Genetic Determinants Affecting Macrolide Resistance Conferred by MEGA in Streptococcus pneumoniae Público

Abstract

Macrolide antibiotic resistance in Streptococcus pneumoniae due to efflux is a major clinical problem and may lead to failure of macrolide treatment of pneumococcal disease. Specifically, macrolide resistance due to efflux is the result of a predicted dual component efflux pump encoded by mefE/mel of the mobile genetic element MEGA. A separate operon, orfs3-6, frequently associates with mefE/mel. orfs3-6 were found to influence macrolide resistance levels of clinical isolate GA17457, but did not have any significant effect on the expression of a PmefE-lacZ reporter fusion as determined by a β-galactosidase assay. GA17457 was compared to another clinical isolate, GA16242, with nearly three-fold higher macrolide resistance (13 µg/mL compared to 43 µg/mL respectively). A two-fold increase in β-galactosidase expression was observed in the GA16242 reporter construct TS8011 when compared to the GA17457 reporter construct XZ7042. A novel element containing a putative transposase and an uncharacterized open reading frame (orfsAB) was identifieddownstream of MEGA in GA 16242. However, deletion of this region did not influence macrolide resistance or mefE/mel in a β-galactosidase expression assay. Results of these studies confirm a role for orfs3-6 in the macrolide resistance conferred by MEGA but not in the transcriptional regulation of mefE/mel. Additionally, the comparison of two strains with different MEGA insertion sites identified significant differences in macrolide resistance and mefE/mel expression, but did not elucidate the cause for these observations.

Table of Contents

Table of Contents

Introduction...1

1. History...1
2. Pathology...2
3. Treatment and Vaccine...2
4. Antibiotic Resistance...4
5. Efflux Pumps...5
6. Macrolides...6
7. mef & mef Containing Elements...7
8. Distribution of mef and Other Macrolide Resistance Elements...9
9. MefE and Mel Function...11
10. orfs3-6 and the SOS response...12

Methods...16
1. Strains...16
2. Plasmids and Deletion Construction...16
3. RTPCR...18
4. Antimicrobial Susceptibility Testing...18
5. Dose Response Curve...19
6. β-galactosidase Expression Assay...19
7. Statistics...20

Results...21
1. Effect of orfs3-6 on Macrolide Susceptability...21
2. Optimal Erythromycin Concentration for Induction...22
3. Effect of orfs3-6 on P mefE-lacZ...22
4. Differences in Macrolide Resistance By Insertion Site...23
5. P mefE-lacZ Expression in a High-Level Resistance Isolate...24

Discussion...26

Figures and Tables...32

References...41

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	MS
Submission	Master's Thesis
Language	English
Research Field	Biology, Genetics Biology, Molecular Biology, Microbiology
Palavra-chave	MEGA macrolide antibiotic resistance Streptococcus pneumoniae mefE orfs3-6 transcriptional expression
Committee Chair / Thesis Advisor	Stephens, David S, Emory University
Committee Members	Shafer, William M, Emory University Rather, Phil N, Emory University Klugman, Keith P, Emory University Moran Jr., Charles, Emory University

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