NORMAL MICROBIAL FLORA

&

ALSO IT’S ROLE IN HUMAN DEFENSE MECHANISM

By

Shahbaz Ahmad

CONTENTS

Introduction

What is Normal Flora?

Why know about Normal Microbial Flora?

Classification of Normal Flora

Role of Normal Flora in Human Defense Mechanism

References

Introduction

In a healthy human, the internal tissues, e.g. blood, brain, muscle,

etc., are normally free of microorganisms. However, the surface

tissues, i.e., skin and mucous membranes are constantly in contact

with environmental organisms and become readily colonized by

various microbial species. The mixture of organisms regularly

found at any anatomical site is referred to as the normal flora. The

normal flora of humans consists of a few eukaryotic fungi and

protists, but bacteria are the most numerous and obvious microbial

components of the normal flora. A healthy fetus in utero is free

from microorganisms. During birth the infant in exposed to vaginal

flora. Within a few hours of birth oral and nasopharyngeal flora

develops and in a day or two resident flora of the lower intestine

appears.

In the past, the role that microorganisms played in the normal

functioning of the body was not appreciated. In the early 1900’s when Dr. Metchnikoff was credited with the discovery of the

importance of intestinal flora, other physicians felt that the colon was totally unnecessary and often surgically removed them from their patients (1) Gordon R. (1993). The colon was described as ‘a

poisonous cess-pit infecting the body with rheumatism, tuberculosis, cancer and other diseases’.

Nowadays we know that normal flora is a dynamic and complex mixture of microbes that have diverse functions including

digestion of essential nutrients, maturation of intestinal physiology, stimulation of immune system, systemic effects on blood lipids and

the inhibition of harmful bacteria.

What is Normal Flora?

The term “normal microbial flora” denotes the population of

microorganisms that inhabit the skin and mucous membranes of

healthy normal persons. It is doubtful whether a normal viral flora

exists in humans.

The skin and mucous membranes always harbor a variety of

microorganisms that can be arranged into two groups.

a. The resident flora consists of relatively fixed types of

microorganisms regularly found in a given area at a given

age; if disturbed, it promptly reestablishes itself.

b. The transient flora consists of nonpathogenic or potentially

pathogenic microorganisms that inhabit the skin or mucous

membranes for hours, days, or weeks; it is derived from the

environment, does not produce disease, and does not

establish itself permanently on the surface. (2)

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Although the term ‘normal flora’ is commonly used, it is really a

misnomer. Microbial flora has spatial and temporal complexity

that differs by individual, body niche, age, geographic location,

health status, diet and type of host. Even within the same

individual, the composition of the microbial flora can vary

according to changes in diet, stress, sexual behavior, medication,

hormonal changes and other host-related factors. With this caveat

in mind, the field of ‘normal flora’ can be examined for common

predominant types of flora present within body niches and shared

functional traits. (3) Lynne V. McFarland (2000)

Why know about Normal Microbial Flora?

There are many reasons to acquire knowledge of the normal human

microbial flora. Four specific examples include:

1. An understanding of the different microorganisms at

specific locations provides greater insight into the possible

infections that might result from injury to these body sites.

2. A knowledge of the normal micro-biota in an infected part

of the body gives the physician-investigator a better

perspective concerning the possible source and significance

of microorganisms isolated from an infection site.

3. A knowledge of the normal micro-biota helps the physician

investigator understand the causes and consequences of

colonization and growth by microorganisms normally

absent at a specific body site.

4. An increased awareness of the role that these normal

micro-biota play in stimulating the host immune response

can be gained. This awareness is important because the

immune system provides protection against potential

pathogens. (4) Prescott (2002)

Classification of Normal Flora

Three types of symbiotic relationships are commensalism,

mutualism, and parasitism. Within each category the association

may be either ecto-symbiotic or endo-symbiotic. In ecto-symbiosis

one organism remains outside the other. In endosymbiosis one

organism is present within the other.

