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Gut Microbiome Lynda Britton, Ph.D., MLS(ASCP)CM, SM Professor of Clinical Laboratory Science [email protected]
Objectives
• Define the gut microbiome and the testing of it.• Describe the effects of the gut microbiome on
various disease states and conditions.• Discuss causes and treatment of dysbiosis.
Microbiome• Humans have 10 x more bacteria
than human cells: hundred trillion• Changes responsible for digestive
disorders, skin diseases, gum disease, diabetes, cancer, obesity, etc.• Humans = ~ 20,000 genes• Microbiome = 10 million bacterial
genes
DefinitionsTerm Definition
Richness the number of distinct members ("species") in the community
Diversity a measure of the richness and evenness characteristics of a community, oftencalculated as a specific "diversity index"
Dysbiosis a term used to refer to a microbiota community associated with a diseasedstate that can be differentiated from the microbiota community associatedwith a healthy control state
Colon Ecosystem
• 100 trillion, majority large intestine• 1 gram = 100 billion organisms• 107 and 1012 species per 1 g of stool
• Majority belong to Firmicutes & Bacteroidetesphyla• Many uncultivatable--described molecularly
Essential Functions• Fermentation of indigestible food
components• Synthesis of essential vitamins• Removal of toxic compounds• Outcompetition of pathogens• Strengthening of the intestinal barrier• Stimulation and regulation of the
immune system• Nutrient metabolism• Xenobiotic and drug metabolism
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Host-microorganism Balance
• Promotes health• Prevents disease• Cancer, neurological, ophthalmological,
premature newborn, extra-intestinal, intestinal—inflammatory bowel disease• Metabolic disorders: non-alcoholic fatty liver
disease, type 2 diabetes, insulin resistance, obesity
Bacterial Phyla
Firmicutes
Bacteroidetes
Actinobacteria
Protebacteria
Other
Bacteroidetes vs FirmicutesBacteroidetes• Bacteroides ssp.• Gram negative• Genes linked to
carbohydrate metabolism
Firmicutes• Clostridium ssp.• Gram positives• Contain genes associated
with nutrient transporters• More efficient absorption
of calories
Actinobacteria Phylum
• Contains Bifidobacteria• Minor but essential component
• Vitamin production, protection against pathogens, regulation of immune system and lactose utilization
• Cross-feeding other members specializing on production of butyrate
• Butyrate essential for colon epithelial cells with anti-inflammatory and anti-cancer properties
How Studied:
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Next Generation Sequencing MethodsTechniques used in next generation sequencing
Accur-acy Advantages Disadvantages
454 Pyrosequencing 99.9%Less amount of sample, long read lengths, large number of samples can be easily read
Homopolymer errors Expensive
Shot gun Sequencing 98% Short reads in short time Assembly process is computationally expensive
Illumina Sequencing (Sequencing by synthesis) 98% Accurate, quicker, reliable and cheap Expensive
Pacific Bio Sequencing (single molecule real-time sequencing)
99.9% Fast and provides long read length Expensive equipment
Ion Torrent Sequencing (Ion semiconductor) 98% Fast and less expensive equipment Multiple monomer errors
SOLiD (Sequencing by Ligation) Sequencing 99.9% Less expensive when compared to
other methodsSlow and difficult to sequence palindromes
Metagenomics and Pyrosequencing
• 16S rRNA• 80% of the bacteria found by molecular tools in the
human gut uncultured• 106 concentration• Lacks sensitivity to delineate different close species• Neglects some gram negatives
Pyrosequencing• Tool that facilitates genotyping and
quantitative examination of sequence variation• Advantages for microbiology:
• Rapid and reliable high-throughput screening • Accurate identification of unknown mutations • Sequence context provides built-in quality control
of assay • No need for expensive labels and dyes • Significant time and cost savings
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Culturomics
• 3rd shift in understanding• MALDI-TOF• Diversity of culture conditions—70 different
• Atmosphere• Time• Temperature• Filtration• Antibiotics
• 15% concomitantly detected by both metagenomics and culture
CulturomicsMicrobiome2018 6:94
Taxonogenomics• Targets proteomic, phenotypic, and genotypic traits• Species considered novel:
• MALDI-TOF score <2 • 16S rRNA gene sequence similarity < 98.7%
• Genome of new species sequenced and comparison with close species:• DNA G+C content, size, gene content, percent of coding
sequences,, number of genes coding for RNA, mobile genetic elements, transmembrane helices, signal peptides
Ability to grow, resistance, sporulation, biochemical
Microbiome 2018 6:94
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Vironomics
• Difficult to study: no shared gene• Cell culture• Electron microscopy• PCR• Metagenomics
• Eukaryotic and prokaryotic viruses• Giant viruses—visible in light microscopy—not
filtered—can be infected by other viruses
Fungal Diversity
• ID—MALDI-TOF• 18S rRNA internal transcribed spacer • Smaller numbers, poor data base• Includes microsporidia• Candida most common• Others food contamination or
additives?
