Differential effect of rpoB mutations on antibacterial activities of rifampicin and KRM-1648 against Staphylococcus aureus

T. A. Wichelhaus,*, V. Schäfer, V. Brade and B. Böddinghaus

Institute of Medical Microbiology, University Hospital, Paul-Ehrlich-Strasse 40, 60596 Frankfurt am Main, Germany


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The in vitro antibacterial activities of the rifamycin derivatives rifampicin and KRM-1648 against 150 Staphylococcus aureus isolates were determined. The MICs of rifampicin and KRM-1648 for 90% of rifampicin-susceptible S. aureus isolates (n = 100) were 0.016 and 0.001 mg/L, respectively. In rifampicin-resistant S. aureus isolates (n = 50), different levels of resistance to rifamycins were associated with mutations at different sites in rpoB. Mutations at some sites were associated with high-level resistance to both rifamycins, while certain mutations were associated with the activity of KRM-1648 being <= 100-fold better than that of rifampicin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rifampicin is a valuable antibiotic in combination therapy, especially for deep-seated staphylococcal infections, owing to its excellent pharmacokinetic properties and bactericidal activity.1 Rifampicin resistance, however, is frequent among methicillin-resistant Staphylococcus aureus (MRSA) in several countries.2 Previous studies provide evidence that mutations in rpoB, the gene encoding the ß-subunit of RNA polymerase, are responsible for rifampicin resistance.3,4 This study was aimed at determining the antibacterial activities of rifampicin and a newly developed rifamycin, KRM-1648, against S. aureus in vitro, as well as investigating the relationship between genetic alterations in the rpoB gene and the level of resistance to both rifamycins.


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

A total of 150 non-duplicate S. aureus isolates were analysed, including 50 rifampicin-susceptible, methicillin-susceptible (MSSA) isolates, 50 rifampicin-susceptible MRSA isolates and 50 rifampicin-resistant MRSA isolates. Thirty of the rifampicin-resistant isolates were of clinical origin and were provided by laboratories in the USA, France, Italy, Poland, Slovenia and Germany, and the other 20 were generated in vitro. S. aureus strain ATCC 29213, the ‘northern German’ epidemic MRSA and the ‘southern German’ epidemic MRSA were used as reference strains.

Antimicrobial susceptibility testing

The MICs of rifampicin (Sigma, Deisenhofen, Germany) and KRM-1648 (Kaneka, Osaka, Japan) were determined for each isolate by the agar dilution method as described previously.4

In vitro generation of mutants resistant to rifamycins

Aliquots (100 µL) from overnight cultures of rifampicin-susceptible strains of the ‘northern German’ epidemic MRSA, the ‘southern German’ epidemic MRSA and ATCC 29213, concentrated to about 1011 cfu/mL as a final inoculum, were plated on Mueller–Hinton agar containing inhibitory concentrations of rifampicin and KRM-1648 (0.25 and 0.008 mg/L, respectively). The number of cfu was determined after 24 h of growth at 37°C. Mutation rates were determined by dividing the number of cfu on antibiotic-supplemented agar by the number of cfu on antibiotic-free agar. The study was performed three times.

Genotyping

All MRSA isolates were analysed by pulsed-field gel electrophoresis (PFGE) as described previously.4 DNA isolation, PCR amplification and sequencing were performed as described previously.5


    Results
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 Materials and methods
 Results
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MICs of rifamycin-susceptible S. aureus

The MIC90s of rifampicin and KRM-1648 were 0.016 and 0.001 mg/L, respectively. No differences between MSSA and MRSA were demonstrated with respect to MIC50s and MIC90s.

Characterization of rifamycin resistance emerging in vivo and in vitro

The genetic background of rifamycin resistance emerging in vivo in MRSA was analysed using 30 clinical isolates from six countries. Restriction digestion followed by PFGE of these isolates revealed 19 different genotypes. The isolates were characterized with regard to the MICs of rifampicin and KRM-1648, as shown in the TableGo. With rifampicin, two levels of resistance, low (MIC <= 4 mg/L) and high (MIC >= 8 mg/L), were evident, while with KRM-1648, low, intermediate and high-level resistance could be differentiated (FigureGo). The most common mutation, capable of conferring low-level resistance on its own, was His481->Asn. This substitution was found in 28 of the 30 clinical isolates, 21 of which had different additional mutations resulting in high-level resistance to rifampicin.


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Table. Correlation of in vivo and in vitro mutations in the rpoB gene and the MICs of rifampicin and KRM-1648
 


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Figure. Correlation between MICs of rifampicin and KRM-1648 for 50 S. aureus isolates. Vertical dashed lines indicate the breakpoints set by NCCLS with respect to rifampicin.

