dr.bsma
09-10-2009, 01:13 PM
Oral Microbiology
Introduction
Bacteria, viruses and fungi; diversity, structure, prokaryotes and eukaryotes
Commensals, opportunists, pathogens
The mouth as a microbial habitat
Host factors affecting colonisation
Microbiology began in the mouth: Antony van Leeuwenhoek developed and used the first microscope to examine material collected from teeth, and described motile “animalcules”.
Microbes include higher and lower organisms, although Oral Microbiology concerns mainly bacteria, some viruses and few fungi:
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/sizes.gif
Structure and Morphology of Microorganisms
Viruses
Viruses are not cells, and have no cell membrane or cytoplasm. Viral particles (virions) consist of nucleic acid surrounded by a protective protein coat, although considerable variety is seen in their morphology and structure. Their genetic material may be DNA or RNA, but one viral type will only contain one type of nucleic acid, providing one of the major differential characteristics used in virology. Viruses do not possess the machinery for synthesising macromolecules, and so are completely dependent on a host cell for their replication. Upon entry of viral nucleic acid into a host cell, the viral components are synthesised within the host cell, viral particles are assembled and finally released by lysis or budding from the host cell membrane. Viruses are not sensitive to antibiotics.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/virusshp.gif Prokaryotes and Eukaryotes
There are fundamental differences in eukaryotic and prokaryotic cell structure and gene expression; the defining difference is the presence of a nuclear membrane surrounding the genetic material of eukaryotes, but not prokaryotes.
Comparison of eukaryotic and prokaryotic cells:
Characteristic Prokaryotes Eukaryote Animal
Eukaryote Plant
nuclear membrane
no
yes
yes
plasma membrane
yes
yes
yes
cell wall
yes
no
yes
ribosomes
yes
yes
yes
endoplasmic reticulum
no
yes
yes
Golgi complex
no
yes
yes
lysosomes
no
yes
yes
peroxisomes
no
yes
yes
nucleolus
no
yes
yes
mitochondria
no
yes
yes
chloroplasts
no
no
yes
9+2 cilia/flagella
no
yes
yes
microtubules
no
yes
yes
actin filaments
no
yes
yes
chromosome
single
multiple
multiple
Protozoa
Protozoa are a diverse group of eukaryotic organisms, usually unicellular, exhibiting a great variety of structures and life styles. They range in size from 1mm to several millimetres. Most are free living (found in soil and water), and most are aerobic. However, some can grow anaerobically or microaerophilically. In the mouth a few species have been isolated (eg. Entamoeba gingivalis, Trichomonas tenax, Hamblia spp.) but their true prevalence and importance in the oral cavity is unclear.
Fungi
Eukaryotic, multinucleate or multicellular organisms with a thick cell wall. There are many growth forms (eg mushrooms) but those responsible for most human disease grow as branched filaments (many hyphae form a mycelium; eg. Aspergillus), or as yeasts (eg. Candida) which are characteristically single celled.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/filament.gif Bacteria
Genetic information is carried on long double stranded circular molecules of DNA, but the complete genome may also comprise plasmids (some of which can transfer antibiotic resistance from one bacterium to another). The cytoplasm is packed with ribosomes but no other organelles. Cellular respiration takes place at the cytoplasmic membrane. The cell wall composition allows separation of most bacteria into two major groups, Gram-positive and Gram-negative, depending on their retention of specific basic dyes. External to the cell wall may be structures such as polysaccharide capsules, fimbriae and flagella. These structures may function as antigens, agents of attachment and/or may protect the bacterial cell from host defences.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/bactwall.gif
Host-Microbe Relationships
Microbes utilise the environment provided by the host to gain their primary requirements which are nutrients, or, in the case of viruses, nuclear synthetic machinery. The results of this interaction, in terms of damage to the host, vary and form the basis for broad categorisation of host-microbe symbiotic associations:
commensalism - microbe derives benefit, host derives neither benefit nor harm
mutualism - microbe and host derive benefit from association, which may be essential
parasitism - one symbiont benefits at the expense of the other
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/sympass.gif Pathogenesis is not the normal and inevitable consequence of these associations, and the healthy mouth is one of the best examples of a host supporting enormous numbers of microorganisms with no deleterious effect. In fact, the host can benefit from the presence of a resident microbiota, because it, for example, excludes exogenous pathogens and ‘primes’ the immune system. Perturbations in the balance between host and microbes can result in pathogenesis: this perturbation may be caused by alterations in host defence or may be caused by changes in the microbiota (for example, after antibiotic therapy).
Intracellular/Extracellular Modes of Life
Organisms such as viruses, because of their dependence on host synthetic machinery, are obligately intracellular. They will not replicate outside a host cell but obviously can survive and be transmitted extracellularly. A few bacterial species (Chlamydia, Rickettsia) are also obligately intracellular and take their nutrients directly from those available within the cell. Organisms which have evolved an extracellular mode of existence gain nutrients primarily from tissue fluids (in the mouth, saliva and gingival crevicular fluid are rich sources of nutrients).
Intracellular existence provides the microorganism with protection from the external environment, making them inaccessible to, for example, antibodies and antibiotics. While organisms which are of importance in the oral cavity are almost all extracellular, some important periodontal pathogens may transiently invade host epithelial cells. Extracellular organisms are constantly exposed to components of the host defence mechanisms, and have evolved varied strategies for evading these.
The Mouth as a Habitat
Host defences associated with the tooth surfaces:-
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/gcfetc.gif The host defences associated with oral mucosal surfaces:-
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/hostdef.gif Specific and non-specific host defence factors in the mouth.
