Detection of Methicillin Resistance in Coagulase-Negative Staphylococci Initially Reported as Methicillin Susceptible Using Automated Methods

Detection of Methicillin Resistance in Coagulase-Negative Staphylococci Initially Reported as Methicillin Susceptible Using Automated Methods Karam Ramotar, Maria Bobrowska, Peter Jessamine, and Baldwin Toye

Reliable detection of methicillin resistance in coagulasenegative staphylococci (CNS) is required for appropriate therapy of serious infections from these pathogens. To determine the most accurate method of measuring methicillin resistance in CNS initially reported as methicillin susceptible by automated methods, we compared mecA detection by polymerase chain reaction (PCR) with phenotypic methods. One hundred eighty-eight blood culture isolates of CNS that were initially reported as susceptible to methicillin using commercial methods (Vitek or MicroScan) were tested by agar dilution, disk diffusion, oxacillin salt agar screen plate, and a multiplex PCR assay using primer sets for mecA and 16S rRNA. Sixteen isolates (8.5%) previously reported as methicillin susceptible by automated methods contained the mecA gene. MICs of these isolates ranged from 0.5 mg/mL to $128 mg/mL. Ten of these isolates had MICs equal to or below the NCCLS break-

point of 2 mg/mL. Six of the 10 isolates (4 with MICs of 0.5 mg/mL and 2 with MICs of 2 mg/mL) did not grow on any of the oxacillin screen plates after 48 h of incubation at 30°C or 35°C. All six isolates were induced to grow in the presence of oxacillin at 128 mg/mL by serial passaging on plates containing increasing concentrations of antibiotic. Retesting with MicroScan and Vitek detected methicillin resistance in 7 and 10 isolates, respectively. Disk diffusion testing with incubation for 48 h proved to be the next best method after PCR for detection of methicillin resistance (15 of 16 isolates). Commercial automated methods and some methods recommended by National Committee for Clinical Laboratory Standards may not detect methicillin resistance in CNS that carry the mecA gene and have MICs just below breakpoint. © 1998 Elsevier Science Inc.

INTRODUCTION

1987). Approximately 80% of hospital strains are methicillin resistant and are usually treated with vancomycin (Schwalbe et al. 1987; York et al. 1996). The preferred treatment of methicillin-susceptible strains is with a penicillinase-resistant penicillin. However, many clinicians still favor vancomycin to treat serious infections caused by methicillin-susceptible strains because of concerns regarding the accuracy of present testing methods. In contrast, CDC recommendations for preventing the spread of vancomycin-resistant enterococci strongly discourage the use of vancomycin in such patients (CDC 1995). Thus, improved compliance with these recommendations would require the availability of susceptibility testing methods that are reliable in detecting methicillin resistance (Bignardi et al. 1996; Gerberding et al. 1991; Woods et al. 1986; York et al. 1996).

The coagulase-negative staphylococci (CNS) are a leading cause of nosocomial infections, particularly bacteremia in immunocompromised patients (Herwaldt et al. 1992) and infections involving indwelling prosthetic devices (Sattler et al. 1984; Younger et al. From the Departments of Pathology and Laboratory Medicine (KR, BT) and Medicine (MB, BT), Ottawa General Hospital; Departments of Laboratory Medicine (PJ) and Medicine (PJ), Ottawa Civic Hospital; and University of Ottawa, Ottawa, Ontario (KR, PJ, BT), Canada. Address reprint requests to Karam Ramotar, Division of Microbiology, Ottawa General Hospital, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. This study was presented at the 96th general meeting of the American Society for Microbiology, New Orleans, LA, May 1996. Received 12 September 1997; accepted 9 December 1997.

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K. Ramotar et al.

