Microbial Physiology 6 2013
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MICR540 !Microbial Physiology 6 ! Johnson 2013 Microbial Physiology 6 Engineering Microbes for the Future Introduction Dental caries is a slow demineralization of the tooth caused by a loss of hydroxypatite crystals. It has been around since the beginning of recorded history. Before the Iron Age, between 2-4% of teeth showed decay. During the Iron Age and through the Roman period, caries increased to about 10%. Some children from the middle-ages had extensive caries, likely caused by pacifiers made of honeycomb wrapped in linen. Caries increased dramatically toward the end of the seventeenth century and, except for a reprieve during world war II, have continued to rise in the Western world until a decade ago. Underdeveloped countries from the 50's and 60's showed a marked change from low to high caries after exposure to a Western diet. Historically, there have been some hot-spots for caries, particularly in the United States. In the Northeast, molasses was a major export and swallowed copiously during the 1700's. A visitor to this region wrote: The inhabitants of this province (Massachusetts) are formed by symmetry, handsome and have delicate complexions . but, both sexes have universally and even proverbially bad teeth, which must probably be occasioned by their eating so much molasses. By the Revolutionary War most 18 year olds from this region failed the physical exam for military duty. It required that the person had two matching front teeth for biting open a container of powder! Military records highlight dental deterioration during the 20th century. During World War II, 8.8% of recruits did not meet the requirement of 6 opposing teeth. A recent by the World Health Organization estimates that 5 billion people world-wide (>70% of the world's population) presently suffer from tooth decay. Why are bacteria implicated? Several lines of evidence point to bacteria as the major cause of caries: i) germ-free animals do not develop caries; ii) bacteria can demineralize the enamel in vitro or in vivo and iii) bacteria are found within the enamel and dentin of carious lesions. !There is good data indicating that Streptococcus mutans is the major offending organism. This organism sticks to the enamel, sets up shop and begins to ferment any carbohydrate it can use to lactic acid, bringing the pH below the critical level of 5.5, where the enamel begins to dissolve. S. mutans itself is quite stable at this pH. If one has more than 1 million S.. mutans per ml of saliva, one is at high risk for dental caries. Why is sucrose special? !History has shown qualitatively, that sucrose is an important factor in forming caries. More recent scientific studies within the last hundred years have shown a direct correlation between sucrose and caries, long before S. mutans was identified as an agent. The Table 1 MICR540 !Microbial Physiology 6 ! Johnson 2013 shows and example of such a correlation in a rat model. In vitro experiments showed that S. mutans can ferment starch, lactose, glucose and mannitol to pH 5.0 and below. How then does sucrose serve so well as a cariogenic substrate for S. mutans? The answer is that S. mutans can rapidly convert sucrose into a sticky long-chain polysaccharide capsule. This capsule acts as a glue to stick to the tooth surface and plaque mass. It is also a fuel source during when nutrients are not available !Sucrose is a disaccharide made of glucose and fructose. The link between them is a dihemiacetal bond, which has a high free energy of hydrolysis. The more negative the values are in the Table, the greater the energy is when the bonds are split. S. mutans can use this high energy to make the sticky glucose polymer dextran (-2 kcal/mol) or the fructose polymer levan (-4.6 kcal/mol). Susceptibility !There is little question that susceptibility is not equal in all individuals. There are host factors that favor as well as inhibit the development of caries. For example, it is obvious that teeth that contain deep pits and fissures, such as those often present in molars, can be particularly susceptible to plaque formation. There are also salivary components. Active salivary components Antibacterial proteins !Saliva contains a host of antibacterial proteins Buffers !Saliva has phosphate and bicarbonate buffers, but bicarbonate is more important. As salivary flow increases, bicarbonate increases but phosphate decreases. Bicarbonate also has a dissociation constant in the range where plaque acids reacts rapidly by losing CO2. Pellicle components !Teeth have a naturally occurring non-bacterial film from salivary proteins that regularly forms on teeth. It may be brushed away, but it will reform within minutes. This serves as a base for plaque formation. The salivary pellicle may also increase the resistance of the enamel to acid. 2 MICR540 !Microbial Physiology 6 ! Johnson 2013 Immunoglobulins !Saliva contains secretory IgA, which is resistant to oral proteolytic enzymes. In at least one