Antibiotikaresistenz und Pathogenität in den Gram-negativen Bakterien Pseudomonas aeruginosa und Klebsiella pneumoniae

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.Der dramatische Anstieg von Infektionen durch multiresistente, gramnegative Bakterien ist ein weltweites Problem, welches möglicherweise eine der größten Herausforderungen moderner Medizin darstellt. Bakterielle Krankheitserreger besitzen verschiedenste Mechanismen, um der Aktivität einer Vielzahl antimikrobieller Verbindungen zu widerstehen und zeigen eine alarmierende Zunahme von multi- oder sogar pan-resistenten Isolaten. Die Ziele der vorliegenden Arbeit waren i) die molekularen Mechanismen der Fluorchinolonresistenz im opportunistischen Erreger Pseudomonas aeruginosa zu erklären und ii) die generelle Genexpression von Klebsiella pneumoniae zu beschreiben und mit den klinisch relevanten Phänotypen der Biofilmbildung, Virulenz und Antibiotikaresistenz zu korrelieren. In diesem Zusammenhang untersuchten wir den quantitativen Einfluss von Mutationen und Veränderung der Expression von Effluxpumpen auf die Fluorchinolonresistenz in Pseudomonas aeruginosa durch eine Kombination von Resequenzierung, quantitativer realtime-PCR und Transkriptom-Sequenzierung (RNA-Seq) anhand einer Sammlung von 172 klinischen Isolaten. Diese umfassenden Daten zeigten die dominierende Rolle bestimmter Mutationen in gyrA und parC, während die Kombination mit weiteren Mutationen (zum Beispiel in gyrB und parE) oder verstärkter Efflux zwar eine additive Wirkung hatte, aber höchstwahrscheinlich nicht zum hohen Resistenzniveau in der Klinik beiträgt. Darüber hinaus nutzen wir die Möglichkeiten hoch-auflösenden Transkriptom-Profilings mittels RNA-Seq um die generelle Gentranskription 37 klinischer K. pneumoniae Isolate unterschiedlichster Herkunft aufzuklären und identifizierten eine große Anzahl von 3346 Genen, die in allen Isolaten exprimiert wurden. Während dieses Kern-Transkriptom weitgehend homogen zwischen Isolaten des gleichen Sequenztypen war, variierte es deutlich zwischen Gruppen unterschiedlicher Sequenztypen. Diese detaillierten Informationen über differentiell exprimierte Gene wurde mit den klinisch relevanten Phänotypen der Biofilmbildung, bakterieller Virulenz und Antibiotikaresistenz verknüpft. Dieses erlaubte die Identifizierung eines Biofilm-spezifischen Genexpressionsprofil in der Gruppe der ST258-Isolate, welche hauptverantwortlich für die Verbreitung der KPC-Carbapenemase sind, als ein Sequenztyp-spezifisches Merkmal. Außerdem ergab die Analyse, dass die Antibiotikaresistenz durch das Auftreten nur weniger, dominanter Resistenzdeterminanten erläutert werden kann

Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen.

Funder: Raymond and Beverly Sackler FoundationFunder: Isaac Newton TrustFunder: Newnham College, University of CambridgeFunder: Medical Research CouncilMutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.This work was primarily funded by an EU Horizon 2020 grant “PIGSs” (727966) and a ZELS BBSRC award “Myanmar Pigs Partnership (MPP)” (BB/L018934/1). G.G.R.M., E.L.M., and L.A.W. were supported by a Sir Henry Dale Fellowship to L.A.W. jointly funded by the Wellcome Trust and the Royal Society (109385/Z/15/Z). N.H. was supported by a Challenge grant from the Royal Society (CH16011) and an Isaac Newton Trust Research Grant [17.24(u)]. G.G.R.M. was also supported by a Research Fellowship at Newnham College. S.B. is supported by the Medical Research Council (MR/V032836/1). PIC North America provided part of the funds for the sequencing of the isolates from the USA. A.J.B. and M.M. were funded by Medical Research Council and Biotechnology and Biological Sciences Research Council studentships respectively, and M.M. was co-funded by the Raymond and Beverly Sackler Fund. We would like to acknowledge Susanna Williamson at the APHA for providing samples, Oscar Cabezón for sampling of the wild boar population in Spain, Mark O’Dea for access to sequence data from Australian isolates, the PIGSs and MPP consortiums for providing samples and helpful discussions, Julian Parkhill and John Welch for helpful discussions, and two anonymous reviewers for their valuable suggestions for improving the manuscript. This research was funded in whole or in part by the Wellcome Trust. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript (AAM) version arising from this submission.info:eu-repo/semantics/publishedVersio

Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella.

Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for non-hypervirulent strains which cause the majority of infections. The insect Galleria mellonella larva is a widely used alternative model organism for bacterial pathogens. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of G. mellonella, to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors specific to ST258, reflecting strain-dependent fitness effects. Our analysis also identified a role for the metalloregulatory protein NfeR (YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae, and provides a framework for using the highly flexible and easily scalable G. mellonella infection model to dissect molecular virulence mechanisms of bacterial pathogens