Human Microbiome Microbiota Faecal transplant.pdf

Human Microbiome
Dr Vivek Patil
PG JR
Dept. of Microbiology
AIIMS Raipur

Contents
 Introduction
 Microbiology of Normal Flora
 Role of Normal Flora
 The human microbiomeproject
 Faecal microbiota transplant

History of microbiome research
Biomedcentral.com

Defining ‘Microbiome’
 “The ecological community of commensal, symbiotic &
pathogenic microorganisms that literally share our body space &
have been all but ignored as determinants of health & disease.”
(Lederberg 2001)
 The microbiome comprises all of the genetic material within a
microbiota (HMP operational definition)

Microbiome vs Microbiota
Microbiome vs Microbiota
 Microbiota : Living microorganisms found in a defined environment
 Microbiome : Collection of genomes
Microbe
community
Structural
elements
Metabolites Environment

Resident flora
 Constant populations which cannot be completely
removed permanently.
 Close association with particular area. (e.g. E.coli in
intestine)
 Harmless
 Re-establish themselves, when disturbed.
 Prevent permanent colonisation of the body by other
organisms
AnanthNarayan

Transient flora
 Temporary for short interval
 Produce disease when resident flora get disturbed
e.g. pneumococcus and meningococcus in
nasopharynx
 Patients may acquire MDR organisms from hospital
environment & HCW
 e.g. MRSA in nose and skin
MDR GNB (Klebsiella, E.coli, Pseudomonas,
Acinetobacter) in respiratory tract.
 Eliminated by proper hand hygiene
Ananthnarayan

Factors affecting the Microbiome
 Intrinsic factors:
1. Body environment : temperature, pH, oxygen concentration,
pressure, osmolarity, and nutrient source(sebum in skin; mucous in
gut: source of C)
2. Genetics, ethnicity, gender, age
 Extrinsic factors: Diet, Lifestyle, Medication, Geographic location,
Climate, Seasonality, Mode of delivery
 Mode of delivery: Vaginal vs Caesarean different similar
to adult around age 3 yrs.
 Bifidobacterium stimulates immunity decreased in old age
Naturereview article

Factors shaping neonatal microbiome
(From Tamburini S, Shen N, Wu HC, Clemente
JC. The microbiomein early life: implications for health outcomes. Nat Med.2016;22:713-722.)

 North-Central India, which was primarily consuming a plant-based
diet, was found to be associated with Prevotella and also showed
an enrichment of BCAA and lipopolysaccharide biosynthesis
pathways.
 In contrast, the gut microbiome of the cohort from Southern India,
which was consuming an omnivorous diet, showed associations
with Bacteroides, Ruminococcus, and Faecalibacterium and had
an enrichment of SCFA biosynthesis pathway & BCAA transporters.

The relative abundancesof the six
dominant bacterial phyla in different
body sites
Unravellingthe effects of the
environmentand host genotype on
the gut microbiome. Nat Rev
Microbiol. 2011;9:279-290.

Different composition but Same function!!!
(From Human Microbiome
Project Consortium)

Oral cavity
 Mouth of an infant at birth is not sterile and it contains mother’s
vaginal flora i.e. micrococci, streptococci, coliforms & lactobacilli
 Diminish in 2-5 days after birth and are replaced
 GPC & GNC predominate; anaerobe: aerobe 100:1
 Anaerobes: Peptostreptococcus, Veillonella, Actinomyces,
Fusobacterium
 Aerobes: Streptococcus & Neisseria
Topley and ASM Manual of clinical microbiology

 Oral streptococci includes: S. salivarius (saliva & tongue)
Tooth surface: S. sanguis & S. mutans
Oral mucosa: S. vestibularis & S. sanguis
S. pneumoniae & B-haemolytic streptococci (grp A,C,F,G)
S. pyogenes can transiently colonise healthy individuals

 20% of individuals are colonized with S. aureus.
 Other GPCs :Peptostreptococcus anaerobius, Micromonas,
Abiotrophia, Gemella spp.
 GNCs: Veillonella spp (15%), Neisseria, Moraxella, Kingella,
Cardiobacterium, Eikenella corrodens.
 GPBs : Actinomyces is predominant, Rothia
(A. israelli, A. naeslundii, A. odontolyticus, A. meyeri, A. georgiae)
They have fimbrie, adhere to mucosa, forms slime, entrap bacteria,
prevent their removal.