 
The TableGo also provides a survey of the amino acid mutational changes found in mutants generated in vitro together with the corresponding MICs of the two rifamycins. The mutants were derived from the ‘southern German’ epidemic MRSA and were picked randomly from agar plates containing rifampicin 0.25 mg/L. In contrast to mutants from clinical sources, sequence analysis revealed eight different mutations that conferred low-level resistance in vitro. Mutants with high-level rifampicin resistance again demonstrated the differential effect of some amino acid changes on the antibacterial activities of the rifamycins. Amino acid substitutions Ala477->Asp and Arg484->His conferred high-level resistance against rifampicin, but resulted in only an intermediate level of resistance against KRM-1648.

Mutation frequency in vitro

The average mutation frequencies for rifampicin and KRM-1648 were 3.2 x 10–9 and 2.4 x 10–9, respectively. Of 120 mutants, 27% showed low-level resistance to rifampicin and 73% showed high-level resistance.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Sequence analysis provided conclusive evidence that missense mutations in rpoB cause rifamycin resistance in MRSA. Specific mutation sites within rpoB were associated with different levels of resistance to rifamycin. For example, certain mutations conferred high-level resistance to rifampicin but only intermediate resistance to KRM-1648 (TableGo, FigureGo). Compared with rifampicin, KRM-1648 displayed <=100-fold better activity against most of the isolates analysed. Additional clinical studies are needed to determine whether ‘low-level resistance’ to KRM-1648 is true resistance in clinical terms. Such a differential impact of some amino acid changes on the antibacterial activities of rifamycins has also been demonstrated for Mycobacterium tuberculosis.6

There were discrepancies between the distribution of mutations that emerged in vivo and those produced in vitro. Low-level resistance in clinical isolates, as shown in this study, was solely attributable to the amino acid mutational change His481->Asn, whereas eight different mutational changes conferring low-level resistance were demonstrated in laboratory mutants. High-level resistance in vivo is attributable mainly to multiple mutations, indicating a step-by-step mechanism in resistance development. In contrast, six single mutations were identified in vitro that conferred high-level resistance on their own. Another curious feature is that the amino acid substitution His481->Asn, which is capable of conferring low-level resistance, was identified in 93% of clinical isolates, although in vitro experiments demonstrate a 73% probability of selecting for high-level resistant mutants. Two explanations may be given for these discrepancies: (i) the clonal spread of MRSA and the frequent exposure of epidemic MRSA to rifampicin chemotherapy may explain the occurrence of distinct and multiple mutations in vivo; or (ii) some in vitro mutations might be associated with a loss of fitness and so, consequently, would be less likely to be selected in vivo. This would explain the reduced variety of mutations in vivo.

The data obtained in the present study suggest that KRM-1648 is a potent antimicrobial against S. aureus, showing good activity even against a subset of rifampicin-resistant isolates. Clinical studies are needed to elucidate the effectiveness of KRM-1648 against S. aureus, especially MRSA.


    Acknowledgments
 
We thank Denia Franck for her technical assistance. This research was partially supported by grants from the Marie Christine Held and Erika Hecker Foundation.


    Notes
 
* Corresponding author. Tel: +49-69-6301-6438; Fax: +49-69-6301-5767; E-mail: wichelhaus{at}em.uni-frankfurt.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Yao, J. D. & Moellering, R. C. (1999). Antibacterial agents. In Manual of Clinical Microbiology, 7th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 1474–504. American Society for Microbiology, Washington, DC.

2 . Schmitz, F. J., Krey, A., Geisel, R., Verhoef, J., Heinz, H. P. & Fluit, A. C. (1999). Susceptibility of 302 methicillin-resistant Staphylococcus aureus isolates from 20 European university hospitals to vancomycin and alternative antistaphylococcal compounds. SENTRY Participants Group. European Journal of Clinical Microbiology and Infectious Diseases 18, 528–30.[ISI][Medline]

3 . Aubry-Damon, H., Soussy, C. J. & Courvalin, P. (1998). Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 42, 2590–4.[Abstract/Free Full Text]

4 . Wichelhaus, T. A., Schäfer, V., Brade, V. & Böddinghaus, B. (1999). Molecular characterization of rpoB mutations conferring cross-resistance to rifamycins on methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 2813–6.[Abstract/Free Full Text]

5 . Wichelhaus, T. A., Schulze, J., Hunfeld, K. P., Schäfer, V. & Brade, V. (1997). Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistant Staphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases 16, 893–7.[ISI][Medline]

6 . Yang, B., Koga, H., Ohno, H., Ogawa, K., Fukuda, M., Hirakata,Y. et al. (1998). Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 42, 621–8.[Abstract]

Received 31 May 2000; returned 16 August 2000; revised 2 October 2000; accepted 19 October 2000