Microbiome profile associated with malignant pleural effusion.

INTRODUCTION:There is ongoing research into the development of novel molecular markers that may complement fluid cytology malignant pleural effusion (MPE) diagnosis. In this exploratory pilot study, we hypothesized that there are distinct differences in the pleural fluid microbiome profile of malignant and non-malignant pleural diseases. METHOD:From a prospectively enrolled pleural fluid biorepository, samples of MPE were included. Non-MPE effusion were included as comparators. 16S rRNA gene V4 region amplicon sequencing was performed. Exact Sequence Variants (ESVs) were used for diversity analyses. The Shannon and Richness indices of alpha diversity and UniFrac beta diversity measures were tested for significance using permutational multivariate analysis of variance. Analyses of Composition of Microbiome was used to identify differentially abundant bacterial ESVs between the groups controlled for multiple hypothesis testing. RESULTS:38 patients with MPE and 9 with non-MPE were included. A subgroup of patients with metastatic adenocarcinoma histology were identified among MPE group (adenocarcinoma of lung origin (LA-MPE) = 11, breast origin (BA-MPE) = 11). MPE presented with significantly greater alpha diversity compared to non-MPE group. Within the MPE group, BA-MPE was more diverse compared to LA-MPE group. In multivariable analysis, ESVs belonging to family S24-7 and genera Allobaculum, Stenotrophomonas, and Epulopiscium were significantly enriched in the malignant group compared to the non-malignant group. CONCLUSION:Our results are the first to demonstrate a microbiome signature according to MPE and non-MPE. The role of microbiome in pleural effusion pathogenesis needs further exploration

The Host-Microbiota Axis in Chronic Wound Healing

Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management

Microbiota fingerprints within the oral cavity of cetaceans as indicators for population biomonitoring

The composition of mammalian microbiota has been related with the host health status. In this study, we assessed the oral microbiome of 3 cetacean species most commonly found stranded in Iberian Atlantic waters (Delphinus delphis, Stenella coeruleoalba and Phocoena phocoena), using 16S rDNA-amplicon metabarcoding. All oral microbiomes were dominated by Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria bacteria, which were also predominant in the oral cavity of Tursiops truncatus. A Constrained Canonical Analysis (CCA) showed that the major factors shaping the composition of 38 oral microbiomes (p-value < 0.05) were: (i) animal species and (ii) age class, segregating adults and juveniles. The correlation analysis also grouped the microbiomes by animal stranding location and health status. Similar discriminatory patterns were detected using the data from a previous study on Tursiops truncatus, indicating that this correlation approach may facilitate data comparisons between different studies on several cetacean species. This study identified a total of 15 bacterial genera and 27 OTUs discriminating between the observed CCA groups, which can be further explored as microbiota fingerprints to develop (i) specific diagnostic assays for cetacean population conservation and (ii) bio-monitoring approaches to assess the health of marine ecosystems from the Iberian Atlantic basin, using cetaceans as bioindicators.The authors would like to acknowledge to Jorg Becker and Joao Sobral for the sequencing services provided at the Instituto Gulbenkian de Ciencia. This work was supported by the project CetSenti RECI/AAGGLO/0470/2012 (FCOMP-01-0124-FEDER-027472), by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), by the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 -Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, funded by national funds through the Fundacao para a Ciencia e a Tecnologia (FCT) I.P. and the European Regional Development Fund (ERDF), through the COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI). The work was supported as well through CESAM UID/AMB/50017/2013 co-funded by the FCT/MEC and FEDER within PT2020 and Compete 2020 (C.E.), FCT grants SFRH/BD/30240/2006 (M.F.), and SFRH/BPD/82407/2011 (A.L.) and NIH RCMI Grant No. 2U54MD007600 (F.G.V.)

