Human microbiota and

microbiota-based therapies

Shir-Ly Huang, PhD (黃雪莉)

sl.huang@nycu.edu.tw
Professor, Institute of Microbiology & Immunology
Professor, Institute of Food Safety and Health Risk Assessment

National Yang Ming Chiao Tung University

Taipei 112, Taiwan

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Microbiota (微生物相) 、
Microbiome (微生物體，微生物群系)

• A microbiota is an "ecological community

of commensal, symbiotic and pathogenic microorganisms
found in and on all multicellular organisms” studied to date
from plants to animals.

• The term microbiome describes either the

collective genomes of the microorganisms that reside in an
environmental niche or the microorganisms themselves.
• “The microorganisms that exist in a particular environment
or in the human body.”

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 Normal human microbiota
• Protection from pathogens
• Nutrition: Vit. K, B etc.
• Immunity development: commensal microbiota
stumulates the production of cross-reactive Abs, preventing
infection by related pathogens

Shir-Ly Huang* et al., 2015. Characterization of fungal and

bacterial components in gut/fecal microbiome.
Current Drug Metabolism 16, 272–283.

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 Human microbiota
• The residential microbes are
• Bacteria Prokaryotes
• Archaea
• Protists
Eukaryotes
• Fungi
• Viruses

The proportion of bacterial cells represented in the human

body is estimated to be ~90%, and of all genes, >99%.

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The hierarchy of biological classification’s eight major taxonomic ranks
Domain: Bacteria
Woese, Kandler & Wheelis,
[1]

A domain contains one or more kingdoms. 1990

Phyla (門)
Acidobacteria
Actinobacteria
Aquificae
Armatimonadetes
Bacteroidetes
Caldiserica
Bacteria Chlamydiae
Chlorobi
Chloroflexi
Chrysiogenetes
Cyanobacteria
* Deferribacteres
Deinococcus-Thermus
Dictyoglomi
Elusimicrobia
Fibrobacteres
Firmicutes
eight taxonomic ranks Fusobacteria
Gemmatimonadetes
Lentisphaerae
Nitrospirae
Planctomycetes
* Proteobacteria
Spirochaetes

* Synergistetes
Tenericutes
Thermodesulfobacteria
Thermotogae
Verrucomicrobia 7
The main phyla of gut biogeography of the
bacterial microbiota

Mazmanian et al., Nat. Rev. Microbiol., 14:20, 2016

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The change of gut microbiota composition

Age Bacterial composition in gut Diversity

Infant1 Actinobacteria and Bifidobacterium are the major Low

Firmicutes + Bacteroidetes >90%
Adult2
(Firmicutes > Bacteroidetes)
High
Firmicutes + Bacteroidetes >90%
The elders3
(Firmicutes~ 40% : Bacteroidetes~ 57% )
Low
1Torroni et al., 2012; 2Rajilic-Stojanovic et al., 2007; 3Claesson et al., 2011
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The similarities and differences in fecal microbial taxa
between AAs (African American) and NAs (Native African)

The solid circle encloses the taxa

that were detected in NA and
the dotted circle those identified
in AA.

The overlap between the 2

circles contains taxa common to
both populations. Much of the
shared taxa were significantly
(independent Kruskal-Wallis
tests) more enriched in one
group than in the other,
indicated by the boxed text in
AAs on the left and underlined
text in NAs on the right.

Ou et al., Am. J. Clin. Nutr., 98:111, 2013

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 Human Microbiome Project
(HMP)

(2008~2013)
A total budget of $115 million

It was a US NIH initiative with the goal of

identifying and
characterizing the microorganisms which are
found in association with both healthy and
diseased humans.

(oral, skin, urogenital, gut, nasal/lung)

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Structure, function and diversity of the healthy human microbiome
Nature 486:207, 2012
The Human Microbiome Project Consortium*

• Even healthy individuals differ remarkably in the microbes that occupy

habitats such as the gut, skin and vagina. Much of this diversity remains
unexplained, although diet, environment, host genetics and early microbial
exposure have all been implicated.
• Microbiome function varies among individual might indicating pathways of
particular importance in maintaining community structure in the face of
personalized immune, environmental or dietary exposures.