 Other GPBs Bifidobacterium, Corynebacterium,
Eubacterium, Lactobacillus, Propionibacterium
B. denticum (dental plaque), Lactobacillus & streptococci
(tooth caries),
 GNB : Anaerobes like Fusobacterium, Bacteroides,
Porphyromonas, Prevotella, Selenomonas

 Haemophilus spp (<5%) mc species is H. parainfluenza & non
capsulated strains of H. influenza
 Actinobacillus actinomycetomcomitans & Treponema spp present
in gingival crevices is associated with periodontal disease
 Enterobacterales, Psuedomonas & Acinetobacter in small numbers.
Increased transiently in hospitalised patients
 Parasites: Trichomonas tenax & Entamoeba gingivalis

 Oral cavity is principle entry point of human body, spread to other
sites occurs
 Nitrates Nitrites NO (antimicrobial,
vasodilator) shown by Lactobacilli
 Streptococcus salivarius Bacteriocins; inhibits GNB & prevents
periodontitis
 Dental caries: Streptococcus mutans, Streptococcus sobrinus, and
Lactobacillus acidophilus
Carbohydrates Low pH tooth demineralisation
& Cavitation
reduction
Fermentation

Nares & Nasopharynx
 Nares: S. aureus, CoNS, Corynebacterium,
Peptostreptococcus, Fusobacterium spp.
 Increased carriage of S. aureus in preadolescent
 Nasopharynx: streptococci & Neisseria spp.
 S. salivarius, S. parasanguis & S. pneumoniae
 Colonisation with N. meningitidis in young adults of
military (10-95)% mc Neisseria spp in them are N.
subflava, N. sicca, N. mucosa, N. lactamica
 GNCB: M. catarrhalis (sinusitis, bronchitis), H. influenza,
Cardiobacterium hominis (damaged heart valve)

Trachea, larynx, bronchi & lungs
 Few organisms, transient
 Long term colonisation from upper airways occurs when
ciliated epithelial cells are damaged (influenza / COPD)

Esophagus
 The esophagus is colonized by bacteria that are
introduced from the oropharynx by swallowing or from
the stomach by reflux.
 As in the oral cavity, Streptococcus is the dominant
genus in the healthy esophageal microbiome
 In Barrett’s esophagus and esophageal carcinoma,
gram negative anaerobes predominates
 In a diseased state, colonisation with Candida & viruses
(HSV, CMV)

Stomach
 Inhospitable environment due to HCL, pepsinogen
 Discovery of H. pylori by Marshal & Warren (1882)
 Lactobacillus spp
 Streptococcus spp
 Helicobacter pylori
 Veillonella
 Clostridium
 H. pylori can modulate gastric environment & alter the habitat of
resident flora
 Increased risk for gastric cancer

Intestine
 Bacteria to host cell ratio 1:1
 Majority in colon
 Main phyla are Firmicutes & Bacteroides (90%)
Others : Actinobacteria, Proteobacteria, Fusobacteria
 Fermicutes: 200 genera
Bacillus, Lactobacillus, Enterococcus, Clostridium, Ruminococcus
among them, S. aureus & C. perfringens are pathogenic
 Bacteroidota: Bacteroides, Prevotella
 Actinobacteria: Bifidobacterium
 Proteobacteria: Enterobacter, Helicobacter, Shigella, Salmonella,
Escherichia coli

 Changes in 3 stages of life
1. Birth to weaning
2. Weaning to normal diet
3. Old age
 Birth : Vaginal delivery -- Vaginal & fecal
Caesarean -- Skin & environment
 Facultative anaerobes are first to colonise, they create anaerobic
conditions & promote growth of obligatory anaerobes Bifidobacterium,
Bacteroides spp. within 2 weeks
 At 3 days, naturally born infants have more Bifidobacteria than
Caesarean born babies
 Exclusively breastfed babies have more stable & less diverse microbiota
 After the start of solid food, Firmicutes are increased

