Genomic analysis and reconstruction of cefotaxime resistance in Streptococcus pneumoniae

Abstract

To identify non-penicillin-binding protein (PBP) mutations contributing to resistance to the third-generation cephalosporin cefotaxime in Streptococcus pneumoniae at the genome-wide scale. The genomes of two in vitro S. pneumoniae cefotaxime-resistant isolates and of two transformants serially transformed with the genomic DNA of cefotaxime-resistant mutants were determined by next-generation sequencing. A role in cefotaxime resistance for the mutations identified was confirmed by reconstructing resistance in a cefotaxime-susceptible background. Analysis of the genome assemblies revealed mutations in genes coding for the 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 fully explain the levels of resistance. Thirty-one additional 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. Mutations in spr1333, spr0981, spr1704 and spr1098, encoding a peptidoglycan N-acetylglucosamine deacetylase, a glycosyltransferase, an ABC transporter and a sortase, respectively, were implicated in resistance by transformation experiments and allowed the reconstruction of the full level of resistance observed in the parent resistant strains. This whole-genome analysis coupled to functional studies has allowed the discovery of both known and novel cefotaxime resistance genes in S. pneumoniae.