A High-Resolution Combined Scanning Laser- and Widefield Polarizing Microscope for Imaging at Temperatures from 4 K to 300 K

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as for example birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a $^4$He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via Faraday effect or imaging of structural features, such as twin-walls in tetragonal SrTiO$_3$. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism and superconductivity.Comment: 14 pages, 11 figures. The following article has been accepted by Review of Scientific Instruments. After it is published, it will be found at http://aip.scitation.org/journal/rs

Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin

Adhesion of bacterial cells to fibronectin (FN) is thought to be a pivotal step in the pathogenesis of invasive infectious diseases. Viridans group streptococci such as Streptococcus gordonii are considered commensal members of the oral microflora, but are important pathogens in infective endocarditis. S. gordonii expresses a battery of cell-surface adhesins that act alone or in concert to bind host receptors. Here, we employed molecular genetic approaches to determine the relative contributions of five known S. gordonii surface proteins to adherence to human FN. Binding levels to FN by isogenic mutants lacking Hsa glycoprotein were reduced by 70 %, while mutants lacking CshA and CshB fibrillar proteins showed approximately 30 % reduced binding. By contrast, disruption of antigen I/II adhesin genes sspA and sspB in a wild-type background did not result in reduced FN binding. Enzymic removal of sialic acids from FN led to reduced S. gordonii DL1 adhesion (>50 %), but did not affect binding by the hsa mutant, indicating that Hsa interacts with sialic acid moieties on FN. Conversely, desialylation of FN did not affect adherence levels of Lactococcus lactis cells expressing SspA or SspB polypeptides. Complementation of the hsa mutant partially restored adhesion to FN. A model is proposed for FN binding by S. gordonii in which Hsa and CshA/CshB are primary adhesins, and SspA or SspB play secondary roles. Understanding the basis of oral streptococcal interactions with FN will provide a foundation for development of new strategies to control infective endocarditis

Understanding the Matrix:The Role of Extracellular DNA in Oral Biofilms

Dental plaque is the key etiological agent in caries formation and the development of the prevalent chronic oral inflammatory disease, periodontitis. The dental plaque biofilm comprises a diverse range of microbial species encased within a rich extracellular matrix, of which extracellular DNA (eDNA) has been identified as an important component. The molecular mechanisms of eDNA release and the structure of eDNA have yet to be fully characterized. Nonetheless, key functions that have been proposed for eDNA include maintaining biofilm structural integrity, initiating adhesion to dental surfaces, acting as a nutrient source, and facilitating horizontal gene transfer. Thus, eDNA is a potential therapeutic target for the management of oral disease–associated biofilm. This review aims to summarize advances in the understanding of the mechanisms of eDNA release from oral microorganisms and in the methods of eDNA detection and quantification within oral biofilms

Effects of glucose and lactate on <em>Streptococcus </em><em>mutans </em>abundance in a novel multispecies oral biofilm model

The oral microbiome plays an important role in protecting oral health. Here, we established a controlled mixed-species in vitro biofilm model and used it to assess the impact of glucose and lactate on the ability of Streptococcus mutans, an acidogenic and aciduric species, to compete with commensal oral bacteria. A chemically defined medium was developed that supported the growth of S. mutans and four common early colonizers of dental plaque: Streptococcus gordonii, Actinomyces oris, Neisseria subflava, and Veillonella parvula. Biofilms containing the early colonizers were developed in a continuous flow bioreactor, exposed to S. mutans, and incubated for up to 7 days. The abundance of bacteria was estimated by quantitative polymerase chain reaction (qPCR). At high glucose and high lactate, the pH in bulk fluid rapidly decreased to approximately 5.2, and S. mutans outgrew other species in biofilms. In low glucose and high lactate, the pH remained above 5.5, and V. parvula was the most abundant species in biofilms. By contrast, in low glucose and low lactate, the pH remained above 6.0 throughout the experiment, and the microbial community in biofilms was relatively balanced. Fluorescence in situ hybridization confirmed that all species were present in the biofilm and the majority of cells were viable using live/dead staining. These data demonstrate that carbon source concentration is critical for microbial homeostasis in model oral biofilms. Furthermore, we established an experimental system that can support the development of computational models to predict transitions to microbial dysbiosis based on metabolic interactions. 38376204</strong

Simbiotics: a multi-scale integrative platform for 3D modeling of bacterial populations

Simbiotics is a spatially explicit multiscale modeling platform for the design, simulation and analysis of bacterial populations. Systems ranging from planktonic cells and colonies, to biofilm formation and development may be modeled. Representation of biological systems in Simbiotics is flexible, and user-defined processes may be in a variety of forms depending on desired model abstraction. Simbiotics provides a library of modules such as cell geometries, physical force dynamics, genetic circuits, metabolic pathways, chemical diffusion and cell interactions. Model defined processes are integrated and scheduled for parallel multithread and multi-CPU execution. A virtual lab provides the modeler with analysis modules and some simulated lab equipment, enabling automation of sample interaction and data collection. An extendable and modular framework allows for the platform to be updated as novel models of bacteria are developed, coupled with an intuitive user interface to allow for model definitions with minimal programming experience. Simbiotics can integrate existing standards such as SBML, and process microscopy images to initialize the 3D spatial configuration of bacteria consortia. Two case studies, used to illustrate the platform flexibility, focus on the physical properties of the biosystems modeled. These pilot case studies demonstrate Simbiotics versatility in modeling and analysis of natural systems and as a CAD tool for synthetic biology

