Detection of methicillin resistance in Staphylococcus aureus isolated from pediatric patients: is the cefoxitin disk diffusion test accurate enough?

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ORIGINAL PAPERS

Detection of methicillin resistance in Staphylococcus aureus isolated from pediatric patients: is the cefoxitin disk diffusion test accurate enough?

Mimica M.J.^I; Berezin E.N.^I; Carvalho R.L.B.^II; Mimica I.M.^II; Mimica L.M.J.^II; Sáfadi M.A.P.^I; Schneider E.^II; Caiaffa-Filho H.H.^{II, III}

^IDivision of Pediatric Infectious Diseases, Department of Pediatrics

^IILaboratory of Microbiology, Department of Pathology Santa Casa University Hospital

^IIILaboratory of Medical Investigation - LIM 03, Hospital das Clinicas-FMUSP; São Paulo, SP, Brazil

^{Address for correspondence}

ABSTRACT

We evaluated the performance of several methods for the detection of methicillin resistance in Staphylococcus aureus using 101 clinical S. aureus isolates from pediatric patients in a tertiary hospital in Brazil; 50 isolates were mecA-positive and 51 were mecA-negative. The Etest and oxacillin agar screening plates were 100% sensitive and specific for mecA presence. Oxacillin and cefoxitin disks gave sensitivities of 96 and 92%, respectively, and 98% specificity. Alterations of CLSI cefoxitin breakpoints increased sensitivity to 98%, without decreasing specificity. Our results highlight the importance of a continuing evaluation of the recommended microbiological methods by different laboratories and in different settings. If necessary, laboratories should use a second test before reporting a strain as susceptible, especially when testing strains isolated from invasive or serious infections. With the new (2007) CLSI breakpoints, the cefoxitin-disk test appears to be a good option for the detection of methicillin resistance in S. aureus.

Key-Words: MRSA, disc diffusion, cefoxitin, mecA.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a major epidemiological and clinical problem over the last decades. These strains have spread worldwide, causing nosocomial and, more recently, community-based infections [1]. This has led to the overuse of glycopeptides, and to the emergence of vancomycin-resistant S. aureus [2]. In this setting, rapid and accurate detection of methicillin resistance would help ensure correct use of antibiotics and appropriate epidemiological control of MRSA. Methicillin resistance in S. aureus is primarily mediated by overproduction of PBP2a protein, an altered penicillin-binding protein with lower affinity for beta-lactam antibiotics than PBP2, the main physiological methicillin target. PBP2a is encoded by the mecA gene, a component of a larger DNA fragment designated the mec region. Phenotypic expression of resistance may vary depending on culture conditions, such as temperature or osmolarity of the medium, despite genetic homogeneity [3].

This heterogeneous resistance phenotype may complicate the detection of MRSA by conventional susceptibility methods. Oxacillin-disk diffusion has been the traditional method for methicillin-resistance routine screening; but recently good accuracy of the cefoxitin disk for predicting methicillin resistance has been reported [4-11], and CLSI (Clinical and Laboratory Standards Institute) has recommended that cefoxitin should be preferred over oxacillin for the detection of mecA-mediated resistance [4]. However, the choice of the best phenotypic method for detecting methicillin resistance in S. aureus remains controversial. Good accuracy of other methods, such as Etest and oxacillin-agar screening plate, has also been demonstrated [12-15]. Detection of the gene is considered the reference method [3], but this is not feasible in most laboratories throughout the world.

Our main objective was compare oxacillin and cefoxitin-disk tests, Etest and oxacillin-agar screening plates for detection of methicillin resistance in S. aureus, using PCR for mecA as the "gold standard" comparison assay.

Materials and Methods

Strains

We studied 101 pediatric clinical isolates of S. aureus (isolated from different anatomical sites of different pediatric patients) that were collected from April 2004 to June 2005 and identified by biochemical procedures. Staphylococcus aureus ATCC 25923, ATCC 29213 and ATCC 33591 were used as quality-control strains.

