organ&#45;wide detection of pathogenic bacteria in infected murine tissue using tomography

Whole&amp;#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

Infections occur in tissues with complex anatomy and compartmentalized physiology. The diverse microenvironments that co-exist in the infected tissue can determine the fates of local pathogen subsets and their contributions to overall disease outcome1,2,3. However, comprehensive 3D mapping of microbial pathogens in cm-sized tissues remains challenging4. Imaging of the brain and other organs is a highly active research field with constantly improving experimental strategies5, but many methods still lack the sub-&#181;m resolution that would be required to identify &#181;m-sized bacterial pathogens confidently. In contrast, serial two-photon (STP) tomography6 enables automated multi-color, deformation-free imaging of entire tissues with sub-&#181;m in-plane resolution, yielding full volumetric datasets. This method combines repeated physical sectioning of the tissue using a vibratome with intermittent two-photon imaging of the emerging block faces with infrared light. STP tomography has been widely used for mapping thin axons in the brain to establish connectivity maps7,8,9,10.
STP tomography also enables 3D mapping of individual microbial pathogen cells (Salmonella, Toxoplasma) in the entire infected tissues11,12 using a tomograph. Second-harmonic generation reveals collagen sheaths around arteries and in fibrous bands such as the trabeculae of spleen, thus providing anatomical context. In vivo injected fluorescent antibodies can be used to stain host cells to reveal interactions between individual pathogen cells and infiltrating immune cells such as neutrophils. Here, the pipeline involving processing of the tissue, imaging, stitching of imaging tiles with illumination correction, stacking of images in three dimensions, and segmentation using machine-learning tools, is described. This pipeline yields 3D positions of individual pathogens cells and microcolonies within their host context. Counting the number of individual cells within microcolonies remains difficult because of resolution limits, but such numbers can be estimated based on the integrated brightness of the microcolony. The pipeline can be readily adapted to other infection models if recombinant GFP- or YFP-expressing pathogens are available.

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Bacteria infections are a major threat to human health. Antimicrobial resistance is increasing, and vaccines are still lacking for several major pathogens. To identify novel avenues for control strategies, we need to know more about how pathogens respond to the host immune system, and to antibiotics in vivo in the infected tissues.Surviving bacteria can cause relapses if antimicrobials fail to eradicate an infection. Using serial tomography, we show that typhoid fever causing salmonella can persist during therapy in a specific part of the spleen. Inflammation is weak in this region, which disrupts the critical synergy between antibiotics and inflammation for effective clearance.Bacteria are micrometer-sized objects. It is difficult to localize such small objects in their tissue context. Serial two-photon tomography can detect one single bacterium in centimeter-sized organs, and determine its replication rate.We can also detect immune cells, such as neutrophils, and determine their spatial relationship with the bacteria. We study the activities of bacteriostatic antibiotics. These antibiotics can cure infections, although they can only inhibit bacterial growth without actually killing the pathogen.We also determine the spreading of bacterial mutants that lack certain virulence factors, and the impact of immune defects on infection and treatment.

Organ&#45;Wide Detection of Pathogenic Bacteria in Infected Murine Tissue Using Tomography

organwide-detection-pathogenic-bacteria-infected-murine-tissue-using

Research

JoVE Journal

Immunology and Infection

265 Aufrufe.  University of Basel. Nehmen Sie zunächst die aufgetauten Proben von Salmonellen-infiziertem Mausgewebe mit einer Pinzette auf. Setze es schnell in den Boden einer Kunststoffform ein, die flüssige Agarose enthält. Sobald die Polymerisation abgeschlossen ist, öffnen Sie die Kunststoffform mit einem Skalpell von den Ecken aus.Nachdem Sie eine Klinge auf ein Mikrotom gelegt haben, übertragen Sie die Probe mit behandschuhten Fingern in eine Probenbox. Positionieren Sie die Probe so unter dem Mikrotom, dass...

Serie: Organweiter Nachweis pathogener Bakterien in infiziertem murinem Gewebe mittels Tomographie

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...