Mutation frequencies for resistance to fusidic acid and rifampicin in Staphylococcus aureus

Alex J. O'Neill, Jonathan H. Cove and Ian Chopra*,

Antimicrobial Research Centre and Division of Microbiology, University of Leeds, Leeds LS2 9JT, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Frequencies at which mutants resistant to fusidic acid and/or rifampicin arose in vitro were determined in Staphylococcus aureus strains including methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-intermediate resistant S. aureus (VISA) and hetero-VISA. The concentrations of fusidic acid (30 and 15 mg/L) and rifampicin (16 and 1 mg/L) used for selection were equal to the expected maximum and minimum serum concentrations after an oral regimen of rifampicin 900 mg od, together with fusidic acid 500 mg tds. Resistant mutants arose at a frequency of around 10-8 for selections with rifampicin, but were undetectable (frequency <10-11) for selections with fusidic acid. Mutants were not recovered (frequency <10-11) after selections in the presence of both fusidic acid and rifampicin at 30/16 and 15/1 mg/L. Our results suggest that these antibiotics, when used in combination, could have a wider role in the management of staphylococcal infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vancomycin has become the drug of choice for treatment of methicillin-resistant Staphylococcus aureus (MRSA).1,2 However, adverse side effects associated with its use1 and the emergence of MRSA with decreased susceptibility to vancomycin (vancomycin-intermediate resistant Staphylococcus aureus; VISA)24 are leading to interest in alternative therapies for these infections.2 Fusidic acid and rifampicin are two older antibiotics that could have a wider role in the management of MRSA infections, including those caused by VISA. Both antibiotics are potent anti-staphylococcal agents with good tissue penetration and are available in parenteral and oral formulations.1,5 Furthermore, the incidence of resistance to these antibiotics in clinical isolates of S. aureus, including MRSA, is low,6 suggesting that the agents might be effective in treatment.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Nine S. aureus strains (Table IGo) 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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Table I. Origin and antimicrobial susceptibility of S. aureus strains

 
Antibiotics and growth media

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Frequencies of mutational resistance to fusidic acid and rifampicin

Mutants were not detected (frequency <10-11) after selection with fusidic acid 15 or 30 mg/L (Table IIGo), 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 IIGo). 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 IIGo), even after 96 h incubation.


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Table II. Mutation frequencies of S. aureus strains
 
Susceptibility to rifampicin of spontaneous rifampicin-resistant mutants

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 IIGo 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).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infections caused by MRSA and VISA/hetero-VISA derived from MRSA are a threat to patient care, and established drugs to which these strains are still susceptible may have a role in chemotherapy. Rifampicin and fusidic acid fall into this class since resistance to these antibiotics among staphylococci, including MRSA, is relatively rare.6

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.


    Acknowledgments
 
We thank Drs A. P. MacGowan, A. Johnson and F. C. Tenover for providing bacteria. Smith and Nephew Medical, Hull, UK, supported this work.


    Notes
 
* Corresponding author. Tel: +44-113-233-5604; Fax: +44-113-233-5638; E-mail: i.chopra{at}leeds.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Kucers, A. & Bennett, N. (1987). The Use of Antibiotics, 4th edn. Heinemann Medical Books, London.

2 . Hiramatsu, K. (1998). Vancomycin resistance in staphylococci. Drug Resistance Updates 1, 135–50.[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]

4 . 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, 1020–7.[Abstract/Free Full Text]

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, 235–40.[Abstract]

6 . 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, 689–98.[Abstract/Free Full Text]

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, 1–50.[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. 297–316. American Society for Microbiology, Washington, DC.

9 . 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, 3163–6.[Abstract/Free Full Text]

10 . Martinez, J. L. & Baquero, F. (2000). Mutation frequencies and antibiotic resistance. Antimicrobial Agents and Chemotherapy 44, 1771–7.[Free Full Text]

Received 19 September 2000; returned 25 November 2000; revised 2 January 2001; accepted 24 January 2001