NORMAL FLORA

OF DIGESTIVE
SYSTEM
Rio Risandiansyah, S.Ked, M.P, Ph.D
Outline

■ Normal flora of the gastrointestinal tract

■ Function and roles of normal flora in health
■ Alteration of normal flora population and type
■ Probiotics: Definition, types, and functions
ANATOMY AND
PHYSIOLOGY OF
THE DIGESTIVE
SYSTEM
What constitutes the Digestive
system?
■ Anatomy of the digestive system
– Oral cavity
– Alimentary Tract
– Liver, Pancreas
■ Normal flora of the skin, oral cavity,
esophagus to anus, liver and
gallbladder.
 BACTERIAL
NORMAL FLORA
OF THE GIT
Normal Flora

■ Normal flora =
– “common predominant types of flora present within body niches and shared
functional traits”.
– “the population of microorganisms that inhabit the skin and mucous
membranes of healthy normal persons”
■ Can be divided into two groups:
1. Resident flora = relatively fixed types of microorganisms regularly found and would
reestablish itself if disturbed
2. Transient flora = nonpathogenic or potentially pathogenic microorganisms that
inhabit the sin or mucous membranes for hours, days, or weeks
■ Members of the normal flora outweigh human body cells 9 to 1.
Human Microbiome Project

■ 2007  National Institutes of Health launched

Human Microbiome Project.
■ Microbiome = ‘genomic information of bacterial
symbionts in a certain environment’; or simply
‘microbial communities in an environment’
■ Aims: To characterize microbial communities found at
multiple human body sites and to look for
correlations between changes in the microbiome and
human health.
Human Microbiome Project

Method:
■ Sampling of 200+
subjects.
■ Culture-independent
analysis, using 16S
rRNA PCR.
■ 16S rRNA, area of the
gene which can
taxonomically
determine organisms
(operational taxonomic
unit = OTUs).
Prior to HMP:
Culture
dependent
method

(McFarland, 2000)
Normal Flora of
the Skin

After HMP:
• Different patterns of bacteria population
reside in different areas of the skin.
• Staphylococcal bacteria dominant in
plantar heel, popliteal fossa and occiput
area.
• Propionibacteria and Corynebacteria
dominant in face area.

Notes:
Proteobacteria = Gram negative bacteria
Actinobacteria = Gram positive bacteria

(Grice and Segre, 2011

Normal Flora of the Skin

■ Staphylococcus and Corynebacterium spp. are most abundant in

moist areas  umbilicus, axilla, inguinal, gluteal crease, sole of the
foot, popliteal fossa and antecubital fossa.
■ Dry areas are occupied by a mixture of bacteria, mostly from phyla
Actinobacteria, Proteobacteria, Firmicutes and Bacteroides.
■ Skin has higher diversity compared to gut or oral microbiome.
■ However, the population is less stable over time (high temporal
variability).
■ Variability probably influenced by environmental factors, host immune
levels, and pathophysiology.
Normal Flora of the Mouth

■ Discovered 280 different species reside in oral cavity  some predict

to be over 500 species.
■ Dominated by bacteria from 6 different phyla: Firmicutes,
Bacteriodetes, Proteobacteria, Actinobacteria, Spirochaetes, and
Fusobacteria  96%
■ Remaining 4%  Euryarchaeota, Chlamydia, Chloroflexi, SR1,
Synergistetes, Tenericutes and TM7.
Firmicutes Phyla

■ Mostly Gram Positive.

■ Includes Streptococcus
spp., Enterococcus spp.,
Peptostreptococcus spp.
and Lactobacillus spp.
■ In the oral cavity,
Streptococcus spp. Is
most dominant.
■ Other classes include
Clostridia 
Veillonellaceae, a gram
negative basil.
Normal Flora of the Mouth

■ Notable species in other Phyla:

