Diversity of Bifidobacteria within the Infant Gut Microbiota

Background The human gastrointestinal tract (GIT) represents one of the most densely populated microbial ecosystems studied to date. Although this microbial consortium has been recognized to have a crucial impact on human health, its precise composition is still subject to intense investigation. Among the GIT microbiota, bifidobacteria represent an important commensal group, being among the first microbial colonizers of the gut. However, the prevalence and diversity of members of the genus Bifidobacterium in the infant intestinal microbiota has not yet been fully characterized, while some inconsistencies exist in literature regarding the abundance of this genus. Methods/Principal Findings In the current report, we assessed the complexity of the infant intestinal bifidobacterial population by analysis of pyrosequencing data of PCR amplicons derived from two hypervariable regions of the 16 S rRNA gene. Eleven faecal samples were collected from healthy infants of different geographical origins (Italy, Spain or Ireland), feeding type (breast milk or formula) and mode of delivery (vaginal or caesarean delivery), while in four cases, faecal samples of corresponding mothers were also analyzed. Conclusions In contrast to several previously published culture-independent studies, our analysis revealed a predominance of bifidobacteria in the infant gut as well as a profile of co-occurrence of bifidobacterial species in the infant’s intestine

A Microbiomic Approach to the Characterization of the Impacts and Influences of Viral, Bacterial, and Harmful Algal Bloom Toxins on the Bottlenose Dolphin

As apex predators that display high site fidelity Tursiops truncatus (bottlenose dolphin) are indicators of marine ecosystem health. Bottlenose dolphins, additionally, display pathogenesis and immune response similar to that of humans. Humans and coastal bottlenose dolphins, in particular, are constantly exposed to the same industrial, agricultural and domestic toxins and pathogens, contaminants and pollutants. Thus, studies on the bottlenose dolphin are also valuable in bridging the gap between ocean and human health. Bottlenose dolphins are susceptible to viral bacterial and toxin infection. Infection in the bottlenose dolphin manifests itself in the form of mass stranding events, unusual mortality events, chronic infection, clinically expressed disease, and unapparent/sustained infections. The focus of this dissertation project is the role and characterization of microorganisms impact on bottlenose dolphin stranding events. In accordance with the three main contributors of bottlenose dolphin stranding events this dissertation employed molecular techniques and next generation sequencing technology for viral, bacterial and harmful algal bloom toxin assessment on dolphins and model organisms. The viromics portion of this dissertation, for instance, took a random hexamer approach towards purified viral RNA and DNA, contributing 86 viruses from BND serum not previously reported in the BND. The bacteriomics portion of this project, additionally, took a 16sPCR approach towards purified bacterial nucleic acid associated with BND skin, demonstrating differences in bacterial diversity and abundance in coastal and offshore BND ecotypes. The effects of harmful algal bloom domoic acid exposure to zebrafish, finally, took a 16sPCR and qPCR approach to illustrate shifts in zebrafish gut microbiome and changes in transcriptional regulation, respectively

A systematic review on omics data (metagenomics, metatranscriptomics, and metabolomics) in the role of microbiome in gallbladder disease

Microbiotas are the range of microorganisms (mainly bacteria and fungi) colonizing multicellular, macroscopic organisms. They are crucial for several metabolic functions affecting the health of the host. However, difficulties hamper the investigation of microbiota composition in cultivating microorganisms in standard growth media. For this reason, our knowledge of microbiota can benefit from the analysis of microbial macromolecules (DNA, transcripts, proteins, or by-products) present in various samples collected from the host. Various omics technologies are used to obtain different data. Metagenomics provides a taxonomical profile of the sample. It can also be used to obtain potential functional information. At the same time, metatranscriptomics can characterize members of a microbiome responsible for specific functions and elucidate genes that drive the microbiotas relationship with its host. Thus, while microbiota refers to microorganisms living in a determined environment (taxonomy of microorganisms identified), microbiome refers to the microorganisms and their genes living in a determined environment and, of course, metagenomics focuses on the genes and collective functions of identified microorganisms. Metabolomics completes this framework by determining the metabolite fluxes and the products released into the environment. The gallbladder is a sac localized under the liver in the human body and is difficult to access for bile and tissue sampling. It concentrates the bile produced in the hepatocytes, which drains into bile canaliculi. Bile promotes fat digestion and is released from the gallbladder into the upper small intestine in response to food. Considered sterile originally,recent data indicate that bile microbiota is associated with the biliary tract’s inflammation and carcinogenesis. The sample size is relevant for omic studies of rare diseases, such as gallbladder carcinoma. Although in its infancy, the study of the biliary microbiota has begun taking advantage of several omics strategies, mainly based on metagenomics, metabolomics, and mouse models. Here, we show that omics analyses from the literature may provide a more comprehensive image of the biliary microbiota. We review studies performed in this environmental niche and focus on network-based approaches for integrative studies