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The proportion of bacterial cells
represented in the human body is
estimated to be ~90%,
and of all genes, >99%.

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 Factors shaping the gut microbiome

Host genetics Food Pharmaceuticals Microbial exposure

• Inflammatory
Gut microbiome bowel Disease
• Ulcerative colitis
• Colon cancer
• Metabolic disease
Beneficial species  Pathobionts • Autoimmune Diseases
(Diabetes, RA, Lupus)
• Brain function
• Depression
• ….

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Human microbiota & diseases

• Commensals pathogenesis
• Antibiotics  colitis  FMT therapy
• Gut microbe-targeted therapy
• Commercial microbial products

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Commensals protects the body against
colonization by pathogenic bacteria

Sassone-Corsi et al., J. Immunology 194.9 (2015): 4081-4087. 60

• Staphylococcus epidermidis secretes a protease that inhibits biofilm
formation and nasal colonization by S. aureus
Iwase, Tadayuki et al., Nature 465.7296 (2010): 346.

• Staphylococcus lugdenensis produces an antibiotic peptide that

inhibits S. aureus colonization in a mouse model of skin infection
Zipperer et al., Nature 535.7613 (2016): 511.

• Escherichia coli strain Nissle 1917 outcompetes pathogenic E. coli

O157:H7 for carbohydrates and Salmonella Typhimurium for iron
Deriu et al., Cell Host & Microbe 14.1 (2013): 26-37.
Maltby et al., PLOS ONE 8.1 (2013): e53957.

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 Neisseria genus members
• The genus Neisseria is composed of 17 species
that may be isolated from humans and six
species that colonize various animals
Tø njum et al., Neisseria. Infectious Diseases (2017): 1553–1564.e1.

• Neisseria: a significant component of the human

microbiome is a large genus of bacteria that
colonize the mucosal surfaces of many animals
Liu et al., Microbiology 161.7 (2015): 1297-1312.

• Over 12 species of commensal Neisseria colonize

many regions of the human mucosa, including
the nasopharynx, endocervix, and rectum.
Diallo et al., Journal of Infection 72.6 (2016): 667-677.
Liu et al., Microbiology 161.7 (2015): 1297-1312.
Tø njum et al., Neisseria. Infectious
• N. gonorrhoeae (淋病雙球菌) gonorrhea (淋病) Diseases(2017): 1553–1564.e1.

Hypothesis: commensal Neisseria antagonize N. gonorrhoeae in vitro and in vivo

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N. elongata interacts physically with
N. gonorrhoeae and kills the pathogen in vitro

N. gonorrhoeae

N. elongata

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The commensal Neisseria kills N. gonorrhoeae through
a DNA-dependent mechanism

• N. elongata kills N. gonorrhoeae

through released DNA which
recombine the genome. N.
gonorrhoeae is killed when it
takes up DNA whose methylation
state is different from its own.

• The potential of developing a

nontraditional DNA-based
method for treating gonorrhea in
the context of N. gonorrhoeae
which has rapidly developed
resistance to virtually all
antibiotics.

Kim et al., Cell Host & Microbe 26 (2019): 228

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Bacterial DNA uptake system
• Pathogen employs the DNA uptake system to acquire
antibiotic resistance genes and evade the immune response,
thereby suggesting that this system may be the unique weak
point of the pathogen.
• Several other pathogens are naturally competent for DNA
transformation:
• Bacteriodes
• Streptococcus pneumonia
• Acinetobacter baylyi
• Haemophilus influenzae
• Helicobacter pylori
• Vibrio cholerae.
Whether members of these genera antagonize each other
using a similar DNA-based killing mechanism?
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Staphylococcus aureus (金黃色葡萄球菌)
• an opportunistic human pathogen
• a significant cause of morbidity and mortality worldwide
• causing diseases ranging from superficial skin and soft tissue infections
to severe life-threatening diseases (endocarditis and pneumonia)
• The enormous health burden caused by attributed to its ability to
acquire resistance against several classes of antibiotics.

