Abstract
Resistance to oxazolidinone antibiotics, including linezolid, in Gram-positive bacteria is mediated by single-nucleotide polymorphisms (SNPs) in the 23S ribosomal RNA. A G2576U change (encoded by a G2576T mutation in the rRNA genes) is found in most resistant clinical isolates of enterococci and staphylococci; a variety of changes have been found in resistant mutants selected in vitro. Pyrosequencing® can be used to detect SNPs known to confer oxazolidinone resistance, including the G2576T change. Most bacteria have more than one rRNA gene copy and Pyrosequencing can also be used for allele quantification, i.e., to estimate the proportions of mutant vs wild-type alleles. The number of mutated rRNA gene copies correlates roughly with the level of oxazolidinone resistance displayed by resistant isolates. This chapter summarizes the Pyrosequencing assays that have been developed in our laboratory for analyzing oxazolidinone-resistant enterococci and staphylococci.
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References
Ford, C. W., Hamel, J. C., Stapert, D., et al. (1997) Oxazolidinones: new antibacterial agents. Trends Microbiol. 5, 196–200.
Shinabarger, D. (1999) Mechanism of action of the oxazolidinone antibacterial agents. Expert. Opin. Investig. Drugs 8, 1195–1202.
Ford, C. W., Zurenko, G. E., and Barbachyn, M. R. (2001) The discovery of linezolid, the first oxazolidinone antibacterial agent. Curr. Drug Targets Infect. Disord. 1, 181–199.
Zurenko, G. E., Todd, W. M., Hafkin, B., et al. (1999) Development of linezolid-resistant Enterococcus faecium in two compassionate use program patients treated with linezolid. Proc. abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, p. 118.
Gonzales, R. D., Schreckenberger, P. C., Graham, M. B., Kelkar, S., DenBesten, K., and Quinn, J. P. (2001) Infections due to vancomycin-resistant Enterococcus faecium resistant to linezolid. Lancet 357, 1179.
Auckland, C., Teare, L., Cooke, F., et al. (2002) Linezolid-resistant enterococci: report of the first isolates in the United Kingdom. J. Antimicrob. Chemother. 50, 743–746.
Johnson, A. P., Tysall, L., Stockdale, M. W., et al. (2002) Emerging linezolid resistant Enterococcus faecalis and Enterococcus faecium isolated from two Austrian patients in the same intensive care unit. Eur. J. Clin. Microbiol. Infect. Dis. 21, 751–754.
Halle, E., Padberg, J., Rosseau, S., Klare, I., Werner, G., and Witte, W. (2004) Linezolid-resistant Enterococcus faecium and Enterococcus faecalis isolated from a septic patient: report of first isolates in Germany. Infection 32, 182–183.
Tsiodras, S., Gold, H. S., Sakoulas, G., et al. (2001) Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 358, 207–208.
Wilson, P., Andrews, J. A., Charlesworth, R., et al. (2003) Linezolid resistance in clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 51, 186–188.
Meka, V. G., Pillai, S. K., Sakoulas, G., et al. (2004) Linezolid resistance in sequential Staphylococcus aureus isolates associated with a T2500A mutation in the 23S rRNA gene and loss of a single copy of rRNA. J. Infect. Dis. 190, 311–317.
Prystowsky, J., Siddiqui, F., Chosay, J., et al. (2001) Resistance to linezolid: characterization of mutations in rRNA and comparison of their occurrences in vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 45, 2154–2156.
Klein, H. L. (1995) Genetic control of intrachromosomal recombination. Bioessays 17, 147–159.
Marshall, S. H., Donskey, C. J., Hutton-Thomas, R., Salata, R. A., and Rice, L. B. (2002) Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis. Antimicrob. Agents Chemother. 46, 3334–3336.
Ruggero, K. A., Schroeder, L. K., Schreckenberger, P. C., Mankin, A. S., and Quinn, J. P. (2003) Nosocomial superinfections due to linezolid-resistant Enterococcus faecalis: evidence for a gene dosage effect on linezolid MICs. Diagn. Microbiol. Infect. Dis. 47, 511–513.
Sinclair, A., Arnold, C., and Woodford, N. (2003) Rapid detection and estimation by Pyrosequencing of 23S rRNA genes with a single nucleotide polymorphism conferring linezolid resistance in enterococci. Antimicrob. Agents Chemother. 47, 3620–3622.
Lobritz, M., Hutton-Thomas, R., Marshall, S., and Rice, L. B. (2003) Recombination proficiency influences frequency and locus of mutational resistance to linezolid in Enterococcus faecalis. Antimicrob. Agents Chemother. 47, 3318–3320.
Engemann, J. J., Joyce, M. J., Harrell, L. J., et al. (2003) Outbreak of linezolid-resistant Enterococcus faecium bloodstream infections on an oncology ward. Proc. abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, p. 359.
Woodford, N., Tysall, L., Auckland, C., et al. (2002) Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment polymorphism analysis. J. Clin. Microbiol. 40, 4298–4300.
Werner, G., Strommenger, B., Klare, I., and Witte, W. (2004) Molecular detection of linezolid resistance in Enterococcus faecium and Enterococcus faecalis by use of 5′ nuclease real-time PCR compared to a modified classical approach. J. Clin. Microbiol. 42, 5327–5331.
Nyren, P. (2007) The history of Pyrosequencing. In: Pyrosequencing Protocols, (Marsh, S., ed.), Humana Press, Totowa, NJ, pp. 1–14.
Johnson, A. P. and Woodford, N. (1998) Plasmid analysis. Methods Mol. Med. 15, 51–62.
Fakhrai-Rad, H., Pourmand, N., and Ronaghi, M. (2002) Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum. Mutat. 19, 479–485.
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Woodford, N., North, S.E., Ellington, M.J. (2007). Detecting Mutations That Confer Oxazolidinone Resistance in Gram-Positive Bacteria. In: Walker, J.M., Marsh, S. (eds) Pyrosequencing® Protocols. Methods in Molecular Biology™, vol 373. Humana Press. https://doi.org/10.1385/1-59745-377-3:103
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DOI: https://doi.org/10.1385/1-59745-377-3:103
Publisher Name: Humana Press
Print ISBN: 978-1-58829-645-0
Online ISBN: 978-1-59745-377-6
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