Lectures 5-6: Microbiomes & Health

Microbiota

Ecological community of microorganisms

Commensal, symbiotic, pathogenic

Microbiome

Combined genetic material of the microorganisms in a particular environment

Human microbiome

10^14 commensal bacteria

10% human cells, 90% microbes

Microbiomes: hair, oral cavity, nostril, skin, vagina, stomach, colon, oesophagus

1 million+ genes in human microbiome

Human genome = 23,000 genes

99% genes are in microbial cells

Gut commensals

Abundance of microbiological matter in the intestine

2 kg of body weight/bacteria living on or in us

60% dry weight of faeces

Respiratory microbiota composition

Actinobacteria

Firmicutes

Proteobacteria

Bacteroidetes

Skin microbiota composition

Actinobacteria

Bacteroidetes

Cyanobacteria

Firmicutes

Proteobacteria

Oral microbiota composition

Firmicutes

Proteobacteria

Bacteroidetes

Actinobacteria

Fusobacteria

GI tract microbiota composition

4 dominant phyla:

Bacteroidetes (most abundant)

Firmicutes - lactobacillus, staphylococcus, streptococcus

Proteobacteria

Actinobacteria - corynebacterium, propionibacterium

Gut microbiota & digestion

Metabolic activities resemble an organ

Breakdown of complex carbohydrates

Production of short chain fatty acids (SCFAs)

Synthesis of vitamins - B12, K & folate

Influences calories harvested

Defends against harmful microorganisms

Influences susceptibility to GI infections

Present approaches to studying microbiota

Lungs are difficult to sample so there is minimal data

Next generation sequencing

Metagenomics

16S rRNA sequencing

Metatranscriptomics

Replaced early studies focused on culture-based approaches

Influences of intestinal microbiota composition

Immunity, lifestyle, antibiotics

Breast feeding, diet, exercise, disease, aging, drugs, geography, birth mode

Establishment of gut microbiome

Early childhood is critical

Amniotic liquid (placenta)

Cesarean or vaginal delivery

Antibiotic exposure

Breast milk and/or formula feeding

Maternal factors ie. nutrition, BMI, weight gain during pregnancy, microbiota

Bacteria exposure in vaginal birth

Lactobacillus

Bifidobacteria

Bacteria exposure in caesarean birth

Staphylococcus

Proponiobacterium

Bacteria exposure in milk consumption

Bifidobacterium

Lactobacillus

Veilonella

Acidic environment

Bacteria exposure in solid food introduction

Bacteroides

Clostridiales

Breastmilk vs formula feeding

Breast-fed infant has high population of bifidobacteria

First bifidobacterium isolated from healthy breast-fed infant in 1900 by Tissier

Bifidobacteria = one of first colonisers

Proteobacteria amounts

Healthy - 4.5%

Neonate - 16%

Gastric bypass - 9.7%

Metabolic disorders - 13.2%

Inflammation & cancer - 14.9%

High community stability in healthy host

Low community stability in diseased host

Germfree mice study

Absence of microbiota contributes to under-developed immunity

Germfree mice = defects in gut-associated lymphoid tissue (GALT) → decreased number of lymphoid follicles (Peyer’s Patches)

Decreases in:

Mucus/AMP secretion

Number of intestinal epithelial lymphocytes

Number of T cells & B cells in spleen

Expression of TLRs on intestinal epithelium

Intestinal epithelium

Largest mucosal surface (400m²)

Physical, biochemical & immunological barrier

Single layer of epithelial cells (IECs)

Enterocytes, paneth cells, endocrine cells, goblet cells

Enterocytes

Absorptive cells

Paneth cells

Produce AMPs

Goblet cells

Secretes mucin

Result of barrier defects

Microbial dysbiosis & dysregulated immune response

Decreased AMP, mucus, PRRs, TJ

Leaky gut, irritable bowel disease, inflammation, allergy, infection, food intolerance

Mucus layer

Contains glycoproteins (mucins), digestive enzymes, antimicrobial proteins & antibodies

Separates epithelium from gut lumen

Limits pathogen invasion

Thinner mucus layer in Germfree mice

Antimicrobial peptides (AMPs)