12:1887 (2021)

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 Staphylococcus chromogenes secretes a molecule that inhibits S. aureus growth

similar niches:
compete for space & nutrients

Coagulase-negative staphylococci vs. S. aureus

S. chromogenes

SH 6-thioguanine (6-TG) Inhibit de novo

(a purine analogy) purine biosynthesis

co-infection in mice with S. aureus USA300

(or use pure 6-TG)

necrotic skin lesions

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 Antibiotics

Clostridium difficile infection (CDI)

Fecal microbiota transplant (FMT)

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Clostridium difficile (難治梭狀桿菌)
• Normal flora in intestine
• Antibiotics use
C. difficile is resistant to antibiotics (such as
clindamycin, cephalosporins, and
fluoroquinolones)
overgrow in colon
produce toxins
(which cannot be treated by an antibiotics)
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Health care-associated C. difficile infection

Arjun Gupta et al., JAMA, 2018. 73

 Clostridium difficile infection (CDI)
• Antibiotic-associated diarrhea: Acute diarrhea generally developing 5 to 10
days after initiation of antibiotic treatment (particularly clindamycin, penicillins,
cephalosporins, fluoroquinolones); may be brief and self- limited or more
protracted
• Pseudomembranous colitis: Most severe form of C. difficile disease with
profuse diarrhea, abdominal cramping, and fever; whitish plaque
(pseudomembranes) over intact colonic tissue seen on colonoscopy

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Microbiota Diversity in Patients before and after Infusion of
Donor Feces, as Compared with Diversity in Healthy Donors.

Microbiota diversity is expressed as Simpson’s

Reciprocal Index of diversity in fecal samples
obtained from nine patients before and 14 days
after the first infusion of donor feces, as
compared with their donors.

The index ranges from 1 to 250, with higher

values indicating more diversity.

The box-and-whisker plots indicate interquartile

ranges (boxes), medians (dark horizontal lines in
the boxes), and highest and lowest values
(whiskers above and below the boxes).

van Nood et al., N. Engl. J. Med., 368:407, 2013

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 Fecal Microbiota Transplantation (FMT)
(糞菌移植)
(4:30 min)
 About 500,000 American are
infected each year.
 Up to 20% of nursing facility
residents may carry
C. difficile.
 3% to 5% of the healthy
population carries C. difficile
bacteria with no symptoms.
 FMT Techniques:
-Nasal Gastric Tube
-Endoscope
-Colonoscopy
-Enema https://www.youtube.com/watch?v=Awn3haOpfcI

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 Fecal microbiota transplant (FMT)
• FMT was first described in 1958 and since then about 500
cases have been published in literature in various small
series and case reports. This procedure has been reported
mainly from centers outside of the United States and
acceptance of the practice has been difficult.
• 2014, the US Food and Drug Administration (FDA) labeled
FMT as a biological drug; as a result, guidelines are
required to help establish it as a mainstream treatment.
More US experience needs to be reported to popularize
this procedure here and form guidelines.
Clin Exp Gastroenterol 7: 1(2014)

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 Risk of Fecal Microbiota Transplantation
Example 1: (in 2019)
U.S. Food and Drug Administration (FDA): the potential risk of
transmission of multi-drug resistant organisms (MDROs) by FMT
and the resultant serious adverse reactions that may occur
• Two immunocompromised adults who received investigational FMT
developed invasive infections caused by extended-spectrum
beta-lactamase (ESBL)-producing Escherichia coli (E. coli). One of the
individuals died.
• FMT used in these two individuals were prepared from stool obtained
from the same donor, whose feces were not tested for ESBL-producing
gram-negative organisms prior to use. After these adverse events
occurred, stored preparations of FMT from this stool donor were tested
and found to be positive for ESBL-producing E. coli identical to the
organisms isolated from the two patients.
• MDRO testing of donor stool and exclusion of stool that tests positive for
MDRO. FDA scientists have determined the specific MDRO testing and
frequency that should be implemented.
https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/important-safety-alert-regarding-use-fecal- 83
microbiota-transplantation-and-risk-serious-adverse
 Risk of Fecal Microbiota Transplantation
Example 2: (in 2020)
U.S. FDA: six patients who received the company’s FMT product
for Clostridium difficile infection not responsive to standard
therapies and who developed infections caused by
enteropathogenic Escherichia coli (EPEC) or Shiga toxin-
producing Escherichia coli (STEC).

