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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 \n \n S. pneumoniae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> mutants. Glutamine synthetase catalyses the transformation of glutamate and ammonium into glutamine and its chemical inhibition is shown to sensitize \n \n S. pneumoniae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> to antibiotics. A mutation affecting the ribosomal-binding site of a putative ribonuclease J2 is also shown to confer low-level resistance. 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