Twincore - Zentrum für Experimentelle und Klinische Infektionsforschung Institut für Molekulare Bakteriologie

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1 Twincore - Zentrum für Experimentelle und Klinische Infektionsforschung Institut für Molekulare Bakteriologie 0 HELMHOLTZ I ZENTRUM FÜR INFEKTIONSFORSCHUNG Technische Universität Braunschweig Institut für Mikrobiologie Helmholtz Zentrum für Infektionsforschung Abteilung Molekulare Bakteriologie Analysis of antibiotic resistance determinants in Pseudomonas aeruginosa - a transcriptomic approach Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von aus Ariane Alwine Khaledi Braunschweig

2 I Table of Contents List of Figures V List of Tables VII List of Abbreviations VIII 1 Introduction Pseudomonas aeruginosa as a model organism Pseudomonas aeruginosa as an opportunistic nosocomial pathogen Antibiotics: past and present ß-lactams - inhibitors of cell wall biosynthesis Antibiotic resistance in Pseudomonas aeruginosa Chromosomally encoded ß-lactam resistance mechanisms affecting antibiotic influx and efflux ß-lactamases - hydrolyzing enzymes How antibiotics affect bacteria - from specific targets to global networks Recent progress in next generation sequencing Aimsofthethesis 17 2 Materials and Methods Bacterial strains and growth conditions Clinical strain collection Plasmids and Oligomers DNAtransfertechniques Transformation of chemically competent E. coli 24

3 u Electroporation of P. aeruginosa Plasmid transfer by diparental mating (conjugation) Antibiotic susceptibility testing E-test Broth dilution RNA-sequencing - sample preparation Culturing conditions and harvesting RNA-extraction and DNA removal rrna removal by hybridization using oligo(dt) 25 beads rrna removal by hybridization using streptavidin tagged magnetic beads rrna removal by hybridization using MICROBExpress Enzymatic rrna removal by terminator-5'-phosphate-dependent exonuclease rrna removal by duplex-specific nuclease (DSN) RNA clean up and concentration by precipitation and chloroform-phenol purification cdna library construction RNA-sequencing - raw data processing Detection of acquired resistance enzymes Phylogeny Data display: the Bactome database SNP matrix calculation Stop matrix generation Gene expression matrix generation Phenotype-genotype group comparisons Statistics for Overall correlation Carbapenemase activity assay Carbapenemase assay with activity rescue Constructions of mexs and oprd knock-out mutants 36

4 III 2.13 OprD antibody generation Antibody purification OprD protein detection by Western Blot Outer membrane protein visualization LC-MS/MS sample preparation LC-MS/MS data acquisition, data analysis and database searching Fluorescence detection by flow cytometry Metabolie isotope analysis Amino acid extraction 41 3 Results An optimized protoeol for cost efficient high-throughput transcriptome sequencing off. aeruginosa mrna enrichment strategies Transcriptome sequencing of clinical P. aeruginosa isolates Coverage and relative transcript abundance of clinical P. aeruginosa isolates Phylotyping by 214 genes identified a clonal outbreak Nature and distribution of acquired ß-lactamases A systematic approach for comprehensive gene sequence and expression data analysis Identifying intrinsic ß-lactam resistance markers via unbiased phenotype-genotype correlation studies Penicillin binding proteins show high sequence variability, but their role in ß-lactam resistance remains unclear The majority of ß-lactam non-susceptible clinical isolates harbor dominant genetic resistance determinants Impact of sub-inhibitory antibiotic concentrations on bacterial cells Sub-inhibitory antibiotic treatment enhanced ROS produetion Transcriptional responses upon sub-inhibitory antibiotic stress 84

5 IV Metabolie changes induced by sub-inhibitory antibiotic stress 89 4 Discussion The increasing threat of bacterial antibiotic resistance Antibiotic treatment leads to resistance selection Antibiotic concentration reservoirs promote adaptation Limitations of culture-based diagnostics Transcriptome sequencing of drug resistant clinical isolates Inexpensive whole transcriptome sequencing provided reasonable Single nucleotide coverage throughout the whole P. aeruginosa genome In depth taxonomical profiling could distinguish between actual clonal outbreaks and sequence type related strains The acquired ß-lactam resistome Unbiased global correlation studies as an approach to detect novel phenotype specific genetic markers Evaluating RNA-sequencing for direct resistance prediction High-throughput target Screening as a future clinical outlook The impact of low-level antibiotic concentration reservoirs Common transcriptional changes cope with oxidative stress Sub-inhibitory antibiotic exposure affects the respiratory chain and leads to fundamental metabolic changes A need for avoiding trace-antibiotic reservoirs and targeting bacterial stress responses References Danksagung 1 7 Lebenslauf 127

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