Azithromycin resistance mutations in Streptococcus pneumoniae as revealed by a chemogenomic screen

Abstract

We report on the combination of chemical mutagenesis, azithromycin selection and next-generation sequencing (Mut-Seq) for the identification of small nucleotide variants that decrease the susceptibility of Streptococcus pneumoniae to the macrolide antibiotic azithromycin. Mutations in the 23S ribosomal RNA or in ribosomal proteins can confer resistance to macrolides and these were detected by Mut-Seq. By concentrating on recurrent variants, we could associate mutations in genes implicated in the metabolism of glutamine with decreased azithromycin susceptibility among S. pneumoniae mutants. Glutamine synthetase catalyses the transformation of glutamate and ammonium into glutamine and its chemical inhibition is shown to sensitize S. pneumoniae to antibiotics. A mutation affecting the ribosomal-binding site of a putative ribonuclease J2 is also shown to confer low-level resistance. Mut-Seq has the potential to reveal chromosomal changes enabling high resistance as well as novel events conferring more subtle phenotypes.

Keywords: Azithromycin; Glutamine; Resistance; Streptococcus pneumoniae; chemical mutagenesis; macrolide; next generation sequencing.

Fig. 1.

Impact of mutations on gene expression. Fold change in expression of glnP (black bars), glnR (white bars) and glnA (grey bars) in mutants M14 and M21 or in S. pneumoniae R6 transformants harbouring a mutation upstream of spr0443 (glnR; from M6), a mutation in spr0189 (ribosomal protein L4^G71R) alone or in combination with a mutation in spr1121 (GlnQ^T46S). Expression changes are relative to R6 wild-type. **P ≤0.01; ***P ≤0.001.

Fig. 2.

Impact of mutations on growth. Growth of S. pneumoniae harbouring different sets of mutations detected by Mut-Seq in the absence of AZM (a) or in the presence of 0.125 µM AZM (b) For R6 transformants, the gene affected by the mutation is indicated in superscript. R6, S. pneumoniae R6 wild-type.

Fig. 3.

The inhibition of glutamine synthetase sensitizes S. pneumoniae to different antibiotics. S. pneumoniae R6 wild-type growth inhibition curves of AZM (a) tetracycline (b) and ciprofloxacin (c) in the absence (grey) or presence (black) of the GlnA inhibitor MSO at 4 µM. Data is expressed as the relative growth compared to non-treated R6 wild-type after overnight incubation.