Next Generation Sequencing (NGS) Data Analysis

Over the last 5–10 years, optical coherence tomography (OCT) and atomic force microscopy (AFM) have been individually applied to monitor the morphological and mechanical properties of various single-species biofilms respectively. This investigation looked to combine OCT and AFM as a multi-scale approach to understand the role sucrose concentration and age play in the morphological and mechanical properties of oral, microcosm biofilms, in-vitro. Biofilms with low (0.1% w/v) and high (5% w/v) sucrose concentrations were grown on hydroxyapatite (HAP) discs from pooled human saliva and incubated for 3 and 5 days. Distinct mesoscale features of biofilms such as regions of low and high extracellular polymeric substances (EPS) were identified through observations made by OCT. Mechanical analysis revealed increasing sucrose concentration decreased Young's modulus and increased cantilever adhesion (p < 0.0001), relative to the biofilm. Increasing age was found to decrease adhesion only (p < 0.0001). This was due to mechanical interactions between the indenter and the biofilm increasing as a function of increased EPS content, due to increasing sucrose. An expected decrease in EPS cantilever contact decreased adhesion due to bacteria proliferation with biofilm age. The application OCT and AFM revealed new structure-property relationships in oral biofilms, unattainable if the techniques were used independently. Oral biofilms are complex microbial communities 1 , embedded in a matrix of extracellular polymeric substances (EPS) 2,3. EPS accounts for up to 90% of a biofilms' total mass 4,5 , consisting of polymers, mainly extracellular DNA (eDNA), polysaccharides, proteins and lipids of bacterial and salivary origin 2,6. Its' role is to provide a protective sheath 7 , encapsulating the multi-species bacterial complex, proliferating into distinct morphotypes, such as bulbous micro-colonies, maintaining its structural integrity 7. Oral biofilm formation is a hierarchical process 8 , consisting of surface attachment by primary bacterial colonizers, such as streptococci 9. This is followed by subsequent attachment of secondary, later and bridging colonizers, such as Actinomyces, Veillonella and fuso-bacterium respectively 8,9. A biofilms hierarchical nature 8 is an important feature to consider when contemplating how to analysis of their morphological and mechanical properties. During formation and proliferation, a biofilms' structural range extends from single molecular EPS constituents to bulbous micro-colonies and finally, fully formed mesoscale surface coverings 4. Applying techniques that span a range of length scales, are capable of in-vitro analysis and preserve biofilm structural and mechanical integrity are vital in the development of structure-property relationships. Traditionally, binding assays such as fluorescence staining have enabled researchers to extensively investigate a biofilms microbiology with increasing detail since the 1980′s 10,11. Since then, characterizing this multi-layered and multi-colonial bacterial community has led to the biological mapping of this complex, hierarchical structure 12. From a structural perspective, common characterization methods include scanning electron microscopy (SEM) 13–15 and confocal laser scanning microscopy (CLSM) 15,16. These techniques only provide basic morphology and quantitative live – dead ratios respectively 16. While these techniques can yield 3D structural information, they can be damaging to the specimen 17 and do not provide any mechanical information. Over the last 5 years, investigators have increasingly utilized atomic force microscopy (AFM) to obtain mechanical information from a variety of single-species biofilms, under several in-vitro conditions 18–20. Through non-destructive indentation, low applied force and known indenter geometry, mechanical properties such as elastic modulus, Young's modulus and adhesion can be obtained from generated force-displacement curves 21 .

Leptospirosis is the most emerging zoonotic disease of epidemic potential caused by pathogenic species of Leptospira. the bacterium invades the host system and causes the disease by interacting with the host proteins. Analyzing these pathogen-host protein interactions (pHpIs) may provide deeper insight into the disease pathogenesis. For this analysis, inter-species as well as intra-species protein interactions networks of Leptospira interrogans and human were constructed and investigated. the topological analyses of these networks showed lesser connectivity in inter-species network than intra-species, indicating the perturbed nature of the inter-species network. Hence, it can be one of the reasons behind the disease development. A total of 35 out of 586 PHPIs were identified as key interactions based on their sub-cellular localization. two outer membrane proteins (GpsA and MetXA) and two periplasmic proteins (Flab and GlyA) participating in pHpIs were found conserved in all pathogenic, intermediate and saprophytic spp. of Leptospira. Furthermore, the bacterial membrane proteins involved in pHpIs were found playing major roles in disruption of the immune systems and metabolic processes within host and thereby causing infectious disease. thus, the present results signify that the membrane proteins participating in such interactions hold potential to serve as effective immunotherapeutic candidates for vaccine development.