IDENTIFICATION OF VACCINE RESPONSIVENESS BIOMARKERS THROUGH KINOME ANALYSIS IN PIGLETS

Individual variability in immune responses to vaccination can result in vaccinated individuals who fail to develop protective immunity. These “vaccine low-responders” remain at risk for infection and compromise the protection achieved through herd immunity. Biomarkers of vaccine unresponsiveness could enable rapid identification of susceptible low-responders while discerning mechanisms of vaccine-induced immune responses. To investigate biomarkers of vaccine unresponsiveness, piglets (n=117) were vaccinated with a commercial Mycoplasma hyopneumoniae bacterin, and vaccine-induced serum IgG titers were quantified 35 days following vaccination. High (HR) and low (LR) vaccine responders within the 80th and 20th percentile of serum IgG titers were stratified, respectively, and split into discovery (n=12) and validation (n=8) cohorts. Within the discovery cohort, kinome analysis conducted on peripheral blood mononuclear cells collected from HR and LR revealed multiple differential phosphorylation events before and 6-days following vaccination. Differential phosphorylation events before vaccination were enriched in cytokine signaling pathways, a result supported by the quantification of higher plasma interferon-gamma (IFNγ) and interleukin-1beta (IL-1β) in LR compared to HR before vaccination. Additionally, LR had lower birth weight than HR, thus establishing significant associations between vaccine responsiveness and kinase signaling, plasma cytokines, and birth weight. Analysis of the validation cohort verified the differential phosphorylation events identified within the discovery cohort, but there were no differences in birth weight or plasma cytokines between LR and HR. In a second trial, piglets (n=67) from a different facility were vaccinated with the same Mycoplasma hyopneumoniae bacterin to further evaluate plasma cytokines and birth weight as biomarkers of vaccine unresponsiveness. Piglets in the second trial all seroconverted, and serum IgG titers varied less than the first trial. While the second trial found no associations between vaccine unresponsiveness and either birth weight or plasma cytokines, it revealed piglets had age- and litter-dependent differences in plasma IFNγ and IL-1β concentrations within the first 2-months of life. Collectively, these data suggest that though plasma cytokines or birth weight can be associated with vaccine unresponsiveness, their temporal and individual variability can make them inconsistent biomarkers. Phosphorylation biomarkers offered consistent discrimination of HR and LR and provided insight into potential mechanisms regulating vaccine-induced immunity

Host-Microbiome Interaction and Cancer: Potential Application in Precision Medicine

It has been experimentally shown that host-microbial interaction plays a major role in shaping the wellness or disease of the human body. Microorganisms coexisting in human tissues provide a variety of benefits that contribute to proper functional activity in the host through the modulation of fundamental processes such as signal transduction, immunity and metabolism. The unbalance of this microbial profile, or dysbiosis, has been correlated with the genesis and evolution of complex diseases such as cancer. Although this latter disease has been thoroughly studied using different high-throughput technologies, its heterogeneous nature makes its understanding and proper treatment in patients a remaining challenge in clinical settings. Notably, given the outstanding role of host-microbiome interactions, the ecological interactions with microorganisms have become a new significant aspect in the systems that can contribute to the diagnosis and potential treatment of solid cancers. As a part of expanding precision medicine in the area of cancer research, efforts aimed at effective treatments for various kinds of cancer based on the knowledge of genetics, biology of the disease and host-microbiome interactions might improve the prediction of disease risk and implement potential microbiota-directed therapeutics. In this review, we present the state of the art of sequencing and metabolome technologies, computational methods and schemes in systems biology that have addressed recent breakthroughs of uncovering relationships or associations between microorganisms and cancer. Together, microbiome studies extend the horizon of new personalized treatments against cancer from the perspective of precision medicine through a synergistic strategy integrating clinical knowledge, high-throughput data, bioinformatics and systems biology

3Ts in Gastrointestinal Microbiome Era: Technology, Translational Research and Transplant

We have entered a new era where some concepts of the complex community of microorganisms (microbiota comprising bacteria, fungi, viruses, bacteriophages and helminths) are being re-discovered and re-visited. Microbiota and human interaction is not new; they have shared a long history of co-existence. Nevertheless, the opportunities to understand the role of these microorganisms in human diseases and to design a potential treatment were limited. At present, thanks to development of innovative and cutting-edge molecular biological and microbiological technologies as well as clinical informatics and bioinformatics skills, microbiome application is moving into clinics. Approaches to therapy based on prebiotics, probiotics and lately on fecal microbiota transplantation has revolutionized medicine. Microbiota outnumbers our genes and is now regarded as another organ of the body. The gastrointestinal tract and gut microbiota display a well-documented symbiotic relationship. Disruption of intestinal microbiota homeostasis—called dysbiosis—has been associated with several diseases. Whether dysbiosis is a cause or consequence of disease initiation and progression still needs to be investigated in more depth. The aim of this book is to highlight recent advances in the field of microbiome research, which are now shaping medicine, and current approaches to microbiome-oriented therapy for gastrointestinal diseases. Dr. Rinaldo Pellicano Dr. Sharmila Fagoonee Guest Editor

The ELDERMET biobank: Isolation and characterization of the intestinal microbiota from elderly Irish subjects

