Dissemination of Tn916-related integrative and conjugative elements in Streptococcus pneumoniae occurs by transformation and homologous recombination in nasopharyngeal biofilms

Dissemination of >20 kb Tn916-related integrative and conjugative elements (ICEs) in Streptococcus pneumoniae (Spn) are responsible for the spread of tetracycline and macrolide resistance. Though prototype Tn916 has been shown to horizontally transfer via conjugation in a number of bacterial species, the molecular mechanism by which Tn916-related ICEs, such as Tn2009 (23.5 kb), Tn6002 (20.8 kb), and Tn2010 (26.3 kb), disseminate in Spn remains unknown. In the present study, we utilized an Spn dual-strain nasopharyngeal biofilm model to investigate the mechanism for horizontal transfer of Tn916-related ICEs. Conjugative gene expression studies, mutation experiments with key conjugative and transformation genes, ICE circular intermediate quantification, conjugative mating assays, Tn916-related ICE-containing recombinant whole genome sequencing, and recombinant SNP analyses were performed, revealing that transformation and homologous recombination of large donor DNA fragments facilitated the horizontal transfer of Tn916-related ICEs in Spn.