Agence Française de Sécurité Sanitaire des Aliments, Laboratoire dEtudes et de Recherches Avicoles et Porcines, Unité de Mycoplasmologie-Bactériologie, BP 53, 22440 Ploufragan, France
Received 9 January 2002; returned 15 April 2002; revised 27 May 2002; accepted 20 June 2002
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Abstract |
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Introduction |
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Materials and methods |
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Following each passage, aliquots of the two cultures retained were plated on to Frey agar to obtain single colonies prior to MIC determinations and genetic analyses. MICs were determined by a dilution method4 on Frey agar. The MIC was defined as the lowest concentration for which no visible growth was observed. A strain was considered susceptible to enrofloxacin when the MIC was 0.5 mg/L and resistant when the MIC was >2 mg/L, according to the breakpoints given for this antibiotic.5 For genetic analysis, chromosomal DNA from all strains was prepared by cellular lysis according to the method of Kellog & Kwok.6 Amplification of gyrA, gyrB, parC and parE QRDRs was performed and all purified PCR products were sequenced directly on both strands as described previously.2
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Results and discussion |
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In two mutants derived from the reference strain MG ATCC 15302, the first mutation was a SerArg substitution at position 83 in the GyrA QRDR (mutants ATm6-2 and ATm3/4-3: Table 1). For two other mutants (ATm7-4 and ATm1-9), two mutations appeared during the same passage. The mutant ATm7-4 showed Glu-87
Gln and Cys-467
Phe substitutions in GyrA and ParE, respectively, at the fourth passage, and the mutant ATm1-9 showed Glu-87
Gly and Glu-84
Gly changes in GyrA and ParC, respectively, during the ninth passage (Table 1). The observation of two mutations occurring during the same passage cannot be due to a mixture of populations in the culture since individual colonies were used for genetic analysis.
For most mutants derived from the field strain MG 41-91, the first mutation was an Asp-426Asn change in the GyrB QRDR (Table 2). However, one mutant (41m16/19-6) harboured a Ser-83
Arg substitution in GyrA, like mutants ATm6-2 and ATm3/4-3, above.
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Influence of mutations on resistance level
Two other mutations were found in the GyrA QRDR at position 83: SerAsn or Ile. They were associated with small increases in resistance (two- and two- to four-fold, respectively) unlike the mutation Ser
Arg, which led to a 16-fold increase for mutant 41m16/19-6 (Table 2). Furthermore, this Ser
Arg mutation was responsible for a 32-fold increase in the MIC of enrofloxacin for mutants ATm6-2 and ATm3/4-3, compared with the parental strain (Table 1). The specific effects of different mutations on resistance level could be explained by structural differences between the three amino acids, as suggested by Kim et al.8 Mutations were also found at positions 81, 84 and 87 in the GyrA QRDR in mutants derived from MG ATCC 15302. However, these mutations led to smaller increases in resistance (two- to four-fold) than the Ser-83
Arg mutation (Table 1). Mutation of Ser-80
Leu or Trp in the ParC QRDR also seems to play a role in enrofloxacin resistance. Mutants 41m18-9, 41m16/19-8 and 41m15-9, which carry one of these mutations, exhibited eight- to 16-fold increases in their enrofloxacin MIC (Table 2). As seen for the GyrA QRDR, mutations observed at other positions (64, 81 and 84) led to smaller increases in resistance level (two- to four-fold) (Tables 1 and 2).
Four mutations were found in the GyrB and ParE QRDRs at three and four different positions, respectively (Tables 1 and 2). These mutations had only a slight influence on resistance: the substitutions AspAsn, at positions 426 in GyrB and 420 in ParE QRDRs, respectively, were responsible for only a two-fold increase in the MIC of enrofloxacin (Table 2). Mutant ATm7 harboured the same alterations in passage 8 (ATm7-8) and in passage 10 (ATM7), but had different MICs (Table 1). This might be explained by the appearance of alterations elsewhere in the topoisomerases genes (outside the QRDR) or in unrelated gene loci (e.g. drug efflux systems).
In conclusion, DNA gyrase seems to be the primary target of enrofloxacin in M. gallisepticum. Our data also suggest that the position of the modified amino acid plays an important role in resistance to quinolones in M. gallisepticum, as was observed in M. hominis.10 Furthermore, the nature of the amino acid also has an influence on the resistance level since Ser-83Arg and Ser-80
Trp observed in the GyrA and ParC QRDRs, respectively, have a greater impact on the resistance level than other mutations described at the same position. It would be interesting to determine whether such mutations could be found in field strains of M. gallisepticum with reduced susceptibility or resistance to enrofloxacin.
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Acknowledgements |
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Footnotes |
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References |
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2
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Reinhardt, A. K., Bébéar, C. M., Kempf, I., Kobisch, M. & Gautier-Bouchardon, A. V. (2002). Characterization of mutations in DNA gyrase and topoisomerase IV involved in quinolone resistance of Mycoplasma gallisepticum mutants obtained in vitro. Antimicrobial Agents and Chemotherapy 46, 5903.
3 . Freundt, E. A. (1983). Culture media for classic mycoplasmas. In Methods in Mycoplasmology (Tully, J. G. & Razin, S., Eds), pp. 12735. Academic Press, New York, NY, USA.
4 . Bébéar, C. & Robertson, J. (1996). Determination of minimal inhibitory concentration. In Molecular and Diagnostic Procedures in Mycoplasmology (Tully, J. G. & Razin, S., Eds), pp. 18997. Academic Press, New York, NY, USA.
5 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Testing for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA, USA.
6 . Kellog, D. E. & Kwok, S. (1990). Detection of human immunodeficiency virus. In PCR Protocols: A Guide to Methods and Amplification (Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J., Eds), pp. 33943. Academic Press, San Diego, CA, USA.
7 . Heisig, P. (1996). Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 87985.[Abstract]
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Kim, J. H., Cho, E. H., Kim, K. S., Kim, H. Y. & Kim, Y. M. (1998). Cloning and nucleotide sequence of the DNA gyrase gyrA gene from Serratia marcescens and characterization of mutations in gyrA of quinolone-resistant clinical isolates. Antimicrobial Agents and Chemotherapy 42, 1903.
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Bébéar, C. M., Charron, A., Bové, J. M., Bébéar, C. & Renaudin, J. (1998). Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis. Antimicrobial Agents and Chemotherapy 42, 202431.
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Bébéar, C. M., Renaudin, H., Charron, A., Gruson, D., Lefrançois, M. & Bébéar, C. (2000). In vitro activity of trovafloxacin compared with those of five antimicrobials against mycoplasmas including Mycoplasma hominis and Ureaplasma urealyticum fluoroquinolone-resistant isolates that have been genetically characterized. Antimicrobial Agents and Chemotherapy 44, 255760.
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