The human gastrointestinal (GI) tract is colonized by a dense and diverse bacterial community, the commensal microbiota, which plays an important role in the overall health of individuals. This microbiota is relatively stable throughout adult life, but may fluctuate over time with aging and disease. The adaptation of the gut microbiota to our changing life-style is probably the reason for the large inter-individual variation observed among different people. Since the gut microbiota plays an essential role in interactions with host metabolism, it is of utmost importance to explore this relationship. The elderly intestinal microbiota has been the subject of a number of studies in recent years. The results presented in this thesis have further contributed to the expansion of knowledge related to gut microbiota research highlighting the combined effect of culture based and molecular methods as powerful tools for understanding the true impact of microbes. The degree of correlation between measurements from both methods suggested that a single method is capable of profiling intestinal Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae populations. Bacteriocins have shown great promise as alternatives to traditional antibiotics. In this respect, the isolation and characterisation of bacteriocinogenic strains are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine. The selection pressure applied on the bacterial population during antibiotic usage is the driving force for the emergence of antibiotic resistant bacteria. Identification of antibiotic resistant isolates opens up the possibility of using such probiotics to offset the problems caused by antibiotics to the gut microbiota and to improve the intestinal microbial environment. Future work is required to explore the culture collection housing thousands of bacterial isolates as a valuable source of potential probiotics for use for the elderly Irish community

Gut microbiota perturbation induced by emerging dietary habits: an in vitro colon model to predict their effects by a microbiomic and metabolomic approach

Over the past 30 years, unhealthy diets and lifestyles have increased the incidence of noncommunicable diseases and are culprits of diffusion on world’s population of syndromes as obesity or other metabolic disorders, reaching pandemic proportions. In order to comply with such scenario, the food industry has tackled these challenges with different approaches, as the reformulation of foods, fortification of foods, substitution of ingredients and supplements with healthier ingredients, reduced animal protein, reduced fats and improved fibres applications. Although the technological quality of these emerging food products is known, the impact they have on the gut microbiota of consumers remains unclear. In the present PhD thesis, the recipient work was conducted to study different foods with the substitution of the industrial and market components to that of novel green oriented and sustainable ingredients. So far, this thesis included eight representative case studies of the most common substitutions/additions/fortifications in dairy, meat, and vegetable products. The products studied were: (i) a set of breads fortified with polyphenol-rich olive fiber, to replace synthetic antioxidant and preservatives, (ii) a set of Gluten-free breads fortified with algae powder, to fortify the protein content of standard GF products, (iii) different formulations of salami where nitrates were replaced by ascorbic acid and vegetal extract antioxidants and nitrate-reducers starter cultures, (iv) chocolate fiber plus D-Limonene food supplement, as a novel prebiotic formula, (v) hemp seed bran and its alkalase hydrolysate, to introduce as a supplement, (vi) milk with and without lactose, to evaluate the different impact on human colonic microbiota of healthy or lactose-intolerants, (vii) lactose-free whey fermented and/or with probiotics added, to be introduced as an alternative beverage, exploring its impact on human colonic microbiota from healthy or lactose-intolerants, and (viii) antibiotics, to assess whether maternal amoxicillin affects the colon microbiota of piglets

Interactive functional networks in microbiota

The aim of this thesis was to elucidate how various microbial communities work, with a focus on next generation sequencing data. The introduction in chapter 1 focuses on the history of biology, how the field of systems biology arose, and how the rise of nucleic acid sequencing has shaped a completely new field (among others), the microbiome research. In chapter 2, an overview is given how the microbiota can be studied, in connection to metabolic syndrome and its sub-pathologies, including obesity, type II diabetes, elevated blood pressure, and dyslipidemia. We summarize which different methodologies (16S rRNA amplicon sequencing, metagenomics, metatranscriptomics) can be used to investigate the microbiome with different foci, and how as a next step the microbiome can be modelled, in vitro and in silico. Chapter 3 describes the genome and transcriptome of the rat gut commensal Romboutsia ilealis CRIBT. We characterized genomic properties, including those related to metabolism and sporulation abilities. The transcriptome allowed us to investigate the organism’s carbohydrate degradation abilities, including its potential regulation. Chapter 4 is an investigation of an in vitro fermentation system, inoculated with human faecal material and the potential prebiotic Isomalto/malto-polysaccharides. The metatranscriptome of this system gave an insight into which genes are involved in the carbohydrate degradation, and which different types of organisms are involved and potentially need to cooperate for a full utilization of this carbohydrate. In chapter 5, the cow rumen microbiota is investigated under different feeding regimes. The metatranscriptome of the cow rumen microbiota showed distinct patterns depending on the ratio of starch or cellulose enriched feed components, namely maize vs. grass silage. The increase in starch led to a decrease in methane emissions of the cow rumen microbiota, which was reflected in the metatranscriptomics data. Most notably, lower expression levels of genes encoding for proteins involved in methanogenic pathways of the rumen archaeon Methanobrevibacter smithii was observed. The last chapter, the general discussion, mainly discusses the research described in this thesis with a focus on the relevant issues with modelling microbial communities, as well as overall scientific integrity in relationship with microbiome research.</p