study, salivary IgA was higher in children with no caries than those with caries. Saliva also contains IgG and IgM from the gingival sulcular fluid. These immunoglobulins could potentially intercept bacteria and decrease their plaque formation. Several antigenic determinants on S. mutans have been used as baits to develop immunity (see section on vaccines) Antimicrobial agents and treatments Anions !Anions include Fluoride and Sodium Lauryl sulfate (detergent). Cations: !Cations such as Zn++ or cationic detergents such as Chlorhexidine are attracted to the bacterial cell walls because of the substances' positive charge and the negative charge of the bacterial cell wall. Gram positive bacteria are more sensitive to cations since they are more negatively charged. S.mutans are Gram positive bacteria and are therefore very sensitive to cations. Non-ionic Agents !There are two types of phenol like substances. One is triclosan which is a non- charged agent and the other is Listerine™ which is a combination of the phenol-related essential oils thymol and eucalyptol. (Thymol is also a constituent in the chlorhexidine varnish Cervitec). Triclosan is normally not the only antimicrobial agent in different vehicles. To augment the efficacy the surface coating copolymer, polyvinylmethyl ether and maleic acid, commercially known as Gantrez™, is added. Enzymes !A glucanhydrolase has been isolated by some Korean chemical engineers that prevents adherence to S. mutans to glass, even in the presence of sucrose. Specific Treatments and Prevention Fluoride !Fluoride has been proven to reduce the incidence of caries. It has been added to drinking water, to table salt, milk and toothpastes, as well as in a variety of mouthwashes, gels and varnishes for topical use, and in tablets. Fluoride is also found naturally in water, tea and fish. !Fluoride is thought to increase the resistance of tooth enamel to acid by replacing the hydroxyl group of hydroxyapatite, producing fluorapatite. 3 MICR540 !Microbial Physiology 6 ! Johnson 2013 !If fluoride is present in the saliva, it will promote remineralization of fresh enamel lesions. Since fluorapatite is more resistant to demineralization by acid, fluoride helps reduce enamel solubility when in the mineral structure. !High concentrations of topical fluorides also reduce the acid production of cariogenic bacteria. Fluoride inhibits an enzyme required for transporting sucrose into the cell. !Fluoride does not generally cause a dramatic decrease in salivary S. mutans levels after “normal” use. On the other hand, lower numbers of cavities occur, decreasing the number of sites for easy colonization. Populations with optimal fluoride concentration in the drinking water are somewhat less colonized by S. mutans, compared to similar populations with less fluoride in the water. Chlorhexidine !Chlorhexidine, originally used as a skin disinfectant, has remarkable anti-plaque activity. An early study found that no plaque accumulated in volunteers who rinsed their mouths with a 0.2% rinse twice a day for 3 weeks. There was an 85-90% reduction in salivary flora and tooth surfaces remained bacteria free. !Chlorhexidine is highly positively charged and sticks to oral surfaces, where it is slowly released in an active form. It is this feature, and not superior antibacterial properties, which make it effective. Its positive charges also help it stick to bacteria for the kill. Chlorhexidine has an unpleasant, bitter taste that may interfere with taste perception. It also stains teeth yellow-brown over time, but this can be removed by dental polishing. Brushing and Flossing !While brushing and flossing do decrease smooth surface caries, studies have shown that they are not effective against fissure and pit caries. The reason is these that pits cannot be effectively reached and cleaned with a brush. There is also some evidence that S. mutans can survive on a brush for some period after brushing. Whether reinfection can occur with this brush and whether flossing can spread the organism from one tooth to another is a subject of debate. Regular profession debridgement !A study in the early >70s in Sweden showed that biweekly dental treatment including debridement and topical fluoride could markedly decrease dental caries. Other studies that included professional cleaning with a non-fluoridated paste were less successful. One reason for this is the rapid rate of recolonization on the tooth surface. !Thus, fluoride"s antimicrobial and remineralization features may be required to enhance the debridement to change plaque flora from cariogenic to non-cariogenic. Sealants 4 MICR540 !Microbial Physiology 6 ! Johnson 2013 !Sealants are a breakthrough in preventive treatment of caries prone areas such as pits and fissures. Leakage is minimal, and microbes trapped below the sealant don=t have enough nutrients to survive. Therefore, sealants can be placed above an incipient lesion and it won"t progress. Bacteria can populate the sealant, but their acid products cannot penetrate it.