Normal colonisation in breast-fed term babies
Carlsson et al JCM, 1975
E. Coli,
streptococci
bifidobacteria
bacteroid
es
Lactobacilli
Enterobacteriaceae
0 5d 10 d 30 d 3-4 m 6m 1yr
Creates
anaerobic
environment

 At old age, decreased diversity, Bifidobacteria , Fermicutes
Increased Enterobacteriaceae, Bacteroidetes (>65years)
 Largest microbiota in large intestine: slow flow rate, mild acidic to
neutral pH
Large intestine contains several microenvironments like epithelial
surface & inner mucin layer mainly contain Obligate anaerobes
Mucin layer: Akkermansia muciniphila
Gut lumen: Ruminococcus spp. Depending upon dietary fiber intake

 Small intestine: short transit time, high bile
Mainly facultative anaerobes Proteobacteria, Bacteroides,
Streptococci, Lactobacilli, Enterococci spp.
 Entamoeba spp: E. dispar, E. coli, E. hartmanii (large intestine)
 Intestinal flagellates: Pentatrichomonas hominis, Retortamonas
intestinalis, Chilomastix mesnili & Enteromonas hominis

Role in health
 Regulate digestion- processing nutrients & metabolites; SCFA, bile
acids, amino acids
 Maintain intestinal epithelial integrity, immunity, prevent bacterial
invasion
 Supress pathogenic colonisation & growth

Genitourinary tract
 Relatively sterile except in female urethra & vagina
 Microbicidal activity of bladder epithelium & flushing action of urine
 Ureters, kidneys, prostate & cervix are sterile
 Female urethra: lactobacilli, streptococci, CoNS
E. coli & Enterococcus spp are transient, can cause UTI

 Anaerobes : Lactobacillus spp. (L.acidophilus,
L.fermentum, L.casei, L.cellobiosus)
Bifidobacterium spp., Porphyromonas, Prevotella,
Peptococcus spp., Propionibacterium, Mobiluncus(in small
numbers), Treponema spp.
 Facultative anaerobes : CoNS, Streptococci,
Corynebacterium spp, Viridans group of streptococci ,
Gardnerella vaginalis, Neisseria spp., Haemophilus

 Mycoplasma (M. hominis, M. genitalium, M. fermentans, M.
primatum, M. spermatophilum, M. penetrans)
M. hominis & U. urealyticum have pathogenic potential

Skin
 Anaerobes >100 folds>> aerobes ; gram positive > gram negatives
 Staphylococcus, Micrococcus, Corynebacterium, Peptococcus,
Finegoldia, Peptostreptococcus & Propionibacterium
 S. epidermidis is most common; other CoNS are S. hominis, S.
hemolyticus, S. warneri, S. capitis, S. saprophyticus, S. caprae, S.
saccharolyticus, S. pasteuri, S. lugdunensis, S. simulans & S. xylosus

 Micrococcus spp (20%), most common is Micrococcus
luteus
Other GPCs are Dermacoccus, Kocuria kristinae, Kocuria
rosea, K. varians & kytococcus
 S. pyogenes are well suited for dry skin surfaces
 Anaerobic cocci survive in hair follicles & skin glands

 Corynebacterium spp ; C. striatum, C. minutissimum, C.
psuedodiphtheriticum, C. xerosis, C.urealyticus (groin), C. jeikeium
(apocrine glands)
 Propionibacterium acnes (sebaceous gland), Propionibacterium
avidum (axilla & perineum)
 Other gram positive includes Dermabacter, Brevibacterium,
Turicella otitidis
 Transient members are Clostridium perfringens, Acinetobacter,
Burkholderia.

Site Anaerobes Aerobes others
Mouth Anaerobic cocci
Actinomyces
Fusobacterium
Bifidobacterium
Prevotella
Spirochetes
Viridans streptococci Trichomonas tenax
Entamoeba gingivalis
Nasopharynx Prevotella species
Anaerobic cocci
Fusobacterium
Streptococci (α and
non-hemolytic)
Neisseria spp
Diphtheroids
Staphylococcus
epidermidis
Haemophilus
Meningococcus
Pneumococcus
Staphylococcus
aureus Gram-
negative bacilli
Yeasts
Gastro-intestinal tract Lactobacillus
Anaerobic cocci
Bacteroides fragilis
Fusobacterium
Bifidobacterium
Prevotella
Clostridium
Helicobacter pylori
Enterobacteriaceae
& other GNB
Enterococci,
non hemolytic),
S. Agalactiae
Diphtheroids
Candida species
Staphylococcus
aureus
Entamoeba spp
intestinal
flagellates