Genome characterization and taxonomy of <em>Actinomyces acetigenes</em> sp. nov., and <em>Actinomyces stomatis</em> sp. nov., previously isolated from the human oral cavity

\ua9 2023, The Author(s).Background: Actinomyces strains are commonly found as part of the normal microflora on human tissue surfaces, including the oropharynx, gastrointestinal tract, and female genital tract. Understanding the diversity and characterization of Actinomyces species is crucial for human health, as they play an important role in dental plaque formation and biofilm-related infections. Two Actinomyces strains ATCC 49340 T and ATCC 51655 T have been utilized in various studies, but their accurate species classification and description remain unresolved. Results: To investigate the genomic properties and taxonomic status of these strains, we employed both 16S rRNA Sanger sequencing and whole-genome sequencing using the Illumina HiSeq X Ten platform with PE151 (paired-end) sequencing. Our analyses revealed that the draft genome of Actinomyces acetigenes ATCC 49340 T was 3.27 Mbp with a 68.0% GC content, and Actinomyces stomatis ATCC 51655 T has a genome size of 3.08 Mbp with a 68.1% GC content. Multi-locus (atpA , rpoB, pgi , metG , gltA , gyrA, and core genome SNPs) sequence analysis supported the phylogenetic placement of strains ATCC 51655 T and ATCC 49340 T as independent lineages. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) analyses indicated that both strains represented novel Actinomyces species, with values below the threshold for species demarcation (70% dDDH, 95% ANI and AAI). Pangenome analysis identified 5,731 gene clusters with strains ATCC 49340 T and ATCC 51655 T possessing 1,515 and 1,518 unique gene clusters, respectively. Additionally, genomic islands (GIs) prediction uncovered 24 putative GIs in strain ATCC 49340 T and 16 in strain ATCC 51655 T, contributing to their genetic diversity and potential adaptive capabilities. Pathogenicity analysis highlighted the potential human pathogenicity risk associated with both strains, with several virulence-associated factors identified. CRISPR-Cas analysis exposed the presence of CRISPR and Cas genes in both strains, indicating these strains might evolve a robust defense mechanism against them. Conclusion: This study supports the classification of strains ATCC 49340 T and ATCC 51655 T as novel species within the Actinomyces, in which the name Actinomyces acetigenes sp. nov. (type strain ATCC 49340 T = VPI D163E-3 T = CCUG 34286 T = CCUG 35339 T) and Actinomyces stomatis sp. nov. (type strain ATCC 51655 T = PK606T = CCUG 33930 T) are proposed

Hierarchical rose-petal surfaces delay the early-stage bacterial biofilm growth

A variety of natural surfaces exhibit antibacterial properties; as a result significant efforts in the past decade have been dedicated towards fabrication of biomimetic surfaces that can help control biofilm growth. Examples of such surfaces include rose petals, which possess hierarchical structures like the micro-papillae measuring tens of microns and nano-folds that range in the size of 700 ±100 nm. We duplicated the natural structures on rose-petal surfaces via a simple UV-curable nanocasting technique, and tested the efficacy of these artificial surfaces in preventing biofilm growth using clinically relevant bacteria strains. The rose-petal structured surfaces exhibited hydrophobicity (contact angle~130.8º ±4.3º) and high contact angle hysteresis (~91.0° ±4.9°). Water droplets on rose-petal replicas evaporated following the constant contact line mode, indicating the likely coexistence of both Cassie and Wenzel states (Cassie-Baxter impregnating wetting state). Fluorescent microscopy and image analysis revealed the significantly lower attachment of Staphylococcus epidermidis (86.1± 6.2% less) and Pseudomonas aeruginosa (85.9 ±3.2% less) on the rose-petal structured surfaces, compared with flat surfaces over a period of 2 hours. Extensive biofilm matrix was observed in biofilms formed by both species on flat surfaces after prolonged growth (several days), but was less apparent on rose-petal biomimetic surfaces. In addition, the biomass of S. epidermidis (63.2 ±9.4% less) and P. aeruginosa (76.0 ±10.0% less) biofilms were significantly reduced on the rose-petal structured surfaces, in comparison to the flat surfaces. By comparing P. aeruginosa growth on representative unitary nano-pillars, we demonstrated that hierarchical structures are more effective in delaying biofilm growth. The mechanisms are two-fold: 1) the nano-folds across the hemispherical micro-papillae restrict initial attachment of bacterial cells and delay the direct contacts of cells via cell alignment, and 2) the hemispherical micro-papillae arrays isolate bacterial clusters and inhibit the formation of a fibrous network. The hierarchical features on rose petal surfaces may be useful for developing strategies to control biofilm formation in medical and industrial contexts