268 Methicillin resistance in staphylococci is mediated by the mecA gene, which codes for the production of a low-affinity penicillin binding protein, PBP 2a (Chambers 1987; Hartman and Tomasaz 1984; Stratton et al., 1990). Other mechanisms of resistance include hyperproduction of b-lactamase, production of PBPs with altered binding capacities, or other unidentified factors (Delencastre et al. 1991; Jorgensen 1991). A number of phenotypic methods has been recommended for detection of methicillin resistance. These include disk diffusion using a 1-mg oxacillin disk, microbroth dilution or agar dilution in medium supplemented with 2% NaCl, or an oxacillin salt agar screen plate (Cormican et al. 1996; Woods et al. 1986). The National Committee for Clinical Laboratory Standards (NCCLS) now recommends that methicillin resistance in CNS is most reliably detected using an oxacillin salt agar screen plate with 48 h of incubation at 35°C (NCCLS M7-A4, M100-S7, 1997). DNA hybridization techniques (Archer and Pennell 1990; Kolbert et al. 1995; Ligozzi et al. 1991) or PCR provide more sensitive methods for detection of methicillin resistance. PCR for the mecA gene may be the most sensitive method, but it is not always practical in the clinical diagnostic laboratory (Geha et al. 1994; Murakami et al. 1991; Predari et al. 1991; Tokue et al. 1992; Ubukata et al. 1992; Unal et al. 1994; Vannuffel et al. 1995). This study compares various phenotypic methods to PCR for mecA for the detection of methicillin resistance in CNS initially reported as methicillin susceptible by automated methods. The objective was to determine whether these phenotypic methods could accurately detect all methicillin-resistant CNS, as determined by PCR for mecA.

MATERIALS AND METHODS Bacterial Isolates Between January 1995 and February 1996, 188 CNS isolated from blood cultures at the Ottawa General and Civic Hospitals were collected and stored at 270°C in brain heart infusion with 10% glycerol. Isolates were thawed and subcultured onto tryptic soy agar containing 5% sheep blood. Fresh subcultures using multiple colonies were made before testing. All of the isolates used in this study were initially reported as methicillin susceptible when tested by the laboratories using routine automated methods: Vitek GPS-SA Gram-Positive general susceptibility card (Software: version VTK-R03.01, bioMerieux Vitek Inc., Hazelwood, MO) or MicroScan Pos Combo Type 6 panels (Baxter Diagnostics Inc., Deerfield, IL) read by the AUTOSCAN-4 instrument

(Software: Version 20.1). Fifty-seven isolates had been tested initially by MicroScan and 131 isolates by Vitek.

Testing for Methicillin Resistance Isolates were tested for mecA by PCR and for methicillin resistance by automated methods (Vitek and MicroScan), and by phenotypic methods using oxacillin as the class drug. Reference strains used in this study were Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, and S. aureus ATCC 29247. PCR for mecA was considered the gold standard for detection of methicillin resistance.

Automated Methods All isolates were retested using the Pos BP Combo Type 6 panels (Dade MicroScan Inc., Deerfield, IL). Panels were incubated overnight at 37°C and read by the AUTOSCAN-4 instrument (Software: Version 20.35). Testing was also performed using the Vitek GPS-SA Gram-Positive general susceptibility card (Software: Version VTK-R03.01). In both instances, testing was performed according to the respective manufacturer’s recommendations.

Disk Diffusion Disk diffusion testing was performed according to NCCLS guidelines (M2-A5, 1993a) using a 1-mg oxacillin disk on Mueller-Hinton agar without salt (Becton-Dickinson Microbiology Systems, Cockeysville, MD). Plates were incubated in ambient air at 35°C and read at both 24 and 48 h. Interpretive zone diameters for oxacillin were as follows: #10 mm was resistant and $13 mm was susceptible. Intermediate zone sizes were between 10 and 13 mm. Any growth, including light growth within the 10-mm diameter zone around the disk was taken to indicate resistance.

Oxacillin Salt Agar Screen Plate Oxacillin salt agar screen plate testing was performed using Mueller-Hinton agar base supplemented with 4% NaCl and containing 6 mg/mL oxacillin. Isolates were tested on commercially available (Becton-Dickinson Microbiology Systems, Cockeysville, MD) and in-house prepared screen plates (BBL™ agar base, Becton-Dickinson Microbiology Systems). Plates were inoculated as described by NCCLS M7-A3 (1993b) and were incubated at 30°C or 35°C in ambient air. Plates were read at both 24 and 48 h. Any growth was considered as a positive test result.

