Novel insights into fibre and lipid substrates on the microbiome

Since the turn of the millennium, advances in sequencing technology have uncovered a plethora of microorganisms that live with and inside us. There are some indications that a considerable part of the environmental influence on human health and disease is mediated by this microbial community and it is recognised that diet massively dictates the structure and function of these symbionts. Identifying dietary components, therefore, that impact this internal community can reveal novel insights into how these symbionts affect and promote human health and well-being. This thesis investigated the effects of dietary fibre and lipid components on the human gut microbiota using in vitro, in vivo and in silico methods. Attempts were made to characterise the effects of these dietary components on gut microbiota composition from an alternative perspective in relation to human health; how they impact colonisation resistance against pathogens, influence bacterial taxa associated with metabolic disease and how they affect an overlooked proportion of the microbiota, the human gut virome. An extensive literature review was undertaken in Chapter 1 to examine how diet may impact colonisation resistance against invading pathogens, exploring how different food components and metabolites either protect or promote gastrointestinal pathogen colonisation. In Chapter 2 and Chapter 3, it was found that dietary fibres have differential effects on colonisation resistance against pathogens by employing an ex vivo model of the distal colon and assessing pathogen growth following 24-hour faecal fermentation. Glucose was used as a positive control to compare the success of pathogen growth in the presence of various pre-digested dietary fibres. Pathogen abundance was evaluated by 16S rDNA sequencing, qPCR and the effects on metabolic activity by short chain fatty acid (SCFA) analysis. We found that SCFA concentrations were increased in the presence of pathogens. The same ex vivo model was implemented, as described in Chapter 4 to study the effects of various dietary fibres and lipids on gut microbiota, using faecal samples collected from subjects with various stages of metabolic syndrome. Effects of microbial composition and diversity were assessed by 16S rDNA sequencing and SCFA analysis was performed for some of the treatments. Dietary fibre supplementation increased bacterial taxa negatively associated with metabolic disease including Bacteroides and Faecalibacterium. Fish oil treatment exhibited a decrease in pro-inflammatory Enterobacteriaceae and an increase in Bifidobacterium and Veillionella. A pilot study, described in Chapter 5, using faecal samples taken from a randomised controlled trial (RCT) on a dietary oily fish intervention was used to study dietary relationships with the human gut virome. The purpose was to evaluate how diet might influence this overlooked proportion of the microbiota and if oily fish intake selects for viral taxa negatively associated with autoimmune disorders. Metagenomic shotgun sequencing was performed on faecal samples taken from the RCT at baseline and at 8 weeks for 2 different fish types (sardine & tuna) eaten at either one portion or two portions of fish per week; these data were compared with a no fish intake group. The analysis was limited to diversity and viral load assessments, owing to the fact that a large fraction of our sequences had no matches in the viral database. There was a trend towards oily fish intake having a transient association with the human gut virome, which may be an artefactual signal from the high degree of inter-individual variation observed in humans. In conclusion, these results highlight the potential of dietary fibre and lipid food components in modulating microbiota composition. Further research is required to assess whether these changes in microbiota composition have a beneficial effect on human health

Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms

peer-reviewedStreptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties

Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy

peer-reviewedWith the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10−3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Effect of diet on pathogen performance in the microbiome

Intricate interactions among commensal bacteria, dietary substrates and immune responses are central to defining microbiome community composition, which plays a key role in preventing enteric pathogen infection, a dynamic phenomenon referred to as colonisation resistance. However, the impact of diet on sculpting microbiota membership, and ultimately colonisation resistance has been overlooked. Furthermore, pathogens have evolved strategies to evade colonisation resistance and outcompete commensal microbiota by using unique nutrient utilisation pathways, by exploiting microbial metabolites as nutrient sources or by environmental cues to induce virulence gene expression. In this review, we will discuss the interplay between diet, microbiota and their associated metabolites, and how these can contribute to or preclude pathogen survival

Strains used in this study and host range of phages ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03.

<p>Strains used in this study and host range of phages ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03.</p

Bacterial challenge test.

<p>The optical density (OD_600nm) was measured after 24 h of contact between <i>E</i>. <i>coli</i> strain DPC6051 and phage ɸAPCEc01 (a), phage ɸAPCEc02 (b), phage ɸAPCEc03 (c), and a cocktail of the three phages (d). *** p<0.001, * p<0.05.</p

One-step growth curves of phages ɸAPCEc01 (a), ɸAPCEc02 (b), and ɸAPCEc03 (c) with <i>E</i>. <i>coli</i> strain DPC6051 in LB broth at 37°C.

<p>One-step growth curves of phages ɸAPCEc01 (a), ɸAPCEc02 (b), and ɸAPCEc03 (c) with <i>E</i>. <i>coli</i> strain DPC6051 in LB broth at 37°C.</p

Transmission electron micrographs of <i>E</i>. <i>coli</i> phages ɸAPCEc01 (a), ɸAPCEc02 (b), and ɸAPCEc03 (c).

<p>The thin arrows in micrograph c indicate the 3 flexible fibres attached to the distal end of the phage tail. The terminal baseplate spike in c is illustrated by the thick arrow.</p