Description: The genome sequence was generated for four Streptococcus pneumoniae cefotaxime resistant isolates either selected for resistance in vitro (S. pneumoniae R6M1 and R6M3) or by serial whole genome transformation of the sensitive S. pneumoniae R6 with gDNA derived from cefotaxime-resistant mutants R6M1 and R6M2. Analysis of the genome assemblies revealed mutations in genes coding for the penicillin-binding proteins (PBPs) 2x, 2a and 3, of which pbp2x was the only mutated gene common to all mutants. The transformation of altered PBP alleles into S. pneumoniae R6 confirmed the role of PBP mutations in cefotaxime resistance but these were not sufficient to entirely explain the levels of resistance observed among cefotaxime resistant strains. In addition to PBPs, thirty-one other genes were found to be mutated in at least one of the four sequenced genomes. Non-PBP resistance determinants appeared to be mostly lineage-specific however since the peptidoglycan GlcNAc deacetylase spr1333 was the only non-PBP gene to be mutated in more than one mutant. A role in cefotaxime resistance for spr1333 mutations in the presence of altered PBPs was confirmed by transformation and enabled explaining the level of resistance of the R6M2 mutant. The analysis for lineage-specific mutations at different steps of selection of cefotaxime resistance in the R6M1 and R6M3 mutants revealed three other genes with a possible role in resistance. Mutations in spr0981 and spr1704, respectively coding for a glycosyltransferase and an ABC transporter, were specific to R6M3 and transformation experiments confirmed that the level of resistance of this mutant was due to a combination of mutations within pbp2x, spr1704, spr1333 and spr0981. Finally, a non-sense mutation in the sortase gene spr1098 was found to account for the non-PBP cefotaxime resistance level of R6M1. Sortase A is a conserved transpeptidase responsible for the covalent attachment of LPXTG-motif-containing proteins to the cell wall and it is possible that the cefotaxime resistance phenotype conferred by the non-sense mutation in spr1098 comes from the mis-localization of one (or more) of these proteins. This is the first report about the role of sortase A mutation in cefotaxime resistance.

Data type: Other

Sample scope: Monoisolate

Release date: 2012-08-02