JAC Advance Access published online on June 19, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn266
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Original research |
In vitro activity of retapamulin against Staphylococcus aureus isolates resistant to fusidic acid and mupirocin
Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL), HPA Centre for Infections, London NW9 5EQ, UK
* Corresponding author. Tel: +44-20-8327-7255; Fax: +44-20-8327-6264; E-mail: neil.woodford{at}hpa.org.uk
Received 27 May 2008; returned 4 June 2008; revised 1 June 2008; accepted 5 June 2008
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Abstract |
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Objectives: We determined the in vitro activity of retapamulin, a novel pleuromutilin antibiotic, against 664 Staphylococcus aureus isolates from the UK, including many resistant to fusidic acid and/or highly resistant to mupirocin.
Methods: MICs were determined on Mueller–Hinton agar in accordance with the CLSI guidelines. Susceptibility was categorized using CLSI criteria, where available; otherwise the European Committee for Antimicrobial Susceptibility Testing (EUCAST)/BSAC criteria were used (for mupirocin and fusidic acid). Mutations in the rplC gene, which encodes ribosomal protein L3, were sought by PCR and DNA sequencing.
Results: The S. aureus included 488 (73%) methicillin-resistant isolates (oxacillin MICs >2 mg/L), 336 isolates (51%) resistant to fusidic acid (MICs >1 mg/L) and 254 (38%) with high-level mupirocin resistance (MICs >256 mg/L); 103 (16%) isolates were resistant both to fusidic acid and to high levels of mupirocin. Retapamulin inhibited 663 (99.9%) isolates at 
0.25 mg/L. A single methicillin-resistant S. aureus isolate, also with high-level mupirocin resistance, required a retapamulin MIC of 2 mg/L, but its reduced susceptibility to retapamulin was not associated with any mutation in ribosomal protein L3.
Conclusions: Retapamulin demonstrated excellent activity in vitro against S. aureus isolates, irrespective of their level of resistance to other antibacterials. These results support the EUCAST epidemiological cut-off value for retapamulin of 0.5 mg/L against S. aureus.
Key Words: S. aureus , MRSA , mechanisms
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Introduction |
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Pleuromutilin antibiotics inhibit protein synthesis through interaction with the 50S ribosomal subunit1–3 and have long been used in veterinary, but not in human, medicine. Retapamulin is a novel, semi-synthetic member of this class. It was licensed in the USA (‘Altabax’) in 2007 for the topical treatment of impetigo caused by Staphylococcus aureus (methicillin-susceptible isolates only) and Streptococcus pyogenes. Retapamulin is also licensed in Europe (‘Altargo’) for the treatment of impetigo and secondarily infected open wounds caused by these organisms.
Methicillin-resistant S. aureus (MRSA) is not a frequent cause of impetigo, but the few cases in clinical trials of retapamulin were clinical successes (100%: 8/8). However, retapamulin is not recommended for use in MRSA infections since, for unknown reasons, its efficacy was reduced in patients with MRSA infection of open wounds (http://www.emea.europa.eu/humandocs/PDFs/EPAR/altargo/H-757-PI-en.pdf).
We determined the activity of retapamulin in vitro against current S. aureus isolates from the UK. We included many isolates resistant to fusidic acid and/or highly resistant to mupirocin, and a large number of MRSA isolates.
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Materials and methods |
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Bacterial isolates
Six hundred and sixty-four isolates of S. aureus were recovered from storage either at –70°C or at room temperature; these had been submitted to the HPA Centre for Infections for reference investigation from laboratories throughout the UK. Four hundred and eighty-eight (73%) isolates were MRSA (oxacillin MICs >2 mg/L).
MICs were determined following the CLSI guidelines on Mueller–Hinton agar. The agents tested were retapamulin, fusidic acid, mupirocin, oxacillin, erythromycin, clindamycin and gentamicin. Susceptibility was categorized using CLSI criteria, where available; the European Committee for Antimicrobial Susceptibility Testing (EUCAST)/BSAC criteria were used for mupirocin and fusidic acid. ‘Wild-type’ susceptibility to retapamulin and mupirocin was defined using epidemiological cut-off values of 0.5 mg/L, as recommended by EUCAST (http://www.srga.org/eucastwt/MICTAB/index.html).
Investigation of mechanisms of reduced pleuromutilin susceptibility
Mutations in rplC, which encodes ribosomal protein L3, were sought in isolates with reduced retapamulin susceptibility.4,5 Primers used to amplify and sequence an 822 bp fragment, including the entire rplC gene, were: 5'-AAC CTG ATT TAG TTC CGT CTA and 5'-GTT GAC GCT TTA ATG GGC TTA.5 Selected PCR products, from isolates with reduced susceptibility and from fully susceptible isolates (used as controls), were sequenced with dye terminator chemistry using a CEQ8000 Genetic Analyser (Beckman–Coulter, High Wycombe, UK). The sequences were compared with GenBank depositions using BLAST algorithms.
