Standardized mupirocin susceptibility testing is needed because of increasing mupirocin use for nasal decolonization (19
), reports that expanding the use of mupirocin results in greater rates of resistance (24
), and the understanding that both mupirocin-low-level-resistant (MICs = 8 to 256 μg/ml) and high-level-resistant (MICs ≥ 512 μg/ml) S. aureus
strains must be differentiated when clinical outcome studies are performed (18
). We have shown with a three-phase collaborative study that both mupirocin disk diffusion testing and broth dilution testing using CLSI testing methods (3
) accurately predict in vitro
susceptibility and both low-level and high-level resistance. As demonstrated in the present study, accurate disk readings are achieved with a full 24-h incubation and by the use of transmitted light to read the zone diameters. In addition, no significant broth medium effects were noted with MIC testing when CAMHBs from three different sources were used. Finally, quality control ranges are recommended for disk and broth testing based on CLSI M23-A3 methods (2
The 200-μg mupirocin disk test differentiates S. aureus
strains with high-level mupirocin resistance (no zone of inhibition) from susceptible and low-level-resistant strains (any zone of inhibition) compared to tests that base such differentiations on detection of the presence or absence of mupA
by PCR (Table ). The sensitivity and specificity of this approach are 98 and 99%, respectively. When the results from the 200-μg disk are compared to the broth dilution MIC results, there is a 100% correlation between MICs of ≥512 μg/ml and no zone of inhibition and MICs of ≤512 μg/ml and a zone of inhibition of >18 mm (Fig. ). In addition, as suggested by Cookson, the use of a 5-μg mupirocin disk can be used to detect S. aureus
stains with low- and high-level resistance, i.e., nonsusceptible isolates (Fig. ) (8
). Our three-laboratory study validated the ability of a 5-μg disk to differentiate mupirocin-susceptible strains (≥12-mm zone diameter and ≤4-μg/ml MIC) from mupirocin-resistant strains (≤11-mm zone diameter and ≥8-μg/ml MIC) with 100% sensitivity and specificity compared to the broth dilution MIC results.
In our study, three strains with mupirocin MICs of >512 μg/ml and no zone of inhibition around the 200-μg disk were mupA
negative by PCR. The same three strains also had no zone of inhibition with the 5-μg disk. High-level-mupirocin-resistant, mupA
-negative strains have been detected previously (1
). These strains could carry a novel mechanism of resistance or, one could postulate, could have multiple base changes in the native, chromosomal isoleucyl-tRNA synthetase gene, elevating MIC values from the low-level to high-level resistance ranges. It has been shown that single base changes in this chromosomal gene result in mupirocin MICs of 8 μg/ml, while two mutations result in MICs of >32 μg/ml (1
). Since repeated passage of S. aureus
in the presence of subinhibitory levels of mupirocin has resulted in mupA
-negative, high-level-resistant mutants, it is tempting to favor the latter mechanism as an explanation for the nonagreement of the results obtained with these three strains (23
In addition, three strains of S. aureus
positive by PCR and had MICs of <4 μg/ml and inhibitory zone diameters of 30, 32, and 33 mm with the 200-μg disk and 23, 24, and 26 mm with the 5-μg disk. As noted by others (18
-positive strains with low-level-resistant or susceptible MIC values occur because (i) the mupA
gene was located on the chromosome rather than a plasmid (low-level resistance) or (ii) a frameshift mutation in the mupA
gene is present and resulted in an inactive gene product.
We expect that the strains tested in this multilaboratory study are representative of current S. aureus community and hospital strains. With 622 strains tested in phase 2 and only 3 (0.5%) appearing to be falsely resistant by PCR, we predict that this finding is rare today. False resistance might lead to the use of an alternative topical drug that has in vitro activity but no FDA approval for decolonization or the use of agents for which clinical trial data document in vivo efficacy for nasal decolonization, such as neomycin sulfate-polymyxin B sulfate (Polysporin)-bacitracin (Neosporin), bacitracin zinc-polymyxin B sulfate, or retapamulin. A false-susceptible finding would subject the patient to decolonization with mupirocin where the isolate is resistant and unlikely to be eradicated. Fortunately, false-susceptible results in our population of strains are equally rare (<0.5% of strains tested).
