Unique miRomics Expression Profiles in Tannerella forsythia-Infected Mandibles during Periodontitis Using Machine Learning.

T. forsythia is a subgingival periodontal bacterium constituting the subgingival pathogenic polymicrobial milieu during periodontitis (PD). miRNAs play a pivotal role in maintaining periodontal tissue homeostasis at the transcriptional, post-transcriptional, and epigenetic levels. The aim of this study was to characterize the global microRNAs (miRNA, miR) expression kinetics in 8- and 16-week-old T. forsythia-infected C57BL/6J mouse mandibles and to identify the miRNA bacterial biomarkers of disease process at specific time points. We examined the differential expression (DE) of miRNAs in mouse mandibles (n = 10) using high-throughput NanoString nCounter® miRNA expression panels, which provided significant advantages over specific candidate miRNA or pathway analyses. All the T. forsythia-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, along with a significant increase in alveolar bone resorption (ABR) (p < 0.0001). We performed a NanoString analysis of specific miRNA signatures, miRNA target pathways, and gene network analysis. A total of 115 miRNAs were DE in the mandible tissue during 8 and 16 weeks The T. forsythia infection, compared with sham infection, and the majority (99) of DE miRNAs were downregulated. nCounter miRNA expression kinetics identified 67 downregulated miRNAs (e.g., miR-375, miR-200c, miR-200b, miR-34b-5p, miR-141) during an 8-week infection, whereas 16 upregulated miRNAs (e.g., miR-1902, miR-let-7c, miR-146a) and 32 downregulated miRNAs (e.g., miR-2135, miR-720, miR-376c) were identified during a 16-week infection. Two miRNAs, miR-375 and miR-200c, were highly downregulated with >twofold change during an 8-week infection. Six miRNAs in the 8-week infection (miR-200b, miR-141, miR-205, miR-423-3p, miR-141-3p, miR-34a-5p) and two miRNAs in the 16-week infection (miR-27a-3p, miR-15a-5p) that were downregulated have also been reported in the gingival tissue and saliva of periodontitis patients. This preclinical in vivo study identified T. forsythia-specific miRNAs (miR-let-7c, miR-210, miR-146a, miR-423-5p, miR-24, miR-218, miR-26b, miR-23a-3p) and these miRs have also been reported in the gingival tissues and saliva of periodontitis patients. Further, several DE miRNAs that are significantly upregulated (e.g., miR-101b, miR-218, miR-127, miR-24) are also associated with many systemic diseases such as atherosclerosis, Alzheimer's disease, rheumatoid arthritis, osteoarthritis, diabetes, obesity, and several cancers. In addition to DE analysis, we utilized the XGBoost (eXtreme Gradient boost) and Random Forest machine learning (ML) algorithms to assess the impact that the number of miRNA copies has on predicting whether a mouse is infected. XGBoost found that miR-339-5p was most predictive for mice infection at 16 weeks. miR-592-5p was most predictive for mice infection at 8 weeks and also when the 8-week and 16-week results were grouped together. Random Forest predicted miR-592 as most predictive at 8 weeks as well as the combined 8-week and 16-week results, but miR-423-5p was most predictive at 16 weeks. In conclusion, the expression levels of miR-375 and miR-200c family differed significantly during disease process, and these miRNAs establishes a link between T. forsythia and development of periodontitis genesis, offering new insights regarding the pathobiology of this bacterium.

Oral Spirochete Treponema denticola Intraoral Infection Reveals Unique miR-133a, miR-486, miR-126-3p, miR-126-5p miRNA Expression Kinetics during Periodontitis.

