CHAPTER 2: MICROBE-HOST AND MICROBE-MICROBE INTERACTIONS
1. MICROBE-HOST INTERACTIONS
THE HUMAN MICROBIOME – A MUTUALISTIC INTERACTION
TOPIC 1: THE HEALTHY MICROBIOTA
1. Introduction
Definitions
• Microbiota
- Sum of all microbes that reside in or on a host or a specified part of
a host. Includes bacteria, archaea, eukaryotes (funghi) and viruses.
• Metagenome
- Genes and genomes of the microbiota. Provides information about
the functional genetic potential of the population.
• Microbiome
- The totality of microbes, their genetic information, their products
and the milieu in which they interact.
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,An historical perspective
• Traditionally:
- Normal microbiota: black box – many (99%) bacteria unculturable. We cannot easily cultivate them in
the lab, not strongly studied.
- Focus on role of specific microbes in disease, we could already cultivate them. Till 20 years ago
microbes in the body will mostly seen as something negative.
• In the past 15 years:
- Field of micobiota research has exploded
- DNA-based analyses: enormous new data sets
- Appreciation of the beneficial influence of microbiota on human health.
• Man is Superorganism
- Ratio microbes/human cells: 1,3/1
- number of bacteria in reference man: 3,9. 1013
- number of human cells 3.1013
- Ratio microbial genes/human genes: 100
- 25 000 human genes, 2-20 million microbial genes
COMPOSITION HMP & FGMP
2. Microbiome composition of ‘healthy’ humans
The human microbiome project
• By US National Institutes of Health ($175M; 2007 - …) - ~250 researchers
• Study of compositional range of the normal microbiome of healthy individuals
- Healthy: they had a whole list of exclusion criteria what healthy means
• 4,788 samples – 300 US individuals – 18 body habitats (5 major areas)
• 16S rRNA gene analysis
- Determine gene level classification of the microbes and species classification, not go to the strain level
• shotgun metagenomic sequencing (subset of 681 samples)
- determined the whole genomes of microbes that were present
- the strain level classification and the function potential
Variation in microbiome composition
• Variation among anatomical sites (A)
• Interpersonal variation (B)
• Temporal variation (C)
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,(A) Microbiome: effect of anatomical site
• Abundance: size of the circle, intensity of the colour: indicate prevalence, in which percentage of the
population is a certain gene present.
• <10 of 50 phyla represented
- This means that many microbes not occupier in our body
• No taxa universally present among all habitats and individuals
- They didn’t find a core microbiome
• Each body habitat in most subjects characterized by few signature taxa making up plurality of the
community
- Signature clades at genus level form on average from 17% to 84% of their respective body habitats
- Anatomical site: primary determinant of microbiome composition. The variation is the largest among
anatomical sites.
• Microbial alpha-diversity is dependent on anatomical site:
- The alpha diversity is the diversity within a body site
- e.g. oral cavity: high diversity; vagina: low
(B) Microbiome: interpersonal variation
• Substantial interpersonal variation in
microbiome composition
- Inter-individual variation in the
microbiome proved to be specific,
functionally relevant and personalized.
• E.g. variation in relative abundance of
Streptococcus species in the oral cavity
(tongue)
- The height of the bares: The relative
abundance of Streptococcus species
highly variable, the height of the bares
is different
- The colour of the bares: different
species within the genes of
Streptococcus species
- Variation because selective pressure is acting on pathways differentially present among Streptococcus
species? Probably because the environmental condition on the tongues of the different individuals will
also be different. They will be adapted to grow under different conditions.
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,• Other examples of relevant interpersonal variation:
- Bacteroides fragilis (role in priming T-cell response): >0,1% in 16% of Samples
o prime T-cell responses in animal models via the capsular polysaccharide A18, and in the HMP
stool
o this taxon was carried at a level of at least 0.1% in 16% of samples (over 1% abundance in 3%).
The majority of the humans doesn’t have Bacteroides fragilis in their gut.
- Staphylococcus aureus (cause of MRSA): 29% nasal and 4% skin carriage
o cause of methicillin-resistant S. aureus (MRSA) infections,
o had 29% nasal and 4% skin carriage rates, roughly as expected. The majority of the humans
doesn’t have Staphylococcus aureus in their nose of in their skin.
(C) Microbiome: temporal variation
• HMP: 131 individuals sampled at additional time
point
➔ Within-subject variation over time
consistently lower than between-subject
variation
➔ Uniqueness of each individuals microbiome is
stable over time
• -Day-to-day variation of gut microbiome (stool) of
two healthy individuals donor A an donor B. Over
a period of more than 350 days.
- clear distinction between individuals (look at
the different abundance and colours)
- temporal stability
- only strong perturbations caused changes:
o travelling/dietary changes, infections, antibiotics, …
Taxonomic versus functional diversity
The metagenome was sequenced and they tell more about the functions that are present.
