies compared with those with the mec gene probe No. (%) of isolates in the following susceptibility testing
Susceptibility testing methodology
error category:
Very major
Broth microdilution Oxacillin screen plate Vitek MicroScan Agar dilution Agar dilution with 4% NaCl
error
Major error
5 (2.0) 0 (0) 36 (14.2) 17 (6.7) 2 (0.8) 0 (0)
2 (0.8) 0 (0) 1 (0.4) 2 (0.8) 1 (0.4) 6 (2.4)
The results of susceptibility testing by agar dilution compared with those obtained with the mec gene probe are given in Table 2. When agar that was not supplemented with NaCl was used, one of the oxacillin-susceptible isolates appeared to be resistant and two oxacillin-resistant isolate appeared to be susceptible. However, when 4% NaCl was added to the agar dilution plates, six of the oxacillin-susceptible isolates appeared to be resistant and no oxacillin-resistant isolates appeared to be susceptible. Of the methods that we tested the one that gave the largest number of very major errors was broth microdilution, with five very major errors and two major errors (Table 1). For isolates for which MICs were 2 p,g/ml when they were tested either by agar dilution or by broth microdilution, MICs were reduced at least fourfold by the addition of clavulanic acid. None of these isolates hybridized with the mec gene probe or grew on the oxacillin agar screen plates. DISCUSSION The isolates used in this study were collected from 14 hospitals across Canada and New York State to ensure genetic diversity. Because efficiency plating studies were not conducted on the isolates, we cannot say with certainty that TABLE 2. Results of susceptibility testing of 252 oxacillinsusceptible and 254 oxacillin-resistant S. aureus determined with the mec gene probe No. of S. aureus isolates MIC (~ml)
(Rg/nl) 1,024
mcBroth
Agar dilution with 4% NaCl Oxacillin Oxacillin Oxacillin Oxacillin Oxacillin Oxacillin susceptible resistant susceptible resistant susceptible resistant
microdilutiona
A dilution' Agar
192 39 19 2 0 0 0 0 0 0 0 0
198 50 3 1 0 0 0 0 0 0 0 0
a Methods were
4 1 0 3 3 12 34 104 63 17 9 4
1 1 0 2 1 12 12 31 59 114 3 18
according to NCCLS guidelines (14).
19 98 129 5 1 0 0 0 0 0 0 0
0 0 0 0 0 3 3 29 138 81 0 0
VOL. 30, 1992
DETECTION OF OXACILLIN RESISTANCE IN S. AUREUS
the expression of resistance to oxacillin was homogeneous or heterogeneous in nature. In this study, the presence of the mec gene, as determined with a DNA probe, was used as the standard against which the other methods that we evaluated were compared. The mec gene, which resides on the chromosomal DNA, is responsible for the production of penicillin-binding protein 2a. This protein has a low binding affinity for beta-lactam antibiotics and is responsible for the resistance to these agents seen in both heterogeneous and homogeneous isolates. The presence of the mec gene does not necessarily mean that the organism will express resistance and may require induction by exposure to beta-lactam antibiotics (18). Archer and Pennell (1) have also shown that the presence of the mec gene correlates 100% with the detection of methicillin resistance in S. aureus when it is compared with the methicillin spread plate technique. De Lencastre et al. (3) found that the mec gene probe was more accurate than Kirby-Bauer and agar dilution testing for detecting methicillin resistance in 108 strains of S. aureus. Recently, polymerase chain reaction (PCR) technology with primers complementary to sequences in the mec gene has been used to detect methicillin resistance in both S. aureus and coagulase-negative staphylococci (13, 20). The results of those studies showed that PCR is more accurate than conventional susceptibility testing and has the same advantage as DNA probes, in that cryptically resistant isolates were also detected. As yet, routine use of both probes and PCR in clinical laboratories for the detection of methicillin resistance is not a feasible proposition. However, the introduction of new rapid probes such as the Accuprobes (GeneProbe, San Diego, Calif.) and the proposed future automation of PCR may allow these techniques to be used on a routine basis. The fact that PCR and mec gene probes are able to detect strains that do not exhibit resistance in routine susceptibility tests would make them both ideal for use as standard techniques when determining the ability of other systems to detect methicillin resistance. The use of the oxacillin screen plate as an acceptable method for the detection of oxacillin resistance has also been suggested by NCCLS (14). This method has been shown to have an excellent sensitivity (98 to 100%) for detecting