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

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

Diversity of antimicrobial resistance genes in Bacteroides and Parabacteroides strains isolated in Germany

Objectives: Bacteroides spp. are normal constituents of the human intestinal microflora, but they are also able to cause severe diseases. The aim of this study was to determine the diversity of antibiotic resistance genes found in phenotypically resistant Bacteroides and Parabacteroides strains. Methods: A total of 71 phenotypically resistant Bacteroides spp. from human clinical specimens were screened for the antibiotic resistance genes cfiA, tetQ, tetM, tet36, cepA, cfxA, nim, ermG, ermF, bexA, blaVIM, blaNDM, blaKPC, blaOXA-48 and blaGES. The presence of these genes was compared with phenotypic resistance to ampicillin/sulbactam, cefoxitin, ceftolozane/tazobactam, piperacillin/tazobactam, imipenem, meropenem, meropenem/vaborbactam, clindamycin, moxifloxacin, tigecycline, eravacycline and metronidazole. Results: tetQ was the most frequently detected gene, followed by cfiA, ermF, cfxA, ermG, cepA, nim and bexA. None of the strains were positive for tetM, tet36, blaVIM, blaNDM, blaKPC, blaOXA-48 or blaGES. Resistance to the tested β-lactams was mainly linked to the presence of the cfiA gene. Clindamycin resistance correlated with the presence of the genes ermG and ermF. The bexA gene was found in six strains, but only two of them were resistant to moxifloxacin. Tigecycline and eravacycline showed good activities despite the frequent occurrence of tetQ. The nim gene was detected in six isolates, five of which were resistant to metronidazole. Conclusion: The findings of our study support the general belief that antimicrobial resistance within Bacteroides should be taken into consideration. This underlines the necessity of reliable routine antimicrobial susceptibility test methods for anaerobic bacteria and the implementation of antimicrobial surveillance programmes worldwide

Two Distinct Clinical Courses of Human Cowpox, Germany, 2015

Here we present two cases of human infection with cowpox virus with distinct clinical courses. A series of clinical photographs documents lesion progression over time. In the first case—an unvaccinated young veterinary assistant—a pustule was treated locally with cortisone. The lesion turned into a large ulcer accompanied by severe lymphadenitis. Based on her close contact to a sick stray cat, infection with cowpox virus was assumed and confirmed by virus isolation, PCR, and serology. The clinical course took up to eleven months until healing of the wound was complete. Transmission of cowpox virus from the cat was likely because a skin swab was PCR-positive and the cat had a high titer of anti-orthopoxvirus antibodies. In contrast, a rather mild clinical course of cowpox was confirmed in a 49-year-old male farmer vaccinated against smallpox. Only a small eschar developed, and wound closure was complete after 6 weeks

Phenotype.

<p>Phenotype.</p

Ultrastructural features of infective endocarditis.

<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

Surface analysis of infected valves.

<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

Clinical characteristics.

<p>Clinical characteristics.</p

Metagenome analysis of 8 valves obtained from patients with acute infective endocarditis.

<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

Species-specific qPCR primers.

<p>Species-specific qPCR primers.</p