The antifungal activity of mupirocin

R. O. Nicholasa,*, Valerie Berrya, Pamela A. Hunterb and Judy A. Kellyc

a SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Road, Collegeville, PA 19426, USA b Burnthouse, Burnthouse Lane, Cowfold, Sussex RH13 8DH c Schering Health Care, The Brow, Burgess Hill, West Sussex RH15 9NE, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The antibacterial agent mupirocin (pseudomonic acid A) is used as a topical agent in the treatment of superficial infections by Gram-positive bacteria, particularly Staphylococcus aureus. However, we demonstrate here that the compound also inhibits the growth of a number of pathogenic fungi in vitro, including a range of dermatophytes and Pityrosporum spp. It inhibited the incorporation of amino acids and precursors of RNA, but not that of acetate, by Trichophyton mentagrophytes. It also inhibited the isoleucyl-tRNA synthetase from Candida albicans, indicating a mechanism of action similar to that in bacteria. When administered topically, mupirocin was efficacious in a T. mentagrophytes ringworm model in guinea pigs. These results suggest that mupirocin could have clinical utility for superficial infections caused by dermatophytes.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mupirocin (pseudomonic acid A, Bactroban, SmithKline Beecham) is a well established antibacterial agent marketed as a topical agent for the treatment of patients with superficial skin infections. It is highly active against staphylococci and most streptococci, but generally much less active against Gram-negative bacteria,1 probably because of poor penetration of the drug through the bacterial outer membrane. 2,3 The antibiotic has a novel mechanism of antibacterial action blocking protein synthesis through inhibition of isoleucyl-tRNA synthetase.3

Although initial indications were that mupirocin had little antifungal effect in vitro, 1 there has been some evidence to suggest it has anti-candida activity in vivo.4,5 Here we present data on the antifungal action of mupirocin with special reference to its activity as a topical agent for superficial fungal infections and its mechanism of action.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Fungi and media

All fungi were from the SmithKline Beecham culture collection and were maintained on slopes of Sabouraud's dextrose agar (SDA, Lab M, Bury, UK) and grown in Sabouraud's liquid medium (SDB, Lab M) or synthetic Yeast Nitrogen Base (YNB, Difco, West Molesey, UK) unless stated otherwise. Pityrosporum (Malasezzia) ovale and Pityrosporum orbiculare were cultivated on a medium containing glycerol monostearate, olive oil and Tween 80.6 To facilitate incorporation studies, Trichophyton mentagrophytes was also grown in the synthetic glucose- glutamic acid- salts medium (GGS) of Kole & Bose,7 solidified, where necessary, with 2% agar. Candida albicans,Cryptococcus neoformansand Aspergillus niger were incubated at 37°C and other species at 30°C.

Sensitivity testing

MICs were determined in liquid and solid media, using doubling dilutions of mupirocin (calcium salt). For agar diffusion tests, mupirocin (500 and 100 mg/L) was placed in 8 mm wells cut in seeded agar plates. Plates were incubated at 30°C or 37°C for 1–2 days depending on the fungal species. The ability of L-amino acids to antagonize the effect of mupirocin was tested by adding 30 µL of mupirocin (2000 mg/L) to wells containing 30 µL of 0.1 M L-amino acid in distilled water.

Incorporation of radiolabelled precursors of macromolecules by T. mentagrophytes

Growth from a 10 day old culture on SDA was homogenized in phosphate buffered saline (PBS), washed and resuspended in PBS, and 0.5 mL diluted in 100 mL GGS medium (in 250 mL flasks). The flasks were shaken at 30°C for 48 h. Portions (20 mL) were then dispensed into 50 mL flasks and shaken at 30°C. Mupirocin was added to give a final concentration of 600 or 2000 mg/L. After incubation for 5 min, carrier-free 14C-radiolabelled precursor (7.4 kBq; Amersham International, Amersham, UK) was added to each flask.

Samples (2.0–5.0 mL) were removed from each flask at intervals and added to 5 mL ice-cold 10% trichloroacetic acid (TCA), mixed thoroughly and kept on ice for 1–2 h. before filtering through glass fibre (GF/C Whatman, Maidstone, UK) discs. The filters were washed twice with TCA and once with 1% acetic acid. Finally, the filters were dried and the radioactivity determined by scintillation counting.

Ringworm infection model

Female guinea pigs (Dunkin Hartley, Tuck, Southend, UK 200–250 g), were shaved on both flanks and depilated (Nair, Carter-Wallace, New York, NY, USA), and a 1.5 cm2 area was lightly scarified. A spore-mycelial suspension of T. mentagrophytes 569A was prepared as described above. Both flanks were infected by applying the suspension directly on to the surface of the skin. Animals were assigned to groups of six per treatment.

