Threatened corals provide underexplored microbial habitats

Contemporary in-depth sequencing of environmental samples has provided novel insights into microbial community structures, revealing that their diversity had been previously underestimated. Communities in marine environments are commonly composed of a few dominant taxa and a high number of taxonomically diverse, low-abundance organisms. However, studying the roles and genomic information of these “rare” organisms remains challenging, because little is known about their ecological niches and the environmental conditions to which they respond. Given the current threat to coral reef ecosystems, we investigated the potential of corals to provide highly specialized habitats for bacterial taxa including those that are rarely detected or absent in surrounding reef waters. The analysis of more than 350,000 small subunit ribosomal RNA (16S rRNA) sequence tags and almost 2,000 nearly full-length 16S rRNA gene sequences revealed that rare seawater biosphere members are highly abundant or even dominant in diverse Caribbean corals. Closely related corals (in the same genus/family) harbored similar bacterial communities. At higher taxonomic levels, however, the similarities of these communities did not correlate with the phylogenetic relationships among corals, opening novel questions about the evolutionary stability of coral-microbial associations. Large proportions of OTUs (28.7–49.1%) were unique to the coral species of origin. Analysis of the most dominant ribotypes suggests that many uncovered bacterial taxa exist in coral habitats and await future exploration. Our results indicate that coral species, and by extension other animal hosts, act as specialized habitats of otherwise rare microbes in marine ecosystems. Here, deep sequencing provided insights into coral microbiota at an unparalleled resolution and revealed that corals harbor many bacterial taxa previously not known. Given that two of the coral species investigated are listed as threatened under the U.S. Endangered Species Act, our results add an important microbial diversity-based perspective to the significance of conserving coral reefs.

Introduction to Molecular Analysis of Ectomycorrhizal Communities

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

Cross-Ocean distribution of Rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters

Bacterial surface colonization is a universal adaptation strategy in aquatic environments. However, neither the identities of early colonizers nor the temporal changes in surface assemblages are well understood. To determine the identities of the most common bacterial primary colonizers and to assess the succession process, if any, of the bacterial assemblages during early stages of surface colonization in coastal water of the West Pacific Ocean, nonnutritive inert materials (glass, Plexiglas, and polyvinyl chloride) were employed as test surfaces and incubated in seawater off the Qingdao coast in the spring of 2005 for 24 and 72 h. Phylogenetic analysis of the 16S rRNA gene sequences amplified from the recovered surface-colonizing microbiota indicated that diverse bacteria colonized the submerged surfaces. Multivariate statistical cluster analyses indicated that the succession of early surface-colonizing bacterial assemblages followed sequential steps on all types of test surfaces. The Rhodobacterales, especially the marine Roseobacter clade members, formed the most common and dominant primary surface-colonizing bacterial group. Our current data, along with previous studies of the Atlantic coast, indicate that the Rhodobacterales bacteria are the dominant and ubiquitous primary surface colonizers in temperate coastal waters of the world and that microbial surface colonization follows a succession sequence. A conceptual model is proposed based on these findings, which may have important implications for understanding the structure, dynamics, and function of marine biofilms and for developing strategies to harness or control surface-associated microbial communities.

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Impacto ambiental do regulador de crescimento vegetal paclobutrazol.

Capítulo 1. Paclobutrazol - Regulador de Crescimento Vegetal. Capítulo 2. Xenobióticos e seus Impactos Ambientais. Capítulo 3. Efeito de Paclobutrazol na Microbiota do Filoplano de Mangueiras. Capítulo 4. Efeito do Paclobutrazol sobre a Microbiota do Solo. Capítulo 5. Degradação do Paclobutrazol em Solos Tropicais. Capítulo 6. Análise do Polimorfismo de Bactérias Degradadoras do Paclobutrazol. Capítulo 7. Avaliação de Risco em decorrência da Exposição Perinatal ao Paclobutrazol: análise de alguns indicadores físicos e neurocomportamentais. Capítulo 8. Toxicidade do Paclobutrazol em Ambiente Aquático.

