Normal oral microbial flora

oral microbiology

normal oral microbial flora lecture no.1
the normal microbial flora of oral cavity comprises of bacteria, fungi, protozoa and rarely viruses. some of microorganisms are usually present in normal cavity known resident flora like ?-hemolytic streptococci, lactobacilli etc, on contrary certain microbes stay for restricted period in the oral cavity of few individuales, they called transient flora like pneumonia, meningococci, haemophilus influenzae.

advantage of resident flora
1- prevent the colonization of pathogenic microbes through bacterial interference on nutrient and receptor sites.
2- production of important nutrient like vitamin k, riboflavin and biotin.
3- production of digestive enzymes like amylase, lipase and protease aid in digestion and absorption of nutrients.
it needs to understand that at birth of new born, mouth is sterile. within few hours of birth sterptococcus salivarius start growing in the mouth. in the first month other forms of streptococcus, staphylococcius, neisseria, veillonella, lactobacilli, actinomycetes, fusobacteria etc, start colonizing in the oral cavity. eruption of teeth gives hard surface in the mouth, these hard surfaces may be colonized with sterptococcus mutans, streptococcus sanguis and actinomyces viscousus. on completion of first year of life, oral microbial flora is almost same as that of adult.
the mouth is similar to other sites of body in having a natural microflora with a characteristic composition and existing for most part in harmonious relationship with the host, this relationship can break down in the mouth and disease can occur. this is usually associated with:
• major changes to the biology of the mouth from exogenous sources (examples include: antibiotic treatment or the frequent intake of fermentable carbohydrates in the diet) or from endogenous changes such as alterations in the integrity of the host defences following drug therapy, which perturb the natural stability of the microflora.
• the presence of microorganisms at sites not normally accessible to them for example, when oral bacteria enter the blood stream following tooth extraction or other traumas and are disseminated to distant organs, where they can cause abscesses or endocarditis. bacteria with the potential to cause disease in this way are termed ‘opportunistic pathogens’ and many oral microorganisms have the capacity to behave in this manner.
indeed, most individuals suffer at some time in their life from localized episodes of disease in the mouth caused by imbalances in the composition of their resident oral microflora, the commonest clinical manifestations of such imbalances are dental caries and periodontal diseases, both of which are highly prevalent in industrialized societies and are now on the increase in developing countries other acute and chronic infections occur but less frequently. dental caries is the dissolution of enamel or root surfaces (demineralization) by acid produced primarily from the metabolism of fermentable carbohydrates in the diet by bacteria colonizing the tooth surface (dental plaque). dental plaque is also associated with the etiology of periodontal diseases in which the host mounts an inappropriate inflammatory response to an increased microbial load (due to plaque accumulation) around the gingival, resulting in damage to the supporting tissues of the teeth.

oral bacteria can be classified primarily as gram positive and gram negative organisms and secondarily as either anaerobic or facultative anaerobic according to their oxygen requirements. some of oral microbes are most closely associated with disease than others while a proportion of the organisms are uncultivable.


a- gram positive coccus

1- streptococcus sp.
2- peptostreptococcus sp.
3- staphyloccus sp.
4- micrococccus sp.
5- enterococcus sp.

oral streptococcus
streptococcus genus makes up the most common gram - positive cocci, facultative anaerobic bacteria in the oral cavity. the name streptococcus was given because the bacteria belonging to this genus always arrange themselves into chains. in clinical bacteriology, members of streptococcus are divided into three categories based on their ability to induce hemolytic:
- ?-hemolytic streptococcus, include all species that can form a ?-hemolytic zone, a broad and completely transparent hemolytic zone around colonies grown on blood agar plates including s. pyogenes and s. agalactiae..
- ?-hemolytic streptococcus (also known as streptococcus viridans), include all streptococcus species that form a partial hemolytic (grass-green) zone around their colonies when grown on blood agar plates such as s. sanguis, s. mitis, and
s. vestibularis.
- ?-hemolytic streptococcus (also known as non hemolytic streptococcus) no hemolytic around the colony like enterococcus fecalis.