Specific Factor
Main function
Non-specific factor
Main function
Intra-epithelial lymphocytes
Cellular barrier to bacteria ± antigens
Saliva flow
physical removal of organisms
Langerhans cell
sIgA
Prevents adhesion & metabolism
Mucin/agglutinins
physical removal of organisms
IgG, IgA, IgM
prevent adhesion, opsonise, complement
Lysozyme-protease-anion system
cell lysis
Complement
activates neutrophils, bactericidal
Lactoferrin
iron sequestration
Neutrophils, macrophages
phagocytosis
Apo-lactoferrin
Cell killing
Sialoperoxidase system
Neutral pH: hypothiocyanite
Acid pH: hypocyanous acid
Histatins
antibacterial and antifungal
Growth and Death of Oral Microorganisms
Reproduction and growth of bacteria
Measurement of growth
Growth curves
Environmental factors affecting growth and survival
Introduction to sterilisation and disinfection
Growth of a biological system or of a living organism, or part of one, may be defined as an increase in mass or size (in any direction) accompanied by the synthesis of macromolecules, leading to the production of a newly organised structure. Measurement of the growth of some organisms is dictated by the nature of the organism; actinomycetes and fungi exist as hyphae, which increase in length by extension of the zone behind the hyphal tip. A fungal colony will increase in diameter with most growth occurring at the margin. Yeasts and some bacteria reproduce by budding. Growth when applied to bacteria normally refers to an increase in the number of individual cells and so is a measure of population density, denoting an increase in number beyond that in the original inoculum. Bacterial growth can be very rapid (a culture of Escherichia coli can double in size in 20 minutes in a rich medium) and this characteristic is especially important in vivo when nutrients may be extremely scarce.
Measurement of Growth
Growth may be estimated as an increase in the number of bacteria, cell mass, or any cellular constituent. When measuring populations counting methods can be divided into two broad groups: total counts, including both living and dead bacteria, and viable counts in which only cells able to grow in the conditions provided are counted. Both types of procedures are used commonly to enumerate and evaluate oral bacteria. Total counts give much higher numbers of organisms in plaque, not only because dead cells are counted, but also because many of the organisms in plaque cannot yet be cultivated in the lab (eg many oral spirochaetes, which can be seen by microscopy and their nucleic acids can be detected, are non-cultivable).
Total counts
1. Direct counts by microscopy using:
counting chambers
a known volume of bacterial suspension filtered onto the surface of a membrane filter, stained with acridine orange and viewed and counted using epi-fluorescence microscopy
2. opacity (turbidity) of bacterial suspensions measured spectrophotometrically
3. separate bacteria by filtration or centrifugation and then weigh: weighing after drying the bacteria (dry weight) is a more accurate assessment than wet weight
4. measurement of cell constituents, eg. N (proportional to protein), ATP, muramic acid.
Viable counts
Dilutions of bacteria may be spread onto the surface of agar (spread plates) or incorporated into molten, cooled agar medium which is then poured into Petri dishes (pour plates). In both cases colonies are counted after incubation (counts are expressed as colony forming units, cfu's, per ml of bacterial suspension). The medium used will determine which organisms are enumerated. For example, Blood agar plates support the growth of most oral commensals and pathogens. Specific agents can be used to inhibit certain groups of bacteria, to enable only a target population togrow. Such media are termed selective (see below), and are valuable in detecting species present at a low concentration in a sample.
Growth of Bacteria in vitro
In an appropriate medium and given suitable physical conditions a bacterial culture will grow at a characteristic rate (unrestricted growth) as long as growth is not influenced by, for example, the concentration of nutrients becoming limiting or by a build-up of toxic metabolic products. In batch culture (eg. a tube or flask of broth) nutrient supply is limited and growth cannot continue indefinitely. When the growth of such a culture is followed over a period of time three distinct phases of growth are seen.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/expgrow.gif lag phase: when a suspension of bacteria is inoculated into a fresh medium growth may not occur immediately. The length of the delay, or lag, in growth depends on the organism, the growth phase of the inoculum, the composition of the new medium and other factors. During this period the viable bacteria in the inoculum undergo physiological readjustment enabling them to utilise substrates in the new medium.
exponential phase: bacteria enter a period of balanced growth, during which all constituents of cells increase by the same proportion over the same time interval and the population doubles in a definite and constant time (the generation time).
stationary phase: eventually the bacteria stop growing, because some nutrient becomes exhausted or because a metabolic end product reaches toxic concentrations. Balanced growth is not possible and cells no longer have a constant composition. Also, cells in the stationary phase are smaller than those in exponential phase because cell division continues after the synthesis of most macromolecules has slowed down.
Growth of Bacteria in vivo
Most of our knowledge of microorganisms has been gained by working with them in vitro in pure culture. However, in vivo in the mouth, growth is probably very different. It is unlikely that the very rapid growth rates achieved in the lab are reached in natural habitats. Microorganisms in the mouth will be exposed to varying conditions of starvation and plenty, and also have to contend with the adverse influences of the host environment and defensive responses. Additionally, the ability to adhere to oral surfaces is an important characteristic of oral organisms. Once adhered they will grow to form a complex community called a biofilm, in which they may function quite differently from cells in a pure suspension.
Antimicrobial agents seek to either kill microorganisms or control their growth by extending the lag phase as long as possible. Such agents may not be as active in vivo as laboratory studies would suggest: some rely on growth of the organisms to be effective (eg. penicillin requires active cell wall synthesis) and so are less effective against slow growing organisms in vivo, and organisms within biofilms are protected from external agents.
Environmental Factors Influencing Growth
Microorganisms in their natural environments and in the laboratory are subjected to a wide variety of environmental influences, which combine to determine whether growth can occur and the rate at which it can occur. Organisms which are best adapted to the environment will grow best and will consequently be selected from a mixed population. For example, as organisms in subgingival plaque grow and the periodontal pocket deepens, conditions become increasingly anaerobic so the bacteria which come to predominate in periodontal pockets do not require oxygen for metabolism.
Temperature: Temperature primarily affects the enzymes of a microorganism: a rise in temperature increases enzyme activity and allows a faster growth rate, until key enzymes are denatured. The temperatures at which these events occur vary widely amongst microbes, which all have characteristic maximum, minimum and optimum temperatures for growth. Organisms which inhabit the human body as commensals and/or pathogens are mesophiles, and grow most rapidly within the range 20·C to 45·C, with growth optima between 35·C and 40·C.
pH: Most bacteria have an optimum pH for growth in the range 6.5 - 7.5 with limits somewhere between 5 and 9. Acidophilic bacteria can grow at a low pH, and such organisms are very important in Oral Microbiology as the causative agents of caries: lactobacilli and mutans streptococci produce acid as end products of metabolism of dietary sugars, and are able to survive and grow in the acidic conditions created (aciduric). The organisms found in periodontal disease are usually not aciduric as they tend to rely for growth on protein/peptide breakdown and this produces slighlty alkaline end products.
