In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling

Genomic diversity of nontypeable strains confers phenotypic heterogeneity. Multiple strains of can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH ( ool ox for ), with standardized modules for fluorescent or bioluminescent labeling, adapted to requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.