Genome sequencing of linezolid resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance
- Jie Feng1,
- Andréanne Lupien1,
- Hélène Gingras1,
- Jessica Wasserscheid2,
- Ken Dewar2,
- Danielle Légaré1 and
- Marc Ouellette1,3
- E-mail: marc.ouellette{at}crchul.ulaval.ca
Abstract
Linezolid is part of a novel class of antibiotics with resistance already being reported. We applied the method of whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step by step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants and notably at G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes spr1021 and spr1887 were correlated with increased expression of these genes and neighboring genes of the same operon. Gene inactivation supported a role for these ABC transporters in resistance to linezolid and other antibiotics. The hypothetical protein spr0333 contains an RNA methyltransferase domain and mutations within that domain were found in all S. pneumoniae linezolid resistant strains. Primer extension experiments indicated that spr0333 methylates G2445 of the 23S rRNA and mutations in spr0333 abolished this methylation. Reintroduction of a non-mutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 orthologue was also mutated in a linezolid resistant clinical Staphylococcus aureus isolate. Whole genome sequencing and comparative analyses of S. pneumoniae resistant isolates was useful for discovering novel resistance mutations.
Footnotes
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- Received November 20, 2008.
- Accepted April 2, 2009.
- Copyright © 2009, Cold Spring Harbor Laboratory Press