Oxygen: Bacteria vary widely in their requirements for oxygen, ranging from obligate aerobes through facultative anaerobes and microaerophiles to obligate anaerobes. Because oxygen and its derivatives are toxic and can lethally damage certain cellular components, aerobic and facultative organisms have evolved protective enzyme systems: superoxide dismutase (SOD) eliminates superoxide radicals and hydrogen peroxide can be removed by catalase and peroxidase enzymes. In general, anaerobes lack protective mechanisms.
Aerobic bacteria use oxygen as the terminal electron acceptor in respiration, and obligate aerobes have an absolute requirement for oxygen to grow.
Microaerophilic (eg. Campylobacter spp.) organisms require a low concentration of oxygen for growth, and are sensitive to atmospheric concentrations.
Facultative anaerobes, such as streptococci and Neisseria, use oxygen but also grow in its absence although growth is usually slower without oxygen.
An obligately anaerobic organism is one whose energy generating and synthetic pathways do not require molecular oxygen, and which demonstrates a high degree of adverse sensitivity to oxygen. Because of their extreme sensitivity, obligate anaerobes must be cultivated in the absence of atmospheric oxygen and a low redox potential (Eh) must be maintained in the growth medium (Eh is a measure of the tendency of a solution to give up or receive electrons). These conditions can be acheived using specialised techniques, such as incubation in anaerobic jars or cabinets. These cultivation techniques, and anaerobic sampling methods, are essential in Oral Microbiology when examining samples from, for example, periodontal pockets or abscesses which contain high numbers of obligately anaerobic bacteria.
Control of microorganisms
Control of the growth and spread of microorganisms is acheived in three main ways (apart from by developments in sanitation, water purification etc.).
Chemotherapy: most successful antimicrobial agents are antibacterial, with target sites in the cell wall, the bacterial ribosome, nucleic acid synthetic pathways or the cell membrane. They may be bactericidal (kill bacteria) or bacteriostatic (inhibit growth, thereby limiting numbers of infecting organisms to levels which the host defences can control). There are far fewer antifungal drugs available; because fungi are eukaryotic, there are fewer target sites within fungal cells which differ sufficiently from host cells to ensure non-toxicity of the antifungal agent. Similarly, development of antiviral drugs is difficult because interference with viruses is often impossible without damage to host cells.
Immunisation: vaccines have been of enormous importance in controlling, and in some cases (eg. smallpox) eradicating, significant diseases. In Oral Microbiology the diseases to be controlled are often caused by too many different organisms to make vaccination a realistic option, but much work has been done on a vaccine based on Streptococcus mutans to control dental caries.
Sterilisation and disinfection: excluding sources of infection from equipment, dressings, medicines, water supplies etc. is of paramount importance in infection control within hospitals/clinics/practices.
Sterilisation means the process of killing or removing all viable organisms. Sterilisation may be acheived by:
heat - moist heat, more often used than dry heat, is used within autoclaves where saturated steam under pressure ensures sufficient killing and penetration of heat into materials to be sterilised. The usual sterilisation cycle of 121·C for 15 minutes is sufficient to kill all vegetative bacterial cells and the heat resistant endospores of clostridia and Bacillus spp.
irradiation - gamma irradiation is used to sterilise needles, syringes, gloves, vaccines and heat-sensitive items and equipment. Free radicals are produced by the irradiation and these attack target sites such as DNA.
chemical agents - the gases, ethylene oxide and formaldehyde, are alkylating agents which damage proteins and nucleic acids. Many chemical agents are capable of disinfecting but few are capable of rendering articles sterile.
filtration - passing fluids through nitrocellulose membranes with pore sizes of 0.6 or 0.22mm removes microbial cells as well as particles and pyrogens. Filtration may also be used to isolate very small numbers of organisms from large volumes of fluid, for example when looking for pathogens in water supplies.
Disinfection is a process which kills most, but not all, viable organisms. It may employ a chemical agent which kills pathogens, but does not kill viruses or endospores, or a physical process such as boilong water to reduce the viable microbial load. Antiseptics, a particular group of disinfectants, reduce the number of organisms on the skin.
Pasteurisation eliminates pathogens and reduce the total numbers of viable microbes, but does not affect endospores. The original regime of 62·C for 30 min has been replaced by the “Flash” method of about 71·C for 15 seconds to pasteurise many bulk fluids.
Metabolism and Metabolic Interactions
· Metabolism, growth requirements, energy generation
Metabolic adaptability
· Metabolic interactions in vivo in the oral environment
· Introduction to principles of microbial interactions and succession
Metabolism (the totality of chemical changes performed by a cell or organism) can be divided into catabolic pathways, which involve the breakdown of larger organic molecules into smaller, and anabolic pathways, or the biosynthetic reactions of an organism. Metabolic pathways fulfil three major requirements of the cell:
Energy generation in the form of ATP - a growing bacterial cell must synthesise about 2.5 million molecules of ATP per second.
Generation of reducing power in the form of reduced coenzymes, such as nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+).
Generation of precursor metabolites, from which all biosynthetic pathways begin such as pyruvate, phosphoenol pyruvate, oxalacetate, acetyl coenzyme A, fructose-6-phosphate etc. These metabolites are formed during the course of catabolic pathways and are withdrawn for biosynthesis.
Nutritional Requirements
The metabolic pathways utilised by bacteria, and therefore their nutritional requirements, are very varied. Six elements (C,O, N, H, P and S) comprise 95% of the dry weight of a bacterial cell. So to synthesize cell materials, bacteria need sources of these elements as well as a variety of inorganic ions (P, K, Mg, Ca, Fe, traces of Zn, Mn). Additionally, a source of energy to support synthetic activities is essential. Most/all bacteria colonising humans utilise an organic compound as a source of both carbon and energy.