Based on different parts of the human body normal flora of the

human body is classified into the following categories:-

a. Conjunctiva

b. Outer ear

c. Stomach

d. Skin

e. Urethra

f. Vagina

g. Large intestine

h. Small intestine

i. Mouth & oropharynx

j. Nose

k. Upper respiratory tract

Table Bacteria commonly found on the surfaces of the human body.

BACTERIUM Skin

Con-

junc-

tiva

Nose Pharynx Mouth Lower

GI

Ant.

ure-

thra

Vagina

Staphylococcus epidermidis (1) ++ + ++ ++ ++ + ++ ++

Staphylococcus aureus* (2) + +/- + + + ++ +/- +

Streptococcus mitis

+ ++ +/- + +

Streptococcus salivarius

++ ++

Streptococcus mutans* (3)

+ ++

Enterococcus faecalis* (4)

+/- + ++ + +

Streptococcus pneumoniae* (5)

+/- +/- + +

+/-

Streptococcus pyogenes* (6) +/- +/-

+ + +/-

+/-

Neisseria sp. (7)

+ + ++ +

+ +

Neisseria meningitidis* (8)

+ ++ +

+

Enterobacteriaceae*(Escherichia

coli) (9) +/- +/- +/- + ++ + +

Proteus sp.

+/- + + + + + +

Pseudomonas aeruginosa* (10)

+/- +/- + +/-

Haemophilus influenzae* (11)

+/- + + +

Bacteroides sp.*

++ + +/-

Bifidobacterium bifidum (12)

++

Lactobacillus sp. (13)

+ ++ ++

++

Clostridium sp.* (14)

+/- ++

Clostridium tetani (15)

+/-

Corynebacteria (16) ++ + ++ + + + + +

Mycobacteria +

+/- +/-

+ +

Actinomycetes

+ +

Spirochetes

+ ++ ++

Mycoplasmas

+ + + +/- +

++ = nearly 100 percent + = common (about 25 percent) +/- = rare (less than

5%) * = potential pathogen

Table Notes

(1) The staphylococci and corynebacteria occur at every site listed.Staphylococcus epidermidis is highly adapted to the diverse

environments of its human host. S. aureus is a potential pathogen. It is a leading cause of bacterial disease in humans. It can be transmitted from the nasal membranes of an asymptomatic carrier

to a susceptible host.

S. epidermidis. Scanning EM. CDC.

(2) Many of the normal flora are either pathogens or opportunistic pathogens, The asterisks indicate members of the normal flora a that may be considered major pathogens of humans.

S. aureus. Gram stain.

(3) Streptococcus mutans is the primary bacterium involved in plaque formation and initiation of dental caries. Viewed as an opportunistic infection, dental disease is one of the most prevalent

and costly infectious diseases in the United States.

Streptococcus mutans. Gram stain. CDC

(4) Enterococcus faecalis was formerly classified as Streptococcus faecalis.The bacterium is such a regular a component of the

intestinal flora, that many European countries use it as the standard indicator of fecal pollution, in the same way we use E. coli in the

U.S. In recent years, Enterococcus faecalis has emerged as a significant, antibiotic-resistant, nosocomial pathogen.

Vancomycin Resistant Enterococcus faecalis. Scanning E.M.

CDC

(5) Streptococcus pneumoniae is present in the upper respiratory tract of about half the population. If it invades the lower respiratory

tract it can cause pneumonia. Streptococcus pneumoniae causes 95 percent of all bacterial pneumonia.

Streptococcus pneumoniae. Direct fluorescent antibody stain.

CDC.

(6) Streptococcus pyogenes refers to the Group A, Beta-hemolytic

streptococci. Streptococci cause tonsillitis (strep throat), pneumonia, endocarditis. Some streptococcal diseases can lead to rheumatic fever or nephritis which can damage the heart and

kidney.

Streptococcus pyogenes. Gram stain.