Limitations
• Conflicting results• Duration, doses, interventions• Technology—16S vs sequencing vs culture • Sequencing
• More expensive• Increased resolution, more specific & functional
classification, new genes and genomes
Microorganisms. 2019 Mar 3;7(3). pii: E68. doi: 10.3390/microorganisms7030068
Leaky GutIncreased Intestinal Permeability
• Intestinal lining is weakened• Enables bacteria and toxic products to enter
bloodstream• Immune system’s attacks on these bacteria could
injure the body’s own tissues—autoimmune• Associated with persistent inflammation and chronic
illnesses• LPS leakage increases inflammation
Frontiers in Immunology, May 2017
Diseases • Crohn’s, HIV, irritable
bowel syndrome, celiac disease, and type 1 diabetes
• Allergies, asthma, acne, obesity, mental illness
• No clinical trial to prove cause and effect
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Probiotics
• Live microorganisms • Confer a health benefit to the host when
administered in adequate amounts and components (strains of bacteria)• Dead bacteria and bacterial molecular components
may also exhibit probiotic properties• May improve mental health• Reduce absorption of mercury and arsenic
Probiotics
• May correct changes caused by antibiotics or cesarean birth
• Inhibit expansion of gram negative pathogens• May lower serum lipids• Improve liver function and hepatic fat accumulation• Prevent and/or reduce duration and complaints of
rotavirus or antibiotic-associated diarrhea• Improve insulin sensitivity and inflammation
Probiotics• Alleviation of complaints of lactose intolerance• Reduction of concentration of cancer-promoting
toxins• Beneficial effects for inflammation and other
complaints:• inflammatory diseases of Gi tract• Helicobacter pylori• bacterial overgrowth
• Normalization stool consistency in IBD
Probiotics• Prevent or alleviate allergies and atopic diseases in
infants• Prevention of respiratory tract infections and
urogenital infections• Hypocholesterolemic effect to prevent cancer• Improvement of mouth flora and caries• Prevention or prevention or therapy of ischemic
heart disease• Amelioration of autoimmune diseases
Prebiotics
• Non-viable food component-fermentable dietary fibers• Confers a health benefit to the host • Modulates intestinal microbiota • Improves intestinal permeability
• Decrease plasma LPS and cytokines• Decrease ALT and AST levels• Reduce accumulation of fat
Synbiotics
• Combination of prebiotics and probiotics• Stimulate growth or activate
metabolism of healthy bacteria• Reduce TNFα, CRP, endotoxin, fasting
glucose, AST• Affects untested in RTCs
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Fecal Microbiota Transplants (FMT)
• Treatment of Clostridioides difficile• Ingested through gastric tube• Enema • Capsulized—freeze dried
Fermented Dairy Products Study
• 150 healthy adults—30 days yogurt drink• 16S rRNA sequencing• Increased Bifidobacterium genus, Adlercreutzia
equolifaciens and Slackia isoflavoniconvertens• Low levels of Bifidobacterium associated with
adverse clinical conditions • Abundance can be increased by consuming
fermented dairy products with live cultures
Nutrients. 2019 Mar 4;11(3)
Other Fermented Foods More Fermented Foods
Biodiversity Hypothesis• Contact with natural environments enriches the
human microbiome• Promotes immune balance• Protects from allergy and inflammatory disorders• Outer layer (soil, natural waters, plants, animals)
and inner layer (gut, skin, airways)• Determinants of immunotolerance key for
prevention and more effective treatment• Biodiversity loss with global climate change
Impact on Immunity
• Germ-free mice have poorly developed IS• GM critical to modulate Th2-biased immune
response• Differentiation of Th1, Th17, and regulatory T ()
Tregcells• Treg controls inflammatory response and microbes
promote them
Am J Transplant. 2018 Mar;18(3):550-555
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Obesity• Worldwide• Dietary, environmental, educational, genetic• Antibiotic exposure • Obese lower diversity•↓ in Bacteroidetes and ↑ Firmicutes• Efficiency of microbes to harvest energy• Fat accumulation from altered metabolism• Levels of short chain fatty acids• GM strong association in mice but not human
Obesity and GM
• Produces short-chain fatty acids that regulates energy harvest• Modulates signaling pathways of energy
metabolism• Promotes absorption of monosaccharides
• Accumulation of triglycerides in adipocytes• Insulin resistance