resistance to oxacillin in both S. aureus and coagulasenegative staphylococci (6), and the test is inexpensive and easy to perform. However, the plates must be carefully quality controlled, since the source of Mueller-Hinton agar can have a substantial effect on the results (6). In the present evaluation, the oxacillin screen plate with Mueller-Hinton II agar was the only method that was 100% concordant with the presence of the mec gene. Neither MicroScan nor Vitek was able to reliably detect oxacillin resistance in the strains that we tested. Although strains for which the oxacillin MIC was >256 p,g/ml, as determined by broth microdilution, were correctly characterized by both systems, there were 17 resistant isolates for which the MICs were lower that were not detected by either system (Table 1). There were a significant number of indeterminate results with the Vitek system because of the "flagged" results. A flagged result occurs when minimum growth is not obtained in the oxacillin well but the isolate is resistant to two or more of the antibiotics erythromycin, tetracycline, clindamycin, or gentamicin. In this case, the strain is reported to be susceptible to oxacillin and the flag alerts the user that the isolate is a "Possible ox, b-lactam, cephlsprn, penem resistant organism-NCCLS" and instructs the user to confirm the result with an oxacillin screen
1987
plate. Although the flag alerts the user to confirm the result, it remains an area where strains may be miscategorized and errors made. Agar dilution with 4% NaCl detected all of the oxacillinresistant strains tested, while agar dilution without NaCl did not detect two of the oxacillin-resistant strains tested. However, the addition of the salt resulted in the interpretation of six oxacillin-susceptible strains as resistant. This was possibly because the results were difficult to interpret because of haze formation on the plates (14). Even though it is recommended by NCCLS (14), we found that the broth microdilution method gave unacceptable results (five very major errors) compared with the mec gene probe and oxacillin screen plate methods. It is also tedious to perform broth microdilution, and the endpoint MICs can be difficult to determine. In this study, using isolates of S. aureus from across Canada and New York State, we found that the abilities of both the MicroScan and the Vitek systems to detect oxacillin resistance in S. aureus were not adequate and that the concomitant use of a secondary method such as an oxacillin screen plate was required for all isolates. The results also suggest that the mec gene probe and oxacillin agar screen plate are accurate alternate methods to broth microdilution for the detection of oxacillin resistance in S. aureus. ACKNOWLEDGMENTS
We gratefully acknowledge Barbara Corber and Alice Au Yeung for excellent secretarial assistance and Randy Smith for technical assistance.
1. 2.
3. 4. 5. 6.
7.
8.
9.
10.
REFERENCES Archer, G. L., and E. Pennell. 1990. Detection of methicillin resistance in staphylococci by using a DNA probe. J. Clin. Microbiol. 34:1720-1724. Chambers, H. F. 1988. Methicillin-resistant staphylococci. Clin. Microbiol. Rev. 1:173-186. De Lencastre, H., A. Figueiredo, C. Urban, J. Rahal, and A. Tomasz. 1990. Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1079. Dickgiesser, N., and B. Kreiswirth. 1986. Determination of aminoglycoside resistance in Staphylococcus aureus by DNA hybridization. Antimicrob. Agents Chemother. 29:930-932. Hackbarth, C. J., and H. Chambers. 1989. Methicillin-resistant staphylococci; genetics and mechanisms of resistance. Antimicrob. Agents Chemother. 33:991-994. Hindler, J. A., and N. L. Warner. 1987. Effect of source of Mueller-Hinton agar on detection of oxacillin resistance in Staphylococcus aureus using a screening methodology. J. Clin. Microbiol. 25:734-735. Kloos, W. E., and D. W. Lambe, Jr. 1991. Staphylococcus, p. 222-237. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. Kornblum, J., B. Kreiswirth, S. J. Projan, H. Ross, and R. Novick. 1990. Agr A polycistronic locus regulating exoprotein synthesis in Staphylococcus aureus, p. 373-401. In R. P. Novick (ed.), Molecular biology of the staphylococci. VCH Publishers, New York. Kreiswirth, B., S. Lofdahl, M. Betley, M. O'Reilly, P. Schlivert, M. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature (London) 305:709-712. Kreiswirth, B. N., A. McGeer, J. Kornblum, A. E. Simor, W. Eisner, R. Poon, J. Righter, L. Campbell, and D. E. Low. 1990. The use of variable gene probes to investigate a multihospital
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15.
