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Gut Microbiome Lynda Britton, Ph.D., MLS(ASCP)CM, SM Professor of Clinical Laboratory Science lbritt1@lsuhsc.edue

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?