– Mycoplasma hominis, Mycoplasma salivarium and Mycoplasma
faucium (found in saliva of 97% individuals).
– Bacteria with the genera of Actinomyces, Mycobacterium,
Bifidobacterium  found primarily in dental caries and dental
plaque.
– Genera Gardnerella also found in oral cavity, which is isolated
primarily from the vagina.
– Bacteria in Actinobacteria phyla includes Propionibacterium spp.,
Bifidobacterium spp., and Corynebacterium spp.
– Treponema is also found, however difficult to cultivate.
Role of microbiome in dental
plaque and caries
■ Dental plaque = adherent dental deposit composed of entirely normal
flora bacteria; prevalent and densest biofilm.
■ Proteins and glycoproteins from saliva and other oral secretions 
dental pellicle  location of biofilm formation
■ Plaque has two layers: (1) in relation to gingival line (supra and sub),
and (2) layers of the bacteria species involved.
■ Initially colonized by Gram positive bacteria  ionic and hydrophobic
interactions  Streptococcus sanguis, but also other Streptococci,
lactobacilli, and Actinomyces.
■ Late colonizers (after 2 – 4 days) consist of anaerobic Gram negative
bacteria  Porphyromonas, Prevotella, Fusobacterium, Veillonella, and
Treponema.
Role of microbiome in dental
plaque and caries
■ Caries = disintegration of the teeth beginning at the surface and
progressing inwards.
■ (1) Demineralization of enamel by acid products of plaque bacteria
glycolytic metabolism, (2) dentin and cementum is decomposed due
to bacterial digestion of protein matrix.
■ S. mutans  initiate caries, also other Streptococci, lactobacilli and
actinomycetes.
■ Bacteria glycolysis requires proper substrate which are dietary
monosaccharides and disaccharides, in particular sucrose which
adheres to the teeth.
Normal Flora of Digestive System
Normal Flora of the Digestive
System
■ Isolated from 10 habitats in the digestive system divided into four
groups:
– Group 1 = buccal mucosa (BM), keratinized gingiva (KG), and
hard palate (HP)
– Group 2 = saliva (Sal), tongue (TD), tonsils (PT) and throat (Th)
– Group 3 = sub and supra gingival plaque (SubP and SupP,
respectively)
– Group 4 = stool
■ Phyla Firmicutes and Bacterioides was found in communities of all ten
sites.
■ Juxtaposed body sites had strikingly different microbial community.
■ Differences was consistent throughout test subjects.
Phyla distribution in 10 body
habitats
Microbiome distribution in 10
body habitats
Most microbes
vary based on
habitat
FUNCTIONS OF
NORMAL FLORA
Functions of Normal Flora:

1. Digestion of metabolizable substrates

2. Colonization resistance
3. Production of vitamins
4. Development of attachment sites
5. Induces development of immune system
6. Production of exogenous enzymes
7. Stimulation of intestinal transit
8. Maturation and turn-over of intestinal cells.
Digestion of metabolizable
substrates
■ Non-absorbable dietary carbohydrates  metabolized by normal flora
in colon
■ This results in short chain fatty acids (SCFA), mainly acetic acid,
propionic acid, and butyric acid.
– Acetic acid and propionic acid  source of energy and stimulates
salt and water absorption.
– Butyric acid  source of energy for colonic epithelial cells.
■ Oxalobacter formigenes  breakdown of oxalic acid
Colonization resistance

■ Colonization resistance  1st line defense against invasion against

exogenous, pathogenic organisms or indigenous opportunistic
organism.
■ Colonization resistance  effective natural barrier against C. difficile,
Salmonella, Shigella, Pseudomonas, Pathogenic E. coli strains,
Candida albicans, etc.
■ Disruption of normal flora enables colonization of transient flora,
which may or may not cause a diseases (depending on the
pathogenicity).
■ In colonization with C. difficile (which causes diarrhea, colitis, toxic
megacolon), inserting protective flora is shown to prevent the disease
from developing.
Colonization resistance:
Production of bacteriocin
■ Some normal flora able to produce antimicrobial peptides
(Bacteriocin) or other substance which inhibits colonisation.
■ Bacteriocin  class of broad spectrum inhibitory antimicrobial protein.
■ Bacteriocin  produced by Lactobacilli (reuterin)
■ In vitro  inhibitis Salmonella, Shigella, Clostridium and Listeria.
Colonization resistance:
Production of hydrogen peroxide
■ Lactobacilli also found to produce hydrogen peroxide (H 2O2).
■ H2O2 causes lipid peroxidation  increases bacterial membrane
permeability  nuclear acid destruction in strains that do not produce
catalase
■ Also protective towards infection caused by Chlamydia trachomatis,
Gardnerella vaginalis, Ureaplasma urealyticum.
Colonization resistance: Adverse
microenvironment
■ Production of acids as an end product of carbohydrate metabolism 
inhibitory against Gram positive and Gram negative bacteria
■ Lactobacili and bifidobacteria  produces acid  unfavourable pH for
growth
■ Nutrient competition  some normal flora may have more efficient
utilization of nutrients (such as monomeric glucose)  reduce growth
of competing pathogenic strains.
 Colonization
resistance:
Attachment
interference
■ Colonization of bacteria
require bacterial
attachment to receptors
in cells.
■ Attachment can be
achieved by adhesin
(adhesion molecules),
by pili/fimbriae or
flagella.
■ Attachment is part of
virulence factor.
(Pizarro-Cerda and Cossart, 2006)
 COLONIZATION
RESISTANCE:
ATTACHMENT
INTERFERENCE