Methicillin Resistance in CNS

Agar Dilution MIC Testing Oxacillin MICs were determined as described by NCCLS M7-A3 (1993b) using the agar dilution method. Oxacillin was tested within the range of 0.125 mg/mL to 128 mg/mL using Mueller-Hinton agar base (Oxoid, Unipath Ltd., Hampshire, UK) supplemented with 2% NaCl. Organisms were inoculated onto antibiotic containing medium using a Cathra Systems replicator (MCT Medical Inc., St. Paul, MN) to yield a final inoculum of 104 CFU/spot. Plates were incubated in ambient air at 35°C and read at both 24 and 48 h. The MIC was defined as the lowest antibiotic concentration showing no growth.

PCR for mecA Four to five well isolated colonies obtained after overnight growth on tryptic soy agar were resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 5 mM EDTA [pH 8.0]). Lysostaphin was added at a final concentration of 20 mg/mL and the suspension was incubated at 37°C for 30 min. Proteinase K was then added to a final concentration of 1 mg/mL and the suspension was incubated at 50°C for 60 min, followed by incubation at 100°C for 10 min. Processed samples were used for PCR immediately or stored at 4°C until they were tested. A multiplex PCR assay was used to detect the mecA gene in the CNS isolates. A 1339-bp mecA fragment (nucleotides 478 –1816) was coamplified with a 479-bp 16S rRNA fragment (nucleotides 911–1390) as the PCR internal control. The primer pairs selected to amplify the mecA fragment and the 16S rRNA fragment have been described elsewhere (Geha et al. 1994; Tokue et al. 1992). The PCR amplification mixture consisted of reaction buffer (final concentration, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl [pH 8.3]), 0.2 mM each of dATP, dCTP, dTTP, and dGTP, 1 mM mecA primer pair, 0.1 mM 16S rRNA primer pair, 1.25 U AmpliTaq DNA polymerase (Roche Molecular Systems, Inc., Branchburg, NJ), and 1.0 mL of CNS DNA in a total volume of 50.0 mL. DNA amplification was carried out in a GeneAmp PCR System 9600 thermal cycler (PE Applied Biosystems, Foster City, CA) for 25 cycles. The cycling conditions consisted of 25 cycles of 94°C for 30 s, 55°C for 30 s, and 94°C for 60 s. A reagent blank (containing all of the components of the reaction mixture with water instead of target DNA), a positive control (S. aureus ATCC 29247), and a negative control (S. aureus ATCC 25923) were included with every PCR run. PCR assays for mecA were only considered to be valid if CNS target DNA was positive for the amplified 16S rRNA fragment and if the controls gave the expected results.

269 Amplified products were detected by agarose gel electrophoresis using 2% Nusieve gels, which were stained with ethidium bromide. A fX174/HaeIII fragments molecular mass marker (Life Technologies, Gaithersburg, MD) was run with each gel. Preand post-PCR areas in the laboratory were physically separated and the workflow was unidirectional. Dedicated pipettes, equipment, and supplies, including aerosol-resistant pipette tips and frequent changes of gloves, were used.

PCR for the Coagulase Gene A coagulase gene PCR assay was performed on selected CNS isolates as described by Goh et al. (1992). S. aureus ATCC 25923 and S. epidermidis ATCC 14990 were run as positive and negative controls, respectively.