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SKULNICK ET AL.
outbreak of MRSA, p. 521-530. In R. P. Novick (ed.), Molecular biology of the staphylococci. VCH Publishers, New York. Kreiswirth, B. N., S. J. Projan, P. M. Schlievert, and R P. Novick. 1989. Toxic shock syndrome toxin 1 is encoded by a variable genetic element. Rev. Infect. Dis. 11:S83-S89. Maple, P. A. C., J. M. T. Hamilton-Miller, and W. Brumfitt. 1989. Worldwide antibiotic resistance in methicillin-resistant Staphylococcus aureus. Lancet i:537-540. Murakami, K., W. Minamide., K. Wada, E. Nakamura., H. Teraoka, and S. Watanabe. 1991. Identification of methicillinresistant staphylococci by polymerase chain reaction. J. Clin. Microbiol. 29:2240-2244. National Committee for Clinical Laboratory Standards. 1990. 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. National Committee for Clinical Laboratory Standards. 1990. Performance standards for antimicrobial disk susceptibility tests, 4th ed. Approved standard M2-A4. National Committee for Clinical Laboratory Standards, Villanova, Pa. Projan, S. J., S. Carlton, and R. P. Novick. 1983. Determination
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17.
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of plasmid copy number by fluorescence densitometry. Plasmid 9:182-190. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J. Mol. Biol. 113: 237. Ryffel, C., F. H. Kayser, and B. Berger-Bachi. 1992. Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci. Antimicrob. Agents Chemother. 36:25-31. Thormsberry, C. 1984. Methicillin-resistant (hetero-resistant) staphylococci. Antimicrob. Newsl. 1:4347. Tokue, Y., S. Shoji, K. Satoh, A. Watanabe, and M. Motamiya. 1992. Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillinresistant Staphylococcus aureus. Antimicrob. Agents Chemother. 36:6-9. Townsend, D. C. E., N. Ashdown, S. Bolton, J. Bradley, G. Duckworth, E. C. Moorhouse, and W. B. Grubb. 1987. The international spread of methicillin resistant Staphylococcus aureus. J. Hosp. Infect. 9:60-71.
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 1985-1988
0095-1137/92/081985-04$02.00/0 Copyright © 1992, American Society for Microbiology
Evaluation of Commercial and Standard Methodology for Determination of Oxacillin Susceptibility in Staphylococcus aureus MARTIN SKULNICK,l* ANDREW E. SIMOR,1'2 DANIEL GREGSON,3'4 MOHAN GLEN W. SMALL,1 BARRY KREISWIRTH,5 DEBORAH HATHOWAY,3 AND DONALD E. LOW1'2
PATEL,'
Department of Microbiology, Mount Sinai Hospital,1 and University of Toronto, 2 Toronto, Ontario MSG JX5, and Department ofMicrobiology, St. Joseph's Hospital, 3 and Department ofMedicine, University of Western Ontario, 4 London, Ontario N6A 4LB, Canada, and The Public Health Research Institute, New York; New York 10O165 Received 23 January 1992/Accepted 15 May 1992
Agar dilution with and without 4% NaCI, broth microdilution with 2% NaCI, the dried MicroScan Rapid Positive MIC 1 panel (Baxter Health Care Corp., West Sacramento, Calif.), the Vitek GPS-SA card (Vitek Systems, Hazelwood, Mo.), and the oxacillin agar screen plate were compared with a DNA probe encoding the mec gene for their abilities to detect oxacillin resistance in 506 clinical isolates of Staphylococcus aureus. The results of testing for the mec gene showed that there were 254 oxacillin-resistant and 252 oxacillin-susceptible isolates of S. aureus. There were 14.2% very major errors with Vitek (a resistant isolate was interpreted as susceptible) and 6.7% very major errors with MicroScan. Fewer major errors were seen: 0.8% with MicroScan (a susceptible isolate was interpreted as resistant) and 0.4% with Vitek. No very major errors but 2.4% major errors occurred by agar dilution with 4% NaCl supplementation, whereas there were 0.8% very major and 0.4% major errors without 4% NaCl supplementation. By broth microdilution there were 2.01% very major and 0.8% major errors. The results of the oxacillin agar screen plate method were 100l% concordant with those of the mec gene probe method. cooled on ice, and 50 ,ul of 1 N HCl and 0.2 ml of Soak II (0.5 M Tris, 3 M NaCl [pH 7.2]) were added. The sample was then applied to the manifold and spotted onto a nitrocellulose membrane (Schleicher & Schuell) under vacuum. The DNA was fixed to the nitrocellulose membrane by baking in a vacuum oven for 1.5 h at 80°C. Strains RN6354 (10) and RN6390 (8) were spotted as positive and negative controls, respectively. Prehybridization and hybridization were performed in the presence of 5x SSC (0.75 M NaCl plus 0.075 M sodium citrate) at 65°C, and the membranes were washed at the same temperature in the presence of lx SSC (9, 11). The filters were dried and exposed to X-ray film (Fuji RX) for 16 h at room temperature, and the film was developed on the following day. A 1.1-kb BglII-XbaI fragment unique to the methicillin resistance gene was cloned into an Escherichia coli pUC plasmid, forming pGO164 (1). This mec-specific plasmid construct was nick translated with [a-32P]dATP and was used as the hybridization probe to determine the presence or absence of the resistance determinant (17). Probing with the pUC plasmid alone did not allow hybridization of pUC to S. aureus DNA. Broth microdilution. Broth microdilution testing was performed according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (14). Dynatech MIC-2000 panels (Dynatech Laboratories Inc., Alexandria, Va.) containing oxacillin (range, 0.5 to 1,024 ,ug/ml) in cation-supplemented Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) and 2% NaCl were used. The panels were inoculated with 10 pul of the test suspension to give a final inoculum of 5 x 104 CFU per well. All panels were incubated at 35°C for 24 h. MicroScan. Testing was performed with the dried Micro-
Oxacillin-resistant Staphylococcus aureus has become important nosocomial pathogens (12, 21). The accurate and rapid detection of these organisms is important for the early implementation of appropriate infection control precautions and for the institution of effective antimicrobial therapy. The detection of oxacillin resistance, however, is complicated by the fact that resistance in most strains is heterogeneous (5, 18, 19). This has resulted in the development of several laboratory techniques that are used to enhance the expression of this resistance in vitro (2, 14, 15). In the study described here, we examined the abilities of several standard and commercial testing methodologies compared with that of a probe encoding for the mec gene to accurately detect oxacillin resistance in S. aureus. MATERIALS AND METHODS Strains. A total of 506 clinical isolates of S. aureus collected from 14 hospitals across Canada and the Public Health Research Institute, New York, N.Y., were tested. The isolates were stored frozen at -70°C in skim milk before testing. At the time of testing, each isolate was subcultured twice onto Columbia sheep blood agar to ensure viability and purity. All of the isolates were identified as S. aureus by tube coagulase and DNase testing (7). DNA probe. S. aureus DNA was extracted by the method of Projan et al. (16). The DNA was denatured and then spotted onto a 96-well manifold (Schleicher & Schuell, Keene, N.H.) (4). A 10-,ul aliquot of DNA was added to 0.2 ml of Soak I (0.5 M NaOH, 1.5 M NaCl), and the solution was incubated at 95°C for 10 min. The tubes were then *
Corresponding author. 1985
1986
SKULNICK ET AL.
Scan Rapid Positive MIC 1 panel (Baxter Health Care Corp., West Sacramento, Calif.) according to the manufacturer's instructions. The panels were then processed by using the MicroScan Walkaway system. Vitek. Susceptibility testing with the Vitek GPS-SA card (Vitek Systems, Hazelwood, Mo.) was performed according to the manufacturer's instructions. A Vitek 120 readerincubator was used to process the cards. Agar dilution. The MIC for each isolate was determined by agar dilution according to guideline M7-A2 of NCCLS (14) by using BBL Mueller-Hinton II agar (Becton Dickinson, Cockeysville, Md.). Additional susceptibility tests with Mueller-Hinton II agar supplemented with 4% NaCl and Mueller-Hinton II agar with 4 p,g of clavulanic acid (SmithKline Beecham Pharmaceuticals, Oakville, Ontario, Canada) per ml were performed. For each series of tests, serial twofold dilutions of oxacillin (Sigma Chemical Co., St. Louis, Mo.) ranging from 0.5 to 1,024 p,g/ml were tested, but for plates containing clavulanic acid, a range of 0.5 to 16 ,ug of oxacillin per ml was used. The inoculum was prepared directly from overnight subcultures of each isolate by touching several colonies into 5 ml of normal saline to achieve a 0.5 McFarland turbidity standard. These inocula were then further diluted 1:10 in saline and were loaded into a multipoint