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Influence of the Intestinal Microbiota Composition on the Individual Susceptibility Towards Enteric Infections in Healthy Individuals and Hematological Patients

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Influence of the Intestinal Microbiota Composition on the Individual Susceptibility Towards Enteric Infections in Healthy Individuals and Hematological Patients Influence of the intestinal microbiota composition on the individual susceptibility towards enteric infections in healthy individuals and hematological patients Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von M. Sc. Lisa Osbelt geb. am 15.08.1992 in Dannenberg (Elbe) Gutachter: Prof. Dr. Thomas Fischer Prof. Dr. Bärbel Stecher eingereicht am: 17.06.2020 verteidigt am: 14.12.2020 Die Wissenschaft nötigt uns, den Glauben an einfache Kausalitäten aufzugeben. ~ Friedrich Nietzsche Summary Summary Variations in the composition of the intestinal microbiota influence the susceptibility towards colonization and infection with multi-drug resistant (MDR) enterobacteria in human and mice. However, the role of specific members and their metabolites contributing to protection against initial colonization remains poorly understood. In this thesis, I aimed to identify factors derived from the microbiota that influence the individual infection susceptibility towards enteric pathogens. For this purpose, human feces samples from patients and healthy volunteers as well as isogenic mouse lines harboring distinct microbiota communities were used to identify potential factors in humans and to test these factors subsequently in relevant mouse models. By inoculation of feces samples of different healthy donors with MDR K. pneumoniae, I observed highly variable degrees of colonization resistance with up to 100,000-fold changes in pathogen growth between protected and susceptible individuals. Microbiome analysis revealed that low pH, high species richness and a high relative abundance of facultative anaerobic bacteria as well as high levels of short-chain fatty acid (SCFA) are key factors for colonization resistance during homeostasis. In line with these findings, I found a lower degree of colonization resistance in stool samples of patients’ cohorts undergoing chemotherapy and after stem cell transplantation to be associated with higher pH, low species richness and low levels of SCFA. Strikingly, differences in colonization susceptibility could be transferred by humanization of germ-free (GF) animals with human stool samples as the animals showed the same response to colonization as their respective donors supporting the predictive value of the in vitro assay to anticipate colonization susceptibility. Similarly, treatment of mouse lines with chemotherapeutic drugs induced massive changes on the microbiome as previously observed in the human cohorts and the severity of mucositis was impacted by microbiota composition. In the second part of the thesis, the influence of natural variation in microbiota compositions on the individual infection susceptibility was explored using the enteric pathogen C. rodentium. Infection of genetically identical mouse lines with variable microbiota compositions led to highly variable colonization even in absence of antibiotic intervention. I found the same differences in formerly GF animals harboring the respective donors microbiota and also in vitro by co-culturing cecal bacteria from resistant and susceptible animals with C. rodentium showing that the phenotype is fully dependent on differences in the microbiota. Further microbiome and metabolome analysis revealed higher abundance of butyrate-producing bacteria and increased levels of butyrate in resistant mice. Supplementation of susceptible animals with butyrate significantly lowered the levels of colonization, highlighting that commensal-derived primary and secondary bacterial metabolites are potent modulators of host’s susceptibility to Enterobacteriaceae. In the last part of the thesis, I assessed the probiotic properties of related commensal bacteria to reduce the susceptibility against MDR K. pneumoniae as initial screening of human feces samples associated the presence of commensal Klebsiella species with elevated protection. Strikingly, addition of commensal K. oxytoca strains significantly reduced the growth of MDR K. pneumoniae strains in in vitro and in vivo assays. By supplementation of susceptible animals with specific K. oxytoca strains, we could significantly lower luminal- and tissue-resident MDR K. pneumoniae and protected K. oxytoca treated animals from systemic spread of the pathogen. Mechanistically, commensal K. oxytoca induced faster restoration of anaerobic SCFA producing bacteria after antibiotic treatment and occupied a similar ecological niche acting as a potential new probiotic strain. Altogether, I identified in this thesis microbiome-derived factors contributing to differences in