In vivo, microorganisms rely on the host and their immediate environment for nutrients. It is important to remember that the environment of oral microorganisms is very dynamic, and that growth of one organism will change it. For example, an organism may utilise one nutrient thereby out-competing other less efficient species, whilst also producing metabolic end-products which may support the growth of another organism, or be toxic.
The nutritional requirements of microorganisms in the laboratory are provided by nutrient broths and agars containing peptones, meat extract and yeast extract. Blood and extracts of animal tissues may be required for the cultivation of fastidious organisms such as many found in the mouth. Selective media contain nutrients to enhance the growth of a particular species and ingredients which inhibit the growth of unwanted species. If only Streptococcus mutans are being grown, a selective medium such as MSB agar is used which contains 20% sucrose and 0.2Uml-1 bacitracin. If yeasts are being evaluated, Sabouraud dextrose agar will allow the growth of fungi but inhibit most bacteria through low pH. Obviously, environmental factors such as temperature or oxygen can also be used as part of a selection procedure. Differential media contain reagents or supplements which allow differentiation of various kinds of organisms.
Bacteria do not generally waste metabolic energy: those that grow in environments relatively rich in organic material, like the mouth, have tended to dispense with redundant biosynthetic pathways, and so have to be provided with growth factors when grown in the laboratory. For example, many of the black pigmented Gram-negative anaerobic organisms (eg. Prevotella spp., Porphyromonas gingivalis) require media supplemented with haemin and menadione/vitamin K for growth. Growth media in which each constituent is known are called defined media and are often essential in the laboratory, especially in research. However, media commonly used to grow bacteria in the lab are complex media because many of the essential nutrients are provided by partially degraded protein sources, peptone (protein of meat, casein, or soya hydrolysed by acids or enzymes). Growth factors required by more nutritionally demanding organisms can be provided by complex additions such as yeast extract, beef extract, blood, soil etc.
Metabolic Adaptability
Bacteria and viruses live in intimate contact with a constantly changing environment and, to survive, they must react immediately to utilise available substrates and resist damage by toxic substances or conditions. This involves the immediate shutdown of synthesis of some enzymes and rapid synthesis of others, and bacteria can be amazingly versatile in their metabolism. Also, studies of bacteria and viruses have provided models for gene regulation which have found parallels and uses in studies of eukaryotes.
Bacterial cells have a complement of 3000-4000 genes, but at one time only about a half of these genes are expressed. The flexibility of bacterial gene expression allows the high degree of metabolic economy they display - given a choice between two available substrates, they will first utilise the one which provides most energy with least metabolic effort. Enzyme synthesis may be controlled at a number of places in the synthetic process, at the level of transcription or translation.
Energy Generation
In bacteria, growth on an organic substrate such as glucose can proceed by either oxidative or fermentative metabolism. These processes differ in the pathways involved, the mechanisms of production of ATP and in the end products generated. However, the first steps in the breakdown of glucose are shared in both types of metabolism: 1 mol of glucose is oxidised by glycolysis to yield 2 mol of pyruvate. Bacteria, unlike other organisms, use one of three possible glycolytic pathways. In this conversion of glucose to pyruvate ATP is produced by substrate level phosphorylation (see below).
Oxidative Metabolism. In facultative organisms growing aerobically, and in obligately aerobic organisms (eg. Pseudomonas spp., Microccus spp., Mycobacterium spp.) glucose is oxidised by aerobic respiration. Pyruvate which has been produced by glycolysis is converted to acetyl-CoA, which enters the Tricarboxylic acid cycle (TCA) with the production of reduced coenzymes (NADH or flavoproteins). Electrons and protons from these reduced coenzymes enter the respiratory electron transport chain. The electron carriers in the electron transport chain are oriented in the membrane in such a way that at some stages protons are extruded from the cell. Thus, an electrochemical gradient (protonmotive force) is established. This force is used to produce ATP by oxidative phosphorylation. The terminal electron acceptor in aerobic respiration is oxygen.
Some bacteria grow in the absence of oxygen by anaerobic respiration. This process also involves the passage of electrons along an electron transport chain and production of ATP by oxidative phosphorylation, but the terminal electron acceptor is a molecule other than oxygen, e.g. nitrate, nitrite, sulphate, CO2, fumarate.
Fermentative Metabolism. In facultatives growing anaerobically, and in many obligately anaerobic bacteria, organic substrates are metabolised by fermentation pathways producing large amounts of fermentation end products, e.g. low molecular weight organic acids, alcohols and fatty acids. An electron transport chain is not involved in fermentation and oxygen does not act as the terminal electron acceptor. In fact, there is no external electron acceptor: organic compounds act as both the electron donors and the electron acceptors, during a process of ATP production termed substrate-level phosphorylation. A fermentation must balance so that the average level of oxidation (i.e., the number of moles of C, H and O) is the same in the products as in the substrates. For example, the fermentation of glucose to lactic acid by streptococci:
C6H12O6 ---> 2 CH3CHOHCOOH
Note: respiration of glucose can yield about ten times as much free energy and 18 times as much ATP as does fermentation.
End products of fermentation are very varied (but all fermentations of glucose begin with glycolysis and the formation of pyruvate).
Metabolism of Oral Microorganisms
Oral microorganisms derive nutrients from saliva and GCF. Additionally, exogenous substrates are provided intermittently in the diet. Thus, there is an enormous diversity in substrates available and in the metabolic activities of the organisms which colonise the mouth.
Carbohydrate metabolism (figure) has received much attention because of its role in caries production. End products of such fermentation in the mouth are varied e.g., Streptococcus mutans produces only lactic acid from sugars, some lactobacilli produce lactic acid and ethanol, whereas yeasts convert glucose to ethanol and CO2. The substrates used are also very varied and many of the anaerobes seen in the mouth are able to utilise amino acids as substrates for fermentation; therefore, periodontal organisms are predominantly proteolytic.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/path1.gif On to next section - Microbiology of Dental Caries (http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/micro2.html)
Introduction
Bacteria, viruses and fungi; diversity, structure, prokaryotes and eukaryotes
Commensals, opportunists, pathogens
The mouth as a microbial habitat
Host factors affecting colonisation
Microbiology began in the mouth: Antony van Leeuwenhoek developed and used the first microscope to examine material collected from teeth, and described motile “animalcules”.