miRNAs are major regulators of eukaryotic gene expression and host immunity, and play an important role in the inflammation-mediated pathways in periodontal disease (PD) pathogenesis. Expanding our previous observation with the global miRNA profiling using partial human mouth microbes, and lack of in vivo studies involving oral spirochete Treponema denticola-induced miRNAs, this study was designed to delineate the global miRNA expression kinetics during progression of periodontitis in mice infected with T. denticola by using NanoString nCounter® miRNA panels. All of the T. denticola-infected male and female mice at 8 and 16 weeks demonstrated bacterial colonization (100%) on the gingival surface, and an increase in alveolar bone resorption (p < 0.0001). A total of 70 miRNAs with at least 1.0-fold differential expression/regulation (DE) (26 upregulated and 44 downregulated) were identified. nCounter miRNA expression profiling identified 13 upregulated miRNAs (e.g., miR-133a, miR-378) and 25 downregulated miRNAs (e.g., miR-375, miR-34b-5p) in T. denticola-infected mouse mandibles during 8 weeks of infection, whereas 13 upregulated miRNAs (e.g., miR-486, miR-126-5p) and 19 downregulated miRNAs (miR-2135, miR-142-3p) were observed during 16 weeks of infection. One miRNA (miR-126-5p) showed significant difference between 8 and 16 weeks of infection. Interestingly, miR-126-5p has been presented as a potential biomarker in patients with periodontitis and coronary artery disease. Among the upregulated miRNAs, miR-486, miR-126-3p, miR-126-5p, miR-378a-3p, miR-22-3p, miR-151a-3p, miR-423-5p, and miR-221 were reported in human gingival plaques and saliva samples from periodontitis and with diabetes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed various functional pathways of DE miRNAs, such as bacterial invasion of epithelial cells, Ras signaling, Fc gamma R-mediated phagocytosis, osteoclast differentiation, adherens signaling, and ubiquitin mediated proteolysis. This is the first study of DE miRNAs in mouse mandibles at different time-points of T. denticola infection; the combination of three specific miRNAs, miR-486, miR-126-3p, and miR-126-5p, may serve as an invasive biomarker of T. denticola in PD. These miRNAs may have a significant role in PD pathogenesis, and this research establishes a link between miRNA, periodontitis, and systemic diseases.

Specific microRNA Signature Kinetics in Porphyromonas gingivalis-Induced Periodontitis.

Porphyromonas gingivalis is one of the major bacteria constituting the subgingival pathogenic polymicrobial milieu during periodontitis. Our objective is to determine the global microRNA (miRNA, miR) expression kinetics in 8- and 16-weeks duration of P. gingivalis infection in C57BL/6J mice and to identify the miRNA signatures at specific time-points in mice. We evaluated differential expression (DE) miRNAs in mandibles (n = 10) using high-throughput NanoString nCounter® miRNA expression panels. The bacterial colonization, alveolar bone resorption (ABR), serum immunoglobulin G (IgG) antibodies, and bacterial dissemination were confirmed. In addition, all the infected mice showed bacterial colonization on the gingival surface, significant increases in ABR (p < 0.0001), and specific IgG antibody responses (p < 0.05-0.001). The miRNA profiling showed 26 upregulated miRNAs (e.g., miR-804, miR-690) and 14 downregulated miRNAs (e.g., miR-1902, miR-1937a) during an 8-weeks infection, whereas 7 upregulated miRNAs (e.g., miR-145, miR-195) and one downregulated miR-302b were identified during a 16-weeks infection. Both miR-103 and miR-30d were commonly upregulated at both time-points, and all the DE miRNAs were unique to the specific time-points. However, miR-31, miR-125b, miR-15a, and miR-195 observed in P. gingivalis-infected mouse mandibles were also identified in the gingival tissues of periodontitis patients. None of the previously identified miRNAs reported in in vitro studies using cell lines (periodontal ligament cells, gingival epithelial cells, human leukemia monocytic cell line (THP-1), and B cells) exposed to P. gingivalis lipopolysaccharide were observed in the in vivo study. Most of the pathways (endocytosis, bacterial invasion, and FcR-mediated phagocytosis) targeted by the DE miRNAs were linked with bacterial pathogen recognition and clearance. Further, eighteen miRNAs were closely associated with the bacterial invasion of epithelial cells. This study highlights the altered expression of miRNA in gingiva, and their expression depends on the time-points of infection. This is the first in vivo study that identified specific signature miRNAs (miR-103 and miR-30d) in P. gingivalis invasion of epithelial cells, establishes a link between miRNA and development of periodontitis and helping to better understand the pathobiology of periodontitis.

Global Noncoding microRNA Profiling in Mice Infected with Partial Human Mouth Microbes (PAHMM) Using an Ecological Time-Sequential Polybacterial Periodontal Infection (ETSPPI) Model Reveals Sex-Specific Differential microRNA Expression.

