Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

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.

Figure 1.

Monitoring mRNA expression by qRT-PCR in linezolid-resistant Streptococcus pneumoniae. Two genomic regions are shown, one containing the ABC protein gene spr1021 (A) and one containing the ABC protein genes spr1887 and spr1885 (B). Between spr1887 and spr1885 there is a putative ORF, although it is not clear whether it is real. The spr# is according to the nomenclature of strain R6. The RNAs were normalized with 16S rRNA. The expression ratios shown are the cycle threshold (Ct) ratios between the resistant cells and the parent wild-type cells for each individual gene shown. Possible sites of transcription initiation (arrows) and termination (vertical bars) of the operons are indicated. Values represent an average of three independent experiments.

Figure 2.

Phylogenetic analysis of a class of RNA methyltransferases related to spr0333. Amino acid sequences of the RNA methyltransferase proteins were aligned using the ClustalW algorithm, and evolutionary trees were constructed using the Neighbor-Joining method with Poisson correction as implemented in the MEGA3.1 package for the most conserved part of the proteins. The reliabilities of each branch point were assessed by the analysis of 1000 bootstrap replicates. A similar tree was obtained also when the whole protein was used for carrying the phylogenetic analysis (not shown).

Figure 3.

Mutations and modifications in the 23S rRNA related to linezolid resistance. (A) Secondary-structure model of part of the V domain of the 23S rRNA of S. pneumoniae. (Blue) Mutations in the 23S rRNA linked to linezolid resistance; (boxed residues) positions 2445, 2447, 2503, and 2576 found in this study; (red) new mutations or resistance mechanisms; (shaded) nucleotides that directly interact with linezolid as determined from several structural studies cited in the text. (B) Reverse transcription analysis of the S. pneumoniae 23S rRNA. U, A, C, G correspond with sequencing lanes. Primer extension results of the 23S rRNA from the control susceptible strain 1974 (lane 1), 1974M1 (lane 2), 1974M1 with pJF0333 (lane 3), 1974M1 with vector pDL289 (lane 4) are shown. The block (when present) in the reverse transcription reaction at position G2445 is indicated.