(7) Neisseria and other Gram-negative cocci are frequent

inhabitants of the upper respiratory tract, mainly the pharynx. Neisseria meningitidis, an important cause of bacterial meningitis, can colonize as well, until the host can develop active

immunity against the pathogen.

Neisseria meningitidis. Gram stain.

(8) While E. coli is a consistent resident of the small intestine, many other enteric bacteria may reside here as well,

including Klebsiella, Enterobacter andCitrobacter. Some strains of E. coli are pathogens that cause intestinal infections, urinary

tract infections and neonatal meningitis.

E. coli. Scanning E.M. Shirley Owens. Center for Electron

Optics. Michigan State University.

(9) Pseudomonas aeruginosa is the quintessential opportunistic pathogen of humans that can invade virtually any tissue. It is a leading cause of hospital-acquired (nosocomial) Gram-negative

infections, but its source is often exogenous (from outside the host).

Colonies of Pseudomonas aeruginosa growing on an agar plate.

The most virulent Pseudomonas species produce mucoid

colonies and green pigments such as this isolate.

(10) Haemophilus influenzae is a frequent secondary invader to viral influenza, and was named accordingly. The bacterium was

the leading cause of meningitis in infants and children until the recent development of the Hflu type B vaccine.

Haemophilus influenzae. Gram stain.

(11) The greatest number of bacteria are found in the lower intestinal tract, specifically the colon and the most prevalent bacteria are the Bacteroides, a group of Gram-negative, anaerobic,

non-sporeforming bacteria. They have been implicated in the initiation colitis and colon cancer.

Bacteroides fragilis. Gram stain.

(12) Bifidobacteria are Gram-positive, non-sporeforming, lactic acid bacteria. They have been described as "friendly" bacteria in

the intestine of humans.Bifidobacterium bifidum is the predominant bacterial species in the intestine of breast-fed infants,

where it presumably prevents colonization by potential pathogens. These bacteria are sometimes used in the manufacture of yogurts and are frequently incorporated into probiotics.

Bifidobacterium bifidum. Gram stain

(13) Lactobacilli in the oral cavity probably contribute to acid formation that leads to dental caries. Lactobacillus

acidophilus colonizes the vaginal epithelium during child-bearing years and establishes the low pH that inhibits the growth of pathogens.

Lactobacillus species and a vaginal squaemous epithelial cell.

CDC

(14) There are numerous species of Clostridium that colonize the

bowel.Clostridium perfringens is commonly isolated from feces. Clostridium difficilemay colonize the bowel and cause

"antibiotic-induced diarrhea" or pseudomembranous colitis.

Clostridium perfringens. Gram stain.

(15) Clostridium tetani is included in the table as an example of a bacterium that is "transiently associated" with humans as a

component of the normal flora. The bacterium can be isolated from feces in 0 - 25 percent of the population. The endospores are

probably ingested with food and water, and the bacterium does not colonize the intestine.

Clostridium tetani. Gram stain.

(16) The corynebacteria, and certain related propionic acid bacteria, are consistent skin flora. Some have been implicated as a cause of acne.Corynebacterium diphtheriae, the agent of

diphtheria, was considered a member of the normal flora before the widespread use of the diphtheria toxoid, which is used to

immunize against the disease.

Corynebacterium diphtheriae. No longer a part of the normal

flora.

(5) http://textbookofbacteriology.net/normalflora.html

Role of Normal Flora in Human Defense Mechanism

The presence of Normal Flora is not essential to life, because

“germ-free” animals can be reared in the complete absence of a

normal microbial flora. Yet the resident flora of certain areas plays

a definite role in maintaining health and normal function. Members

of the resident flora in the intestinal tract synthesize vitamin K and

aid in the absorption of nutrients. On mucous membranes and skin,

the resident flora may prevent colonization by pathogens and

possible disease through “bacterial interference.” The mechanism

of bacterial interference is not clear. It may involve competition for

receptors or binding sites on host cells, competition for nutrients,

mutual inhibition by metabolic or toxic products, mutual inhibition

by antibiotic materials or bacteriocins, or other mechanisms.

