Brenda S. Antezana, United States of America

Emory University Medicine

Presenter Of 1 Presentation

EFFICIENT DISSEMINATION OF INTEGRATIVE AND CONJUGATIVE ELEMENTS CONFERRING MULTIDRUG RESISTANCE IN STREPTOCOCCUS PNEUMONIAE IN AN EX VIVO HUMAN NASOPHARYNGEAL BIOFILM (ID 125)

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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Author Of 2 Presentations

EFFICIENT DISSEMINATION OF INTEGRATIVE AND CONJUGATIVE ELEMENTS CONFERRING MULTIDRUG RESISTANCE IN STREPTOCOCCUS PNEUMONIAE IN AN EX VIVO HUMAN NASOPHARYNGEAL BIOFILM (ID 125)

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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STREPTOCOCCUS PNEUMONIAE GENERATES HYDROXYL RADICALS TO RAPIDLY INTOXICATE AND KILL STAPHYLOCOCCUS AUREUS STRAINS (ID 256)

Abstract

Background

Streptococcus pneumoniae (Spn) strains rapidly kills Staphylococcus aureus (Sau) by producing membrane-permeable hydrogen peroxide (H2O2). The exact mechanism by which the H2O2-mediated killing occurs is not well understood.

Methods

An in vitro model that mimicked Spn-Sau contact during colonization of the upper airways and whole genome sequencing was conducted. Different Spn H2O2 mutants were constructed to confirm the Sau killing mechanism.

Results

Sau killing required outcompeting densities of Spn. A collection of MRSA/MSSA strains showed a linear sensitivity (R2=0.95) for Spn killing but the same strains had different susceptibilities when challenged against pure H2O2. WGS of these MRSA/MSSA strains revealed no association between clonal complex and susceptibility, or resistance, to Spn, or H2O2,respectively. A sublethal dose (~1 mM) of pure H2O2 when incubated with TIGR4DspxB eradicated cultures of Sau strains suggesting that Spn converts H2O2 to the hydroxyl radical (·OH). Accordingly, Sau killing was completely blocked by incubating with scavengers of ·OH radicals, DMSO, or thiourea.

Conclusions

Spn produces H2O2 which is rapidly converted to a more potent oxidant, the ·OH radical. Hydroxyl radicals does not affect Spn viability but rapidly intoxicate Sau strains. The target(s) of the ·OH radicals represents an exciting new alternative for the development of therapeutics against Sau infections.

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