Small proteins of innate immune system

Found along intestinal epithelium

Produced by paneth cells in response to infection

Potent & broad spectrum

Antifungal, antibacterial, antiparasitic, antiviral

Decreased production (defensins) in Germfree mice

Intestinal microbiota dysbiosis & disease

Lung, colorectal, pancreatic & oral cancer

Heart disease ie. hypertension & atherosclerosis

IBD ie. Crohn’s disease & ulcerative colitis

Liver disease ie. cirrhosis & hepatitis

Chronic kidney disease

Type 1, type 2 & gestational diabetes

Parkinson’s, Alzheimer’s & depression

Respiratory disease ie. asthma & bronchitis

Bifidobacteria abundance

Indicator of health

Decreases w/ age (diversity in general too)

Hygiene hypothesis

Lack of early childhood exposure to infectious agents & symbiotic microorganisms increases susceptibility to allergic diseases by suppressing the natural development of the immune system

Westernised countries w/ intestinal microflora stability, high antibiotic use, low/absent heminth burden, good sanitation & low orofaecal burden = allergic disorders genes ie. asthma, aczema & rhinitis

Developing countries w/ livestock, intestinal microflora variability, low antibiotic use, high helminth burden & poor sanitation = non-allergic genes

Allergies & gut microbiota

Gut microbiota differs in composition w/o allergies

Higher levels of “bad bacteria” - clostridium difficile, staphylococcuus aureus

Lower levels of “good bacteria” = bacteroides, bifidobacteria

Microbiota trains immune system to respond properly to foreign antigens

Lack of exposure in early life → inadequately trained immune system → overreaction to antigens

Antibiotic use early in life

Salmonella typhimurium & antibiotics

Antibiotic treatment allows it to proliferate & induce inflammation

Pathogenic E. coli & antibiotics

Accumulates after antibiotic treatment in mice & crosses the barrier

Clostridium difficile & antibiotics

Present in low numbers in healthy adult

Antibiotic treatment in hospital patients leads to an increase & sever inflammation

Inflammatory bowel disease (IBD)

Linked w/ lack of breastfeeding, overconsumption of large amounts of sucrose & animal fat, use of antibiotics (especially childhood)

Ulcerative colitis, Crohn’s disease

Adherent-Invasive E. coli (AIEC)

Diet & enhancing gut microbiota

Dietary interventions & probiotic supplements to manipulate commensal bacteria

Introducing transiently colonising immunobiotic strains that produce AMPs

Re-establishing stable & healthy microbiota

Mediterranean vs western diet

Probiotic

Any bacterium that has health promoting activities when administered to human/animal host

Lactobacillus, lactococcus, proponibacterium, streptococcus thermophilus, bifidobacterium, bulgaricus

Probiotic characteristics

Non-pathogenic & non-toxic

Human origin

Resists gastric acid & bile

Attaches to epithelial cells

Temporarily colonises intestinal tract

Adapts to native intestinal microbiots

Beneficial effect on host

Probiotic uses

Irritable bowel syndrome

IBD

Infectious diarrhoea

Antibiotic-related diarrhoea

Taken w/ antibiotics

Faecal microbiota transplantation (FMT)

Intestinal microbiota transferred from healthy donor to patient to introduce or restore a stable microbial community in the gut

Non-profit stool bank expanding safe access to faecal transplants

FMT & Clostridium difficile infection

Dysbiosis via antibiotics use

C. difficile gains a foothold

FMT shows promising results

Recipient bacteria in stool resembles healthy donor within 2 weeks

Persists for up to 4 months

Environmental conditions in lungs

Warm

Moist

Rich in O2

Few cm from oral cavity

Chronic obstructive pulmonary disease (COPD)

Common lung disease associated w/ restricted airflow & breathing problems

Emphysema or chronic bronchitis

History of lung microbiome

Believed to be sterile until 2010 when airway microbiota was discovered

Relationship between pulmonary diseases & lung microbiome 2011-2012

Microbial immigration

Inhalation of bacteria, microaspiration & direct mucosal dispersion

Microbial elimination

Cough, mucociliary clearance, innate & adaptive host defenses

Cannot get rid of bacteria inside of macrophages

Regional growth conditions

Nutrient availability, oxygen tension, temp, pH

Conc. & activation of inflammatory cells

Local microbial competition

Host epithelial cell interactions

Unidirectional travel

Migration of microbes in one way & serially interrupted by widely varying physical & chemical barriers

