Antibiotic resistance and pathogenicity in the Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumoniae

Abstract

The dramatic increase of infections caused by multidrug-resistant Gram-negative bacteria is an emerging global problem and possibly one of the greatest challenges of modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds and there is an alarming increase of multi- or even pandrug-resistant isolates. The aims of this thesis were i) to elucidate the molecular mechanisms of fluoroquinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa and ii) to describe the transcriptomic landscape of Klebsiella pneumoniae to correlate gene transcription with the clinical relevant phenotypes of biofilm formation, virulence and antibiotic resistance. In this context, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa by applying a combination of directed resequencing methods, quantitative real-time PCRs and whole-transcriptome sequencing (RNA-Seq) on a broad and cross-sectional panel of 172 clinical isolates. This comprehensive data showed the role of distinct mutations in the quinolone resistance-determining regions of gyrA and parC. The combination with further mutations (e.g. in gyrB and parE) or enhanced efflux exhibited additive effects Furthermore, we exploited the power of deep transcriptome profiling by RNA-seq to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origin. We identified a large set of 3346 genes which were expressed in all isolates. While these core-transcriptome profiles were largely homogenous among isolates of the same sequence type, they varied substantially between groups of different sequence types. This detailed information on differentially expressed genes was linked with the clinically relevant phenotypes of biofilm formation, bacterial virulence and antibiotic resistance. This allowed the identification of a low biofilm-specific gene expression profile within the group of ST258 isolates, the dominant clonal lineage associated with KPC-carbapenemase spread, as a sequence type-specific trait. Moreover, the analysis revealed that antimicrobial resistance in this panel of clinical isolates can be explained by the occurrence of only a few dominant resistance determinants.