Pre-Pregnancy BMI vs Late Pregnancy Early Infancy GM• Normal weight and obese women more diverse than
over weight•↑ Bacteroides in overweight• Pre-pregnancy BMI GM affects maternal and infant
• Increased risk of childhood obesity• Vaginal birth more GM diversity—obese 2-4 time
more likely to get C-section• Differs in breast milk fed—obese women produce less
milk
PLoS One. 2019 Mar 18;14(3):e0213733
Firmicutes to Bacteroidetes Ratio
• Obese higher Firmicutes and less Bacteroidetes• 20% increase in Firmicutes and 20% decrease in
Bacteroides = addition energy of 150 kcal/day• Inverts after 1 year diet or gastric bypass• No clear trend in F/B ratio and obesity• Much more complex• Association studies weak
Twin Obesity Study• Adult 31 MZ and 23 DZ lean/obese pairs and
mothers• Impact of genotype and early environmental
exposures• Same family shared similar bacteria• MZ and DZ varied the same• Lower proportion of Bacteroidetes and higher
Actinobacteria in obese versus lean EA and AA• Obesity associated with less diversity
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Infant GM Associations: Lack of Bifidobacterium infantis• Necessary to digest HMOs• Pre-pregnancy BMI, C-section & formula feeding• 33.3% of C-section born infants were exclusively fed
human milk• 80% of vaginally born infants of overweight women
and 50% of vaginally born infants of obese women were exclusively fed human milk
• Greater risk for allergies, overweight/obesity, asthma, allergies and other chronic diseases if lack B. infantis
Aug 13, 2018 | Laboratory Management and Operations
B. infantis Importance in Infants• Production of short-chain fatty—provide energy and
help control yeast and fungus growth• Support gut integrity--signals gut cells in infants to
generate proteins that fill gaps between intestinal cells• Keeping undesirable bacteria at bay• Release sialic acid—crucial nutrient for brain
development • Production of folate-- necessary for infant
development and growth and creation of RBCs
Obesity Can Influence GM• When obese diet and lose weight, Bacteroidetes
increases• When obese resume their previous diets and gain
weight, Firmicutes increases• Reflect and cause • Low-grade gut inflammation caused by GM may
increase the risk of obesity and type 2 diabetes• Activation of TLR4 by bacterial LPS causes release of
pro-inflammatory cytokines that promotes insulin resistance
JAMA. 2017 Jan 24;317(4):355-356
Gut inflammation
• Weakens epithelial tight junctions in the gut mucosa--entry of bacterial LPS
• Increased activity of the innate immune system• Leads to insulin resistance and weight gain• Type 2 diabetics have ↓ butyrate-producing species,
leading to low-grade inflammation in the gut• No single spp--but ↑ Akkermansia muciniphila reduces
inflammation in adipose tissue and improves insulin signaling
Type 2 Diabetes
• Gut production of SCFA butyrate associated with improved insulin response in GGT• SCFA propionate increased risk of T2D• Insulin sensitivity ↑ after FMT from lean donors
Type 2 Diabetes Mellitus and GM
• Increased risk of type 2 diabetes in obesity• Also influence type 2 diabetes• More than enhanced absorption of carbohydrates• High ratios of Firmicutes to Bacteroidetes also alter the
production of short-chain fatty acids• Acetate production is increased and butyrate production
decreased• Increased blood levels of acetate cause insulin resistance
and increase production of ghrelin (the appetite-stimulating hormone)
• Lower butyrate levels --low-level inflammation, which evokes insulin resistance
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Evidence in Type 2 Diabetes
• Men with metabolic syndrome had GM eliminated• Randomized to receive their own feces or a lean
donor• Lean recipients increased insulin sensitivity that
decreased over time and varied• Due to more butyrate producing bacteria• GM influenced by genetics
Type 1 Diabetes Mellitus
• 7000 children study linked the use of probiotics during the first month of life to a lower risk of islet autoantibodies• GM may play a role in type 1 diabetes
JAMA Pediatr . 2016;170(1):20-28
GM and Arteriosclerosis and CV Disease
• Metabolite released by GM trimethylamine N oxide• Promote inflammation, arteriosclerosis & immune
response• Directly increases platelet hyperactivity
(thrombosis)• May promote Alzheimer’s disease• Normalization of GM may prevent
Cell. 165, 111–124 (2016)
Differences in Cardiometabolic Diseasnand GM
Mol Metab. 2018 Sep; 15: 70–81.
