Antimicrobial Research Centre and Division of Microbiology, University of Leeds, Leeds LS2 9JT, UK
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Abstract |
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Introduction |
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An issue affecting the potential use of these antibiotics as single agents concerns the apparent high spontaneous chromosomal mutation frequencies for development of resistance in S. aureus.1 Thus the use of either fusidic acid or rifampicin alone for treatment of staphylococcal infections is not recommended, because treatment may fail through emergence of resistant mutants during therapy.1 Therefore, these antibiotics are either administered with another agent, or, more frequently, in the form of a combination of fusidic acid and rifampicin.1,5 Although in vitro mutation frequencies for resistance have been determined for both these drugs in S. aureus,1 the rates of resistance after exposure to the drugs separately, or in combination, at expected serum Cmax and Cmin concentrations, have not been examined. Furthermore, mutation frequencies in MRSA and VISA strains, for which these drugs might prove useful, have not been examined. This paper reports such data.
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Materials and methods |
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Nine S. aureus strains (Table I) were used in this study. The term hetero-VISA denotes strains that produce cultures containing sub-populations of cells that exhibit intermediate resistance to vancomycin.2,3
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Fusidic acid and rifampicin were from Sigma, Poole, UK. IsoSensitest broth and agar were from Oxoid, Basingstoke, UK.
Determination of susceptibility to antibiotics
MICs were determined by agar dilution on IsoSensitest agar with an inoculum in IsoSensitest broth of 106 cfu/spot.7
Determination of spontaneous mutation frequencies for resistance to rifampicin and fusidic acid, alone or combined
Bacteria were grown aerobically at 37°C in IsoSensitest broth until they reached the late logarithmic phase of growth. Aliquots were spread on to selective and non-selective IsoSensitest agar plates and incubated at 37°C. After 24 h, mutation rates were calculated according to Eisenstadt et al.8 with triplicate determinations from each of three independent cultures. In some instances bacteria were concentrated and aliquots (1 mL) were added to IsoSensitest agar before pouring plates. With 10 agar plates, mutations could potentially be detected at frequencies of 1 in 1011 bacteria. For rifampicin the selecting concentrations were 16 and 1 mg/L; those for fusidic acid were 30 and 15 mg/L. These concentrations are the expected Cmax and Cmin in serum1,7 on the basis of an oral regimen of rifampicin 900 mg od or fusidic acid 500 mg tds.5 The response of antibiotic-resistant clinical isolates was compared with that of a clinical methicillin-susceptible S. aureus (MSSA) isolate (7030676) and S. aureus 8325-4, an antibioticsusceptible strain that has been subjected to prolonged subculture in the laboratory.
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Results |
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Mutants were not detected (frequency <10-11) after selection with fusidic acid 15 or 30 mg/L (Table II), although they arose with frequencies between 10-7 and 10-8 with fusidic acid 10 mg/L (data not shown). For rifampicin, resistant mutants occurred with frequencies of around 10-8 in all strains, at both selective concentrations (16 and 1 mg/L) (Table II
). No mutants were recovered (frequencies <10-11) when bacteria were selected with combinations of fusidic acid and rifampicin, at either 15 and 1 mg/L or 30 and 16 mg/L, respectively (Table II
), even after 96 h incubation.
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Previous studies have established that various mutations can arise within rpoB (which encodes the ß-subunit of RNA polymerase) that confer different levels of resist-ance to rifampicin.9 A population analysis of rifampicinresistant mutants arising in the strains described in Table II was performed to determine whether there was bias towards the generation of highly resistant mutants in VISA, hetero-VISA or MRSA types. Twenty rifampicin-resistant mutants of each strain were picked at random from colonies appearing on selection plates containing rifampicin (16 or 1 mg/L). The individual MICs of rifampicin were determined respectively for the 180 rifampicin-resistant mutants. Mutants displayed MICs in the range 16>1024 mg/L. However, the phenotypic distribution patterns were similar for all nine strains, with no evidence for high-level resistance arising more frequently in the VISA, hetero-VISA or MRSA strains than in the clinical MSSA isolate (strain 7030676) or 8325-4 (data not shown).
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Discussion |
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Mutants of S. aureus resistant to fusidic acid and rifampicin can be readily selected in vitro.1,8 As a result of concern that treatment failures will occur because of the emergence of resistant mutants during therapy, these agents are not used as single chemotherapeutic agents for staphylococcal infections.1 However, since the rate of selection of antibiotic-resistant mutants depends upon the concentration of the selecting agent,10 a full risk assessment for emergence of resistance to fusidic acid and rifampicin should include quantification of mutation frequencies at therapeutically achievable drug concentrations.
Although mutants resistant to rifampicin were recovered after single selections at expected serum Cmax and Cmin values, resistant mutants were not selected when rifampicin was combined with fusidic acid at therapeutic concentrations expected during combination therapy. Our observations that resistant mutants are not selected in the presence of therapeutically achievable rifampicin/fusidic acid concentrations are consistent with earlier findings that resistant staphylococci do not readily emerge after dual therapy.1 A recent report indicates that some bacterial populations contain hyper-mutable variants.10 However, we found no evidence that clinical S. aureus isolates exhibited higher mutation frequencies for generation of fusidic acid- or rifampicin-resistant mutants. Furthermore, the rifampicin-resistant derivatives we recovered from VISA, hetero-VISA and MRSA hosts did not show any trend towards the selection of mutants with high-level resistance when compared with MSSA. These findings, together with evidence that rifampicin/fusidic acid combinations interact synergically or additively,1 support a wider role for the use of these agents in controlling or treating staphylococcal disease, provided that the organisms responsible are susceptible to both antibiotics.
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Acknowledgments |
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Notes |
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References |
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2 . Hiramatsu, K. (1998). Vancomycin resistance in staphylococci. Drug Resistance Updates 1, 13550.[ISI]
3 . Howe, R. A., Bowker, K. E., Walsh, T. R., Feest, T. G. & MacGowan, A. P. (1998). Vancomycin-resistant Staphylococcus aureus. Lancet 351, 602.[ISI][Medline]
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Tenover, F. C., Lancaster, M. V., Hill, B. C., Steward, C. D., Stocker, S. A., Hancock, G. A. et al. (1998). Characterisation of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides. Journal of Clinical Microbiology 36, 10207.
5 . Drancourt, M., Stein, A., Argenson, J. N., Roiron, R., Groulier, P. & Raoult, D. (1997). Oral treatment of Staphylococcus spp. infected orthopaedic implants with fusidic acid or ofloxacin in combination with rifampicin. Journal of Antimicrobial Chemotherapy 39, 23540.[Abstract]
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Andrews, J., Ashby, J., Jevons, G., Lines, N. & Wise, R. (1999). Antimicrobial resistance in Gram-positive pathogens isolated in the UK between October 1996 and January 1997. Journal of Antimicrobial Chemotherapy 43, 68998.
7 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 150.[ISI][Medline]
8 . Eisenstadt, E., Carlton, B. C. & Brown, B. J. (1994). Gene mutation. In Methods for General and Molecular Bacteriology, (Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R., Eds), pp. 297316. American Society for Microbiology, Washington, DC.
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O'Neill, A., Oliva, B., Storey, C., Hoyle, A., Fishwick, C. & Chopra, I. (2000). RNA polymerase inhibitors with activity against rifampin-resistant mutants of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 44, 31636.
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Martinez, J. L. & Baquero, F. (2000). Mutation frequencies and antibiotic resistance. Antimicrobial Agents and Chemotherapy 44, 17717.
Received 19 September 2000; returned 25 November 2000; revised 2 January 2001; accepted 24 January 2001