Microbes include higher and lower organisms, although Oral Microbiology concerns mainly bacteria, some viruses and few fungi:
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/sizes.gif
Structure and Morphology of Microorganisms
Viruses
Viruses are not cells, and have no cell membrane or cytoplasm. Viral particles (virions) consist of nucleic acid surrounded by a protective protein coat, although considerable variety is seen in their morphology and structure. Their genetic material may be DNA or RNA, but one viral type will only contain one type of nucleic acid, providing one of the major differential characteristics used in virology. Viruses do not possess the machinery for synthesising macromolecules, and so are completely dependent on a host cell for their replication. Upon entry of viral nucleic acid into a host cell, the viral components are synthesised within the host cell, viral particles are assembled and finally released by lysis or budding from the host cell membrane. Viruses are not sensitive to antibiotics.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/virusshp.gif Prokaryotes and Eukaryotes
There are fundamental differences in eukaryotic and prokaryotic cell structure and gene expression; the defining difference is the presence of a nuclear membrane surrounding the genetic material of eukaryotes, but not prokaryotes.
Comparison of eukaryotic and prokaryotic cells:
Characteristic Prokaryotes Eukaryote Animal
Eukaryote Plant
nuclear membrane
no
yes
yes
plasma membrane
yes
yes
yes
cell wall
yes
no
yes
ribosomes
yes
yes
yes
endoplasmic reticulum
no
yes
yes
Golgi complex
no
yes
yes
lysosomes
no
yes
yes
peroxisomes
no
yes
yes
nucleolus
no
yes
yes
mitochondria
no
yes
yes
chloroplasts
no
no
yes
9+2 cilia/flagella
no
yes
yes
microtubules
no
yes
yes
actin filaments
no
yes
yes
chromosome
single
multiple
multiple
Protozoa
Protozoa are a diverse group of eukaryotic organisms, usually unicellular, exhibiting a great variety of structures and life styles. They range in size from 1mm to several millimetres. Most are free living (found in soil and water), and most are aerobic. However, some can grow anaerobically or microaerophilically. In the mouth a few species have been isolated (eg. Entamoeba gingivalis, Trichomonas tenax, Hamblia spp.) but their true prevalence and importance in the oral cavity is unclear.
Fungi
Eukaryotic, multinucleate or multicellular organisms with a thick cell wall. There are many growth forms (eg mushrooms) but those responsible for most human disease grow as branched filaments (many hyphae form a mycelium; eg. Aspergillus), or as yeasts (eg. Candida) which are characteristically single celled.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/filament.gif Bacteria
Genetic information is carried on long double stranded circular molecules of DNA, but the complete genome may also comprise plasmids (some of which can transfer antibiotic resistance from one bacterium to another). The cytoplasm is packed with ribosomes but no other organelles. Cellular respiration takes place at the cytoplasmic membrane. The cell wall composition allows separation of most bacteria into two major groups, Gram-positive and Gram-negative, depending on their retention of specific basic dyes. External to the cell wall may be structures such as polysaccharide capsules, fimbriae and flagella. These structures may function as antigens, agents of attachment and/or may protect the bacterial cell from host defences.
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Host-Microbe Relationships
Microbes utilise the environment provided by the host to gain their primary requirements which are nutrients, or, in the case of viruses, nuclear synthetic machinery. The results of this interaction, in terms of damage to the host, vary and form the basis for broad categorisation of host-microbe symbiotic associations:
commensalism - microbe derives benefit, host derives neither benefit nor harm
mutualism - microbe and host derive benefit from association, which may be essential
parasitism - one symbiont benefits at the expense of the other
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/sympass.gif Pathogenesis is not the normal and inevitable consequence of these associations, and the healthy mouth is one of the best examples of a host supporting enormous numbers of microorganisms with no deleterious effect. In fact, the host can benefit from the presence of a resident microbiota, because it, for example, excludes exogenous pathogens and ‘primes’ the immune system. Perturbations in the balance between host and microbes can result in pathogenesis: this perturbation may be caused by alterations in host defence or may be caused by changes in the microbiota (for example, after antibiotic therapy).
Intracellular/Extracellular Modes of Life
Organisms such as viruses, because of their dependence on host synthetic machinery, are obligately intracellular. They will not replicate outside a host cell but obviously can survive and be transmitted extracellularly. A few bacterial species (Chlamydia, Rickettsia) are also obligately intracellular and take their nutrients directly from those available within the cell. Organisms which have evolved an extracellular mode of existence gain nutrients primarily from tissue fluids (in the mouth, saliva and gingival crevicular fluid are rich sources of nutrients).
Intracellular existence provides the microorganism with protection from the external environment, making them inaccessible to, for example, antibodies and antibiotics. While organisms which are of importance in the oral cavity are almost all extracellular, some important periodontal pathogens may transiently invade host epithelial cells. Extracellular organisms are constantly exposed to components of the host defence mechanisms, and have evolved varied strategies for evading these.
The Mouth as a Habitat
Host defences associated with the tooth surfaces:-
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/gcfetc.gif The host defences associated with oral mucosal surfaces:-
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/hostdef.gif Specific and non-specific host defence factors in the mouth.