As advocated by de Oliveira et al. (10
), we also validated the use of two mupirocin disk concentrations to identify three phenotypic categories: mupirocin susceptible, mupirocin low-level resistant, and mupirocin high-level resistant (Table ). Using zone/no zone criteria, no zone around both the 5-μg and 200-μg disks indicates mupirocin high-level resistance. No zone around the 5-μg disk and any zone around the 200-μg disk indicate low-level resistance. Any zone of inhibition around both disks indicates mupirocin susceptibility. The accuracies of the two-disk method for the prediction of high-level resistance, low-level resistance, and susceptibility are 100%, 100%, and 99%, respectively, compared to the results of broth dilution MIC testing.
The results from phase 3, using an eight-laboratory study, recommend acceptable limits for quality control of broth microdilution and disk diffusion (Table ). S. aureus
ATCC 29213 and E. faecalis
ATCC 29212, with ranges at low and intermediate MIC levels, respectively, are recommended for use for broth dilution quality control testing. S. aureus
ATCC 25923 is used for quality control testing with the 5- and 200-μg disks. A mupA-
positive strain, S. aureus
ATCC BAA-1708, should grow in a well or tube containing 256 μg/ml of mupirocin and produce no zone of inhibition with the 200-μg disk. This strain is recommended for quality control testing of the CLSI broth dilution and disk tests for the detection of high-level mupirocin resistance (7
Reasons for performing mupirocin susceptibility testing may be to identify individual patients who are at risk for mupirocin decolonization failure or to perform surveillance studies for emerging mupirocin resistance. High-level mupirocin resistance has been associated with mupirocin decolonization failures. In a prospective evaluation of mupirocin decolonization, at 3 days posttreatment, the decolonization rates were lower for patients colonized with high-level-mupirocin-resistant MRSA (27.7%) than for patients colonized with either low-level-mupirocin-resistant MRSA (80%) or mupirocin-susceptible MRSA (78.5%) (25
). In a randomized controlled trial where a combination of nasal mupirocin use, oral doxycycline therapy, and chlorohexidine washes was compared to no decolonization intervention for preventing MRSA infections in a health care setting, nasal colonization with high-level mupirocin-resistant S. aureus
isolates was identified as an independent predictor of decolonization failure in the treatment arm (21
). Studies correlating mupirocin HLR and decolonization treatment failure have been done only with MRSA (14
), although we have no reason to believe that it would be different with MSSA.
The clinical significance of low-level mupirocin resistance is unclear. One reason for this is that isolates with this phenotype are relatively uncommon. In the randomized controlled trial, there were not enough isolates with low-level resistance to evaluate how this affected the mupirocin decolonization outcome. In the study of mupirocin decolonization by Walker et al. described above, patients colonized with low-level-mupirocin-resistant isolates were initially decolonized (80% at the 3-day follow-up), but at the 1- to 4-week follow-up, the decolonization rate for patients colonized with low-level-resistant strains was similar to the rate for patients colonized with high-level-resistant strains, and these rates were lower than those for patients colonized with susceptible strains (25
). However, only 5 of the 40 patients were colonized with low-level-resistant S. aureus
isolates; therefore, these results are difficult to interpret. The availability of standardized and validated mupirocin broth dilution and disk diffusion testing methods should facilitate data collection to understand the clinical significance of low-level mupirocin resistance.
Currently, there are no FDA breakpoints for mupirocin, and this has limited the availability of commercial products for mupirocin susceptibility testing in the United States. However, similar broth-based tests are included on panels from some manufacturers of automated susceptibility testing devices sold outside the United States. Commercial disks are currently available in the United States for research use only. Laboratories in the United States that want to perform mupirocin susceptibility testing could prepare their own reagents and validate the test in-house. If mupirocin use increases, it is likely that mupirocin resistance will increase as well. In this case, it will be important for microbiology laboratories to have access to mupirocin susceptibility testing methods and reagents.