Anna Kopenhagen (Brunswick / DE), Michael Steinert (Brunswick / DE), Miriam Katsburg (Berlin / DE), Etienne Aubry (Berlin / DE), Silver Wolf (Berlin / DE), Torsten Semmler (Berlin / DE), Marcus Fulde (Berlin / DE), Simone Bergmann (Brunswick / DE)
Introduction: Streptococcus canis (S. canis) is an animal pathogen and causes zoonotic infections such as septicemia in humans. In previous studies, the M-protein of S. canis has been characterized as a bacterial surface receptor. S. canis is also known to produce a pore-forming toxin targeting the membrane of eukaryotic cells. Apart from these studies, little is known about other factors directly contributing to cell adherence and bacterial invasion into various host niches.
Goals: The aim of this study is to identify and characterize novel adherence factors that play a role in the pathogenesis of systemic infections, particularly in bloodstream infections.
Materials & Methods: For bacterial infection analyses of human primary endothelial cells (EC) under physiological conditions defining bloodstream infections, a microfluidic system was used to apply a defined shear stress. Bacterial infection was quantified and visualized microscopically after differential immunofluorescence staining followed by confocal laser scanning microscopy. A transposon-mediated mutagenesis library of a clinical S. canis isolate was generated, representing a high genome coverage. During the cell culture infection process, a differential selection of adherent and non-adherent transposants enabled an efficient transposon directed insertion-site sequencing (TraDIS).
Results: S. canis bacteria effectively adhere to EC even at high shear force levels during flow infection. A serial selection process of transposants successfully differentiates between a recovered pool of adherent and of non-adherent bacteria. Bioinformatical analyses of sequence pools resulted in identification of several putative factors contributing to bacterial adherence and cell damage.
Summary: The combination of microfluidic infection of endothelial cells with the TraDIS technique proved to be ideally suited for the analysis of S. canis infections in the vascular system and for the search for new potential adherence factors.