Alternative representation: different taxa in the different body areas. Then every little bar is one individual, so
you see the variation in the individuals. And all the different colours indicate certain metabolic functions.
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,• Unlike taxa, several core functions are ubiquitous among individuals and body habitats at a high
abundance
- You have something as a core metabolic, core function
- i.a. ribosome and translational machinery, nucleotide charging and ATP synthesis, and glycolysis
• Unlike taxa, only few core functions are highly variable among subjects within any body habitat
• Greater variability in niche-specific functions of rare but consistently present pathways
- Niche specific functions: only occur in a certain habitat, these are present in much lower abundance
‘rare’, but still consistently present. Much higher variation between individuals.
• long tail’ of low-abundance genes and pathways probably encodes much of the uncharacterized
biomolecular function and metabolism of these metagenomes
- many genes are present in low abundance, but they are still consistently present, but at different
abundancy between individuals.
Our metagenome is more unique than our genome
• Any 2 humans: 99,9% identical genomes
• e.g. 2 E. coli cells can have genomes that are 40% different
• Strong and stable interpersonal variation in metagenome composition
3. Zoom in on the gut microbiome
Gut microbiome: composition
• 500-1000 species: verry large diversity
• Anaerobes (oxygen depleted conditions): 100 to 1000-fold more abudant than aerobes (need oxygen)
/facultative anaerobes (normally grow in oxygen conditions but can also grow in the absent of oxygen)
• Firmicutes and Bacteroidetes are most abundant phyla ; inverse association
- When you have more of the one, you have less of the other
• Adult intestine 1014 bacterial cells – more than 5 million genes
• Enormous metabolic capacity: considered as additional organ
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,• Three gradients
- Longitudinal: from the proximal part of the gut to the distal part
o Microbial density increases from the proximal to the distal gut: the stomach contains 101
microbial cells per gram of content, the duodenum 103 cells per gram, the jejunum 104 cells per
gram, the ileum 107 cells per gram and the colon up to 1012 cells per gram
o Increasing of diversity: The identity of the microbes are also different.
o Reason why we see this diversity of microbes when you go from the stomach to the colon?
1) Different electron donors = nutrient, will be different from the small intestine
Small intestine: degradation products of food components as our human enzymes will
breakdown. Like the breakdown of carbohydrates into simple sugars, the breakdown to
amino acids and fatty acids. The breakdown product can be absorbed by the small
intestine, but that will also be used by the microbes
Column: there are many types of components in our food (such as fibres, glycans, …) that
actually are not digestible by our own cells but that can be fermented by microbes.
2) Different electron acceptors: presence or absence of oxygen
Small intestine: Because the epithelium is not completely impermeable to oxygen, small
amounts of oxygen will pass towards the guts. On the other hand cells in the small
intestine will produce nitric oxide. The nitric oxide can react with certain reactive oxygen
species to form nitrate. Than nitrate can be used as an electron acceptor by most
facultative anaerobes. So that's why we mainly see facultative anaerobes in the smaller
testing
large intestine: Because the barrier is much more strong, there is no oxygen passing the
epithelium. There is no oxygen or nitrate. That's why you mainly have fermentation, with
production of short chain fatty acids and production of lactate in the large intestine.
Secondary fermenters can use these fermentation products. They use Iron 3+ as an
electron acceptor.
- Axal: from the epithelium towards the lumen
o Microbial density increases along the tissue–lumen axis (with few bacteria adhering to the tissue
or mucus but a large number being present in the lumen) Many bacterial species are present in
the lumen, whereas fewer, but well-adapted species, including several proteobacteria and
Akkermansia muciniphila, adhere and reside within the mucus layer close to the tissue.
- Time: from the birth to the death
o Colonization of the host begins during birth, and the composition of the microbiota changes
throughout host development. In the adult intestine, a total of about 10 14 bacterial cells are
present, which is ten times the number of human cells in the body.
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,3 distinct gut enterotypes
• An enterotype is a classification of living organisms based on the bacteriological composition of their gut
microbiota, groups of microbiomes that are more related to each other, strongly related to the diet
• Well-balanced, defined microbial community compositions of which only a limited number exist
• Not as sharply delimited as, e.g. blood groups; but densely populated areas in a multidimensional space of
community composition.
BENEFITS FOR MICROBES AND HOST
A mutualistic association
• Mutualism: mode of symbiosis which is beneficial for both organisms
• Host offers to the microbiota:
- nutrient-rich environment
- maintained at constant temperature
• Microbiota offers to the host, e.g.:
- metabolism of otherwise indigestible polysaccharides
- production of essential vitamins
- protection against invasion by opportunistic pathogens
- factors required for the development and differentiation of the host’s intestinal epithelium and
immune system (see next)
- key role in maintaining tissue homeostasis (see next, not only in the gut)
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,Microbial impact on host physiology
• intestinal function
- The microbiota has been shown to influence intestinal function in the host, promoting gut-associated
lymphoid tissue (GALT) maturation, tissue regeneration (in particular of the villi) and gut motility
(peristaltic activity), and reducing the permeability of epithelial cells lining the gut, thus promoting
barrier integrityIntestinal vessel formation
• Metabolism
- In the case of host metabolism, the gut microbiota has been shown to facilitate energy harvest from
the diet, to modulate host metabolism (for example, by decreasing energy expenditure) and to
promote host adiposity.