Periodontitis (PD) is a polymicrobial dysbiotic immuno-inflammatory disease. It is more prevalent in males and has poorly understood pathogenic molecular mechanisms. Our primary objective was to characterize alterations in sex-specific microRNA (miRNA, miR) after periodontal bacterial infection. Using partial human mouth microbes (PAHMM) (Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in an ecological time-sequential polybacterial periodontal infection (ETSPPI) mouse model, we evaluated differential mandibular miRNA profiles by using high-throughput Nanostring nCounter® miRNA expression panels. All PAHMM mice showed bacterial colonization (100%) in the gingival surface, an increase in alveolar bone resorption (p < 0.0001), and the induction of a specific immunoglobin G antibody immune response (p < 0.001). Sex-specific differences in distal organ bacterial dissemination were observed in the heart (82% male vs. 28% female) and lungs (2% male vs. 68% female). Moreover, sex-specific differential expression (DE) of miRNA was identified in PAHMM mice. Out of 378 differentially expressed miRNAs, we identified seven miRNAs (miR-9, miR-148a, miR-669a, miR-199a-3p, miR-1274a, miR-377, and miR-690) in both sexes that may be implicated in the pathogenesis of periodontitis. A strong relationship was found between male-specific miR-377 upregulation and bacterial dissemination to the heart. This study demonstrates sex-specific differences in bacterial dissemination and in miRNA differential expression. A novel PAHMM mouse and ETSPPI model that replicates human pathobiology can be used to identify miRNA biomarkers in periodontitis.

Intracerebral but Not Peripheral Infection of Live Porphyromonas gingivalis Exacerbates Alzheimer's Disease Like Amyloid Pathology in APP-TgCRND8 Mice.

The impact of oral microbial dysbiosis on Alzheimer's disease (AD) remains controversial. Building off recent studies reporting that various microbes might directly seed or promote amyloid ß (Aß) deposition, we evaluated the effects of periodontal bacteria (Porphyromonas gingivalis, Treponema denticola) and supragingival commensal (Streptococcus gordonii) oral bacterial infection in the APP-transgenic CRND8 (Tg) mice model of AD. We tracked bacterial colonization and dissemination, and monitored effects on gliosis and amyloid deposition. Chronic oral infection did not accelerate Aß deposition in Tg mice but did induce alveolar bone resorption, IgG immune response, and an intracerebral astrogliosis (GFAP: glial fibrillary acidic protein). In contrast, intracerebral inoculation of live but not heat-killed P. gingivalis increased Aß deposition and Iba-1 (ionized calcium-binding adaptor-1) microgliosis after 8 weeks of bacterial infection but not at 4 days. These data show that there may be differential effects of infectious microbes on glial activation and amyloid deposition depending on the species and route of inoculation, and thereby provide an important framework for future studies. Indeed, these studies demonstrate marked effects on amyloid ß deposition only in a fairly non-physiologic setting where live bacteria is injected directly into the brain.

Community-Based 16S rDNA Fingerprinting Analysis of Geographically Distinct Marine Sediments of Unexplored Coastal Regions of Palk Bay and Gulf of Mannar.

The present study aims to carefully delineate the bacterial community composition in marine sediments from different geographical coastal regions of Palk Bay and Gulf of Mannar that are known for human recreational activities. Bacterial richness in different marine sediments was assessed using 16S rRNA gene-based Denaturing Gradient Gel Electrophoresis (DGGE) which is a widely deployed fingerprinting technique. The DGGE profiles revealed that the bacterial community profiles of sediment from different coastal regions were complex and dynamic. The most dominant phylum present in the marine sediment samples were Proteobacteria followed by Cyanobacteria, Bacteriodetes, Firmicutes, Acidobacteria, and Actinobacteria. Cosmopolitan presence of Thioalkalivibrio sp. was observed in all the marine sediments. Sequencing of the abundant band reveals the presence of Vibrio spp. in all the marine sediments. Comparative illumina data analysis revealed the presence of 51 different Vibrio species in which Vibrio alginolyticus holds the highest abundance (67.2%) followed by V. harveyi (13.5%). This is the one of the very few reports that compared the complex microbial community composition of the marine sediments of different geographical regions of unexplored coastal region. Further in-depth analysis needs to be taken to understand the presence of complex microbial compositions and their functions through high-throughput whole metagenome sequencing and metaproteomic approaches.

