My Scientific Blog - Research and Articles: June 2013

The Science of Normal Flora

General Overview

In a healthy animal, 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, normal microbiota or indigenous microbiota. 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.

Normal flora may be categorized into two types:

1. Resident flora - always present

2. Transient flora - only present for short period of time

The predominant bacterial floras of humans are shown in Table 1. This table lists only a fraction of the total bacterial species that occur as normal flora of humans. A recent experiment that survey the diversity of bacteria in dental plaque revealed that only one percent of the total species found has ever been cultivated. Similar observations have been made with the intestinal flora. Also, this table does not indicate the relative number or concentration of bacteria at a particular site.

Table 1: Bacteria commonly found on the surfaces of the human body

BACTERIUM	Skin	Conjunctiva	Nose	Pharynx	Mouth	Lower Intestine	Anterior urethra	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 % + = common (about 25 %) +/- = rare (less than 5%) * = potential pathogen

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.

(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.

(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.

(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.

(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.

(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.

(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.

(8) While E. coli is a consistent resident of the small intestine, many other enteric bacteria may reside here as well, including Klebsiella, Enterobacter and Citrobacter. Some strains of E. coli are pathogens that cause intestinal infections, urinary tract infections and neonatal meningitis.

(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).

(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.

(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.

(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

(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

(14) There are numerous species of Clostridium that colonize the bowel. Clostridium perfringens is commonly isolated from feces. Clostridium difficile may colonize the bowel and cause "antibiotic-induced diarrhea" or pseudomembranous colitis.

(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.

(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.

Associations between Humans and the Normal Flora

Three types relationships between host and normal flora

1. Commensalism (Commensals) - no harm, no benefit to host

2. Mutualism (Mutualistic): beneficial relationship, both microbe and host benefit

3. Opportunistic (Opportunists): potential pathogens producing infection when host defenses depressed or when normal flora disturbed

1. Commensalism:

· Such a relationship where there is no apparent benefit or harm to either organism during their association is referred to as a commensal relationship.

· Many of the normal flora that are not predominant in their habitat, even though always present in low numbers, are thought of as commensal bacteria.

2. Mutualism

· Both host and bacteria are thought to derive benefit from each other, and the associations are, for the most part, mutualistic.

· The normal flora derives from their host a steady supply of nutrients, a stable environment, and protection and transport.

· The host obtains from the normal flora certain nutritional and digestive benefits, stimulation of the development and activity of immune system, and protection against colonization and infection by pathogenic microbes.

3. Opportunistic:

· Normally commensal flora but become potential pathogen and produces infection when host defenses depressed or when normal flora disturbed

· While most of the activities of the normal flora benefit their host, some of the normal flora are parasitic (live at the expense of their host), and some are pathogenic (capable of producing disease). Diseases that are produced by the normal flora in their host may be called endogenous diseases.

· Most endogenous bacterial diseases are opportunistic infections, meaning that the organism must be given a special opportunity of weakness or let-down in the host defenses in order to infect. An example of an opportunistic infection is chronic bronchitis in smokers wherein normal flora bacteria are able to invade the weakened lung.

Tissue Specificity of Normal Flora

Most members of the normal bacterial flora prefer to colonize certain tissues and not others. This "tissue specificity" is usually due to properties of both the host and the bacterium. Usually, specific bacteria colonize specific tissues by one or another of these mechanisms.

1. Tissue tropism: This is the bacterial preference or predilection for certain tissues for growth. One explanation for tissue tropism is that the host provides essential nutrients and growth factors for the bacterium, in addition to suitable oxygen, pH, and temperature for growth.

2. Specific adherence: Most bacteria can colonize a specific tissue or site because they can adhere to that tissue or site in a specific manner that involves complementary chemical interactions between the two surfaces. Specific adherence involves biochemical interactions between bacterial surface components (ligands or adhesins) and host cell molecular receptors. The bacterial components that provide adhesins are molecular parts of their capsules, fimbriae, or cell walls. The receptors on human cells or tissues are usually glycoprotein molecules located on the host cell or tissue surface.
Some examples of adhesins and attachment sites used for specific adherence to human tissues are described in the table below.