Estudo da prevalência da utilização de protetores bucais em praticantes da arte marcial Muay Thai

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Crianças Respiradoras Bucais e possíveis tratamentos Ortodônticos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

Molecular analysis of virulence mechanisms associated with adherent-invasive Escherichia coli (AIEC)

Crohn's Disease (CD) is a chronic inflammatory bowel disease of unknown etiology. Recent work has shown that a new pathotype of Escherichia coli, Adherent Invasive E. coli (AIEC) may be associated with CD. AIEC has been shown to adhere to and invade epithelial cells and to replicate within macrophages (together this is called the AIEC phenotype). In this thesis, the AIEC phenotype of 84 E. coli strains were determined in order to identify the prevalence of this phenotype within the E. coli genus. This study showed that a significant proportion of E. coli strains (approx. 5%) are capable of adhering to and invading epithelial cells and undergoing intramacrophage replication. Moreover, the results presented in this study indicate a correlation between survival in macrophage and resistance to grazing by amoeba supporting the coincidental evolution hypothesis that resistance to amoebae could be a driving force in the evolution of pathogenicity in some bacteria, such as AIEC. In addition, this study has identified an important regulatory role for the CpxA/R two component system (TCS) in the invasive abilities of AIEC HM605, a colonic mucosa-associated CD isolate. A mutation in cpxR was shown to be defective in the invasion of epithelial cells and this defect was shown to be independent of motility or the expression of Type 1 fimbriae, factors that have been shown to be involved in the invasion of another strain of AIEC, isolated from a patient with ileal CD, called LF82. The CpxA/R TCS responds to disturbances in the cell envelope and has been implicated in the virulence of a number of Gram negative pathogens. In this study it is shown that the CpxA/R TCS regulates the expression of a potentially novel invasin called SinH. SinH is found in a number of invasive strains of E. coli and Salmonella. Moreover work presented here shows that a critical mechanism underpinning AIEC persistence in macrophages is the repair of DNA bases damaged by macrophage oxidants. Together these findings provide evidence to suggest that AIEC are a diverse group of E. coli and possess diverse molecular mechanisms and virulence factors that contribute to the AIEC phenotype. In addition, AIEC may have gone through different evolutionary histories acquiring various molecular mechanisms ultimately culminating in the AIEC phenotype. The gastrointestinal (GI) tract harbors a diverse microbiota; most are symbiotic or commensal however some bacteria have the potential to cause disease (pathobiont). The work presented here provides evidence to support the model that AIEC are pathobionts. AIEC strains can be carried as commensals in healthy guts however, when the intestinal homeostasis is disrupted, such as in the compromised gut of CD patients, AIEC may behave as opportunistic pathogens and cause and/or contribute to disease by driving intestinal inflammation.

Antimicrobial activities and diversity of sponge derived microbes

In this study, marine sponges collected in Irish waters were analysed for their associated microbiota. Of the approximately 240 bacterial isolates obtained from two sponges several showed antimicrobial activity; among them members of genera which have rarely been shown to produce antimicrobial compounds. Differences observed from the sponge-derived groups of isolates in terms of bioactivity suggests that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. More than 60% of fungal isolates obtained from 12 sponge samples proved to be bioactive. One of the isolates, which was closely related to Fusarium oxysporum and showed activity against bacteria and fungi, was investigated for its secondary metabolite genes. At least 5 different NRPS genes, with a sequence similarity as low as 50 % to known genes, were identified highlighting the likelihood that this isolate may be capable of producing novel secondary metabolites. A Micromonospora sp. was isolated from a Haliclona simulans sample collected in Irish waters. The isolate inhibited the growth of Gram positive bacterial test strains in three different antimicrobial assays. Employing preparative layer chromatography the compound responsible for the bioactivity could be isolated. According to LC-MS andNMR data the bioactive compound could indeed be novel. Finally, two deep water sponges were shown to host a remarkably different bacterial and archaeal diversity by application of 454 Pyrosequencing. The L. diversichela –proteobacterial community was dominated by a single ƴ-proteobacterial bacterium whereas the S. normani sample hosted a largely sponge specific microbial community, even more diverse than has been previously reported for shallow water sponges. Organisms potentially involved in nitrification, sulphate reduction and secondary metabolite production were found to be spatially distributed in the sponge. Furthermore, a deep sea specific population was implied.

Functional genomics of motile commensal intestinal bacteria

Flagella confer upon bacteria the ability to move and are therefore organelles of significant bacteriological importance. The innate immune system has evolved to recognise flagellin, (the major protein component of the bacterial flagellar filament). Flagellate microbes can potentially stimulate the immune systems of mammals, and thus have significant immunomodulatory potential. The flagellum-biogenesis genotype and phenotype of Lactobacillus ruminis, an autochthonous intestinal commensal, was studied. The flagellum-biogenesis genotypes of motile enteric Eubacterium and Roseburia species were also investigated. Flagellin proteins were recovered from these commensal species, their amino-termini were sequenced and the proteins were found to be pro-inflammatory, as assessed by measurement of interleukin-8 (IL-8) secretion from human intestinal epithelial cell lines. For L. ruminis, this IL-8 secretion required signalling through Toll Like Receptor 5. A model for the regulation of flagellum-biogenesis in L. ruminis was inferred from transcriptomics data and bioinformatics analyses. Motility gene expression in this species may be under the control of a novel regulator, LRC_15730. Potential promoters for genes encoding flagellin proteins in the Eubacterium and Roseburia genomes analysed were inferred in silico. Relative abundances of the target Eubacterium and Roseburia species in the intestinal microbiota of 25 elderly individuals were determined. These species were found to be variably abundant in these individuals. Motility genes from these species were variably detected in the shotgun metagenome databases generated by the ELDERMET project. This suggested that a greater depth of sequencing, or improved evenness of sequencing, would be required to capture the full diversity of microbial functions for specific target or low abundance species in microbial communities by metagenomics. In summary, this thesis used a functional genomics approach to describe flagellum-mediated motility in selected Gram-positive commensal bacteria. The regulation of flagellum biosynthesis in these species, and the consequences of flagella expression from a host-interaction perspective were also considered.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Functional genomics of commensal Lactobacilli