Specific Factor
Main function
Non-specific factor
Main function
Intra-epithelial lymphocytes
Cellular barrier to bacteria ± antigens
Saliva flow
physical removal of organisms
Langerhans cell
sIgA
Prevents adhesion & metabolism
Mucin/agglutinins
physical removal of organisms
IgG, IgA, IgM
prevent adhesion, opsonise, complement
Lysozyme-protease-anion system
cell lysis
Complement
activates neutrophils, bactericidal
Lactoferrin
iron sequestration
Neutrophils, macrophages
phagocytosis
Apo-lactoferrin
Cell killing
Sialoperoxidase system
Neutral pH: hypothiocyanite
Acid pH: hypocyanous acid
Histatins
antibacterial and antifungal
Growth and Death of Oral Microorganisms
Reproduction and growth of bacteria
Measurement of growth
Growth curves
Environmental factors affecting growth and survival
Introduction to sterilisation and disinfection
Growth of a biological system or of a living organism, or part of one, may be defined as an increase in mass or size (in any direction) accompanied by the synthesis of macromolecules, leading to the production of a newly organised structure. Measurement of the growth of some organisms is dictated by the nature of the organism; actinomycetes and fungi exist as hyphae, which increase in length by extension of the zone behind the hyphal tip. A fungal colony will increase in diameter with most growth occurring at the margin. Yeasts and some bacteria reproduce by budding. Growth when applied to bacteria normally refers to an increase in the number of individual cells and so is a measure of population density, denoting an increase in number beyond that in the original inoculum. Bacterial growth can be very rapid (a culture of Escherichia coli can double in size in 20 minutes in a rich medium) and this characteristic is especially important in vivo when nutrients may be extremely scarce.
Measurement of Growth
Growth may be estimated as an increase in the number of bacteria, cell mass, or any cellular constituent. When measuring populations counting methods can be divided into two broad groups: total counts, including both living and dead bacteria, and viable counts in which only cells able to grow in the conditions provided are counted. Both types of procedures are used commonly to enumerate and evaluate oral bacteria. Total counts give much higher numbers of organisms in plaque, not only because dead cells are counted, but also because many of the organisms in plaque cannot yet be cultivated in the lab (eg many oral spirochaetes, which can be seen by microscopy and their nucleic acids can be detected, are non-cultivable).
Total counts
1. Direct counts by microscopy using:
counting chambers
a known volume of bacterial suspension filtered onto the surface of a membrane filter, stained with acridine orange and viewed and counted using epi-fluorescence microscopy
2. opacity (turbidity) of bacterial suspensions measured spectrophotometrically
3. separate bacteria by filtration or centrifugation and then weigh: weighing after drying the bacteria (dry weight) is a more accurate assessment than wet weight
4. measurement of cell constituents, eg. N (proportional to protein), ATP, muramic acid.
Viable counts
Dilutions of bacteria may be spread onto the surface of agar (spread plates) or incorporated into molten, cooled agar medium which is then poured into Petri dishes (pour plates). In both cases colonies are counted after incubation (counts are expressed as colony forming units, cfu's, per ml of bacterial suspension). The medium used will determine which organisms are enumerated. For example, Blood agar plates support the growth of most oral commensals and pathogens. Specific agents can be used to inhibit certain groups of bacteria, to enable only a target population togrow. Such media are termed selective (see below), and are valuable in detecting species present at a low concentration in a sample.
Growth of Bacteria in vitro
In an appropriate medium and given suitable physical conditions a bacterial culture will grow at a characteristic rate (unrestricted growth) as long as growth is not influenced by, for example, the concentration of nutrients becoming limiting or by a build-up of toxic metabolic products. In batch culture (eg. a tube or flask of broth) nutrient supply is limited and growth cannot continue indefinitely. When the growth of such a culture is followed over a period of time three distinct phases of growth are seen.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/expgrow.gif lag phase: when a suspension of bacteria is inoculated into a fresh medium growth may not occur immediately. The length of the delay, or lag, in growth depends on the organism, the growth phase of the inoculum, the composition of the new medium and other factors. During this period the viable bacteria in the inoculum undergo physiological readjustment enabling them to utilise substrates in the new medium.
exponential phase: bacteria enter a period of balanced growth, during which all constituents of cells increase by the same proportion over the same time interval and the population doubles in a definite and constant time (the generation time).
stationary phase: eventually the bacteria stop growing, because some nutrient becomes exhausted or because a metabolic end product reaches toxic concentrations. Balanced growth is not possible and cells no longer have a constant composition. Also, cells in the stationary phase are smaller than those in exponential phase because cell division continues after the synthesis of most macromolecules has slowed down.
Growth of Bacteria in vivo
Most of our knowledge of microorganisms has been gained by working with them in vitro in pure culture. However, in vivo in the mouth, growth is probably very different. It is unlikely that the very rapid growth rates achieved in the lab are reached in natural habitats. Microorganisms in the mouth will be exposed to varying conditions of starvation and plenty, and also have to contend with the adverse influences of the host environment and defensive responses. Additionally, the ability to adhere to oral surfaces is an important characteristic of oral organisms. Once adhered they will grow to form a complex community called a biofilm, in which they may function quite differently from cells in a pure suspension.
Antimicrobial agents seek to either kill microorganisms or control their growth by extending the lag phase as long as possible. Such agents may not be as active in vivo as laboratory studies would suggest: some rely on growth of the organisms to be effective (eg. penicillin requires active cell wall synthesis) and so are less effective against slow growing organisms in vivo, and organisms within biofilms are protected from external agents.
Environmental Factors Influencing Growth
Microorganisms in their natural environments and in the laboratory are subjected to a wide variety of environmental influences, which combine to determine whether growth can occur and the rate at which it can occur. Organisms which are best adapted to the environment will grow best and will consequently be selected from a mixed population. For example, as organisms in subgingival plaque grow and the periodontal pocket deepens, conditions become increasingly anaerobic so the bacteria which come to predominate in periodontal pockets do not require oxygen for metabolism.
Temperature: Temperature primarily affects the enzymes of a microorganism: a rise in temperature increases enzyme activity and allows a faster growth rate, until key enzymes are denatured. The temperatures at which these events occur vary widely amongst microbes, which all have characteristic maximum, minimum and optimum temperatures for growth. Organisms which inhabit the human body as commensals and/or pathogens are mesophiles, and grow most rapidly within the range 20·C to 45·C, with growth optima between 35·C and 40·C.
pH: Most bacteria have an optimum pH for growth in the range 6.5 - 7.5 with limits somewhere between 5 and 9. Acidophilic bacteria can grow at a low pH, and such organisms are very important in Oral Microbiology as the causative agents of caries: lactobacilli and mutans streptococci produce acid as end products of metabolism of dietary sugars, and are able to survive and grow in the acidic conditions created (aciduric). The organisms found in periodontal disease are usually not aciduric as they tend to rely for growth on protein/peptide breakdown and this produces slighlty alkaline end products.
