Dissemination of Tn 916 -Related Integrative and Conjugative Elements in Streptococcus pneumoniae Occurs by Transformation and Homologous Recombination in Nasopharyngeal Biofilms.

Multidrug resistance in Streptococcus pneumoniae (or pneumococcus) continues to be a global challenge. An important class of antibiotic resistance determinants disseminating in are >20-kb Tn-related integrative and conjugative elements (ICEs), such as Tn, Tn, and Tn. Although conjugation has been implicated as the transfer mechanism for ICEs in several bacteria, including , the molecular basis for widespread dissemination of pneumococcal Tn-related ICEs remains to be fully elucidated. We found that Tn acquisition was not detectable via transformation nor conjugative mating with donor GA16833, yielding a transfer frequency of <10. GA16833 Tn conjugative gene expression was not significantly induced, and ICE circular intermediate formation was not detected in biofilms. Consistently, Tn transfer efficiency in biofilms was not affected by deletion of the ICE conjugative gene However, GA16833 Tn transfer occurred efficiently at a recombination frequency (rF) of 10 in dual-strain biofilms formed in a human nasopharyngeal cell bioreactor. DNase I addition and deletions of the early competence gene or transformation apparatus genes and in the D39 recipient strain prevented Tn acquisition (rF of <10). Genome sequencing and single nucleotide polymorphism analyses of independent recombinants of recipient genotype identified ~33- to ~55-kb donor DNAs containing intact Tn, supporting homologous recombination. Additional pneumococcal donor and recipient combinations were demonstrated to efficiently transfer Tn-related ICEs at a rF of 10 in the biofilms. Tn-related ICEs horizontally disseminate at high frequency in human nasopharyngeal biofilms by transformation and homologous recombination of >30-kb DNA fragments into the pneumococcal genome. The World Health Organization has designated Streptococcus pneumoniae as a priority pathogen for research and development of new drug treatments due to extensive multidrug resistance. Multiple strains of colonize and form mixed biofilms in the human nasopharynx, which could enable exchange of antibiotic resistance determinants. Tn-related integrative and conjugative elements (ICEs) are largely responsible for the widespread presence of macrolide and tetracycline resistance in . Utilizing a system that simulates colonization of donor and recipient strains in the human nasopharynx, efficient transfer of Tn-related ICEs occurred in human nasopharyngeal biofilms, in contrast to conditions of planktonic cells with exogenous DNA. This high-frequency Tn-related ICE transfer between strains in biofilms was due to transformation and homologous recombination, not conjugation. Understanding the molecular mechanism for dissemination of Tn-related ICEs can facilitate the design of new strategies to combat antibiotic resistance.