RESULTS The mecA gene was detected in 16 (8.5%) of the 188 CNS isolates tested (Table 1). This included 7 of the 57 isolates (12.3%) initially tested using MicroScan and 9 of the 131 isolates (6.9%) initially tested using Vitek. All 16 isolates were confirmed to be coagulase negative by PCR testing for the coagulase gene. Retesting using a breakpoint panel and a newer version of the MicroScan software detected methicillin resistance in 7 of the 188 isolates. Five of these had been found to be methicillin susceptible on initial testing with MicroScan and two with Vitek. In contrast, retesting using the same software version of Vitek detected methicillin resistance in 10 of the 188 isolates. Four of these had been previously reported as methicillin susceptible on initial testing with Vitek and six with MicroScan. The oxacillin MICs of the 188 CNS isolates were determined by the agar dilution method, with plates read at 24 h. Additional incubation for an additional 24 h did not show any change in MICs. One hundred seventy of the 188 isolates had oxacillin MICs #0.25 mg/mL, and all of these isolates were mecA negative and methicillin susceptible by all other phenotypic tests. MICs of the remaining 18 isolates ranged from 0.5 mg/mL to $128 mg/mL. Twelve of these isolates had MICs ranging from 0.5 to 2.0 mg/mL. The mecA gene was present in 10 of these 12 isolates (Table 1). Based on the NCCLS breakpoint for oxacillin (#2 mg/mL), these 10 isolates would be considered methicillin susceptible. The other two isolates that were mecA negative both had MICs of 0.5 mg/mL. All six isolates with an oxacillin MIC $4 mg/mL were mecA positive. Disk diffusion testing was performed using Mueller-Hinton agar without salt, as recommended

K. Ramotar et al.

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TABLE 1 Detection of Methicillin Resistance in CNS Initially Reported as Methicillin Susceptible by Automated Methods No. of Isolates Resistant by Oxacillin MIC (mg/mL)

Disk Diffusion

No. of Isolates Tested

MicroScan

Vitek

24 h

#0.25 0.5 1.0 2.0 $4.0

170 6 2 4 6

0 0 1 1 5

0 0 1 3 6

0 0 2 3 6

Total Sensitivity (%)

188

7 44

10 63

11 69

a

48 h

PCR for mecA

0 4a 2 3 6

0 4 2 4 6

15 94

16 100

These four isolates all had intermediate zone sizes at 24 h.

by NCCLS (1993a). All 172 mecA-negative isolates were methicillin susceptible by this method after 24 and 48 h of incubation. Eleven (68.8%) mecA-positive isolates tested as methicillin resistant after 24 h of incubation. On further incubation for another 24 h, four additional isolates that had demonstrated intermediate zone sizes at 24 h became resistant. These four isolates all had MICs of 0.5 mg/mL. Thus, 15 of 16 (93.7%) of the mecA-positive isolates were detected by disk diffusion testing after 48 h of incubation (Table 1). Testing using the oxacillin salt agar screen plate method was carried out at two temperatures using commercially available as well as in-house prepared plates. However, the Mueller-Hinton agar base used in both screen plates was from the same manufacturer. One hundred seventy-six of 188 isolates (MICs of #0.25– 0.5 mg/mL) did not grow on any of these plates after 24 or 48 h of incubation at 30°C or 35°C. The in-house prepared plates performed marginally better at detecting methicillin resistance (Table 2). Both types of plates gave the best results when incu-

bated at 35°C for 48 h. Ten of 16 mecA-positive isolates grew on in-house prepared screen plates, compared to 9 of 16 isolates that grew on commercial plates under these conditions. The poorest results were obtained using the commercial plates with incubation for 24 h at 30°C (1 of 16 isolates) (Table 2). The six mecA-positive isolates (four with MICs of 0.5 mg/mL and two with MICs of 2 mg/mL) that failed to grow on the screen plates under any conditions were serially passaged on Mueller-Hinton agar containing increasing twofold concentrations of oxacillin starting at 0.25 mg/mL. All six isolates were induced to grow on plates containing up to 128 mg/mL oxacillin after a single passage. Similarly, 14 isolates that were mecA negative with oxacillin MICs of #0.125 mg/mL were serially passaged on MuellerHinton agar containing increasing twofold concentrations of this antibiotic starting at 0.03125 mg/mL. All isolates failed to grow on plates with concentrations of oxacillin higher than 0.5 mg/mL. There were no isolates methicillin resistant by a phenotypic method that were negative for mecA by

TABLE 2 Detection of Methicillin Resistance by Oxacillin Agar Screen Plate in CNS Initially Reported as Methicillin Susceptible by Automated Methods No. of Isolates Resistant by the Indicated Method Commercial

In-House

No. of Isolates Tested

30°C, 24 h

30°C, 48 h

35°C, 24 h

35°C, 48 h

30°C, 24 h

30°C, 48 h

35°C, 24 h

35°C, 48 h

#0.25 0.5 1.0 2.0 4.0 $8.0

170 6 2 4 4 2

0 0 0 0 0 1

NTa 0 1 0 3 2

NT 0 0 0 3 2

NT 0 1 2 4 2

0 0 1 0 0 2

0 0 1 1 3 2

NT 0 1 0 3 2

NT 0 2 2 4 2

0 4 2 4 4 2

Total Sensitivity (%)

188

1 6

6 38

5 31

9 56

3 19

7 44

6 38

10 63

16 100

Oxacillin MIC (mg/mL)

a

NT, not tested.