Oxygen: Bacteria vary widely in their requirements for oxygen, ranging from obligate aerobes through facultative anaerobes and microaerophiles to obligate anaerobes. Because oxygen and its derivatives are toxic and can lethally damage certain cellular components, aerobic and facultative organisms have evolved protective enzyme systems: superoxide dismutase (SOD) eliminates superoxide radicals and hydrogen peroxide can be removed by catalase and peroxidase enzymes. In general, anaerobes lack protective mechanisms.
Aerobic bacteria use oxygen as the terminal electron acceptor in respiration, and obligate aerobes have an absolute requirement for oxygen to grow.
Microaerophilic (eg. Campylobacter spp.) organisms require a low concentration of oxygen for growth, and are sensitive to atmospheric concentrations.
Facultative anaerobes, such as streptococci and Neisseria, use oxygen but also grow in its absence although growth is usually slower without oxygen.
An obligately anaerobic organism is one whose energy generating and synthetic pathways do not require molecular oxygen, and which demonstrates a high degree of adverse sensitivity to oxygen. Because of their extreme sensitivity, obligate anaerobes must be cultivated in the absence of atmospheric oxygen and a low redox potential (Eh) must be maintained in the growth medium (Eh is a measure of the tendency of a solution to give up or receive electrons). These conditions can be acheived using specialised techniques, such as incubation in anaerobic jars or cabinets. These cultivation techniques, and anaerobic sampling methods, are essential in Oral Microbiology when examining samples from, for example, periodontal pockets or abscesses which contain high numbers of obligately anaerobic bacteria.
Control of microorganisms
Control of the growth and spread of microorganisms is acheived in three main ways (apart from by developments in sanitation, water purification etc.).
Chemotherapy: most successful antimicrobial agents are antibacterial, with target sites in the cell wall, the bacterial ribosome, nucleic acid synthetic pathways or the cell membrane. They may be bactericidal (kill bacteria) or bacteriostatic (inhibit growth, thereby limiting numbers of infecting organisms to levels which the host defences can control). There are far fewer antifungal drugs available; because fungi are eukaryotic, there are fewer target sites within fungal cells which differ sufficiently from host cells to ensure non-toxicity of the antifungal agent. Similarly, development of antiviral drugs is difficult because interference with viruses is often impossible without damage to host cells.
Immunisation: vaccines have been of enormous importance in controlling, and in some cases (eg. smallpox) eradicating, significant diseases. In Oral Microbiology the diseases to be controlled are often caused by too many different organisms to make vaccination a realistic option, but much work has been done on a vaccine based on Streptococcus mutans to control dental caries.
Sterilisation and disinfection: excluding sources of infection from equipment, dressings, medicines, water supplies etc. is of paramount importance in infection control within hospitals/clinics/practices.
Sterilisation means the process of killing or removing all viable organisms. Sterilisation may be acheived by:
heat - moist heat, more often used than dry heat, is used within autoclaves where saturated steam under pressure ensures sufficient killing and penetration of heat into materials to be sterilised. The usual sterilisation cycle of 121·C for 15 minutes is sufficient to kill all vegetative bacterial cells and the heat resistant endospores of clostridia and Bacillus spp.
irradiation - gamma irradiation is used to sterilise needles, syringes, gloves, vaccines and heat-sensitive items and equipment. Free radicals are produced by the irradiation and these attack target sites such as DNA.
chemical agents - the gases, ethylene oxide and formaldehyde, are alkylating agents which damage proteins and nucleic acids. Many chemical agents are capable of disinfecting but few are capable of rendering articles sterile.
filtration - passing fluids through nitrocellulose membranes with pore sizes of 0.6 or 0.22mm removes microbial cells as well as particles and pyrogens. Filtration may also be used to isolate very small numbers of organisms from large volumes of fluid, for example when looking for pathogens in water supplies.
Disinfection is a process which kills most, but not all, viable organisms. It may employ a chemical agent which kills pathogens, but does not kill viruses or endospores, or a physical process such as boilong water to reduce the viable microbial load. Antiseptics, a particular group of disinfectants, reduce the number of organisms on the skin.
Pasteurisation eliminates pathogens and reduce the total numbers of viable microbes, but does not affect endospores. The original regime of 62·C for 30 min has been replaced by the “Flash” method of about 71·C for 15 seconds to pasteurise many bulk fluids.
Metabolism and Metabolic Interactions
· Metabolism, growth requirements, energy generation
Metabolic adaptability
· Metabolic interactions in vivo in the oral environment
· Introduction to principles of microbial interactions and succession
Metabolism (the totality of chemical changes performed by a cell or organism) can be divided into catabolic pathways, which involve the breakdown of larger organic molecules into smaller, and anabolic pathways, or the biosynthetic reactions of an organism. Metabolic pathways fulfil three major requirements of the cell:
Energy generation in the form of ATP - a growing bacterial cell must synthesise about 2.5 million molecules of ATP per second.
Generation of reducing power in the form of reduced coenzymes, such as nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+).
Generation of precursor metabolites, from which all biosynthetic pathways begin such as pyruvate, phosphoenol pyruvate, oxalacetate, acetyl coenzyme A, fructose-6-phosphate etc. These metabolites are formed during the course of catabolic pathways and are withdrawn for biosynthesis.
Nutritional Requirements
The metabolic pathways utilised by bacteria, and therefore their nutritional requirements, are very varied. Six elements (C,O, N, H, P and S) comprise 95% of the dry weight of a bacterial cell. So to synthesize cell materials, bacteria need sources of these elements as well as a variety of inorganic ions (P, K, Mg, Ca, Fe, traces of Zn, Mn). Additionally, a source of energy to support synthetic activities is essential. Most/all bacteria colonising humans utilise an organic compound as a source of both carbon and energy.