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Results and discussion |
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The susceptibilities of the 664 isolates to oxacillin, erythromycin, clindamycin and gentamicin are summarized in Table 1, while Table 2 shows the MIC distributions of those agents prescribed for topical use versus impetigo. The majority of the isolates were MRSA (488, 73%, which reflects the bias in S. aureus submissions to the national reference laboratories). The isolates studied included 254 (38%) with high-level mupirocin resistance (MICs >256 mg/L) and 49 with low-level resistance (MICs 8–256 mg/L), which may result in slower eradication by mupirocin than for isolates with full susceptibility.6 A total of 324 (49%) isolates were above the epidemiological cut-off value for wild-type mupirocin susceptibility (MICs
0.5 mg/L) set by EUCAST (http://www.srga.org/eucastwt/MICTAB/index.html). Over half of the isolates (336, 51%) were resistant to fusidic acid (MICs >1 mg/L), and 103 (16%) were resistant both to fusidic acid and to high levels of mupirocin.
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Retapamulin inhibited 663 (99.9%) isolates at
0.25 mg/L, with a unimodal MIC distribution, centred at 0.06 mg/L. These data are consistent with the epidemiological cut-off value for wild-type susceptibility of 0.5 mg/L set by EUCAST. A single MRSA isolate in our study, also with high-level mupirocin resistance, required a retapamulin MIC of 2 mg/L. As reduced susceptibility and resistance to pleuromutilins has been associated with mutations in ribosomal protein L3,4,5 the 663 bp rplC gene of this isolate was sequenced alongside that of a fully retapamulin-susceptible MRSA isolate (MIC 0.06 mg/L). The sequence obtained from the fully susceptible isolate was identical to rplC of MRSA252 (GenBank BX571856
[GenBank]
); that of the isolate with reduced susceptibility varied at three positions known to show polymorphism between S. aureus strains (C198T, C492T and T600C). Importantly, each of these changes in the DNA sequence was silent, with no amino acid substitutions in the predicted L3 protein. Reduced susceptibility to pleuromutilins in staphylococci may also involve production of the Cfr methyltransferase, which also confers cross-resistance to phenicols, lincosamides, oxazolidinones and streptogramin A compounds,7–9 or non-target-specific efflux mediated via VgaAv, which also affects streptogramin A compounds (Summary of Product Characteristics, http://www.emea.europa.eu/humandocs/PDFs/EPAR/altargo/H-757-PI-en.pdf). The presence of cfr was not indicated in our isolate, which remained susceptible to linezolid (MIC 2 mg/L), quinupristin/dalfopristin (MIC 1 mg/L) and clindamycin (MIC 0.5 mg/L); the possible contribution of efflux requires further study. Our collection of S. aureus isolates was selected to include large numbers of fusidic acid- and mupirocin-resistant strains since these agents would frequently be considered for the topical treatment of impetigo, the licensed indication for retapamulin in the USA and one of its indications in Europe. Retapamulin showed excellent activity against these resistant isolates in vitro, including against those resistant both to fusidic acid and to high levels of mupirocin. Recently, O'Neill et al.10,11 described an epidemic European fusidic acid-resistant impetigo clone (EEFIC) of S. aureus, which has a fusB determinant and typical fusidic acid MICs of 4 mg/L. We did not determine whether representatives of this clone were included in our study collection. However, retapamulin retained activity in vitro against many S. aureus isolates with high- and low-level forms of fusidic acid resistance (see the trimodal distribution in Table 2). It is likely that those with low-level resistance (MICs 2–16 mg/L) had fusB, whereas high-level resistance (MICs 32 to >256 mg/L) is more likely to be caused by mutations in elongation factor G (EF-G; fusA genotype).11 By inference, it is likely that retapamulin would also retain activity against the EEFIC strain, although this requires formal evaluation.
In summary, retapamulin demonstrated excellent activity in vitro against this collection of S. aureus isolates, which was greatly biased towards MRSA and to isolates with resistance to mupirocin or fusidic acid. Reduced susceptibility (MIC 2 mg/L) was seen in only a single isolate and was not associated with any mutations in ribosomal protein L3; the mechanism(s) underlying the reduced susceptibility of this isolate warrants further investigation. These results support the EUCAST epidemiological cut-off value for retapamulin of 0.5 mg/L against S. aureus.
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Transparency declarations |
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N. W. has received research grants and speaking invites from various pharmaceutical companies. D. M. L. has shares in GSK, within the ambit of a diversified portfolio. None of these poses a conflict of interest with this work. Other authors: none to declare.
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Funding |
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This work was funded by a research grant from GlaxoSmithKline.
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Acknowledgements |
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We would like to thank GlaxoSmithKline for supporting this work and its presentation at the Eighteenth ECCMID (Barcelona, April 2008). Thanks also to Dr Keith Miller (University of Leeds) for disclosing details of the rplC PCR assay before publication.
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References |
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1 . Long KS, Hansen LH, Jakobsen L, et al. Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center. Antimicrob Agents Chemother (2006) 50:1458–62.
2 . Poulsen SM, Karlsson M, Johansson LB, et al. The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome. Mol Microbiol (2001) 41:1091–9.[CrossRef][Web of Science][Medline]
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