Detection of the mecA Gene

A single bacterial colony was obtained from a fresh subculture and was resuspended in 25 µL of sterile water. The suspension was boiled at 95ºC for DNA extraction. One microliter of the DNA samples was added to 19 µL of PCR mixture, consisting of 1U Taq polymerase, 1X polymerase buffer [50 mM KCl, 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl₂], 200 µM dNTPs mixture and 0.5 µM of each primer. Amplification was performed using a Perkin Elmer 2400 thermocycler (Applied Biosystems, California, USA). After an initial denaturation step (three minutes at 94ºC), 30 cycles of amplification were performed: denaturation at 94ºC for one minute, annealing at 56ºC for one minute and DNA extension at 72ºC for one minute. The reaction was finished with a final extension step at 72ºC for seven minutes. The set of primers used (M1, 5'-TGGCTATCGTGTCACAATCG-3' and M2, 5'-CTGGAACTTGTT-GAGCAGAG-3') was described by Vannuffel [16]. The amplified product was a 310-bp DNA fragment that was detected by 1.5% agarose gel electrophoresis, with ethidium bromide staining visualized under UV light.

Susceptibility Tests

The isolates were tested with oxacillin (1 µg) and cefoxitin (30 µg) disks, using Mueller-Hinton agar plates inoculated with a suspension (equivalent to a 0.5 McFarland standard) of the S. aureus clinical isolates. The plates were incubated at 35ºC for 24 hours and inhibition zones were measured. The oxacillin MICs (minimum inhibitory concentration) were determined by Etest (AB Biodisk, Solna, Sweden) using Mueller-Hinton agar plates supplemented with 2% NaCl. The CLSI 2005 criteria [4] were used for interpretation (Table 1). Isolates were also tested with oxacillin agar screening, which was performed by inoculating a direct colony suspension (0.5 McFarland standard) with a swab, spotting an area 10 to 15 mm in diameter, on Mueller-Hinton agar supplemented with 4% NaCl and oxacillin at 6 mg/L. After incubation for 24 hours, any growth was interpreted as a positive result for MRSA.

Results and Discussion

Among the 101 strains included in our study, 50 were mecA-positive and 51 were mecA-negative. The results of the phenotypic tests are shown in Table 2. Etests and oxacilin plates were the most accurate methods. Both were 100% sensitive and specific.

In other studies that used the presence of mecA as the gold standard, the accuracy of these techniques was also very good [12-15].

The oxacillin and the cefoxitin disk tests showed sensitivities of 96% and 92%, respectively, and 98% specificity. Six strains had discrepant results between at least one of the disks and the mecA gene (Table 3). The accuracy of the cefoxitin disk test in our study was different from that previously reported. However, among the four mecA-positive strains giving false-negatives by the cefoxitin test, three had an inhibition zone of 20 mm, and none of the mecA-negative strains had such a large zone diameter. Thus, if there were changes in the cefoxitin breakpoints, the sensitivity of the method could be increased, perhaps without any decrease of specificity. Other authors have also suggested different breakpoints in the interpretative zone diameters of cefoxitin for better detection of methicillin resistance in S. aureus [7,10].

We evaluated our strains in 2005, using the former CLSI cefoxitin breakpoints (R: < 19 mm; S: > 20 mm). But, in 2007 the recommended CLSI breakpoints were changed [17], in order to increase the accuracy of the method. With the new breakpoints (R: < 21 mm; S: > 22 mm), the three strains that had a 20 mm zone would have been correctly diagnosed, increasing the sensitivity to 98%, again without any decrease in specificity, since we did not find any mecA-negative strains with inhibition zones smaller than 22 mm.

Although the number of isolates tested in our study was low and there was a chance of clonality, our results highlight the importance of a continuing evaluation of recommended microbiological methods by different laboratories and in different settings. If necessary and if the evidence supports it, the method should be withdrawn from the recommendations or its breakpoints changed. In addition, laboratories could use a second test before reporting a strain as susceptible, especially when testing strains from invasive or serious infections.

Conclusion

In our study the oxacillin agar screening plate appeared to be a good option for the detection of methicillin resistance in S. aureus, due to its great accuracy and low cost. With the new (2007) CLSI breakpoints, the cefoxitin-disk test may also be a reasonable alternative.

Received on 5 March 2007; revised 8 July 2007.

Financial support: M.J. Mimica received financial support from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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Publication Dates

• Publication in this collection
  04 Sept 2007
• Date of issue
  Aug 2007

History

• Received
  05 Mar 2007
• Reviewed
  08 July 2007