In vivo, microorganisms rely on the host and their immediate environment for nutrients. It is important to remember that the environment of oral microorganisms is very dynamic, and that growth of one organism will change it. For example, an organism may utilise one nutrient thereby out-competing other less efficient species, whilst also producing metabolic end-products which may support the growth of another organism, or be toxic.
The nutritional requirements of microorganisms in the laboratory are provided by nutrient broths and agars containing peptones, meat extract and yeast extract. Blood and extracts of animal tissues may be required for the cultivation of fastidious organisms such as many found in the mouth. Selective media contain nutrients to enhance the growth of a particular species and ingredients which inhibit the growth of unwanted species. If only Streptococcus mutans are being grown, a selective medium such as MSB agar is used which contains 20% sucrose and 0.2Uml-1 bacitracin. If yeasts are being evaluated, Sabouraud dextrose agar will allow the growth of fungi but inhibit most bacteria through low pH. Obviously, environmental factors such as temperature or oxygen can also be used as part of a selection procedure. Differential media contain reagents or supplements which allow differentiation of various kinds of organisms.
Bacteria do not generally waste metabolic energy: those that grow in environments relatively rich in organic material, like the mouth, have tended to dispense with redundant biosynthetic pathways, and so have to be provided with growth factors when grown in the laboratory. For example, many of the black pigmented Gram-negative anaerobic organisms (eg. Prevotella spp., Porphyromonas gingivalis) require media supplemented with haemin and menadione/vitamin K for growth. Growth media in which each constituent is known are called defined media and are often essential in the laboratory, especially in research. However, media commonly used to grow bacteria in the lab are complex media because many of the essential nutrients are provided by partially degraded protein sources, peptone (protein of meat, casein, or soya hydrolysed by acids or enzymes). Growth factors required by more nutritionally demanding organisms can be provided by complex additions such as yeast extract, beef extract, blood, soil etc.
Metabolic Adaptability
Bacteria and viruses live in intimate contact with a constantly changing environment and, to survive, they must react immediately to utilise available substrates and resist damage by toxic substances or conditions. This involves the immediate shutdown of synthesis of some enzymes and rapid synthesis of others, and bacteria can be amazingly versatile in their metabolism. Also, studies of bacteria and viruses have provided models for gene regulation which have found parallels and uses in studies of eukaryotes.
Bacterial cells have a complement of 3000-4000 genes, but at one time only about a half of these genes are expressed. The flexibility of bacterial gene expression allows the high degree of metabolic economy they display - given a choice between two available substrates, they will first utilise the one which provides most energy with least metabolic effort. Enzyme synthesis may be controlled at a number of places in the synthetic process, at the level of transcription or translation.
Energy Generation
In bacteria, growth on an organic substrate such as glucose can proceed by either oxidative or fermentative metabolism. These processes differ in the pathways involved, the mechanisms of production of ATP and in the end products generated. However, the first steps in the breakdown of glucose are shared in both types of metabolism: 1 mol of glucose is oxidised by glycolysis to yield 2 mol of pyruvate. Bacteria, unlike other organisms, use one of three possible glycolytic pathways. In this conversion of glucose to pyruvate ATP is produced by substrate level phosphorylation (see below).
Oxidative Metabolism. In facultative organisms growing aerobically, and in obligately aerobic organisms (eg. Pseudomonas spp., Microccus spp., Mycobacterium spp.) glucose is oxidised by aerobic respiration. Pyruvate which has been produced by glycolysis is converted to acetyl-CoA, which enters the Tricarboxylic acid cycle (TCA) with the production of reduced coenzymes (NADH or flavoproteins). Electrons and protons from these reduced coenzymes enter the respiratory electron transport chain. The electron carriers in the electron transport chain are oriented in the membrane in such a way that at some stages protons are extruded from the cell. Thus, an electrochemical gradient (protonmotive force) is established. This force is used to produce ATP by oxidative phosphorylation. The terminal electron acceptor in aerobic respiration is oxygen.
Some bacteria grow in the absence of oxygen by anaerobic respiration. This process also involves the passage of electrons along an electron transport chain and production of ATP by oxidative phosphorylation, but the terminal electron acceptor is a molecule other than oxygen, e.g. nitrate, nitrite, sulphate, CO2, fumarate.
Fermentative Metabolism. In facultatives growing anaerobically, and in many obligately anaerobic bacteria, organic substrates are metabolised by fermentation pathways producing large amounts of fermentation end products, e.g. low molecular weight organic acids, alcohols and fatty acids. An electron transport chain is not involved in fermentation and oxygen does not act as the terminal electron acceptor. In fact, there is no external electron acceptor: organic compounds act as both the electron donors and the electron acceptors, during a process of ATP production termed substrate-level phosphorylation. A fermentation must balance so that the average level of oxidation (i.e., the number of moles of C, H and O) is the same in the products as in the substrates. For example, the fermentation of glucose to lactic acid by streptococci:
C6H12O6 ---> 2 CH3CHOHCOOH
Note: respiration of glucose can yield about ten times as much free energy and 18 times as much ATP as does fermentation.
End products of fermentation are very varied (but all fermentations of glucose begin with glycolysis and the formation of pyruvate).
Metabolism of Oral Microorganisms
Oral microorganisms derive nutrients from saliva and GCF. Additionally, exogenous substrates are provided intermittently in the diet. Thus, there is an enormous diversity in substrates available and in the metabolic activities of the organisms which colonise the mouth.
Carbohydrate metabolism (figure) has received much attention because of its role in caries production. End products of such fermentation in the mouth are varied e.g., Streptococcus mutans produces only lactic acid from sugars, some lactobacilli produce lactic acid and ethanol, whereas yeasts convert glucose to ethanol and CO2. The substrates used are also very varied and many of the anaerobes seen in the mouth are able to utilise amino acids as substrates for fermentation; therefore, periodontal organisms are predominantly proteolytic.
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http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/path1.gif On to next section - Microbiology of Dental Caries (http://www.dentistry.leeds.ac.uk/OROFACE/PAGES/micro/micro2.html)