Thermal discharge-induced seawater warming alters richness, community composition and interactions of bacterioplankton assemblages in a coastal ecosystem.

Despite accumulating evidence on the impact of global climate warming on marine microbes, how increasing seawater temperature influences the marine bacterioplankton communities is elusive. As temperature gradient created by thermal discharges provides a suitable in situ model to study the influence of warming on marine microorganisms, surface seawater were sampled consecutively for one year (September-2016 to August-2017) from the control (unimpacted) and thermal discharge-impacted areas of a coastal power plant, located in India. The bacterioplankton community differences between control (n = 16) and thermal discharge-impacted (n = 26) areas, as investigated using 16S rRNA gene tag sequencing revealed reduced richness and varied community composition at thermal discharge-impacted areas. The relative proportion of Proteobacteria was found to be higher (average ~ 15%) while, Bacteroidetes was lower (average ~ 10%) at thermal discharge-impacted areas. Intriguingly, thermal discharge-impacted areas were overrepresented by several potential pathogenic bacterial genera (e.g. Pseudomonas, Acinetobacter, Sulfitobacter, Vibrio) and other native marine genera (e.g. Marinobacter, Pseudoalteromonas, Alteromonas, Pseudidiomarina, Halomonas). Further, co-occurrence networks demonstrated that complexity and connectivity of networks were altered in warming condition. Altogether, results indicated that increasing temperature has a profound impact on marine bacterioplankton richness, community composition, and inter-species interactions. Our findings are immensely important in forecasting the consequences of future climate changes especially, ocean warming on marine microbiota.

Nanostructured Surfaces to Promote Osteoblast Proliferation and Minimize Bacterial Adhesion on Titanium.

The objective of this study was to investigate the potential of titanium nanotubes to promote the proliferation of human osteoblasts and to reduce monomicrobial biofilm adhesion. A secondary objective was to determine the effect of silicon carbide (SiC) on these nanostructured surfaces. Anodized titanium sheets with 100-150 nm nanotubes were either coated or not coated with SiC. After 24 h of osteoblast cultivation on the samples, cells were observed on all titanium sheets by SEM. In addition, the cytotoxicity was evaluated by CellTiter-BlueCell assay after 1, 3, and 7 days. The samples were also cultivated in culture medium with microorganisms incubated anaerobically with respective predominant periodontal bacteria viz. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as monoinfection at 37 °C for 30 days. The biofilm adhesion and coverage were evaluated through surface observation using Scanning Electron Microscopy (SEM). The results demonstrate that Ti nanostructured surfaces induced more cell proliferation after seven days. All groups presented no cytotoxic effects on human osteoblasts. In addition, SEM images illustrate that Ti nanostructured surfaces exhibited lower biofilm coverage compared to the reference samples. These results indicate that Ti nanotubes promoted osteoblasts proliferation and induced cell proliferation on the surface, compared with the controls. Ti nanotubes also reduced biofilm adhesion on titanium implant surfaces.

Polymeric antibiofilm coating comprising synergistic combination of citral and thymol prevents methicillin-resistant Staphylococcus aureus biofilm formation on titanium.

Biomaterial associated microbial infections are complicated and mostly lead to revision surgery or removal which are painful to the patients and quite expensive. These infections are difficult to treat with antibiotics as it is often related to biofilm formation. Methicillin resistant Staphylococcus aureus (MRSA) is the leading pathogen in biomaterial associated infections and well known to form biofilm on foreign materials. To reduce the risk of biomaterial associated infections, recent treatment strategies focus on modification of the implant surface to prevent the adhesion of bacteria. Antibiofilm coating is the effective approach than coating with antimicrobials as antibiofilm agents will not create selective pressure thereby excludes possibility of drug resistance. The current study identified and validated the synergistic antibiofilm activity of citral (CIT) and thymol (THY) by crystal violet quantification and microscopic analysis without alteration in growth and metabolic viability of MRSA. Polymeric antibiofilm coating with CIT + THY as active ingredients was formulated and coated on titanium surface by the process of spin coating. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the effective blending of polymeric formulation and the presence of CIT and THY. Atomic force microscopy (AFM) images revealed the homogenous coating and reduced surface roughness and thickness of the coating was measured by surface profilometer. Antibiofilm coating released CIT and THY in a sustained manner for 60 days. Antibiofilm coating effectively inhibited MRSA adherence in vitro and antibiofilm activity of coating was not affected by plasma conditioning. In addition, antibiofilm coating was non-hemolytic and non-toxic to PBMC. Thus, the current study demonstrated the effectual strategy to prevent biomaterial associated infections and proposes the prospective role of antibiofilm coating in clinical applications.