inoculator. This dilution was then inoculated onto the plates to give a final inoculum of 104 CFU. The plates were incubated at 35°C for 24 h before they were examined. The following strains of S. aureus served as controls: ATCC 29213 (oxacillin susceptible), ATCC 43387 (borderline resistant), American MicroScan (Baxter Health Care Corp.) 241 and 242 (both of which are oxacillin resistant). Susceptibility and resistance breakpoints were .2 and 24 ,ug/ml, respectively (14). Oxacillin agar screen plate. Agar screen plates were prepared as directed in NCCLS guidelines (14). Mueller-Hinton II agar was supplemented with 6 ,ug of oxacillin per ml and 4% NaCl. The isolates were spot inoculated onto the agar by using a cotton swab dipped into a 0.5 McFarland suspension of each test isolate, and the agar plates were incubated for 24 h at 35°C. In addition to the screen plate, a growth control plate was used. If growth was evident on the agar screen plate, the isolate was considered oxacillin resistant. RESULTS Of the 506 isolates tested, 254 were oxacillin resistant and 252 were oxacillin susceptible, as determined by the presence of the mec gene. When the strains were tested by using the oxacillin agar screen plate, there was total agreement with the gene probe results. Compared with the mec gene probe, MicroScan and Vitek failed to detect resistance in 17 and 36 oxacillin-resistant isolates, respectively (Table 1), resulting in 14.2% very major errors for Vitek and 6.7% very major errors for MicroScan. However, 20 of the 36 resistant isolates not detected by Vitek were "flagged" by Vitek as being possibly oxacillin resistant because of multiple resistance to other antibiotics, and so the results could be classified as partially correct. There were few major errors, with Vitek and MicroScan having 0.4 and 0.8% major errors, respectively. Both systems incorrectly identified the resistances of nine isolates. The mean times required to generate a final report for MicroScan with oxacillin-resistant and oxacillin-susceptible strains were 5.8 and 5.1 h, respectively, and for Vitek the times were 6.9 and 6.6 h, respectively.
J. CLIN. MICROBIOL.
TABLE 1. Distribution of errors with the various susceptibility testing methodologies compared with those with the mec gene probe No. (%) of isolates in the following susceptibility testing
Susceptibility testing methodology
error category:
Very major
Broth microdilution Oxacillin screen plate Vitek MicroScan Agar dilution Agar dilution with 4% NaCl
error
Major error
5 (2.0) 0 (0) 36 (14.2) 17 (6.7) 2 (0.8) 0 (0)
2 (0.8) 0 (0) 1 (0.4) 2 (0.8) 1 (0.4) 6 (2.4)
The results of susceptibility testing by agar dilution compared with those obtained with the mec gene probe are given in Table 2. When agar that was not supplemented with NaCl was used, one of the oxacillin-susceptible isolates appeared to be resistant and two oxacillin-resistant isolate appeared to be susceptible. However, when 4% NaCl was added to the agar dilution plates, six of the oxacillin-susceptible isolates appeared to be resistant and no oxacillin-resistant isolates appeared to be susceptible. Of the methods that we tested the one that gave the largest number of very major errors was broth microdilution, with five very major errors and two major errors (Table 1). For isolates for which MICs were 2 p,g/ml when they were tested either by agar dilution or by broth microdilution, MICs were reduced at least fourfold by the addition of clavulanic acid. None of these isolates hybridized with the mec gene probe or grew on the oxacillin agar screen plates. DISCUSSION The isolates used in this study were collected from 14 hospitals across Canada and New York State to ensure genetic diversity. Because efficiency plating studies were not conducted on the isolates, we cannot say with certainty that TABLE 2. Results of susceptibility testing of 252 oxacillinsusceptible and 254 oxacillin-resistant S. aureus determined with the mec gene probe No. of S. aureus isolates MIC (~ml)
(Rg/nl) 1,024
mcBroth
Agar dilution with 4% NaCl Oxacillin Oxacillin Oxacillin Oxacillin Oxacillin Oxacillin susceptible resistant susceptible resistant susceptible resistant
microdilutiona
A dilution' Agar
192 39 19 2 0 0 0 0 0 0 0 0
198 50 3 1 0 0 0 0 0 0 0 0
a Methods were
4 1 0 3 3 12 34 104 63 17 9 4
1 1 0 2 1 12 12 31 59 114 3 18
according to NCCLS guidelines (14).