PCR for mecA

Methicillin Resistance in CNS PCR. One mecA-positive isolate with an oxacillin MIC of 2 mg/mL was methicillin susceptible by all phenotypic methods and was the only isolate not detected as methicillin resistant by disk diffusion testing after 48 h of incubation. The six mecA-positive isolates with oxacillin MICs between 4.0 and $128.0 mg/mL were all detected by oxacillin agar screen plate incubated for 48 h at 35°C (in-house prepared plates), by disk diffusion testing, and by Vitek. MicroScan detected five of these six. In contrast, with the exception of disk diffusion (9 of 10 isolates), the phenotypic methods performed poorly at detecting methicillin resistance in the mecA-positive isolates with low oxacillin MICs (0.5–2.0 mg/mL).

DISCUSSION We found that 8.5% of the CNS isolates previously reported as methicillin susceptible by the clinical laboratories contained the mecA gene. The initial reporting was based on the results obtained using automated methods commonly used in routine laboratories. Several isolates initially reported as methicillin susceptible by either MicroScan or Vitek were shown to be methicillin resistant on retesting using the same methods. Retesting by MicroScan was performed using different panels and with updated software, which may explain these results. In contrast, there was no change in testing methodology using Vitek when the isolates were retested by this method, and yet 10 isolates were subsequently found to be methicillin resistant. It is uncertain as to why Vitek initially failed to detect methicillin resistance in these isolates. It may be because retesting was performed by a single technologist as part of a research protocol, whereas the initial testing was performed by various technologists as part of their routine bench assignments. Additionally, minor variations in inoculum size used to inoculate panels for MicroScan or Vitek testing may have contributed to the initial false susceptibility results. This may be especially true of isolates with MICs close to the NCCLS breakpoint. Nonetheless, at least six mecA-positive isolates would still have been reported as methicillin susceptible using Vitek, all of which had oxacillin MICs below breakpoint (#2.0 mg/mL). On comparing the two automated methods, Vitek detected 4, whereas MicroScan detected only 2 of the 10 mecA-positive isolates with the MICs below breakpoint (#2.0 mg/mL). Thus, neither of these automated methods can be relied upon as the sole method for detecting methicillin resistance in CNS. The 16 mecA-positive isolates had oxacillin agar dilution MICs ranging between 0.5 mg/mL and $128 mg/mL, and 10 of these had MICs at or below breakpoint (#2.0 mg/mL). York et al. (1996) reported that

