Figure 1.
Gross morphological features of patient gallstones employed in this study.
A) Representative image of a group of gallstones retrieved from a single patient (GS 2) demonstrating consistent size and visual appearance. B) Cross section of gallstone (GS 1) and C) Cross section of gallstone (GS 4), revealing heterogeneous appearance throughout a single gallstone. D) The sectioning procedure was attempted on 4 patient gallstones of varying size and visual appearance. Gallstone sample GS 1 was selected for use in biofilm analysis experiments based on the success of the sectioning technique and having characteristics consistent with gallstones containing a high percentage of cholesterol.
Figure 2.
Photomicrographs of sectioned human gallstones with surface associated biofilms of Salmonella Typhimurium.
Gallstone surface (pale purple band) with associated biofilm stained with anti-Salmonella polyclonal antibody (brown) visualized at A) 20×B) 100× magnification and C) Differential interference contrast (DIC) micrographs reveal well-preserved surface-associated bacterial communities. D) Congo Red staining indicates abundance of β-amyloid fibrils and/or acidic polysaccharides.
Figure 3.
IF staining controls demonstrating minimal non-specific fluorescence.
Sectioned patient gallstones with A–C) S. Typhimurium biofilm, D–F) S. Typhi biofilm or G–I) no bacteria control. All sections were stained with relevant primary and secondary antibodies and counterstained with DAPI. Micrographs of control gallstones (G–I) incubated in sterile media, exhibit regions of DAPI staining resembling pre-existing bacterial aggregates within the gallstone.
Figure 4.
Visualization of polysaccharide antigens in DAPI counterstained Salmonella biofilms grown on human gallstones.
Upper panel 3A) Biofilms of S. Typhi (I–IV) and S. Typhimurium (V–VIII) grown in LB without bile. Sections were stained for LPS (red: I, V) Vi-ag (green, III) or O-ag capsule (green, VII). Merged images of LPS and Vi-ag/O-ag capsule (IV, VIII) illustrate the heterogeneous detection of these polysaccharides within the biofilm. Lower panel 3B) Representative image of S. Typhi biofilm section grown in LB with 3% bile, counterstained with DAPI and fluorescently labeled for LPS (green) and Vi-antigen (red). Confocal (I) and DIC (II) images demonstrate the intimate association of the biofilm with gallstone surfaces and the abundance of polysaccharides in the extracellular matrix.
Figure 5.
Flagella and CsgA detection in bacterial microcolonies and whole cell lysates.
A) Although minimally detected in the gallstone biofilm extracellular matrix, confocal scanning micrographs demonstrate that flagella (red) and CsgA (green) are readily detectable in bacterial microcolonies grown in matrix inducing conditions (LBNS, 22°C, 5days). B) Western blot detecting CsgA (≈15 kDa) in whole cell lysates of S. Typhimurium wild type JSG210 or δcsgA reveal CsgA production at 22°C but not at 37°C.
Figure 6.
Visualization of flagella in DAPI counterstained gallstone biofilm sections.
A) Confocal micrograph of S. Typhimurium biofilm demonstrating localization of flagella. Magnified CSLM (B) and DIC/CSLM merge (C) indicate that flagella is restricted to the interface of the biofilm and cholesterol gallstone surface.
Table 1.
Antibodies/stains employed in immunohistochemistry and immunofluorescence slide staining.