![]() We extended this approach recently to the respiratory tract of mice, which displays 10 4 to 10 5-fold less bacterial content than the intestine ( Yildiz et al., 2020). Previous studies have used fluorescent based approaches to determine spatial organization of commensal microbiota in the intestine and lung ( Yun et al., 2014 Tropini et al., 2017 Welch et al., 2017) using phylum specific probes. As a complement to these techniques we present here a protocol for RNA-based in situ hybridization to detect commensal or pathogenic bacteria in a sensitive fashion in context of the host organ. Culturomics, that is the isolation and amplification of bacteria by using multiple growth conditions, allows further detailed characterization of bacterial species which were previously considered unculturable ( Sibley et al., 2011 Browne et al., 2016). Complementary analysis of shotgun DNA sequencing allows deeper insight into the physiological state of a given group of living bacteria under changing environmental conditions ( Westermann et al., 2016 Griesenauer et al., 2019 Becattini et al., 2021) since bacterial RNA is rather short-lived. It does, however, not distinguish between living and dead bacteria and resolution of spatial organization of the identified bacteria within a given niche is limited to the choice of organ section used for DNA isolation. This highly sensitive and valuable technique allows quasi unbiased quantification and identification of bacteria from DNA of swabs, lavages or total tissue samples ( Human Microbiome Project Consortium, 2012). We here provide a detailed step-by-step protocol describing the detection of commensal lung bacteria in respiratory tissue.Ĭharacterization of bacterial communities in various ecological niches of human or animal bodies largely relies today on 16S rRNA gene specific next generation sequencing. This technique allows species-specific detection of living bacteria using RNAScope TM technology, while preserving the natural environment of the organ. #Leica acquire download 3.3.5 freeWe recently applied highly sensitive in situ RNA hybridization to detection of commensal bacteria in low abundance respiratory tract samples of mice housed under specific pathogen free conditions. ![]() In situ hybridization (ISH) is a complementary technique to sequencing and culturing to visualize the presence of individual bacterial operational taxonomic unit (OTUs) in context of the colonized organ. Spatial organization of microbiota within a given host environment is critical to the physiological or pathological phenotypes provoked by commensal microbiota. Culturomics, next allowed the isolation and characterization of commensal bacteria in the lab and the establishment of artificial communities of bacteria, which were eventually reintroduced in model organisms. ![]() 16S rRNA gene-specific next generation sequencing from DNA of total organs, swabs or lavages has revolutionized the characterization of bacterial communities in virtually every ecological niche of the body. Microbiology and Molecular Medicine Department, University of Geneva, Geneva, SwitzerlandĬommensal microbes are an integral component of mammalian physiology. ![]()
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