Suppression of the normal flora clearly creates a partial local void

that tends to be filled by organisms from the environment or from

other parts of the body. Such organisms behave as opportunists and

may become pathogens. (2)

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a. Colonization resistance

Colonization resistance is the first line of defense against

invasion by exogenous, pathogenic organisms or

indigenous opportunistic organisms and the normal flora is

responsible for this formidable task. Even though the focus

here is the intestinal tract, it should be remembered that

colonization resistance plays an important role at other

body sites (oral, skin, vagina, etc.). Colonization resistance

is a dynamic phenomenon that may differ dramatically by

microbial species, type of host, diet and other host factors.

Colonization resistance has been found to be an extremely

effective natural barrier against such pathogens as C.

difficile, Salmonella, Shigella, Pseudomonas, pathogenic E.

coli strains, Candida albicans and others.

b. Production of Inhibitory End Products

Normal flora may also produce other metabolic end

products that are inhibitory to other microbes. Most notable

is hydrogen peroxide (H2O2) produced under anaerobic

conditions by several strains of normal flora. The presence

of H2O2 results in peroxidation of lipid membranes,

increased bacterial membrane permeability, destruction of

bacterial nuclear acids in bacterial strains that do not

possess catalase. The vaginal tract is usually predominantly

colonized with lactobacilli and H2O2 producing

Lactobacillus strains have been found in 75%of vagina

samples from healthy women. Vaginal colonization with

Lactobacillus strains that produce H2O2 has been shown to

be protective of infections caused by Chlamydia

trachomatis, Gardnerella vaginalis, Ureaplasma

urealyticum and the development of bacterial vaginosis. In

women with bacterial vaginosis, these strains of H2O2

producing lactobacilli are absent and, instead, high

concentrations of Gardnerella vaginalis and anaerobes are

present.

c. Production of Low pH

Another protective mechanism is the production of a low

pH environment, which may be inhibitory for certain

pathogens. The production of acids as an end product of

carbohydrate metabolism is common in many species of the

normal flora and is inhibitory against Gram-positive and

Gram-negative bacteria. Several pathogens, including

Staphylococcus aureus, Salmonella, E. coli, and Bacillus

cereus, are inhibited by acids produced by normal flora

such as lactobacilli and bifidobacteria. A common end

product of microbial fermentation is short chain fatty acids

(SCFA). The presence of these SCFA have been shown to

be inhibitory to nonindigenous bacteria. Rolfe showed in a

hamster model of C. difficile disease that SCFA levels

inhibited C. difficile growth. As newborn hamsters age,

they start to produce high levels of acetic, butyric and

propionic acids by day 16–19. Growth of C. difficile was

significantly reduced when levels of these SCFA increased.

If normal flora are disrupted, decreased levels of SCFA

result and pathogenic microbes may take advantage of this

decrease and reproduce to levels that induce disease. To

date however, the identification of which specific SCFA or

mixtures of SCFAs are responsible for the inhibition of

pathogens has not been demonstrated.

d. Competition for Nutrients

Competition for nutrients may be another mechanism for

colonization resistance. As it is extremely difficult to assess

the levels of specific nutrients in the interior of the colon,

most of the research on nutrient depletion has been done

using continuous flow culture techniques. Wilson etal.

inoculated normal flora from a mouse into a continuous

flow culture and found one or more of the flora competed

more successfully for monomeric glucose,

Nacetylglucosamine and sialic acid, resulting in

significantly reduced levels of C. difficile. Sweeney et al.

found that even small numbers of an ingested E. coli strain

F-18 could supplant established flora, as this strain utilized

an available nutrient (gluconate) more efficiently than the

other microbes present in the system. However, continuous

flow cultures are extremely dependent on culturing and

incubation parameters and the applicability of these results

is unclear.

e. Production of Inhibitory Enzymes

Normal flora may also produce extracellular enzymes that

are inhibitory or interfere with pathogen attachment.