Exploring the impacts of heavy metals on spatial variations of sediment-associated bacterial communities.

One of the fundamental objectives in modern ecology is to decipher how bacterial communities in natural environment respond to anthropogenic activities. In recent times consequences of marine pollution, especially with heavy metals (HMs) have received increasing attention. However, insights into the response of bacterial communities to HMs in coastal sediments of India remain scarce. Here, we analyzed HMs content in three areas, along the southern coastal region of India. Based on the calculated pollution indices viz., enrichment factor (EF), contamination factor (CF), geo-accumulation index (Igeo) and sediment quality guidelines (SQGs), the studied areas were classified as uncontaminated, moderately contaminated and significantly contaminated. To explore the response of bacterial community to HMs, sediment-associated microbiota was investigated using high-throughput 16S rRNA gene amplicon sequencing. The obtained metataxonomic results revealed that bacterial diversity and community composition varied considerably in significantly contaminated area than moderately contaminated and uncontaminated areas. Proportion of bacterial classes was higher for Gammaproteobacteria, Betaproteobacteria and Actinobacteria, but lower for Alphaproteobacteria and Flavobacteriia in significantly contaminated area. Also, samples of significantly contaminated area were dominated by well-documented metal-resistant bacterial genera such as Ralstonia and Arthrobacter. Canonical correspondence analysis (CCA) showed that spatial variability of bacterial community composition was strongly correlated with HMs content such as Chromium, Cadmium and Nickel. Further analysis using PICRUSt programme indicated that the predictive functional profile also varied considerably in significantly contaminated area. By linking HMs with bacterial compositional variations, the present study highlights the likely influence of HMs in shaping sedimentary microbiota of coastal regions.

sarA-Dependent Antibiofilm Activity of Thymol Enhances the Antibacterial Efficacy of Rifampicin Against Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen which has been listed as a high-priority multi-drug resistance pathogen by the World Health Organization (WHO). Persistent MRSA infections are often associated with biofilm formation and resistance to conventional antimicrobial therapy. Inhibiting the surface adherence and the virulence of the bacterium is the current alternative approach without affecting growth to reduce the possibility of resistance development. Although numerous antibiofilm agents have been identified, their mode of action remains unclear. Combining two drugs with different modes of action will improve the efficiency of the treatment strategy against MRSA. The present study was aimed to decipher the molecular mechanism underlying the antibiofilm activity of thymol against MRSA and assess the ability of thymol to improve the antibacterial activity of rifampicin. Thymol significantly inhibited 88% of MRSA biofilm formation at 100 µg/ml and reduced the surface adherence of MRSA on glass, stainless steel, and titanium surface coated with human plasma as evidenced by microscopic analyses. qPCR analysis of global virulence regulatory genes and biofilm assay with S. aureus wild type, ΔsarA, and Δagr strains revealed the sarA-mediated antibiofilm activity of thymol and inhibition of sarA-controlled virulence factors. Congo red assay and erythrocyte lysis assay further confirmed the reduction in polysaccharide intracellular adhesin and hemolysin. Importantly, thymol enhanced the antibacterial and the biofilm eradication efficiency of rifampicin against MRSA and also reduced the formation of persisters. Thus, the present study reveals the sarA-dependent antibiofilm efficacy of MRSA and suggests thymol as the promising combinatorial candidate in potentiating the antibacterial activity of rifampicin against persistent MRSA infections.