• Homeostasis of other types like bone
- the gut microbiota can influence tissue homeostasis, for example decreasing bone mass by promoting
the function of osteoclasts (which cause bone resorption) and increasing the numbers of pro-
inflammatory T helper 17 (TH17) cells
• Behaviour (gut brain access)
- Much of this is study by using mice where mice doesn’t have gut microbes and with other gut
microbiota. Strong impact of the behaviour.
- the gut microbiota can influence the host’s nervous system, decreasing synaptic connectivity and
promoting anxiety-like behavior and pain perception.
• Important role of comparative studies between germ-free and conventionally raised mice
• Note: timing of colonization of germ-free mice is crucial to recapitulate the phenotypes of conventionally
raised mice. E.g. (early life) colonization before weaning is required to restore behavioural defects
- To understand which cause what, the disease cause the gut microbiota or the gut microbiota causes
the disease?
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,SHAPING FACTORS
4. Factors shaping the gut microbiome
• The composition of the gut microbiota
is influenced by various environmental
factors, including the use of antibiotics,
lifestyle, diet and hygiene preferences.
• The host’s genetic disposition also has
a role:
- -hyperimmunity (owing to over-
representation of pro-
inflammatory mediators such as
interleukin-6 (IL-6), IL-12 or
tumour necrosis factor (TNF))
- immunodeficiency (owing to
mutations in regulatory immune
proteins such as NOD2
(nucleotide-binding
oligomerization domain protein 2)
or IL-10) can influence the gut
microbiota composition. In turn,
dysbiosis affects levels of immune mediators and induces both chronic inflammation and metabolic
dysfunction.
Population level analysis of gut microbiome variation: The Flemish Gut Flora Project
• Effect of host and environmental factors on gut microbiota variation in healthy population?
• Belgian Flemish Gut Flora Project (1106 healthy individuals) + 503 metadata variables (medication, health,
lifestyle, diet, …)
• Core microbiota in the gut of 20 genera (i.e. shared by 99% of the samples) only at the genus level
• 69 factors that correlate with composition and diversity of gut flora: n° 1 = stool consistency (only
correlations, doesn’t know which impact to what, the causation we can’t know)
• However, only 7% of variation explained; not all gut bacteria identified yet
• Medication has the largest effect, illustrate how researchers tried to find links
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, ORIGIN
5. The microbiome in early life: implications for health outcomes
Factors influencing the microbiome in early life
• Prenatal factors
- Culture-based and culture-independent
studies have questioned the idea of
whether the uterus is sterile, and they have
suggested that microbes are present in the
placenta, amniotic fluid, fetal membrane,
umbilical cord blood and meconium.
Maternal gut microbiota could be
translocated to the fetus via the
bloodstream, a hypothesis supported by the
detection of green fluorescent labeled
Enterococcus faecium in the amniotic fluid
and the meconium of orally inoculated
mice.
- A recent study has also reported the
presence of low levels of bacterial biomass
in human placenta and has identified a
nonpathogenic microbiota similar to that of
the oral cavity and the authors hypothesize
that the bloodstream could be the route to
deliver maternal oral bacteria to the fetus.
- However, the presence of bacteria is associated with pregnancy risks, and nearly 25% of preterm
infants are born to mothers that had an intrauterine infection and occult microbial invasion of the
amniotic cavity. The bacteria detected were in many cases common vaginal residents, suggesting that
the uterine microbiota derives from vaginal infection, at least in preterm deliveries.
- Further studies will be required to confirm the existence of a viable intrauterine-resident microbiota,
quantify its variability and determine how it might affect the future development of the newborn.
• Mode of delivery
The first major exposure of the newborn to microbes happens during the birthing process and is highly
dependent on mode of delivery
- Vaginal delivery
o The skin, gut, and oral and nasopharyngeal cavities of vaginally delivered infants are initially
enriched in Lactobacillus spp., which resembles the maternal vaginal microbiota.
- C-section
o In contrast, the skin, mouth and gut of children delivered by C-section lack this inoculum and are
instead colonized by common skin and environmental microbes such as Staphylococcus,
Streptococcus or Propionibacteria
- This initial microbiota evolves over time, adapting to the physicochemical and biological characteristics
of each body site, and is shaped by the availability of different nutrients. Although these differences
gradually decrease between vaginally delivered infants and C-section-born infants, a bacterial signal
remains associated with C-section-delivered infants until 12–24 months of age. This suggests that early
colonization provides a competitive advantage to the bacterial communities associated with each
delivery mode. Furthermore, C-section delivery has also been shown to delay the colonization of the
gut by specific bacterial taxa.
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