Nonalcoholic Fatty Liver Disease (NAFLD)
• Most common cause of chronic liver disease• Cirrhosis or hepatocellular carcinoma• Gut derived endogenous alcohol• Decrease in Firmicutes or abundance of
Bacteroidetes• Insulin resistance accelerates fat accumulation
and inflammation in hepatocytes
NAFLD
• Choline prevents accumulation of lipids in liver• GM converts choline to toxins transported to liver• Choline deficiency leads to liver steatosis• Toxins converted to trimethylamine oxide (TMAO)• Leads to inflammation and liver damage• Cardiovascular disease• Dietary choline influences GM
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Microbiota-Gut-Brain Axis
• Do microbes influence happiness?• What components are critical?• What role do environmental pollutants play?
GM and Neurodevelopmental and Neurodegenerative Disorders• Linked to depression, autism and Parkinson's
disease• Causal or secondary effects?• Different communication routes between tGM and
brain have been identified• Further studies needed to ID all the underlying
mechanisms involved• Transplantation may modulate
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Mood Disorders
• 10% of world population• Bidirectional communication• Neuroimmune and neuroendocrine pathways• IBS patients improve GI with psychotropic
treatment• GM regulates hippocampal serotonergic system
Dementia and GM• Lower presence of Bacteroides and higher of
others• Bacteroides can regulate endothelial cell function• Reduce inflammation
• GM and systemic arteriosclerotic disease suggests common underlying mechanism• disruption of the neuro-inflammatory system may
be caused by gut microbes --could lead to deposition of amyloid β
Scientific Reportsvolume 9, Article number: 1008 (2019)
Exercise and GM
• Independently alters composition and function • 6 weeks of endurance exercise • Inflammatory bacteria ↓• Increased Akkermansia• Decreased phospholipids and cholesterol
Exerc Sport Sci Rev. 2019 Apr;47(2):75-85
Can GM Affect Sleep?
• Levels of bacteria that produce serotonin and gamma aminobutyric acid (GABA)
Consumer Technology Association 2019 Digital Health Summit
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Pharmacokinetics
• Absorption• Probiotics enhance some
• Metabolism• Digoxin inactivated• Lovastatin reduced
• Antidepressants alter GM • Antibacterial
• Alcohol increases gut permeability and endotoxins
Cancer Immunotherapy• Only 30% respond to immune checkpoint inhibitors• High fiber diet improves response 5X
• Fruit, vegetables, and whole grains• Sugar & processed food diet negatively associated
• More diverse gut microbiome• Taking probiotic supplements = less diversity• Taking antibiotics = nonresponse• Akkermansia muciniphila
Autoimmune Diseases and GM
• Enterococcus gallinarum in mice• Moves from gut to lymph nodes, live, spleen• Initiates autoimmunity and inflammation• Suppressing growth eliminated autoantibodies and
T cells• In mice, blocking inflammation (antibiotics and
vaccine) blunted effect on immune system
Science 09 Mar 2018: Vol. 359, Issue 6380, pp. 1156-116
GM Combine to Cause Colon Cancer?
• Induce CC or drive precancerous cells toward CC• Bacterial toxins damage DNA in epithelial cells• Patients with no genetic risk vs familial
adenomatous polyposis• Biofilms of B. fragilis & E. coli• Fusobacterium also implicated
https://www.nytimes.com/2018/02/01/health/colon-cancer-bacteria.html
Can We Alter GM?