oral streptococci can be divided into four main species groups as follows:
1. mutans group (streptococcus mutans, s. sobrinus)
2. salivarius group (streptococcus salivarius, s. vestibularis)
3. anginosus group (streptococcus. constellatus, s. anginosus)
4. mitis group (streptococcus sanguinis, s. gordonii and s. oralis)

a- streptococcus mutans
phylum: firmicutes (firmus: strong, cutis: skin reffering to cell wall)
class: bacilli
order: lactobacillales
family:streptococaceae

is g+ ve coccus, facultative anaerobic, commonly found in oral human cavity and is a significantly contributor to dental caries, the microbe was first describe by kilian clarke in 192 and then recovered from case of infective endocarditic (growth of bacteria on heart valves) after that little attention was paid to this species until 1960s when it was demonstrated that caries could be experimentally induced and transmitted in animals by oral inoculation with the organism. the name ‘mutans’ results from its frequent transition from coccal phase to coccobacillary phase. currently, seven distinct species of human and animal mutans streptococci and eight serotypes (a–h) are recognized, based on the antigenic specificity of cell wall carbohydrates. the term streptococcus mutans is limited to human isolates belonging to three serotypes (c, e and f). streptococcus mutans, s. sobrinus, s. rattus, s. ferus, s. cricetus, and s. macacae are collectively known as mutans streptococci. this bacterium, along with the closely related with species sterptococcus sorbinus can inhibit the mouth both contribute to oral disease often considered together as group called the mutans streptococcus.


habitat and transmission
streptococci make up a large proportion of the resident oral flora. it is known that roughly one-quarter of the total cultivable flora from supragingival and gingival plaque and half of the isolates from the tongue and saliva are streptococci. they are vertically transmitted from mother to child. infective endocarditis caused by these organisms as a result of their entry into the blood stream during intraoral surgical procedures e.g. tooth extraction and sometimes even during tooth-brushing.



colonization characteristics
s. mutans is a facultative anaerobe, but the optimal atmospheric condition for cultures should be anaerobic or contain only a low percentage of oxygen with 5–10% carbon dioxide. s. mutans grows quickly at 37°c and some strains can grow at 45 °c. growth not inhibited by bile or optochin (ethylhydrocupreine hydrochloride), in contrast to pneumococci. s. mutans cultures require nutrient-rich complex media. colonies of s. mutans grown on blood agar after 48 h anaerobic incubation are either regular and smooth or irregular, hard and sticky. the diameter of colonies is 0.5–1.0 mm. zones of ?- or ?-hemolysis can be observed around colonies of most strains, while ?-hemolytic zones can also be observed with the colonies of a few strains.
mitis – salivarius agar (ms) is the commonly used selective medium and both smooth and rough colonies can be observed on the same plate.
on agar containing sucrose, like mitis – salivarius sucrose bacitracin agar (msb), most strains form stacked colonies about 1 mm in diameter with droping-like or myxoid glucan products above or surrounding the colonies. cells tend to clump or attach to the bottom of the tube when incubated in glucose broth and the final ph is usually between ph 4.0 and 4.3.

biochemical reactions
most strains are catalase negative, ferment carbohydrates like mannitol, sorbitol, raffinose, lactose, inulin, salicin, mannose and trehalose to produce acid, but do not ferment arabinose, xylose, glycerol, and melezitose. s. mutans hydrolyzes esculin, but not arginine, hippurate and gelatin. s. mutans does not produce h2o2.

pathogenicity
s. mutans is mainly isolated from the surface of teeth. it synthesizes a variety of extracellular polysaccharides, including water soluble and non-water-soluble glucan and fructan from sucrose. these polysaccharides promote bacterial colonization and are key virulence factors in the formation of dental caries. due to their adhesive capacity, acid production, acid tolerance, and water-soluble glucan production. s. mutans has long been regarded as one of the main oral pathogens in tooth surface and dental caries. it is also involved in other secondary infections such as bacteremia and endocarditis.

s. mutans possess several attributes that contribute to its success as a cariogenic organism:

(1) ability to adhere to the tooth surface and develop plaque communities.
(2) production of glucans and other polysaccharides from excess carbohydrate (often sucrose) in the diet, leading to plaque accumulation. extra polysaccharide (eps) it is significant in enhancing colonization of s. mutans, s. salivarius and s. gordonii, can express a number of different enzymes that are active in the synthesis of glucans (chains of glucose residues) and fructans (fructose polymers) like glycosyltransferases and fructosyltransferases.
(3) production of acids (principally lactic acid), that generate a low ph environment and enrich for aciduric organisms s. mutans can metabolize a variety of sugars, resulting in the production of a number of weak acids, including lactic, formic and acetic acids.