Therapy (right flank) commenced 24 or 48 h after infection, and continued once daily for 7 days. The left flank was untreated and served as a control to detect percutaneous absorption. Animals received mupirocin (calcium salt 2% w/w) topically in a white paraffin wax/Softisan (Huls Ltd., Milton Keynes, UK) or cetomacrogol cream base. Ketoconazole, 2% cream (Nizoral, Janssen, Wantage, UK) was included for comparison. Further groups of animals received the corresponding vehicle as placebo and served as untreated controls.

The degree of infection was graded (on an arbitrary scale from 1 to 8) based on erythema, alopecia, scaling, scabbing and the area involved. Grading continued for approximately 16 days, when the majority of lesions had healed. Skin cells and hair samples were plated on to SDA plates containing penicillin G (100 mg/L) and streptomycin (100 mg/L) to suppress contaminants from the hay and skin, and examined microscopically for fungal growth after incubation for 24 and 48 h at 30°C.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antifungal activity in vitro

In agar diffusion tests, mupirocin 500 mg/L produced zones of inhibition against all the filamentous fungi tested (except Rhizopus oryzae), Pityrosporum canis and Candida albicans (Table I). Activity against R. oryzae,Saccharomyces cerevisiae and Cryptococcus neoformans was only detected in the defined YNB agar.


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Table I. Zones of inhibition (mm diameter) produced by mupirocin against a range of fungi in agar diffusion tests
 
Relatively high concentrations of mupirocin were required to inhibit fungal growth in broth and in agar (Table II), with the notable exception of Pityrosporumspp. MICs for T. mentagrophytes were lower in the defined GGS medium (pH 5.5) than in complex SDB medium (data not shown). Increased activity was seen at a lower pH and an inoculum effect was also observed (data not shown).


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Table II. MIC values (mg/L) for mupirocin against yeasts and filamentous fungi
 
Of the 20 L-amino acids, only L-isoleucine antagonized the inhibition of T. mentagrophytes by mupirocin in GGS agar; no such antagonism was seen with isoleucine or any other amino acids in SDA. Antagonism by L-isoleucine was also observed against other species (C. albicans, S. cerevisiae, P. canis,Hendersonula toruloidea, Paecilomyces varioti, R. oryzae) in defined YNB agar (results not shown). A similar antagonistic effect has been seen with Staphylococcus aureus.8 This is not surprising in view of the established mechanism of action of mupirocin in bacteria, namely the inhibition of the charging of isoleucyl-tRNA at the level of the activation of the amino acid. 3 De Wet et al.,5 in contrast, found that isoleucine did not reverse the inhibitory effect of mupirocin against C. albicans. In their study only aspartic and glutamic acids reversed the effect of mupirocin. They further demonstrated that mupirocin inhibits the uptake of these two acidic amino acids, although the effect on isoleucine uptake was not measured. The differences in response to exogenous amino acids seen in the present study and that of De Wet et al. 5 probably result from the use of different strains and media.

Mechanism of action

When mupirocin (2000 mg/L) was added to a 48 h flask culture of T. mentagrophytes in GGS medium, growth was almost completely inhibited, but at lower concentrations (<=600 mg/L) it had little or no effect on growth. Using this system the effect of mupirocin on the ability of T. mentagrophytes to incorporate various radiolabelled precursors into macromolecular material was monitored. Mupirocin inhibited the incorporation of [14C]L-isoleucine into acid-insoluble material at growth-inhibitory concentrations, with an immediate effect (by 76.1% after 15 min at 2000 mg/L). The incorporation of [14C]phenylalanine and [14C]uridine was also inhibited (by 80.0% and 48.0% respectively). In contrast, the incorporation of [14C]acetate was unaffected (6.6% inhibition). T. mentagrophytes was unable to incorporate thymine. The differential effect of mupirocin on the incorporation of macromolecular precursors by T. mentagrophytes suggests that a specific mechanism is in operation. Mupirocin is known to inhibit protein, RNA and DNA synthesis in bacteria.8 The absence of protein synthesis and in particular of tRNA charging results in a stringent response and the shutdown of RNA synthesis. There is evidence of control of RNA and protein formation during nutritional shutdown in S. cerevisiae, analogous to the stringent response in bacteria. 9

We were unable to obtain an active isoleucyl-tRNA synthetase preparation from cell homogenates of T. mentagrophytes to test the activity of mupirocin. However isoleucyl-tRNA synthetase activity in a cytoplasmic extract of C. albicans isoleucyl-tRNA synthetase was inhibited by mupirocin (I50 = 7.4 µM; data not shown), at a concentration similar to that reported to inhibit S. cerevisiae isoleucyl-tRNA synthetase (Ki = 15 µM10).