• Not resolved• Must create niche• Affected by diet, gut motility, infections, immune
function, antibiotics• Some diets alter GM by providing prebiotics• Mediterranean• Fermented dairy product
• ↑ lactose, amino acids, ↓lipopolysaccharides
Nutrients 2019, 11, 547.
Can We Alter GM?• Still learning what is healthy• No one food or perfect cocktail—baking perfect cake
with 5000 ingredients• Limit sugar and simple carbs• Eat complex grains• Side effects?• Influenced by genes• Resilient and difficult to change from diet unless
complete removal of carbs
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Carboxymethylcellulose (CMC) and Polysorbate 80 (P80)
• Synthetic emulsifiers added to a variety of processed foods • Alter mucus and promote bacterial translocation• Chronic gut inflammatory disease by changing
GM gene expression and composition
Antibiotics and GM Study
• Young healthy men—3 antibiotics for 4 days• Almost complete eradication of gut bacteria• Gradual recovery of most species over 6 months• Missing 9 of their common beneficial bacteria • New and potentially non-desirable bacteria had
colonized the gut• Western populations less diversity than natives
Nature Microbiology: 3(11) · October 2018
Mayo Study
•↑ abundance of Phascolarctobacteriumassociated with successful weight loss•↑ abundance of Dialiste and genes encoding gut
microbial carbohydrate-active enzymes associated with failure• Gut bacteria breakdown food to provide energy• Individualized plans based on GM?
DOI: https://doi.org/10.1016/j.mayocp.2018.02.019
Comparison of Mediterranean and DASH Diet on Cognitive Performance• Mediterranean less decline• A Priori Diet Quality Score [APDQS] less decline• 46% to 52% lower risk• DASH no change• Antioxidants, polyphenols, omega-3• Fruit, vegetable, legumes high• Nuts, fish, alcohol moderate• Meat low
Neurology: March 6, 2019, DOI: https://doi.org/10.1212/WNL.0000000000007243
Mediterranean Diet
• May:• Improve cardiovascular
health• Significantly reduce risk of
stroke• Lower risk of Alzheimer's
disease, cancer, Parkinson's and rheumatoid arthritis
Plant Polyphenols
• Quercetin, catechin & puerarin• Catechin most intense inhibitor• Inhibit growth of Firmicutes & ↑ Bacteroidetes• Change pathway of degrading fructose-
oligosaccharide or energy metabolism• Up-regulated serum leptin• Changed certain genera
Food Funct. 2016 Mar;7(3):1501-7
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Comparison of 2 Diets• New Nordic Diet high fiber/whole grain vs Average
Danish Diet• 2 enterotypes: Prevotella ÷ Bacteroides• High P/B ratio larger weight loss with NND• Low P/B ratio didn’t matter what diet and lost less
body fat• Prevotella higher total short chain fatty acid
production—stimulate appetite hormones• Only 2 of 182 changed ratio over 6 months of diet
International Journal of Obesity volume42, pages580–583 (2018)
Dietary Modification for GERD• 85 LPR sufferers who were treated with PPIs• 99 LPR sufferers who were put on a Mediterranean diet and
drink alkaline water• Americans spend $13 billion a year on PPIs• Affects ~20 % U.S. population• Consuming a Mediterranean diet may be as effective as
PPIs• Avoiding processed foods and sugar, eating real food and
beneficial bacteria from fermented foods or high-quality probiotic
• Apple cider vinegar, baking soda, ginger and slippery elm
JAMA Otolaryngol Head Neck Surg. 2017 Oct; 143(10): 1023–1029
Dangers of Long Term Use of PPIs• Chronic kidney disease: 10,000 participants tracked in 12-
year study• Death from any cause: 350,000 U.S. veterans over 6 years—
20% more likely to die & longer the use , the greater risk • •Heart disease and heart attacks: 3 million study--increased
risk for heart disease and heart attacks • — even if you have no prior history• 16% increased risk of heart attack, and 2X increased risk of
cardiovascular mortality.• Increased risk of bone fractures: 80,000 postmenopausal
women--hip fracture • Vitamin B12 deficiency: use for 2 or more years."
Conclusions
• Much more study needed• Conclusions contradictory depending on methods• Part of individualized medicine?