Site Anaerobes Aerobes Others
Female genital tract Anaerobic cocci
Lactobacillus
Prevotella
Bifidobacterium
Clostridium
Corynebacterium
species
Streptococci (α, non-
hemolytic and
S. agalactiae)
Neisseria (non-
pathogenic species)
Enterococci
Enterobacteriaceae
and
other gram-negative
rods
S. epidermidis
Candida species
Skin Propionibacterium
Anaerobic cocci
Staphylococcus
epidermidis
Diphtheroids
Micrococcus species
Neisseria(Non-
pathogenic species)
non hemolytic)
Staphylococcus
aureus Candida
species
Acinetobacter
species

The Human Microbiome Project
 The Human Microbiome Project (HMP) was a United States National
Institutes of Health (NIH) research initiative to improve
understanding of the microbiota involved in human health and
disease (2007-2016)
 The first phase (HMP1) focused on identifying and characterizing
the microbiomes of healthy human subjects at 5 major body sites
using 16S & metagenomic shotgun sequencing
Human Microbiome Project

 The second phase, known as the Integrative Human
Microbiome Project (iHMP) launched in 2014 with the
aim of characterization of microbiome & human host
from 3 cohorts of microbiome associated conditions
using multiple ‘omics’ strategies
 Study methods included 16S rRNA gene profiling,
whole metagenome shotgun sequencing,
metatranscriptomics, metabolomics

Culture independent molecular approaches to
study host-microbiome interaction
 DNA-based approaches- 16s rRNA, 18s rRNA
Who is there and what can they do?
 RNA-based approaches-
How do they respond?
What pathways are activated?
 Protein-based approaches-
How are they interacting with the host?
What proteins are being produced?
 Metabolite-basedapproaches-
What are the chemical outcomes of their activity?
Metagenomics
Metatranscriptomics
Metaproteomics
Metabolomics
the Human Microbiome, and Health Risk: A Research Strategy (2018)

Noecker C, McNallyCP, Eng A, Borenstein E. High-resolutioncharacterizationof
the human microbiome.
Schemes of Microbiome
Analysis

Fecal Microbiota Transplant
 Process of transferring fecal bacteria and other microbes from a
healthy individual into another individual. FDA approved
for Clostridioides difficile infection
 FMT procedure includes
1. Patient criteria
2. Stool donar criteria
3. Bowel preparation
4. FMT administration
European consensus conference on FMT in clinical practice

Patient eligibility criteria (FDA Approved)
 >18years
 Recurrent CDI not responding to standard vancomycin therapy
 SevereCDI not responding to therapy after 48 hours
 Laboratory confirmed CDI
Stool donor exclusion criteria
 H/O antibiotics 12 wks
 Major GI surgery
 HIV, HBV, HCV, tattooswithin last 6 months

DONAR SELECTION
Stool testing
(1) Clostridioides difficile (PCR or EIA test for Toxin A and B)
(2) Routine culture for enteric bacterial pathogens
(3) Complete Ova and Parasite studies, if pertinent travel history
Serologic testing
(4) anti-HIV type 1 and 2
(5) anti-HAV IgM
(6) HBsAg
(7) anti-HCV
(8) Rapid plasma reagin (RPR)

Specimen preparation
 Fresh stool (30-100 grams)
 Samples prepared within 6-8 hrs of defecation
 The sample is then diluted with 2.5–5 times the volume of the
sample with either normal saline, sterile water or 4% milk.
 Mix the solution with morter & pestle/ blender
 The suspension is then strained through a filter and transferred to an
administration container
 For later use it can be frozen after dilution using 10% glycerol

Administration
(A)Donor stool and normal
saline (1:3) ground in a
blender.
(B)Fecal suspension in 50-mL
syringes.
(C)Infusion using
colonoscopy.

First oral pill for CDI, FDA
APPROVED on 16 APR 23
SER-109

Role of a microbiologist in fecal transplant!

Human Microbiome Microbiota Faecal transplant.pdf

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