https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-
transplantation-and-risk-serious-adverse-events-likely

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 Risk of Fecal Microbiota Transplantation
Example 3: (in 2020)
U.S. FDA: for any use of FMT that is found to be necessary for
clinical care if it involves stool donated after December 1, 2019:
• Donor screening with questions directed at identifying donors who may
be currently or recently infected with SARS-CoV-2
• Testing donors and/or donor stool for SARS-CoV-2, as feasible
• Informed consent that includes information about the potential for
transmission of SARS-CoV-2 via FMT, including FMT prepared from stool
from donors who are asymptomatic for COVID-19

*The virus has been named “SARS-CoV-2” and the disease it causes has been
named “COVID-19.”

https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota- 85
transplantation-and-additional-safety-protections
Ministry of Health Welfare, Taiwan
→Consider FMT as a techniques, but not a
new drug (in June, 2018)
→The clinical indications are:
1. recurrent CDI*
or
2. refractory CDI
with the agreements from donor & patient
*Clostridium difficile infection

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Human microbiota & diseases

• Commensals pathogenesis
• Antibiotics  colitis  FMT therapy
• Gut microbe-targeted therapy
• Commercial microbial products

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 Roberts AB, et al. Nat Med. (2018)

Development of a gut microbe-targeted nonlethal

therapeutic to inhibit thrombosis potential

• Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite

that enhances platelet responsiveness and in vitro thrombosis in animal models.
• TMAO plasma levels predict incident atherothrombotic event risks in human
clinical studies.
• TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA)
moiety-containing nutrients, which are abundant in Western diet.
• A major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D) is used to
develop inhibitors that are potent, time-dependent, and irreversible and that do
not affect commensal viability.
• Nonlethal targeting of gut microbial enzymes linked to host diseases limits
systemic exposure of the inhibitor in the host.

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 Targeting the gut bacterial metabolic pathway
• A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to inhibit
trimethylamine (TMA) lyases and reduce trimethylamine N-oxide(TMAO) levels
in mice fed a high-choline or L-carnitine diet. －Wang Z, et al. Cell. (2015)
• Design and development of potent, mechanism-based, nonlethal inhibitors,
X-methylcholine (X= F, Cl, Br, I).
－Roberts AB, et al. Nat Med. (2018)
X X-methylcholine (XMC)
X: F, Cl, Br, I

Choline

Choline
trimethylamine-lyase

Trimethylamine
(TMA)

FMO3

Trimethylamine N-oxide
(TMAO)
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Choline lyase inhibitors, XMC:
• High affinity choline analog
inhibitors
• Nonlethal to the bacterial growth
• It accumulated within intestinal
microbes to millimolar levels,
a concentration over 1-million-fold
higher than needed for a
therapeutic effect.

Cut C/D: Choline trimethylamine-lyase

FMO3: Flavin-containing monooxygenase 3

Roberts AB et al. Nat Med. (2018) 127

Microbiota-based therapies
• Fecal Microbiota Transplantation (FMT)(live cells and metabolites)
• Probiotics (live bacterial cells)
• Prebiotics (bacterial nutrients)
• Postbiotics (Microbial metabolites or components)

Additive microbiota therapies

• Engineered bacterial strain
• Selective elimination (Bacteriophages/Bacteriocins/Antibiotics)

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 Microbiota-based therapeutics
• FDA-approved microbiome-based intervention: fecal microbiota transplantation
(FMT).
• FMT involves the transfer of an entire microbial community from a healthy donor to
a diseased recipient in order to replace the disease-associated microbiome.
• FMT has been shown to have remarkable efficacy in treating Clostridium difficile
infection (CDI), which most commonly presents after antibiotic treatment.

Wong & Levy,. (2019). MSystems, 4(3).