dental caries (tooth decay)
early colonizers of tooth surface are mainly neisseria sp. and sterp. including
s. mutans, they adhere sufficiently to dental hard tissues. the growth and metabolism of these pioneer sp. changes local environment conditions like ph thereby enabling more fastidious along with s. sorbinus, s. mutans play a major role in tooth decay, metabolizing sucrose to lactic acid using the enzyme glucansucrase, the acidic environment created in the mouth by this process is what causes the highly mineralized tooth enamel to be vulnerable to decay s. mutans is one of a few specialized organisms equipped with receptors that improve adhesion to the surface of teeth sucrose is used by s. mutans to produce a sticky extracellular, dextran – based polysaccharide that allows these adhere forming plaque, s. mutans produces dextran via the enzyme dextransucrase using sucrse as a substrate in the fallowing reaction:
n sucrose (glucose)n + n fructose
sucrose is the only sugar that bacteria can use to form this sticky polysaccharide. however other sugars glucose, fructose, lactose can also digested by s. mutans , but they produce lactic acids as an end product the combination of plaque and acid leads to dental decay due to the role s. mutans plays in tooth decay

b- streptococcus salivarius
s. salivarius is mainly isolated from the oral cavity of humans and animals. it a is part of the normal flora of tongue and saliva microbial communities. moreover, s. salivarius is also detected in fecal and blood samples of endocarditis patients.

colonization characteristics
s. salivarius is a gram-positive coccus, facultative anaerobes, but the optimal atmosphere condition for bacterial cultures should contain a low percentage of oxygen with 5–10% carbon dioxide. s. salivarius grows quickly at 37°c, although it can also grow at 45 °c. cultures require nutrient-rich complex media, such as trypticase yeast – extract cystine agar (tpy). the final ph of glucose broth incubated with s. salivarius usually falls between 4.0 and 4.4. the ability to synthesize extracellular polysaccharides determines whether colonies of s. salivarius are smooth or rough. on agar plates with sucrose (msb), most strains synthesize soluble fructan and form sticky ball-like colonies, a characteristic that can be used to identify s. salivarius. very few strains produce ?- or ?-hemolytic zones when incubated on agar containing horse blood.


biochemical reactions
s. salivarius can ferment glucose, sucrose, maltose, raffinose, inulin, salicin, trehalose, and production of lactic acid. it cannot ferment glycerol, mannitol,, sorbitol, xylose, and arabinose. most strains can hydrolyze esculin and urea but not arginine. s. salivarius can produce acetone from glucose.


2 - peptostreptococcus sp.
kingdom: bacteria

phylum: firmicutes

class: clostridia

order: clostridiales

family: peptostreptococcaceae

genus: peptostreptococcus

peptostreptococcus is the most common gram-positive anaerobic coccus in the human oral cavity. peptostreptococcus anaerobius and peptostreptococcus micros are the most commonly encountered species n this genus. dental plaque and gingival sulcus is the main habitats for p. anaerobius in the oral cavity. moreover, p. anaerobius is often detected in clinical samples of periodontitis, pulpitis, and pericoronitis

peptostreptococcus anaerobius
the cells of p. anaerobius are gram-positive, spherical, approximately 0.5–0.6 ?m in diameter, arranged in pairs or chains, cells in early cultures have been observed to form long chains. the optimal temperature for p. anaerobius growth is 37°c, and cells of this species do not grow well at 25°c or 30°c, and do not grow at all at 45°c. growth is stimulated by 0.02% polysorbate-80. p. anaerobius cells form pinpoint- like or rounded (about 1mm in diameter), raised, white, glossy, non transparent colonies with a smooth surface, without hemolytic zone. colonies formed on the surface of brain heart infusion (bhi) medium without addition of blood are gray. broth cultures of p. anaerobius are usually not muddy, and granulated or viscous precipitates can be observed.





biochemical reaction
p. anaerobius is relatively biochemically inactive, and cells usually do not ferment carbohydrates. in deep glucose agar, p. anaerobius can produce a large amount of gas and generate ammonia from peptone.


3- staphylococcus
staphylococcus resembles streptococci in appearance but is arranged in grape-like clusters and is all catalase-positive (all streptococci are catalase-negative). staphylococcus epidermidis is frequently detected in saliva and dental plaque and thought to be associated with periodontics.


4 - micrococcus
micrococcus species often resemble staphylococci. they are g+ve cocci form regular packets of four (tetrads) or eight cocci and catalase -positive organisms. their colonies can be yellow, red, or orange. found free living in the environment and micrococci are rarely associated with disease.


5 enterococcus fecalis,
gram positive cocci, facultative anaerobes and belong to lancefield group d, in recent years, it has been closely studied due to its high detection rate in infected root canal.