Antifungal activity in vivo

Mupirocin formulated in a Softisan/paraffin-based ointment had a marked therapeutic effect against experimental ringworm in guinea pigs when therapy commenced 24 h after infection, and compared favourably with ketoconazole (Figure 1a). No significant effect was seen with the placebo. Clinical scorings were corroborated by microbiological tests of skin samples and demonstrated that mupirocin had a curative effect (data not shown).



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Figure. Clinical effect of mupirocin in a guinea pig model of T. mentagrophytes infection. Therapy was delayed for (a) 24 h or (b) 48 h after infection and continued until the time indicated by the arrow. Animals were untreated ({circ}), or received either topical placebo (•), mupirocin ({square}) Or ketoconazole ({blacksquare}). Mupirocin was prepared in either (a) a Softisan/paraffin base, or (b) a cetomacrogol cream base.

 
In confirmatory tests, a different formulation— a cetomacrogol cream base— showed equally good activity when therapy started 24 h after infection, but when therapy was delayed for 48 h a slight reduction in efficacy was seen (Figure 1b). Initial suppression of the infection was good, but some recrudescence of infection occurred and this was greater than that seen with ketoconazole.

Although high levels of mupirocin are required to inhibit fungal growth in vitro, it may have clinical utility in situations where relatively high local concentrations can be achieved, as in topical preparations. Interestingly, Rode et al.4 reported eradication of Candida spp. in infected wounds, following treatment of perineal skin infections with 2% mupirocin in a polyethyleneglycol base (Bactroban). In their study the MICs of mupirocin against C. albicanswere reported to be 256–512 mg/L, lower than those reported here.

The in-vivo data presented here suggest that mupirocin could have clinical utility for superficial fungal infections caused by dermatophytes. Its efficacy in infections caused by Pityrosporum spp., such as pityriasis versicolor, may also be worth testing. Preliminary testing indicates that mupirocin is active in the treatment of otitis externa caused by P. canis in the dog (G. Palmer, personal communication).


    Notes
 
* Corresponding author. Tel: +1-610-917-7000; Fax: +1-610-917-7901; E-mail: richard_o_nicholas{at}sbphrd.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Sutherland, R., Boon, R. J., Griffin, K. E., Masters, P. J., Slocombe, B. & White, A. R. (1985). Antibacterial activity of mupirocin (pseudomonic acid), a new antibiotic for topical use. Antimicrobial Agents and Chemotherapy 27, 495–8.[ISI][Medline]

2 . Hughes, J. & Mellows, G. (1978). On the mode of action of pseudomonic acid: inhibition of protein synthesis in Staphylococcus aureus. Journal of Antibiotics 31 , 330–5.[ISI][Medline]

3 . Hughes, J. & Mellows, G. (1980). Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase. Biochemical Journal 191, 209–19.[ISI][Medline]

4 . Rode, H., de Wet, P. M., Millar, A. J. W. & Cywes, S. (1991). Efficacy of mupirocin in cutaneous candidiasis. Lancet 338,578 .

5 . De Wet, P. M., Rode, H., Steyn, L. M. & Grobler, J. (1995). Anti-candidal activity of mupirocin— evidence for uptake via an amino acid transport system. Journal of Antimicrobial Chemotherapy 35, 1107–8.

6 . Faergemann, J. & Bernander, S. (1981). Micro-aerophilic and anaerobic growth of Pityrosporum species. Sabouraudia 19, 117–21.[ISI][Medline]

7 . Kole, H. K. & Bose, S. K. (1984). Mycoversilin, a new antifungal antibiotic. III. Mechanism of action on a filamentous fungus Trichophyton rubrum. Journal of Antibiotics 37, 1238–45.[ISI][Medline]

8 . Hughes, J. & Mellows, G. (1978). Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid. Biochemical Journal 176, 305–18.[ISI][Medline]

9 . Warner, J. R. & Gorenstein, C. (1978). Yeast has a true stringent response. Nature 275, 338–9.[ISI][Medline]

10 . Racher, K. I., Kalmar, G. B. & Borgford, T. J. (1991). Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase. Journal of Biological Chemistry 266, 17158–64.[Abstract/Free Full Text]

Received 29 April 1998; returned 3 July 1998; revised 26 October 1998; accepted 17 November 1998





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