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ABSTRACT: Several mutations were present in the genome of Streptococcus pneumoniae linezolid-resistant strains but the role of several of these mutations had not been experimentally tested. To analyze the role of these mutations, we reconstituted resistance by serial whole genome transformation of a novel resistant isolate into two strains with sensitive background. We sequenced the parent mutant and two independent transformants exhibiting similar minimum inhibitory concentration to linezolid.
Comparative genomic analyses revealed that transformants acquired G2576T transversions in every gene copy of 23S rRNA and that the number of altered copies correlated with the level of linezolid resistance and cross-resistance to florfenicol and chloramphenicol. One of the transformants also acquired a mutation present in the parent mutant leading to the overexpression of an ABC transporter (spr1021). The acquisition of these mutations conferred a fitness cost however, which was further enhanced by the acquisition of a mutation in a RNA methyltransferase implicated in resistance. Interestingly, the fitness of the transformants could be restored in part by the acquisition of altered copies of the L3 and L16 ribosomal proteins and by mutations leading to the overexpression of the spr1887 ABC transporter that were present in the original linezolid-resistant mutant.
Our results demonstrate the usefulness of whole genome approaches at detecting major determinants of resistance as well as compensatory mutations that alleviate the fitness cost associated with resistance.
BMC Genomics 10/2011; 12:512. · 4.07 Impact Factor
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ABSTRACT: Linezolid is an oxazolidinone antibiotic that inhibits the initiation of translation. Although resistance to linezolid is an uncommon event, it has been reported in clinical isolates. The genome sequence of Streptococcus pneumoniae linezolid-resistant mutants recently revealed mutations associated with resistance. A proteomic and transcriptomic screen now reveals a possible increase in the metabolism and transport of carbohydrates in these linezolid-resistant S. pneumoniae mutants. Several glycolytic proteins were shown to be overexpressed in the resistant strains, along with other enzymes and transporters involved in the metabolism of sugars. An increase in energy needs appears to be required to sustain extended levels of resistance to linezolid as the disruption of two ABC transporters putatively involved in the import of carbohydrates leads to a 2-fold sensitization to linezolid. Furthermore, the disruption of the catabolite control protein A, a regulator of the metabolism of sugars whose expression is highly increased in one linezolid-resistant mutant, resulted in a 2-fold increase in linezolid susceptibility. This global scale analysis of gene and protein expression profiling highlights metabolism alterations associated with linezolid resistance in S. pneumoniae.
Journal of Proteome Research 08/2011; 10(10):4439-52. · 5.11 Impact Factor
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ABSTRACT: Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used 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 including, notably, 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 nonmutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells being more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 ortholog 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.
Genome Research 05/2009; 19(7):1214-23. · 13.61 Impact Factor
