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Improvement of mupirocin E-test for susceptibility testing of Staphylococcus aureus

¹Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil ²Departamento de Patologia, Universidade Federal Fluminense, Rio de Janeiro, RJ, Brazil

Correspondence Marcia Giambiagi-deMarval marciagm{at}micro.ufrj.br

Received June 25, 2002
Accepted October 1, 2002

	Abstract

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Interpretation of the mupirocin E-test for low-level mupirocin-resistant Staphylococcus aureus strains has been improved by adding the indicator dye tetrazolium. E-tests were compared with agar dilution methods for assessing mupirocin susceptibility. MICs obtained by the agar dilution method and E-tests showed 89.3 % agreement within 2 log₂ dilution criteria. The agreement between MICs increased to 100 % in the 1 log₂ dilution definition when the indicator dye tetrazolium was added to the E-test. The use of the E-test with tetrazolium reduction is more accurate for determining mupirocin MICs for S. aureus strains.

	Introduction

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Mupirocin is an effective antibiotic for elimination of methicillin-resistant Staphylococcus aureus (MRSA) from nasal colonization and has been used to control the spread of these organisms among patients during outbreaks (Eltringham, 1997). Mupirocin-resistant strains can be found, and they are grouped in two distinct categories: low-level or intermediate resistance (Mup^I), with MICs of 4–256 µg ml^-1, and high-level resistance (Mup^R), with MICs 512 µg ml^-1 (Eltringham, 1997). Mup^R has been associated with the failure to clear the organism in patients. However, it has been suggested that Mup^I nasal isolates may still be controlled with mupirocin therapy (Hudson, 1994), since the ointment used contains a much higher mupirocin concentration (20 000 µg ml^-1).

We have previously shown that the acquisition of mupirocin resistance in the Hospital of the Federal University of Rio de Janeiro was associated with the prior use of mupirocin in the nosocomial environment, indicating that mupirocin should be used solely for the treatment and prevention of serious MRSA infections (Santos et al., 1996).

In this context, the E-test appears to be an excellent tool for determining the level of mupirocin resistance in S. aureus. However, Staphylococcus strains tend to give lower MICs in the mupirocin E-test than in the corresponding mupirocin agar dilution (Simpson et al., 1995). It has been shown that the disc diffusion method can be improved by adding the indicator dye tetrazolium–phenazine methosulfate (Bartlett & Mazens, 1979). Furthermore, the E-test for antimicrobial susceptibility of Helicobacter pylori was more accurate when tetrazolium/egg-yolk agar was employed (Valdez et al., 1998).

In the present paper, we investigate the possibility of improving the S. aureus mupirocin E-test by adding the tetrazolium indicator dye.

	Methods

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Strains and cultivation.

MICs were evaluated for 42 MRSA isolates from patients from the Teaching Hospital of the Federal University of Rio de Janeiro, Brazil. Fourteen strains (Table 1) were selected on the basis of their low-level mupirocin resistance, as characterized by the agar dilution method (Santos et al., 1999). S. aureus strains HU1A (Mup^R; Bastos et al., 1999) and ATCC 2913 [mupirocin-susceptible (Mup^S)] were used as controls. Mueller–Hinton agar (Difco) was used throughout all assays.

Susceptibility agar dilution method.

Agar plates containing double-dilution series of mupirocin (512–1 µg ml^-1) were inoculated with 2 µl (10⁴ c.f.u. per spot) of each strain using a Steers replicator and incubated for 18 h at 35 °C (NCCLS, 2001). MICs were determined as the lowest concentration of antibiotic that inhibited bacterial growth completely.

Susceptibility E-test method.

Mupirocin susceptibility was determined by the E-test according to the manufacturer's guidelines (AB Biodisk). Plates were swabbed in three directions with 0.5 McFarland inocula. The E-test strip was applied to each plate with sterile forceps. Following incubation at 35 °C for 18 h, E-test MIC values were read by two independent operators, as the point of intersection of the zone of inhibition with the E-test strip. Immediately, 100 µl of a dye-indicator solution containing 1 mg 2(p-iodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride and 60 µg phenazine methosulfate ml^-1 (Sigma) (Bartlett & Mazens, 1979) was added on the inhibition ellipse edge along each E-test strip. After 30 min, each plate was examined for colour development and the intersection-point of the zone of inhibition was again read by two independent operators. A magnifying glass was used to confirm the absence of isolated colonies or hazes.