(Haiko and Westerlund-Wikstrom, 2013)

Colonization resistance:
Attachment interference
■ Normal flora may produce enzymes which inhibit pathogen
attachment
■ Normal flora may occupy attachment sites  prevent occupation of
attachment site by pathogenic bacteria
■ Non-enterotoxin producing E. coli  prevents occupation of
enterotoxin strains of E. coli.
■ Precolonization with E. coli in mice  prevents colonization of B.
longum
Colonization resistance summary
Production of Vitamins

■ Intestinal flora produces vitamins including vitamin B5 (panthothenic

acid), biotin (vitamin H), pyridoxine (vitamine B6) and menaquinone
(Vitamine K2).
■ Intestinal may break down vitamins into an absorbale form.
■ Vitamin B12, niacin, riboflavin and thiamin also made by intestinal
flora but not abdorbed in the colon  microbially supplemented foods.
 Production
of Vitamins
■ Recent paper:
– Vitamin B8 (biotin)
– Vitamin B12
(Cobalamin)
– Vitamin B9 (Folate)
– Vitamin B3 (Niacin)
– Vitamin B5
(Pantothenate)
– Vitamin B7 (Pyridoxine)
– Vitamin B2 (Riboflavin)
– Vitamin B1 (Thiamine)
(Magnusdottir et al, 2015)
Production of Vitamins: Pathway still in research
Immune response stimulation

■ Interaction between microbiota and host immune system are

numerous, complex and bidirectional.
■ Immune response must tolerate commensal microbiota
■ Normal flora provides antigenic stimulants to the gut-associated
lymphoid system (GALT) cells  increase IgM and IgG in pathogenic
immune response.
■ Peptostreptococci act as an immune system primer to other bacterial
antigens.
Intestinal Transit

■ Intestinal flora  stimulate peristalsis via

enteric nervous system
■ Mechanism is probably via Gut-Brain axis

(Carabotti et al, 2015)

FACTORS THAT
INFLUENCE
NORMAL FLORA
Age

■ Microecology is
established in early
infancy (<2 years old).
■ Adults have greater
microbial diversity than
adolescents or the elderly.
■ Post menopausal women
increased number of fungi,
clostridia, and lactobacili.
■ Reasons for variation
remains unclear.
Geography

■ Ying et al, 2015: Bacterial communities were predominately

influenced by whether the participants were living in an urban or rural
environment.
■ Also shown in urban japanses compared to rural  urban japanese
where found to have significantly less Bifidobacterium adolescentis,
but more total total anaerobic bacteria, bacilli and Clostridium spp.
■ May be caused by different dietary habits and antibiotic use, but
require further research.
Diet

■ Diet is probably the most understood factor of gut microflora diversity, as shown
in table above.
Diet

■ Little direct studies in humans.

– In infants, breast-fed infants  bifidobacteria.
– Formula-fed infants are colonized with enterbacteria, bacteroides
and clostridia
■ Dietary changes (in mice) lead to significant changes in bacterial
metabolism.
■ High fiber vs low fiber  lower levels of Firmicutes than Bacteroidetes
in high fiber diets.
Stress

■ Frequently quoted as a major influence in intestinal function.

■ Limited evidence on its effects on normal flora  may be misleading.
Antibiotics

■ Antibiotics effects in normal flora population is well known.

■ Used to reduce post-surgical infections by decontaminating the
intestine.
END