Sarah Lohsen, United States of America

Emory University Infectious Diseases

Poster Author Of 2 e-Posters

Online Abstracts Basic Sciences - Conventional and Molecular Microbiology A2 Conventional and Molecular Microbiology

Presenter Of 1 Presentation

INDUCIBLE MEGA-MEDIATED MACROLIDE ANTIBIOTIC RESISTANCE CONFERS HETERORESISTANCE IN STREPTOCOCCUS PNEUMONIAE. (ID 609)

Session Name
Basic Sciences - Conventional and Molecular Microbiology

Abstract

Background

In Streptococcus pneumoniae (Spn), the Macrolide Genetic Assembly (Mega) provides macrolide antibiotic resistance via the efflux pump Mef(E) and the ribosomal protection protein Mel. This resistance has previously been assumed to confer traditional resistance. Heteroresistance is commonly missed during traditional clinical resistance screens, and is highly concerning as resistant sub-populations can persist despite treatment.

Methods

Spn strains containing the Mega element were screened via Etesting and Population Analysis Profiling. In addition to screening wildtype strains, strains with deletions of the 5’UTR of Mef(E) were also queried.

Results

All wildtype Mega-containing Spn strains screened displayed heteroresistance (>eight fold range in MICs) to Mef(E)/Mel-inducing macrolides, but not to non-Mef(E)/Mel-inducing Macrolides or other classes of antibiotics. When macrolide induction uniformly increased mef(E)/mel expression, heteroresistance was eliminated. A deletion of the 5’UTR of mef(E) resulted in a mutant deficient in induction as well as heteroresistance. Only the mef(E)L leader peptide sequence of the 5’UTR is required for induction and heteroresistance.

Conclusions

This study finds that inducibility and heteroresistance are linked for macrolide resistance conferred by the Mega element independent of insertion class. Stochastic variation or epigenetics affecting mef(E)/mel expression inside a population of Spn are possible mechanisms for heteroresistance.

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Author Of 2 Presentations

INDUCIBLE MEGA-MEDIATED MACROLIDE ANTIBIOTIC RESISTANCE CONFERS HETERORESISTANCE IN STREPTOCOCCUS PNEUMONIAE. (ID 609)

Session Name
Basic Sciences - Conventional and Molecular Microbiology

Abstract

Background

In Streptococcus pneumoniae (Spn), the Macrolide Genetic Assembly (Mega) provides macrolide antibiotic resistance via the efflux pump Mef(E) and the ribosomal protection protein Mel. This resistance has previously been assumed to confer traditional resistance. Heteroresistance is commonly missed during traditional clinical resistance screens, and is highly concerning as resistant sub-populations can persist despite treatment.

Methods

Spn strains containing the Mega element were screened via Etesting and Population Analysis Profiling. In addition to screening wildtype strains, strains with deletions of the 5’UTR of Mef(E) were also queried.

Results

All wildtype Mega-containing Spn strains screened displayed heteroresistance (>eight fold range in MICs) to Mef(E)/Mel-inducing macrolides, but not to non-Mef(E)/Mel-inducing Macrolides or other classes of antibiotics. When macrolide induction uniformly increased mef(E)/mel expression, heteroresistance was eliminated. A deletion of the 5’UTR of mef(E) resulted in a mutant deficient in induction as well as heteroresistance. Only the mef(E)L leader peptide sequence of the 5’UTR is required for induction and heteroresistance.

Conclusions

This study finds that inducibility and heteroresistance are linked for macrolide resistance conferred by the Mega element independent of insertion class. Stochastic variation or epigenetics affecting mef(E)/mel expression inside a population of Spn are possible mechanisms for heteroresistance.

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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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