Proteomic profiling unveils citral modulating expression of IsaA, CodY and SaeS to inhibit biofilm and virulence in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the dangerous human pathogens and it is categorized as a high priority multi-drug resistant bacterium by WHO. Biofilm forming ability of MRSA is responsible for persistent infections and also difficult to eradicate using antibiotic therapy as biofilm is much more resistant to antibiotics. Thus, targeting biofilm and virulence has become an alternative approach to attenuate the pathogenicity of bacterium without affecting the growth. Hence, the present study was aimed at evaluation of antibiofilm potential of citral against MRSA and to decode the possible mode of action. Citral inhibited biofilm formation by MRSA without affecting growth at 100 µg/mL. Microscopic analyses evidenced that citral greatly hampered the surface adherence of MRSA. Effect of citral on cellular proteome of MRSA was studied using two-dimensional gel electrophoresis (2DGE) and differentially regulated proteins were identified using nano LC-MS/MS and MALDI-TOF/TOF analysis. Gene ontology and STRING analysis revealed that citral differentially regulated the proteins involved in pleotropic transcriptional repression (CodY), cell wall homeostasis (IsaA), regulation of exotoxin secretion (SaeS), cell adhesion, hemolysis, capsular polysaccharide biosynthesis and pathogenesis. Gene expression analysis and in vitro assays further validated the alteration in synthesis of slime, hemolysin, lipase, staphyloxanthin and oxidant susceptibility. Thus, the present study unveiled the multiple protein targeted antibiofilm potential of citral and portrays citral as a promising therapeutic agent to combat biofilm mediated MRSA infections with less possibility of resistance development.

Deciphering the Antibacterial Mode of Action of Alpha-Mangostin on Staphylococcus epidermidis RP62A Through an Integrated Transcriptomic and Proteomic Approach.

Background: Alpha-mangostin (α-MG) is a natural xanthone reported to exhibit rapid bactericidal activity against Gram-positive bacteria, and may therefore have potential clinical application in healthcare sectors. This study sought to identify the impact of α-MG on Staphylococcus epidermidis RP62A through integrated advanced omic technologies. Methods: S. epidermidis was challenged with sub-MIC (0.875 µg/ml) of α-MG at various time points and the differential expression pattern of genes/proteins were analyzed in the absence and presence of α-MG using RNA sequencing and LC-MS/MS experiments. Bioinformatic tools were used to categorize the biological processes, molecular functions and KEGG pathways of differentially expressed genes/proteins. qRT-PCR was employed to validate the results obtained from these analyses. Results: Transcriptomic and proteomic profiling of α-MG treated cells indicated that genes/proteins affected by α-MG treatment were associated with diverse cellular functions. The greatest reduction in expression was observed in transcription of genes conferring cytoplasmic membrane integrity (yidC2, secA and mscL), cell division (ftsY and divlB), teichoic acid biosynthesis (tagG and dltA), fatty-acid biosynthesis (accB, accC, fabD, fabH, fabI, and fabZ), biofilm formation (icaA) and DNA replication and repair machinery (polA, polC, dnaE, and uvrA). Those with increased expression were involved in oxidative (katA and sodA) and cellular stress response (clpB, clpC, groEL, and asp23). The qRT-PCR analysis substantiated the results obtained from transcriptomic and proteomic profiling studies. Conclusion: Combining transcriptomic and proteomic methods provided comprehensive information about the antibacterial mode of action of α-MG. The obtained results suggest that α-MG targets S. epidermidis through multifarious mechanisms, and especially prompts that loss of cytoplasmic membrane integrity leads to rapid onset of bactericidal activity.

Inhibitory effect of α-mangostin on Acinetobacter baumannii biofilms - an in vitro study.

The present study was designed to investigate the anti-biofilm potential of alpha-mangostin (α-MG) against Acinetobacter baumannii (AB). The biofilm inhibitory concentration (BIC) of α-MG against AB was found to be 2 µg ml-1. α-MG (0.5, 1 and 2 µg ml-1) exhibited non-bactericidal concentration-dependent anti-biofilm activities against AB. However, α-MG failed to disintegrate the mature biofilms of AB even at a 10-fold increased concentration from its BIC. Results from qRT-PCR and in vitro bioassays further demonstrated that α-MG downregulated the expression of bfmR, pgaA, pgaC, csuA/B, ompA, bap, katE, and sodB genes, which correspondingly affects biofilm formation and its associated virulence traits. The present study suggests that α-MG exerts its anti-biofilm property by interrupting initial biofilm formation and the cell-to-cell signaling mechanism of AB. Additional studies are required to understand the mode of action responsible for the anti-biofilm property.

