Institute of Medical Microbiology, University Hospital, Paul-Ehrlich-Strasse 40, 60596 Frankfurt am Main, Germany
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Abstract |
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Introduction |
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Materials and methods |
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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 MuellerHinton 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
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Results |
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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 Table. 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 (Figure
). 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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Mutation frequency in vitro
The average mutation frequencies for rifampicin and KRM-1648 were 3.2 x 109 and 2.4 x 109, respectively. Of 120 mutants, 27% showed low-level resistance to rifampicin and 73% showed high-level resistance.
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Discussion |
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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 His481Asn, 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.
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Acknowledgments |
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Notes |
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References |
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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, 52830.[ISI][Medline]
3
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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, 25904.
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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, 28136.
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, 8937.[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, 6218.[Abstract]
Received 31 May 2000; returned 16 August 2000; revised 2 October 2000; accepted 19 October 2000