Catabolic flexibility affords a bacterium the ability to utilise different sugar sources as carbon for energy. This is important for commensal lactobacilli like Lactobacillus ruminis which can be exposed to a variety of carbohydrates in vivo. However, little is known about the fermentation capabilities, metabolic pathways, genetic diversity or potential survival mechanisms used by L. ruminis in vivo. A combination of in vitro and in silico techniques was used to identify the catabolic pathways of L. ruminis. I also compared 16 L. ruminis strains using a panel of biochemical and survival assays, genetically, whole genome sequencing and RNA sequencing. Multi locus sequence typing revealed that strains clustered according to their host sources. Transcriptome analysis by RNAseq of two motile strains under three growth conditions, including swarming, identified the up-regulation of carbohydrate-related genes under swarming conditions. This suggests that carbohydrate flexibility may have an uncharacterised role in L. ruminis swarming. Following on from the assessment of L. ruminis catabolic flexibility, the porcine diet was supplemented with galactooligosaccharides or L. ruminis ATCC 25644 plus galactooligosaccharides. Supplementation of the porcine diet with galactooligosaccharide had no effect on microbiota diversity. In contrast, the L. ruminis plus galactooligosaccharide treatment significantly reduced the microbiota diversity. Diet is a major factor that affects the diversity of the gut microbiota. In order to get a more thorough understanding of diet and gut health in animals such as racehorses and domesticated herbivores, I determined the core microbiota of animals consuming different feeds. Interestingly, the gut microbiota diversity correlated with the host phylogeny of the animal. The genome of Lactobacillus equi (2.19 Mb), isolated from a healthy Irish thoroughbred was also sequenced and annotated, and comprised 2,263 predicted genes. The large repertoire of predicted carbohydrate-related genes may offer L. equi an advantage in the complex and harsh hindgut environment. In summary, this thesis uses functional genomics to assess the effect that carbohydrates have on commensal lactobacilli and the microbiota as a whole.

Functional metagenomic analysis of the human gut microbiome to identify novel salt tolerance genes

The ability to adapt to and respond to increases in external osmolarity is an important characteristic that enables bacteria to survive and proliferate in different environmental niches. When challenged with increased osmolarity, due to sodium chloride (NaCl) for example, bacteria elicit a phased response; firstly via uptake of potassium (K+), which is known as the primary response. This primary response is followed by the secondary response which is characterised by the synthesis or uptake of compatible solutes (osmoprotectants). The overall osmotic stress response is much broader however, involving many diverse cellular systems and processes. These ancillary mechanisms are arguably more interesting and give a more complete view of the osmotic stress response. The aim of this thesis was to identify novel genetic loci from the human gut microbiota that confer increased tolerance to osmotic stress using a functional metagenomic approach. Functional metagenomics is a powerful tool that enables the identification of novel genes from as yet uncultured bacteria from diverse environments through cloning, heterologous expression and phenotypic identification of a desired trait. Functional metagenomics does not rely on any previous sequence information to known genes and can therefore enable the discovery of completely novel genes and assign functions to new or known genes. Using a functional metagenomic approach, we have assigned a novel function to previously annotated genes; murB, mazG and galE, as well as a putative brp/blh family beta-carotene 15,15’-monooxygenase. Finally, we report the identification of a completely novel salt tolerance determinant with no current known homologues in the databases. Overall the genes identified originate from diverse taxonomic and phylogenetic groups commonly found in the human gastrointestinal (GI) tract, such as Collinsella and Eggerthella, Akkermansia and Bacteroides from the phyla Actinobacteria, Verrucomicrobia and Bacteroidetes, respectively. In addition, a number of the genes appear to have been acquired via lateral gene transfer and/or encoded on a prophage. To our knowledge, this thesis represents the first investigation to identify novel genes from the human gut microbiota involved in the bacterial osmotic stress response.

An investigation into mechanisms of visceral pain in rodents

Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.