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. 2011 Oct 17:12:512.
doi: 10.1186/1471-2164-12-512.

Whole genome analysis of linezolid resistance in Streptococcus pneumoniae reveals resistance and compensatory mutations

Dewan S Billal  1 , Jie Feng, Philippe Leprohon, Danielle Légaré, Marc Ouellette
Affiliations

Whole genome analysis of linezolid resistance in Streptococcus pneumoniae reveals resistance and compensatory mutations

Dewan S Billal et al. BMC Genomics. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
Venn diagram of the mutations identified in the S. pneumoniae R6M1, R6M2, 1974M1 and 1974M2 mutants resistant to LNZ. a The four gene copies of 23S rRNA, b spr0333 codes for a hypothetical protein/rRNA methyltransferase, c spr1021 codes for the nucleotide-binding subunit of an ABC protein, d spr1887 codes for an ABC transporter.
Figure 2
Figure 2
Growth kinetics of S. pneumoniae wild-type (1974), LNZ-resistant mutant (1974M1) and LNZ-resistant transformants. A. Growth curves of S. pneumoniae 1974 transformed with four mutated copies of 23S rRNA alone (T-7); or along with altered versions of spr0333 (T-7spr0333); spr1021 (T-7spr1021); spr1887 (T-7spr1887); or a combination of the three (T-7spr0333, 1021, 1887). B. Growth curves of S. pneumoniae 1974 transformed with four mutated copies of 23S rRNA alone (T-7); or along with altered versions of spr0188 (T-7L3); spr0188 and spr0196 (T-7L3L16); spr0188, spr0196 and spr0333 (T-7L3L16spr0333); or spr0188, spr0196 and spr1887 (T-7L3L16spr1887). Data are expressed as the mean of three independent experiments and the statistical significance of the growth differences are indicated in the text.
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