Table 2: Examples of bacterial specific adherence to host cells or tissue

Bacterium	Bacterial adhesin	Attachment site
Streptococcus pyogenes	Cell-bound protein (M-protein)	Pharyngeal epithelium
Streptococcus mutans	Cell- bound protein (Glycosyl transferase)	Pellicle of tooth
Streptococcus salivarius	Lipoteichoic acid	Buccal epithelium of tongue
Streptococcus pneumoniae	Cell-bound protein (choline-binding protein)	Mucosal epithelium
Staphylococcus aureus	Cell-bound protein	Mucosal epithelium
Neisseria gonorrhoeae	N-methylphenylalanine pili	Urethral/cervical epithelium
Enterotoxigenic E. coli	Type-1 fimbriae	Intestinal epithelium
Uropathogenic E. coli	P-pili (pap)	Upper urinary tract
Bordetella pertussis	Fimbriae ("filamentous hemagglutinin")	Respiratory epithelium
Vibrio cholerae	N-methylphenylalanine pili	Intestinal epithelium
Treponema pallidum	Peptide in outer membrane	Mucosal epithelium
Mycoplasma	Membrane protein	Respiratory epithelium
Chlamydia	Unknown	Conjunctival or urethral epithelium

3. Biofilm formation: Some of the indigenous bacteria are able to construct biofilms on a tissue surface, or they are able to colonize a biofilm built by another bacterial species. Many biofilms are a mixture of microbes, although one member is responsible for maintaining the biofilm and may predominate. The classic biofilm that involves components of the normal flora of the oral cavity is the formation of dental plaque on the teeth. Plaque is a naturally-constructed biofilm, in which the consortia of bacteria may reach a thickness of 300-500 cells on the surfaces of the teeth. These accumulations subject the teeth and gingival tissues to high concentrations of bacterial metabolites, which result in dental disease.

The Distribution and Composition of the Normal Flora

The normal flora of humans is exceedingly complex and consists of more than 200 species of bacteria. The makeup of the normal flora may be influenced by various factors, including genetics, age, sex, stress, nutrition and diet of the individual. Three developmental changes in humans, weaning, the eruption of the teeth, and the onset and cessation of ovarian functions, invariably affect the composition of the normal flora in the intestinal tract, the oral cavity, and the vagina, respectively. However, within the limits of these fluctuations, the bacterial flora of humans is sufficiently constant to a give general description of the situation.

A human first becomes colonized by a normal flora at the moment of birth and passage through the birth canal. In utero, the fetus is sterile, but when the mother's water breaks and the birth process begins, so does colonization of the body surfaces. Handling and feeding of the infant after birth leads to establishment of a stable normal flora on the skin, oral cavity and intestinal tract in about 48 hours.

It has been calculated that a human adult houses about 10¹² bacteria on the skin, 10¹⁰ in the mouth, and 10¹⁴ in the gastrointestinal tract. The latter number is far in excess of the number of eukaryotic cells in all the tissues and organs which comprise a human. The predominant bacteria on the surfaces of the human body are listed in Table 3.

Table 3: Predominant bacteria at various anatomical locations in adults

Anatomical Location	Predominant bacteria
Skin	staphylococci and corynebacteria
Conjunctiva	sparse, Gram-positive cocci and Gram-negative rods
Oral cavity
teeth	streptococci, lactobacilli
mucous membranes	streptococci and lactic acid bacteria
Upper respiratory tract
nasal membranes	staphylococci and corynebacteria
pharynx (throat)	streptococci, neisseria, Gram-negative rods and cocci
Lower respiratory tract	none
Gastrointestinal tract
stomach	Helicobacter pylori (up to 50%)
small intestine	lactics, enterics, enterococci, bifidobacteria
colon	bacteroides, lactics, enterics, enterococci, clostridia, methanogens
Urogenital tract
anterior urethra	sparse, staphylococci, corynebacteria, enterics
vagina	lactic acid bacteria during child-bearing years; otherwise mixed