colonization resistance against enteropathogens in human and mice that may have therapeutic potential. Zusammenfassung Zusammenfassung Die Zusammensetzung der intestinalen Mikrobiota beeinflusst die individuelle Infektionsanfälligkeit mit multiresistenten (MDR) Enterobakterien im Menschen und dem Mausmodell. Allerdings ist die Identität der spezifischen Bakterien und die spezifische Funktion ihrer Stoffwechselprodukte beim Schutz gegen die initiale Besiedelung nicht hinreichend bekannt. In dieser Disputation sollten daher die komplexen Wechselwirkungen zwischen der Mikrobiota, dem Wirt und dem Krankheitserreger näher untersucht werden und diejenigen Faktoren identifiziert werden, welche für eine erhöhte Besiedelungsresistenz verantwortlich sind. Dafür sollten Stuhlproben von Patienten, gesunden Probanden und isogenetischen Mauslinien mit unterschiedlicher Mikrobiota verwendet werden, um mögliche Faktoren in humanen Proben zu identifizieren und daraus abgeleitete Hypothesen dann unter in vivo Bedingungen in Mausmodellen zu testen. Ein Besiedelungsassay mit einem MDR K. pneumoniae Stamm in humanen Stuhlproben gesunder Personen zeigte eine hohe Variabilität in der Besiedelungsfähigkeit mit dem Erreger. Vergleiche des Mikrobioms anfälliger und geschützter Probanden konnten einen niedrigeren pH-Wert, erhöhtes Artenreichtum insgesamt und bei fakultativen Anaerobiern im Speziellen sowie erhöhte Level an kurzkettigen Fettsäuren mit Besiedelungsresistenz gegenüber MDR K. pneumoniae assoziieren. Zusätzlich zeigten Stuhlproben von leukämischen Patienten unter Chemotherapie und nach Stammzelltransplantation eine höhere Besiedelunganfälligkeit mit dem Erreger und waren durch einen erhöhten pH-Wert, niedrige Artenvielfalt und niedrige Level an kurzkettigen Fettsäuren charakterisiert. Diese Unterschiede ließen sich durch die Humanisierung von keimfreien Mäusen in vivo nachstellen und unterstützen somit den prädiktiven Wert der Beobachtungen an Patientenproben. Die Behandlung von zwei Mauslinien in einem von Patienten abgeleiteten Chemotherapie-Modell, induzierte Änderungen in der Mikrobiomzusammensetzung und eine unterschiedlich starke Darmentzündung, welche auf Unterschiede im Mikrobiom zurückzuführen waren. Im zweiten Teil der Arbeit wurden unbehandelte isogenetische Mauslinien mit dem Erreger C. rodentium infiziert und zeigten eine hohe Variabilität in der Infektionsanfälligkeit. Die Unterschiede ließen sich durch Stuhltransplantation in keimfreie Mäuse und Kultivierungsversuche von isoliertem Darmmaterial reproduzieren und somit auf Unterschiede im Mikrobiom zurückführen. Detaillierte Analysen der Mikrobiota von anfälligen und geschützten Tieren identifizierten eine höhere Abundanz von Buttersäure-produzierenden Bakterien und erhöhte Buttersäurelevel als ursächliche Faktoren für den Schutz der resistenten Mauslinie. Supplementierung von anfälligen Tieren mit Buttersäure konnte die Anfälligkeit signifikant reduzieren und unterstreicht die Relevanz von primären und sekundären bakteriellen Metaboliten als potente Modulatoren bei der Wirtsanfälligkeit für Infektionen. Im letzten Teil der Arbeit wurde das probiotische Potenzial von artverwandten, kommensalen Bakterien gegen MDR Erreger näher untersucht. Ein initiales Screening von geschützten Spendern zeigte einen erhöhten Anteil an kommensalen Klebsiellen. Tatsächlich führten K. oxytoca Isolate bei unterschiedlichen in vitro und in vivo Versuchen zu einer signifikanten Reduktion von MDR K. pneumoniae Stämmen. Zusätzlich konnte die Anzahl luminaler und Gewebe-assoziierter MDR Bakterien und die daraus resultierende systemische Verteilung von MDR Bakterien durch die Supplementierung anfälliger Mäuse mit K. oxytoca verhindert werden. Mikrobiota-Analysen zeigten, dass kommensale K. oxytoca Isolate die Wiederherstellung der Besiedelungsresistenz nach Antibiotika-Gabe fördern und die metabolische Nische von K. pneumoniae effektiv blockieren können. Zusammenfassend konnten verschiedene Faktoren und Bakterienstämme in gesunden und medikamentös veränderten Mikrobiomen identifiziert werden, welche zu den Unterschieden in der Infektanfälligkeit zwischen Individuen beitragen könnten. I. Table of contents Summary ...........................................................................................................................................3 Zusammenfassung .............................................................................................................................1 II. List of abbreviations .......................................................................................................................9 III. List of figures .............................................................................................................................. 12 IV. List of tables ..............................................................................................................................
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