Application of Bioinformatics Methodologies in the Fields of Skin Biology and Dermatology

Bioinformatics is a research field that uses computer‐based tools to investigate life sciences questions employing “big data” results from large‐scale DNA sequencing, whole genomes, transcriptomes, metabolomes, populations, and biological systems, which can only be comprehensively viewed in silico. The epidermis was among the earliest targets of bioinformatics studies because it represents one of the most accessible targets for research. An additional advantage of working with the epidermis is that the sample can even be recovered using tape stripping, an easy, noninvasive protocol. Consequently, bioinformatics methods in the fields of skin biology and dermatology generated a fairly large volume of bioinformatics data, which led us to originate the term “skinomics.” Skinomics data are directed toward epidermal differentiation, malignancies, inflammation, allergens, and irritants, the effects of ultraviolet (UV) light, wound healing, the microbiome, stem cells, etc. Cultures of cutaneous cell types, keratinocytes, fibroblasts, melanocytes, etc., as well as skin from human volunteers and from animal models, have been extensively experimented on. Here, we review the development of the skinomics, its methodology, current achievements, and future potentials

Psoriasis: A Study of the Skin Transcriptome and Microbiome

Psoriasis is a complex autoimmune skin disorder characterized by dry, scaly plaques and painful flares. Even though genetic contribution and environmental factors are suspected, the exact trigger of psoriasis is not well understood. The chronic condition of the disease and the lack of effective and definitive treatments are burdens on the patients. Recent emergence of transcriptome and genomic datasets for the host, as well as the taxonomic datasets for the microbiome has enabled the use of bioinformatics approaches to investigate altered gene circuits in psoriasis. As a first step, open source microarray datasets of psoriasis were analyzed in context of other skin conditions. The analysis showed that upregulated genes in the psoriasis transcriptome included those involved in epidermal differentiation complex and antimicrobial processes, while the top downregulated genes were involved in lipid metabolism. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched with significantly altered genes point to the upregulation of both innate and adaptive immune responses. The psoriasis gene signature was distinctive from other inflammatory skin conditions and it resembled the wound healing process in terms of keratinization and immune response signals. On the microbiome side, over-abundance of opportunistic bacteria on the psoriasis microbiome was observed compared to controls. Virulence genes were consistently in high abundance across different body sites. Bacterial invasion of epithelial cells gene pathway was crowded with both significantly altered genes on the host side and high-abundance orthologs on the microbiome side. The findings suggested bacterial involvement in the initiation or maintenance of psoriasis flares. Genetic components also play a role in susceptibility to psoriasis. Human Leukocyte Antigen (HLA) is one of the regions that has previously been associated with psoriasis through Genome Wide Association studies. The Single Nucleotide Polymorphisms (SNPs) typed in the HapMap dataset (11 ethnic populations) within the HLA region have been analyzed using extended haplotype homozygosity based tests to identify positive selection on polymorphisms that have not yet reached fixation. Results showed regional specificity of positive selection signals on the sub-classes of HLA. The positive selection signals in Class I sub-region showed European ancestry specificity with intronic SNPs on a psoriasis related gene PSORS1C1 as well as on TCF19, MUC22, TRIM10, and TRIM15. The region specific selection signals were also seen in the Class III region for the East Asian populations and in the Class II region for African ancestry populations. Similar to single population tests, the cross population tests showed that the significant SNPs were concentrated in the Class II region for African ancestry populations, whereas for European ancestry populations, they were concentrated in the Class I region. The results show how positive selection of a SNP can encourage genetic hitchhiking of the susceptibility SNPs for a disease along with a SNP that is under positive selection. This research thesis bridges large scale transcriptome datasets of the host and operational taxonomy unit abundance datasets of the microbiome, opening up new avenues for drug repositioning studies by pointing out specific host-microbiome genes as drug targets.Ph.D., Biomedical Engineering -- Drexel University, 201