271 when incubation time was prolonged from 24 to 48 h, 12 of 23 mecA-positive isolates that were susceptible at 24 h (MICs #2.0 mg/mL by microbroth dilution) became resistant. In our study, further incubation of the plates for another 24 h showed no change in the MICs of the isolates. Oxacillin salt-agar screen plate testing was performed using plates that were commercially available and prepared in-house. In-house prepared plates performed better than commercial plates to varying degrees, depending on incubation time or temperature. This was despite the fact that the Mueller-Hinton agar base medium was from the same manufacturer. Assuming similar preparation protocols, it is unclear why performances of the plates differ. Although this was not the case in this study, it has been shown that performance of the oxacillin agar screen plate may depend on the source of Mueller-Hinton base media (Hindler and Warner 1987). Incubation of the screen plates for 48 h compared to incubation for 24 h at 30°C or 35°C improved the detection of methicillin resistance among the mecApositive isolates. Screen plates did not detect the four isolates with oxacillin MICs of 0.5 mg/mL under any conditions, even after several repeat testings. The in-house prepared plates incubated for 48 h at 35°C produced the highest sensitivity in detecting methicillin resistance (63%), but still performed poorly compared to disk diffusion testing. Therefore, oxacillin salt-agar screen plates with incubation for 48 h, as currently recommended by NCCLS, would still fail to detect a significant number of mecA-positive strains, especially those with MICs below breakpoint. Disk diffusion was performed on Mueller-Hinton agar without salt supplementation. Incubating Mueller-Hinton plates for 48 h detected 15 of 16 mecA-positive isolates overall, and 9 of 10 that had MICs below breakpoint. This method, therefore, compares favorably to PCR for mecA for detection of methicillin resistance and is similar to the findings of McDonald et al. (1995). Our findings also demonstrate that disk diffusion testing provides better sensitivity for detecting methicillin resistance in CNS when the plates are incubated for 48 h (94%) compared to 24 h (69%). However, NCCLS (1993a) recommends that plates be incubated for 24 h and not 48 h. Cormican et al. (1996) have suggested that methicillin resistance in CNS is best detected using the disk diffusion test with incubation of up to 48 h. In their study, disk diffusion testing using the 1-mg oxacillin disk performed better than the screen plate (sensitivity of 98% versus 91%). These results and our study, however, contrast with those of York et al. (1996), who reported that the screen plate method

272 was superior to the disk test (sensitivity of 100% versus 81% after 48 h of incubation). These discrepancies may represent differences in the source of Mueller-Hinton agar used (Mackenzie et al. 1995) or be due to differences in expression of the mecA gene in the strains that were tested. Our data are similar to those of other studies that have demonstrated that the sensitivity of the standard disk diffusion test is comparable with that of mecA detection (Cormican et al. 1996; McDonald et al. 1995; Olsson-Lilejequist et al. 1993) and better than the oxacillin salt agar screen plate (Cormican et al. 1996) or microbroth dilution MIC methods (Woods et al. 1986). Therefore, it is clear that further studies using a greater number of mecA-positive strains from various geographic locations that have MICs below breakpoint are needed. There were 12 isolates that had oxacillin MICs ranging from 0.5 to 2.0 mg/mL and 10 of these were mecA positive. Others (Cormican et al. 1996; McDonald et al. 1995; York et al. 1996) have suggested that the breakpoint for CNS be reduced to 1.0 mg/ mL. The study of York et al. (1996) found that 12 isolates of CNS with MIC less than 1.0 mg/mL contained mecA and grew on the screen plate. In the study of Cormican et al. (1996), 87% of the strains tested were resistant based on the current breakpoint; reducing the breakpoint to 0.5 mg/mL increased sensitivity to 100%. These investigators suggest that lowering the breakpoint would make detection of methicillin resistance in CNS more reliable. The six mecA-positive isolates that did not grow on the oxacillin salt screen plate and had MICs in the susceptible range (#2 mg/mL) should be considered

K. Ramotar et al. clinically resistant, as they were induced to grow in the presence of 128 mg/mL oxacillin. In contrast, several isolates that were mecA negative with MICs #0.125 mg/mL could not be induced to grow on plates with oxacillin concentrations above 0.5 mg/ mL. mecA is constitutively expressed in most staphylococci that contain the gene, and this correlates well with detection of methicillin resistance. However, not all strains that possess mecA are able to express the gene product, and in these cases, induction by oxacillin is necessary (Chambers 1988; Predari et al. 1991). Although clinical data are required, it would appear that treatment of patients with serious infections caused by such strains with a penicillinase-resistant penicillin would result in clinical failure. PCR for the mecA gene represents the most accurate method for detecting methicillin resistance in CNS. As PCR for mecA is not yet practical for routine testing in all clinical laboratories, other methods for detection of methicillin resistance must be used to supplement automated methods for routine susceptibility testing of clinically significant CNS. The results of this study indicate that disk diffusion testing using a 1-mg oxacillin disk with 48 h of incubation represents the most sensitive phenotypic method to accomplish this.

We thank Wendi Woods for technical assistance and other members of the Microbiology Laboratories at the Ottawa General and Civic Hospitals and the National Defence Medical Centre.

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