Mouth to intestines

Bidirectional travel

Presence of vomiting or esophageal reflux

Mouth to intestines & intestines to mouth

pH of mouth, stomach & duodenum

Mouth: 7.4

Stomach: 2.0

Duodenum: 8.0

Orally introduced microbes have to endure acidic pH of stomach & alkaline pH of duodenum to travel to cecum

Temp of GI tract

37°C throughout 9 m of length

Gradient at point of inhalation to core body temp

Lung vs GI bacterial density

Duodenum has higher density than airways

Trachea, bronchi & gut are lined w/ heavily glycosylated proteins of secreted mucus

Vast majority of lung’s surface area is lined w/ lipid-rich surfactant that has bacteriostatic effects against select bacterial species

Lung vs gut host-bacterial interactions

Gut = higher luminal IgA levels
IgA, MALT

Lungs = more extraluminal interactions between bacteria & host leukocytes
Alveolar macrophages, IgE, BALT

Upper respiratory tract

Primary source of lung microbiome

Mouth, nose, nasal cavity, pharynx & larynx

Microorganism source in lungs

Nasopharyngeal aspiration

Inhalation

Reflux & aspiration

Bloodstream

Primarily oral - humans swallow 2 litres of saliva per day

Gram positive bacteria is less common in lungs

Healthy lung microbiome

Mainly transient microorganisms

Composition is determined by balance between microbial immigration & elimination

Coughing clears lungs (protected by respiratory cilia)

Low microbial density

Diseased lung microbiome

High microbial density

Impaired mucociliary clearance & dysfunctional cilia

Asthma, pulmonary fibrosis, bronchiectasis, cystic fibrosis

Respiratory diseases & lung microbiome

Lung microbiome is altered:

Cystic fibrosis & non-cystic fibrosis bronchiectasis
Chronic obstructive pulmonary disease
Idiopathic pulmonary fibrosis
T_H2-low & eosinophilic asthma

Healthy vs asthmatic lung

Healthy = majority bacteriodetes & firmicutes

Asthmatic = proteobacteria outgrowth & streptococci firmicutes increase

Healthy vs COPD lung

Healthy = majority bacteriodetes & firmicutes

COPD = increase in proteobacteria, streptococci & staphylococci firmicutes

Healthy vs cystic fibrosis lung

Healthy = majority bacteriodetes & firmicutes

Cystic fibrosis = increase in proteobacteria & actinobacteria

Cystic fibrosis (CF)

Shannon diversity = 1.90 (healthy is 4.06)
Reduced diversity compared to healthy

>75% proteobacteria

COPD lungs

Increased proteobacteria - Haemophilus spp. & Moraxella spp.

Exacerbated: decrease in actinobacteria & firmicutes
increase in proteobacteria & firmicutes

Gut-lung axis

Connections between the microbiota

Sampling airways

Sputum - spontaneous or induced saliva/mucus coughed up

Bronchoalveolar lavage (>140 ml)

Protected endobronchial brush

Bronchoscopic biopsy

Sterile tissue sample

Lower airway sampling

Invasive

>10³ cfu = number of colonies

Infrequent - 2 to 3 samplings

Potential contamination of samples via nasal or oral microbiota

Potential therapies for lung disorders

Predict & prognosis - test microbiome, predict disease via imbalance

Prevention - next gen of probiotics, prevent bacterial colonisation to solve drug resistance, microbiome dysregulation & healthcare associated infection

Cancer therapy - inhibiting ERK & PI3K pathways increases via antibiotic phage therapy & IL-17 therapy

Broncho alveolar lavage (BAL) samples

Healthy: contain predominance of bacterial taxa also found in mouth

Diseased: samples have numerous bacterial taxa not in the mouth → selective pressures in lungs for persistence, colonisation & growth