Elucidating the mechanisms of anastomotic leakage:a new point of view

After surgical resection of a part of the colon an anastomosis is made to restore the continuity of the colon. Unfortunately, these anastomoses are often complicated by leakage. This thesis embarks on the quest to elucidate the mechanisms behind anastomotic leakage (AL). New techniques are used to study a long-ignored factor in AL: the intestinal microbiota. We demonstrate that the bacterial composition during the construction of the anastomosis plays a role in the subsequent development of anastomotic leakage. The lack of diversity in the microbial composition is associated with AL. Furthermore, the virulence factors (pathogenic molecules) of bacteria and other micro-organisms play an important role in the development of AL and the associated subsequent tumour recurrence. These virulence factors are a high expression of the collagen degrading GelE gene and the ability to activate the collagen degrading MMP9, together called the “leakage phenotype”. The Western diet causes a change in and reduction of the microbial diversity and a presence of micro-organisms with the “leakage phenotype”. Lastly, the expression of genes in the colon plays a role in AL, with a reduced expression of genes involved in the immune response, angiogenesis and collagen crosslinking. To conclude: the intestinal microbiota, partly influenced by the Western diet, and the colonic gene expression are important factors in the development of AL. In the nearby future, the results from this thesis can be used for therapeutic or preventive measures to prevent anastomotic leakage, partly focused on the manipulation of the intestinal microbiota

Interplay of Staphylococcal and Host Proteases Promotes Skin Barrier Disruption in Netherton Syndrome.

Netherton syndrome (NS) is a monogenic skin disease resulting from loss of function of lymphoepithelial Kazal-type-related protease inhibitor (LEKTI-1). In this study we examine if bacteria residing on the skin are influenced by the loss of LEKTI-1 and if interaction between this human gene and resident bacteria contributes to skin disease. Shotgun sequencing of the skin microbiome demonstrates that lesional skin of NS subjects is dominated by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Isolates of either species from NS subjects are able to induce skin inflammation and barrier damage on mice. These microbes promote skin inflammation in the setting of LEKTI-1 deficiency due to excess proteolytic activity promoted by S. aureus phenol-soluble modulin α as well as increased bacterial proteases staphopain A and B from S. aureus or EcpA from S. epidermidis. These findings demonstrate the critical need for maintaining homeostasis of host and microbial proteases to prevent a human skin disease

Investigating the role of the respiratory microbiome and metabolome in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease of unknown aetiology. There is growing evidence that the lung microbiota may play a role in IPF. However, no study has investigated the functional impact of the short-chain fatty acids (SCFAs) on disease pathogenesis. The aim of this thesis was to characterise the respiratory microbiome and metabolome in healthy controls and subjects with IPF and to investigate the influence of the SCFAs on human primary bronchial epithelial cells (PBECs) and human lung fibroblasts (HLFs) from these individuals. Subjects diagnosed with IPF (n=201) and healthy controls (n=40) were prospectively recruited and underwent bronchoscopy. Bacterial DNA was isolated and 16S rRNA gene sequencing undertaken to characterise bacterial communities. Untargeted 1H nuclear magnetic resonance spectroscopybased metabolomics and targeted gas chromatography-mass spectrometry captured the metabolic profile of these samples. PBECs, differentiated at air-liquid interface, and HLF from healthy controls and subjects with IPF were either left untreated or exposed to the SCFAs. Compared to healthy controls, the IPF microbiota was less diverse (P<0.01), and had increased proportions of Firmicutes (P<0.01), Streptococcus and Staphylococcus (P<0.05). Relative concentrations of the SCFAs were increased in IPF compared to healthy controls, and in IPF, propionate positively correlated with the bacterial burden (rho=0.47, P=8 x 10-5). Treatment of PBECs from IPF subjects but not healthy controls with the SCFAs led to morphological changes, a dose-dependent release of pro-inflammatory mediators in the cell supernatant, and a decrease in transepithelial electrical resistance (TEER) over time. Compared to baseline, exposure of IPF PBECs to 1 mM of propionate led to a 40% reduction in TEER and a significant increase (P<0.05) in the secretion of IL-6. Treatment of HLFs with 5 mM of propionate or butyrate was found to increase cell proliferation at 72 hours. Subjects with IPF display an altered microbiome which is associated with a distinct metabolic signature in the lower airways. Differences in bacterial genera and an increased bacterial burden in IPF results in changes in the SCFAs in the airways. In vitro work demonstrates the potential of these SCFAs to shape immunological responses in the lung, mediating the pathogenesis of fibrosis.Open Acces