6 research outputs found
Surface analysis of infected valves.
No full text<p>For surface analysis using scanning electron microscopy (SEM), infected native (A-C; patient 3) and prosthetic (D-F; patient 7) heart valve tissues were cut into small pieces and fixed. After pretreatment and exposure to osmium tetroxide, tissues were dehydrated and mounted on standard SEM stubs with carbon tape. The cured samples were finally sputter-coated with a platinum layer and evaluated with a scanning electron microscope. (A-C) SEM images of the surface of an infected native valve. (A) Overview. (B) Epithelial cell boundaries are discernable. Inset: note that few bacteria are attached to the smooth surface and that these seem to be often damaged (arrow). (C) At higher magnification scattered bacteria showing apparently intact morphology (arrow) can also be found. (D-F) Ultrastructure images of infected biological prosthetic valve. (D) Overview. (E) The surface is characterized by deep holes and cracks where microbes may be concealed; the surface appears rough. Inset: note that the few bacteria attached to the outside often seem to be damaged (arrow). (F) At higher magnification a number of apparently intact bacteria (arrows) showing different morphologies can be found; note the fibrous structure of the substrate, providing ideal adhesion sites.</p
Ultrastructural features of infective endocarditis.
No full text<p>Using focused ion beam scanning electron microscopy (FIB-SEM), 3D reconstructions of infected phagocytic cells were generated. (A) Transversal cut through a native infected valve (patient 3). Cells cluttered with numerous viable bacteria showed evidence of a process of intracellular bacterial survival. Nuclei of human cells are indicated by n (black or white). Note some cells carrying a large number of bacteria that nearly fill the cytoplasmic space. Survival of these cells would be unlikely. Inset: a further example from another area. (B) Transverse cut through a biological prosthesis infected with <i>Staphylococcus aureus</i> (patient 5). Monocytes cluttered with numerous viable bacteria show evidence of a process in which bacteria escape from a phagocytic vacuole into the cytoplasm; the plasma membrane of some cells is partially disrupted (white arrows), indicating cell death and release of bacterial cargo. Nuclei of monocytes showing intact plasma membrane are indicated by n (black), and the nucleus of a heavily damaged immune cell is indicated by n (white); arrowheads denote phagosomes with intact (black) and disrupted membranes (white); white arrows indicate disrupted plasma membranes. Insets: further examples from another area. (C-E, patient 3) 3View-SEM 3D-reconstruction of a 11.2 μm x 13.3 μm x 3.2 μm (xyz) block at 10 nm x 10 nm x 50 nm (xyz) resolution showing two cells cluttered with numerous viable bacteria (yellow); yellow = bacteria, green = cell membrane. (F-H, patient 5) 3View-SEM 3D-reconstruction of a 24.6 μm x 19.2 μm x 5.7 μm (xyz) block at 10 nm x 10 nm x 50 nm (xyz) resolution showing a monocyte cluttered with numerous viable bacteria (yellow); note that the plasma membrane is partially disrupted (arrows), indicating death of the cell. In addition, several bacteria are located within the extracellular space (red); yellow = bacteria, green = cell membrane, blue = nucleus. (I-K, patient 5) High resolution (2.5 nm x 2.5 nm x 5 nm (xyz)); (F) the nucleus of the cell is highly fragmented as is typical for neutrophilic granulocytes.</p
Metagenome analysis of 8 valves obtained from patients with acute infective endocarditis.
No full text<p>After surgical valve replacement, total DNA was prepared from resected infected heart valve tissue of IE patients, with microbial DNA being additionally enriched. Subsequently, two hypervariable regions of bacterial 16S rDNA were amplified, Illumina paired-end sequencing libraries were constructed, and next-generation sequencing was carried out using Illumina Miseq technology. The quality of reads obtained was controlled using fastqc and adapters, and low-quality bases were trimmed using Trimmomatic. Remaining reads were aligned to NCBI database 16SMicrobial using software blastn in the megablast mode. Local alignment was repeated with minimum identity filters at 99%. Blast results where then analyzed with MEGAN5. Taxonomic classification was summarized at the species level and further analyzed using statistics software R. Species and genus results were standardized as a proportion of all aligned reads, and cut-off levels of at least 0.5% of species/genus-specific reads were applied. The legend shown in panel A also applies to panels B and C. Bacterial species and genera detected are indicated by colored triangles within a white square. The location of the triangles illustrate the underlying analysis used: <b>top</b>—16S rDNA V1-V3 region was amplified and sequenced from total DNA; <b>left</b>—16S rDNA V3-V5 region was amplified and sequenced from total DNA; <b>right</b>—16S rDNA V3-V5 region was amplified and sequenced from microbial enriched DNA; <b>bottom</b>—16S rDNA V1-V3 region was amplified and sequenced microbial enriched DNA. The abundance of genus- or species-specific DNA determined from sequence analysis is indicated in each triangle using a color scale starting from blue (0.5% proportion) to red (100% proportion). Positive culture-dependent identification of bacterial species is highlighted by red squared frames and used in panel C. Matched antibiosis (initial and extended) given prior to surgical intervention is denoted by a white circle. Bacterial genera (B) and species (C) identified in each of the individual patients (numbered) suffering from acute IE are shown. The red-highlighted square frames denote species identified in culture-based microbial analysis. (D) It is shown whether individual bacterial species identified are known and have been previously described as an IE-related pathogen (y = yes) or not (Ø = no). TV = tricuspid valve; MV = mitral valve, AV = aortic valve.</p
