Ultrastructural, metabolic and genetic determinants of the acquisition of macrolide resistance by .

Aim: (Spn) acquires genes for macrolide resistance, MEGA or , in the human host. These genes are carried either in the chromosome, or on integrative conjugative elements (ICEs). Here, we investigated molecular determinants of the acquisition of macrolide resistance.

Methods And Results: Whole genome analysis was conducted for 128 macrolide-resistant pneumococcal isolates to identify the presence of MEGA (44.5%, 57/128) or (100%), and recombination events in Tn-related elements or in the locus encoding competence genes. Confocal and electron microscopy studies demonstrated that, during the acquisition of macrolide resistance, pneumococcal strains formed clusters of varying size, with the largest aggregates having a median size of ~1600 μm. Remarkably, these pneumococcal aggregates comprise both encapsulated and nonencapsulated pneumococci, exhibited physical interaction, and spanned extracellular and intracellular compartments. We assessed the recombination frequency (rF) for the acquisition of macrolide resistance by a recipient D39 strain, from pneumococcal strains carrying MEGA (~5.4 kb) in the chromone, or in large ICEs (>23 kb). Notably, the rF for the acquisition of MEGA, whether in the chromosome or carried on an ICE was similar. However, the rF adjusted to the acquisition of the full-length ICE (~52 kb), compared to that of the capsule locus (~23 kb) that is acquired by transformation, was three orders of magnitude higher. Finally, metabolomics studies revealed a link between the acquisition of ICE and the metabolic pathways involving nicotinic acid and sucrose.

Conclusions: Extracellular and intracellular pneumococcal clusters facilitate the acquisition of full-length ICE at a rF higher than that of typical transformation events, involving distinct metabolic changes that present potential targets for interventions.