Comparative assessment of bacterial diversity associated with co-occurring eukaryotic hosts of Palk Bay origin.

Epibacterial communities of co-occurring eukaryotic hosts of Palk Bay origin (five seaweed species (Gracilaria sp, Padina sp, Enteromorpha sp, Sargassum sp, and Turbinaria sp) and one seagrass [Cymodaceae sp]) were analyzed for diversity and compared using 16S rRNA based Denaturant Gradient Gel Electrophoresis analysis. Diversity index revealed that Turbinaria sp hosts highest bacterial diversity while it was least in Gracilaria sp. The DGGE band profile showed that the epibacterial community differed considerably among the studied species. Statistical assessment using cluster analysis and Non-metric multidimensional scale analysis also authenticated the observed variability. Despite huge overlap, the composition of bacterial community structure differed significantly among the three closely related species namely Sargassum, Turbinaria and Padina. In addition, Enteromorpha and Sargassum, one being chlorophyta and the other phaeophyta showed about 80% similarity in bacterial composition. This differs from the general notion that epibacterial community composition will vary widely depending on the host phyla. The results extended the phenomenon of host specific epibacterial community irrespective of phylogeny and similarity in geographical location.

Ultradeep 16S rRNA sequencing analysis of geographically similar but diverse unexplored marine samples reveal varied bacterial community composition.

BACKGROUND: Bacterial community composition in the marine environment differs from one geographical location to another. Reports that delineate the bacterial diversity of different marine samples from geographically similar location are limited. The present study aims to understand whether the bacterial community compositions from different marine samples harbour similar bacterial diversity since these are geographically related to each other. METHODS AND PRINCIPAL FINDINGS: In the present study, 16S rRNA deep sequencing analysis targeting V3 region was performed using Illumina bar coded sequencing. A total of 22.44 million paired end reads were obtained from the metagenomic DNA of Marine sediment, Rhizosphere sediment, Seawater and the epibacterial DNA of Seaweed and Seagrass. Diversity index analysis revealed that Marine sediment has the highest bacterial diversity and the least bacterial diversity was observed in Rhizosphere sediment. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant taxa present in all the marine samples. Nearly 62-71% of rare species were identified in all the samples and most of these rare species were unique to a particular sample. Further taxonomic assignment at the phylum and genus level revealed that the bacterial community compositions differ among the samples. CONCLUSION: This is the first report that supports the fact that, bacterial community composition is specific for specific samples irrespective of its similar geographical location. Existence of specific bacterial community for each sample may drive overall difference in bacterial structural composition of each sample. Further studies like whole metagenomic sequencing will throw more insights to the key stone players and its interconnecting metabolic pathways. In addition, this is one of the very few reports that depicts the unexplored bacterial diversity of marine samples (Marine sediment, Rhizosphere sediment, Seawater) and the host associated marine samples (Seaweed and Seagrass) at higher depths from uncharacterised coastal region of Palk Bay, India using next generation sequencing technology.

Correlation of matrix metalloproteinases and their inhibitors with hypoxia and angiogenesis in premenopausal patients with adenocarcinoma of the breast.

OBJECTIVES: The present study was designed to correlate the expression of proteins regulating invasion and angiogenesis in patients with adenocarcinoma of the breast. DESIGN AND METHOD: Seventy-five premenopausal breast cancer patients histologically categorized as grades I, II and III were chosen for the study. We analyzed the expression of MMP-2, and -9 and their inhibitors TIMP-2 and RECK together with HIF-1α and VEGF in tumor, adjacent tissues and serum samples by immunohistochemical and Western blot analysis. RESULTS: The breast tumors analyzed in the present study were characterized by increased expression of MMP-2, -9, HIF-1α and VEGF with differential expression patterns of TIMP-2 and downregulation of RECK. CONCLUSIONS: The simultaneous analysis of the expression of these molecular markers is important to understand the intricate network between key molecules involved in invasion and angiogenesis that eventually determines the clinical course of the disease.