A yeast (Saccharomyces boulardii ) has been shown to

produce a protease that destroys toxin A and toxin B

receptor sites in rabbit ileal models for C. difficile disease.

The toxins of C. difficile act by inactivating Rho proteins

that keep the cytoskeleton of the intestinal enterocyte

intact, thereby distorting the cellular morphology leading to

fluid loss and diarrhea. Rho proteins are also involved in

yeast budding processes (reproduction), thus this yeast may

produce the protease to protect itself against soil Clostridia

that may produce similar toxins to C. difficile, but in the

human, the protease may coincidentally protect the host

against infection with C. difficile. (3) Lynne V.

McFarland (2000)

f. Stimulation of the immune system

Normal flora also induces the maturation of the gut-

associated lymphoid system (GALT). The intestinal flora

provides an array of antigenic stimulants to the GALT

cells, affecting both local and systemic levels. Gnotobiotic

mice have been shown to have fewer intraepithelial

lymphocytes, plasma cells and Peyer’s patches than mice

with intact intestinal flora. When gnotobiotic mice are

immunized, the only local immune response is secretory

IgA, however, mice with intact intestinal flora also respond

with IgM and IgG. Intestinal flora may also be involved in

the development of tolerance to antigens. Herias et al. gave

germ-free rats E. coli alone or a mixture of E. coli,

Lactobacillus acidophilus and a strain of an obligate

anaerobe (Peptostreptococcus) and observed two effects.

The peptostreptococci reduced translocation rates of E. coli

and increased serum anti-E. coli antibodies. It may be that

peptostreptococci act as an immune system primer to other

bacterial antigens, thereby leading to a decrease in

translocation. Further evidence that normal flora may act as

an immune primer was found in a study using BALB:c

mice. Pulverer et al. found that normal flora releases low

molecular weight substances that interact with MALT

(mucosa associated lymphoid tissue) and these substances

appear to be essential for an adequate immune response.

Antibiotic decontamination in a mouse model resulted in a

decreased immune response. Thus, normal flora may have

an important role as an immune system primer. (3) Lynne

V. McFarland (2000) 198

g. Antibodies production

The normal flora evokes the Antibodies production. These

Antibodies cross react with pathogens having related or

shared antigens, thus raising the immune status of the host

against the invading pathogen. (6)

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ogy/Lesson-07.pdf

Although normal microbiota offers some protection from

invading pathogens, they may themselves become

pathogenic and produce disease under certain

circumstances, and then are termed opportunistic

microorganisms or pathogens. These opportunistic

microorganisms are adapted to the noninvasive mode of life

defined by the limitations of the environment in which they

are living. If removed from these environmental restrictions

and introduced into the bloodstream or tissues, disease can

result. For example, streptococci of the viridans group are

the most common resident bacteria of the upper respiratory

tract. If large numbers of them are introduced into the

bloodstream (e.g., following tooth extraction or a

tonsillectomy), they may settle on deformed or prosthetic

heart valves and cause endocarditis. (4) Prescott (2002)

References

1. Gordon R. The alarming history of medicine, New York: St.

Martin’s Press, 1993 p. 164.

2. Normal microbial flora of the human body chapter 11 p 196,

196 http://202.120.143.134/Download/1531f207-c8b9-48d9-

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3. Lynne V. McFarland, Microbial Ecology in Health and

Disease 2000; 12: 193–207 Normal flora: diversity and

functions pages, 193, 197, 198

4. Lansing M. Prescott, Microbiology, 5th edition, 2002, p. 699,

701

5. http://textbookofbacteriology.net/normalflora.html

6. Module microbiology notes: Normal flora of the human body

page 80

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son-07.pdf