19 98 129 5 1 0 0 0 0 0 0 0
0 0 0 0 0 3 3 29 138 81 0 0
VOL. 30, 1992
DETECTION OF OXACILLIN RESISTANCE IN S. AUREUS
the expression of resistance to oxacillin was homogeneous or heterogeneous in nature. In this study, the presence of the mec gene, as determined with a DNA probe, was used as the standard against which the other methods that we evaluated were compared. The mec gene, which resides on the chromosomal DNA, is responsible for the production of penicillin-binding protein 2a. This protein has a low binding affinity for beta-lactam antibiotics and is responsible for the resistance to these agents seen in both heterogeneous and homogeneous isolates. The presence of the mec gene does not necessarily mean that the organism will express resistance and may require induction by exposure to beta-lactam antibiotics (18). Archer and Pennell (1) have also shown that the presence of the mec gene correlates 100% with the detection of methicillin resistance in S. aureus when it is compared with the methicillin spread plate technique. De Lencastre et al. (3) found that the mec gene probe was more accurate than Kirby-Bauer and agar dilution testing for detecting methicillin resistance in 108 strains of S. aureus. Recently, polymerase chain reaction (PCR) technology with primers complementary to sequences in the mec gene has been used to detect methicillin resistance in both S. aureus and coagulase-negative staphylococci (13, 20). The results of those studies showed that PCR is more accurate than conventional susceptibility testing and has the same advantage as DNA probes, in that cryptically resistant isolates were also detected. As yet, routine use of both probes and PCR in clinical laboratories for the detection of methicillin resistance is not a feasible proposition. However, the introduction of new rapid probes such as the Accuprobes (GeneProbe, San Diego, Calif.) and the proposed future automation of PCR may allow these techniques to be used on a routine basis. The fact that PCR and mec gene probes are able to detect strains that do not exhibit resistance in routine susceptibility tests would make them both ideal for use as standard techniques when determining the ability of other systems to detect methicillin resistance. The use of the oxacillin screen plate as an acceptable method for the detection of oxacillin resistance has also been suggested by NCCLS (14). This method has been shown to have an excellent sensitivity (98 to 100%) for detecting resistance to oxacillin in both S. aureus and coagulasenegative staphylococci (6), and the test is inexpensive and easy to perform. However, the plates must be carefully quality controlled, since the source of Mueller-Hinton agar can have a substantial effect on the results (6). In the present evaluation, the oxacillin screen plate with Mueller-Hinton II agar was the only method that was 100% concordant with the presence of the mec gene. Neither MicroScan nor Vitek was able to reliably detect oxacillin resistance in the strains that we tested. Although strains for which the oxacillin MIC was >256 p,g/ml, as determined by broth microdilution, were correctly characterized by both systems, there were 17 resistant isolates for which the MICs were lower that were not detected by either system (Table 1). There were a significant number of indeterminate results with the Vitek system because of the "flagged" results. A flagged result occurs when minimum growth is not obtained in the oxacillin well but the isolate is resistant to two or more of the antibiotics erythromycin, tetracycline, clindamycin, or gentamicin. In this case, the strain is reported to be susceptible to oxacillin and the flag alerts the user that the isolate is a "Possible ox, b-lactam, cephlsprn, penem resistant organism-NCCLS" and instructs the user to confirm the result with an oxacillin screen
1987
plate. Although the flag alerts the user to confirm the result, it remains an area where strains may be miscategorized and errors made. Agar dilution with 4% NaCl detected all of the oxacillinresistant strains tested, while agar dilution without NaCl did not detect two of the oxacillin-resistant strains tested. However, the addition of the salt resulted in the interpretation of six oxacillin-susceptible strains as resistant. This was possibly because the results were difficult to interpret because of haze formation on the plates (14). Even though it is recommended by NCCLS (14), we found that the broth microdilution method gave unacceptable results (five very major errors) compared with the mec gene probe and oxacillin screen plate methods. It is also tedious to perform broth microdilution, and the endpoint MICs can be difficult to determine. In this study, using isolates of S. aureus from across Canada and New York State, we found that the abilities of both the MicroScan and the Vitek systems to detect oxacillin resistance in S. aureus were not adequate and that the concomitant use of a secondary method such as an oxacillin screen plate was required for all isolates. The results also suggest that the mec gene probe and oxacillin agar screen plate are accurate alternate methods to broth microdilution for the detection of oxacillin resistance in S. aureus. ACKNOWLEDGMENTS
We gratefully acknowledge Barbara Corber and Alice Au Yeung for excellent secretarial assistance and Randy Smith for technical assistance.
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