Comparison of MICs determined by the agar dilution method and E-test.

Since E-test MICs are determined on continuous antibiotic concentration gradients, E-test MIC values that fell between two-fold dilutions were rounded up to the standard log₂ scale of MICs from agar dilution. Hence, E-test MIC values of 3–4 µg ml^-1 were rounded up to 4 µg ml^-1, those of 6–8 µg ml^-1 to 8 µg ml^-1, 12–16 µg ml^-1 to 16 µg ml^-1 and 24–32 µg ml^-1 to 32 µg ml^-1.

	Results and Discussion

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We observed that the reading of MIC values determined by the E-test could be improved by the addition of the indicator dye tetrazolium. For one S. aureus isolate (HU2D), the MIC was determined as either 8 µg ml^-1 by standard E-test or 16 µg ml^-1 after adding tetrazolium to the E-test plate (Fig. 1).

View larger version (126K): [in this window] [in a new window]   Fig. 1. E-test MICs of mupirocin for a clinical isolate of S. aureus MupI, strain HU2D, without (a) or with (b) tetrazolium reduction.

Therefore, we decided to analyse the MICs of 14 nosocomial strains of S. aureus exhibiting low-level mupirocin resistance by agar dilution, E-test and E-test with tetrazolium (Table 1). The intercepts of the E-test scale were read by two different operators and end-points were determined. For the MICs obtained in the E-test, when seven assays were read independently by two operators, five of the resulting 14 readings (35.7 %) were in agreement. After tetrazolium reduction, determination of the interception point was much clearer and easier. The readings of MICs from the E-test with tetrazolium were more similar than those observed for the conventional E-test. The operators agreed on 13 of 14 readings, thus increasing the agreement from 35.7 to 92.8 % (Table 1).

The coefficients of agreement of MICs between E-test and agar dilution were 89.3 % within 2 log₂ dilutions, 57.2 % within 1 log₂ dilution and 17.9 % at identical dilutions (Table 2). Whenever the MICs did not agree exactly, we observed a clear bias towards E-test values being lower than those obtained by agar dilution. These differences were reduced by the addition of the indicator tetrazolium. There was 100 % agreement between the MICs of E-test with tetrazolium and the agar dilution method by the 1 log₂ dilution definition and 32.2 % of MICs were identical (Table 2). No strain showed differences in the MICs obtained by agar dilution and E-test with tetrazolium, regardless of operators, within the criterion limits of 2 or >2 log₂ dilutions. It should be noted that control strains, exhibiting high-level resistance or sensitivity to mupirocin, also presented lower values of MICs when comparing standard E-test with agar dilution. These results reinforce the advantage of adding the tetrazolium dye for the E-test method.

The need to perform antimicrobial susceptibility tests on Staphylococcus isolates is becoming more pressing. The versatility and feasibility of the E-test make the method an attractive alternative to conventional diffusion and dilution susceptibility tests. Therefore, the purpose of this study was to test the validity of tetrazolium reduction for improving mupirocin E-testing. It was shown that the mupirocin E-test is a reliable method for determining the susceptibility of staphylococci to mupirocin, although there was a tendency for the E-tests to give lower MICs when they did not correspond to the agar MICs (Simpson et al., 1995). It should be emphasized that the high-level mupirocin-resistant control strain HU1A used in this work also presented an MIC with an E-test value lower than that observed for the agar dilution method. This reinforces the advantage of using the tetrazolium dye for the E-test method.

Here, we present an improvement of the mupirocin E-test by tetrazolium reduction and show that this modified technique brings the MICs determined by the E-test closer to the ‘gold standard’ agar dilution method.

	Acknowledgments

 
The authors express their thanks to Professor Walter Oelemann for helpful comments on the manuscript and SmithKline Beecham for kindly supplying the mupirocin powder. This study was supported by grants from CNPq, FAPERJ and MCT/PRONEX (Brazilian Ministry of Science and Technology).

	References

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