Fig 1.
(left) Box-Cox transformed graph of S. aureus bacterial count with glue, glass, plastic microspheres, and cholesterol crystals (p<0.0001). (right) Box-Cox transformed graph of P. aeruginosa bacterial count demonstrating highest count with cholesterol crystals (p<0.0001). P-values were obtained from analysis of variance and adjusted for multiplicity by the Bonferroni method.
Fig 2.
Log-transformed relative response of bacterial colony count with cholesterol crystals and control at 3, 4, 5 and 6 hours of incubation. Left panel, S. aureus; Right panel P. aeruginosa. P-values were obtained from t-tests in repeated measures analyses. For both studies the time effect was significant (P<0.0001). Group effect: P = 0.514 for S. aureus, and P = 0.032 for P. aeruginosa.
Fig 3.
Ex vivo bacterial growth in rabbit arteries.
Bacterial growth graph with bacterial counts in normal control and atherosclerotic rabbit arteries using Box-Cox transformation. P- value was obtained from t-test in a mixed effects model with group and hour as factors.
Fig 4.
Ex vivo bacterial growth in human arterial plaques.
(a) Graphic of bacterial counts of normal control and atherosclerotic human arterial plaques using Box-Cox transformation. P-value was obtained from a two-sample t-test. (b) The arteries embedded in a washer ring exposing the intimal surface and covering the back of the washer. (c) Bacterial count in human normal carotid tissue comparted to (d) bacterial count in atherosclerotic carotid tissue.
Fig 5.
Scanning electron micrograph of bacteria engaging cholesterol crystals.
In vitro S. aureus incubated with cholesterol crystals (top): (a) Cholesterol crystals incubated in broth without bacteria as control (b) Staphylococcus bacteria engulfing and degrading crystal after 2.5 hours incubation. (c) Staphylococcus bacteria noted engaging and punctating the crystal surface at 1 hour incubation. In vitro P. aeruginosa incubated with cholesterol crystals (bottom): (d) Pseudomonas bacteria seen engulfing and eroding crystals forming wedges into the crystal body (e) higher magnification demonstrates the detail of the bacterial and the crystal erosion with loss of crystal sharp edges; (f) another example of eroding crystal with bacteria above and around the crystal.
Fig 6.
Micrographs of ex vivo S. aureus bacteria on human plaques.
(a) Scanning electron micrograph of normal carotid artery with few bacteria attached to intimal surface; (b) atherosclerotic carotid plaque with many bacteria and macrophages (white arrow) attached to cholesterol crystals; (c) bacteria attached and degrading crystal in human plaque; (d) Scanning electron micrograph demonstrating extensive sheets of cholesterol crystals covering the plaque surface with absence of any fibrous cap at this advanced stage of plaque disruption; (e) Example of an intact fibrous cap (arrows) from a human carotid plaque; (f) Light microscopic image of a completely ruptured and eroded plaque with cholesterol crystals filling the base and a remnant insertion site of a fibrous cap (arrow).
Fig 7.
Fluorescence microscopy of bacteria with adherent cholesterol.
At same light intensity confocal microscopy of S. aureus incubated with (a, b) and without (c, d) cholesterol crystals demonstrate binding of BODIPY stain to the bacteria exposed to cholesterol crystals but not in the bacteria that were not exposed to crystals.