EFFICIENT DISSEMINATION OF INTEGRATIVE AND CONJUGATIVE ELEMENTS CONFERRING MULTIDRUG RESISTANCE IN STREPTOCOCCUS PNEUMONIAE IN AN EX VIVO HUMAN NASOPHARYNGEAL BIOFILM
Abstract
Background
Multidrug resistance in Streptococcus pneumoniae (Spn) has been increasingly attributed to dissemination of integrative and conjugative elements (ICEs), such as Tn2009 (23.5kb). The mechanism for Spn ICE dissemination has not been defined.
Methods
Recombination frequency (rF) for Tn2009 was investigated utilizing in vitro transformation or an ex vivo nasopharyngeal biofilm bioreactor. Recombinant lineage and extracellular DNA (eDNA) concentrations were determined by serotype-specific qPCR. Whole genome sequencing (WGS) identified putative junctions for Tn2009 recombination.
Results
In vitro transformation yielded no Tn2009-containing D39 recombinants (rF<10-9) while mutation-mediated streptomycin resistance was obtained (rF 10-6). However, in the bioreactor, Tn2009 transference from donor GA16833Tet/Ery (ST19F) to recipient D39Str (ST2) generated >90% D39Tet/Str recombinants with variably sized donor DNA fragments encompassing intact Tn2009 (rF 10-4), indicating varied recombination junctions. Tn2009 transference was prevented by DNaseI addition (rF<10-7). D39 competence mutants (ΔcomC/D/E) with GA16833 yielded reduced rFs (10-8-10-6) and nearly 100% ST19F recombinants acquiring Str resistance. Similar bacterial densities and eDNA concentrations from each strain were detected. D39ΔcomC with GA16833ΔcomC yielded no recombinants (rF<10-7).
Conclusions
Efficient Tn2009 dissemination among Spn strains occurs in an ex vivo nasopharyngeal biofilm and requires recipient competence development. Further, there is a com-mediated dominance for a specific Spn strain to acquire resistance.
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