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New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis

View Article: PubMed Central - PubMed

ABSTRACT

Aims: In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE.

Material and methods: Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM).

Results: Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified.

Conclusion: The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

No MeSH data available.


Related in: MedlinePlus

Ultrastructural features of infective endocarditis.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 Staphylococcus aureus (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.
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pone.0175569.g002: Ultrastructural features of infective endocarditis.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 Staphylococcus aureus (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.

Mentions: Ultrastructural transverse cuts of native and prosthetic specimens featured bacterial clusters and scattered localization pattern (Fig 2A and 2B). At high resolution, the majority of bacteria were localized within phagocytic immune cells, identified as monocytes and neutrophil granulocytes by size and the characteristic structural features of their nuclei (Fig 2B, 2F–2H). In addition to cells cluttered with numerous viable bacteria and apparently intact morphology, many cells were detected with plasma membranes partly disrupted, indicating cell death (Fig 2B and 2H). Although released bacteria seemed to be phagocytized by intact monocytes, ultrastructural analysis showed evidence of a process of bacterial escape from phagocytic vacuoles into the cytoplasm (Fig 2B), which may represent a mechanism to circumvent host immune response and enable survival and persistence inside infected cells. Using the most recent 3View-FIB-SEM 3D-reconstructions, intracellular and intramural localization of bacteria was observed in infected valve tissue (Fig 2C–2K). Intracellular and intramural localization of infective bacteria may complicate successful culture-based microbial standard diagnostics. Therefore, we examined whether the application of culture-independent, DNA-based metagenome analysis might be an appropriate method to overcome such difficulties.


New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis
Ultrastructural features of infective endocarditis.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 Staphylococcus aureus (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.
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pone.0175569.g002: Ultrastructural features of infective endocarditis.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 Staphylococcus aureus (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.
Mentions: Ultrastructural transverse cuts of native and prosthetic specimens featured bacterial clusters and scattered localization pattern (Fig 2A and 2B). At high resolution, the majority of bacteria were localized within phagocytic immune cells, identified as monocytes and neutrophil granulocytes by size and the characteristic structural features of their nuclei (Fig 2B, 2F–2H). In addition to cells cluttered with numerous viable bacteria and apparently intact morphology, many cells were detected with plasma membranes partly disrupted, indicating cell death (Fig 2B and 2H). Although released bacteria seemed to be phagocytized by intact monocytes, ultrastructural analysis showed evidence of a process of bacterial escape from phagocytic vacuoles into the cytoplasm (Fig 2B), which may represent a mechanism to circumvent host immune response and enable survival and persistence inside infected cells. Using the most recent 3View-FIB-SEM 3D-reconstructions, intracellular and intramural localization of bacteria was observed in infected valve tissue (Fig 2C–2K). Intracellular and intramural localization of infective bacteria may complicate successful culture-based microbial standard diagnostics. Therefore, we examined whether the application of culture-independent, DNA-based metagenome analysis might be an appropriate method to overcome such difficulties.

View Article: PubMed Central - PubMed

ABSTRACT

Aims: In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE.

Material and methods: Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM).

Results: Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified.

Conclusion: The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

No MeSH data available.


Related in: MedlinePlus