Gastroenterology 2015;149:223–237
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Mixture of microorganisms to
reprogram the microbial ecology

Designer microbial consortia are informed by community profiling

studies of clinical samples from healthy and diseased patients.
Clinical isolates from these patient scan then be assembled into a
defined mixture of microorganisms that can reprogram the
microbial ecology within an individual.

Mimee et al., (2016). Advanced drug delivery reviews, 105, 44-54. 130
Microbiota-based therapeutics
• To alter the microbial composition of the gut through exogenous
administration of live microbes by using probiotics.

• One alternative approach to probiotics is prebiotics, which are

compounds consumed with the intention of affecting microbiome
composition or function in a beneficial way.

Image Source: Recover Your Health Today

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Metabolite-based therapeutics (postbiotics)
• Metabolite-based therapeutics, or “postbiotics,” target downstream
signaling pathways of the microbiome and act by mitigating the
negative effects of an excess scarcity, or dysregulation of metabolites
involved in these pathways

• Exogenous administration or inhibition of metabolites has the potential

to counteract and correct the negative effects of dysbiosis

Wong & Levy,. (2019). MSystems, 4(3). 132

Overview of postbiotics

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Aguilar et al., (2018). Trends in Food Science & Technology, 75, 105-114.
Engineered bacterial strain to
produce therapeutic proteins

▲ Engineered microbes have been one strategy for

microbiota-based therapies. Gene circuits are constructed
using libraries of genetic parts to enable microbial production
of therapeutic proteins. Introduction of these
microorganisms into the endogenous microbiota allows for in
situ detection of disease biomarkers and/or drug production.

134
Mimee et al., (2016). Advanced drug delivery reviews, 105, 44-54.
Human microbiota & diseases

• Commensals pathogenesis
• Antibiotics  colitis  FMT therapy
• Gut microbe-targeted therapy
• Commercial microbial products

137
 Akkermansia muciniphila (艾克曼嗜黏蛋白菌)
• the type species for a new genus, Akkermansia, was
proposed in 2004
• Strictly anaerobic bacterium
• Non-motile, Gram-negative
• Widely distributed in human and animal intestines
• Use mucin as main carbon and nitrogen source
• Growth conditions: 37°C for 4 days; 100% N2 Classification
• Brain Heart Infusion Broth (BHI); Kingdom Bacteria
Trypticase soy broth (TSB) with defibrinated sheep blood Phylum Verrucomicrobia
Class Verrucomicrobiae
Order Verrucomicrobiales
BHI medium Content TSB medium Content Family Akkermansiaceae
(pH 7.4 +/- 0.2) (g/L) (pH 7.3 +/- 0.2) (g/L) Genus Akkermansia
Calf Brains, infusion form 200 Tryptone 15 Species A. muciniphila
Beef Hearts, infusion form 250 Soytone 5 Derrien et al., (2004). International
Proteose Peptone 10 NaCl 5 Journal of Systematic and Evolutionary
Microbiology, 54 (5): 1469–1476.
Dextrose 2
NaCl 5
Na2HPO4 2.5 Zhou, K. (2017). Journal of functional foods, 33, 194-201.
https://www.atcc.org/products/baa-835 138
 Mild heat inactivation of Akkermansia muciniphila
improves metabolic benefits in obese and overweight
human subjects

DPPIV: dipeptide peptidase

IV
Anhê, et al. (2019). Bacteria to alleviate metabolic syndrome. Nature medicine, 25(7), 1031-1033.

139
 Commercial Akkermansia products
Prebiotic + Akkermansia muciniphia WB-STR-0001 (gut-lining probiotic)

“Nuture your body’s microbiome and help keep your gut healthy”

Pendulum Akkermansia

Prebiotic + 5 Probiotics
Akkermansia muciniphila WB-STR-0001
Clostridium beijerinckii
Clostridium butyricum
Anaerobutyricum hallii
Pendulum Glucose Control Bifidobacterium infantis

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How the food you eat affects your gut? (Shilpa Ravella)
(5:09)

https://www.youtube.com/watch?v=1sISguPDlhY
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