Hundreds of bacterial species populate the body. The oral cavity provides a unique environment

1.0 Introduction Hundreds of bacterial species populate the body. The oral cavity provides a unique environment that supports a wide range of bacterial species. The highly diverse flora grows in the different surfaces found in the mouth. They are able to grow on both hard and soft tissue by binding to them using adhesion molecules. Fig 1.1 shows the anatomy of the mouth. Fig 1.1 (a) Side view of the oral cavity; (b) Anterior view of the oral cavity (Marieb & Hoehn, 2007) Bacteria can colonise on all the surfaces. These bacteria found in the mouth are commensals, resident bacteria. They have a symbiotic relationship with the host. They have a symbiotic relationship with the host. These bacteria can be opportunistic. The oral cavity is constantly exposed to changes in the environment. Opportunistic bacteria can take advantage of this and cause disease if the conditions are favourable. These changes could be in nutrient levels, ph changes due to saliva secretions and epithelial debris. Their opportunistic behaviour means that the bacterial growth must but kept under control. 5

Oral bacteria colonising on surfaces in the mouth can be removed through different methods, such as saliva or brushing. Bacteria can be allowed to colonise in small crevices, such as the gingival pocket as they are not easily accessible by saliva, the tongue or brushing making them a favourable, and therefore common, site for bacterial growth. Mouthwash has the ability to reach these pockets and remove bacteria either by killing or inhibiting their growth. This means that a useful mouthwash must have strong bactericidal properties that can target many types of bacteria. 1.1 Microbiota found in the oral cavity The oral cavity is the habitat of around 300 bacterial species (Loesche, 1996). Microflora found in the mouth is extremely variable. The bacterial diversity in the oral cavity is due to the various anatomical structures that support different ecological environments. Thus, research to find the composition of oral microflora uses samples from multiple oral sites to reveal the degree of oral microbial diversity (Asikainen & Karched, 2008). For example, Haemophillus spp are found in saliva (Samaranayake. et. al., 2002), Candida albicans adhere to the buccal mucosa (Pizzo et. al., 2001). The characteristic properties of the oral sites depend on the level of substances coming from the host. The subgingival areas have a rich blood supply and as a result they have a higher degree of nutrients and host defence molecules. The supragingival tooth surfaces are constantly exposed to saliva, which acts as a major source to molecules for bacteria. The soft tissues composed of mucosal surfaces have an elevated renewal rate. This makes for a unique environment for bacteria to strive as the shedding of the outer epithelial cells affects the growth of bacteria and their ability to colonise. Bacteria found in the mouth include Firmicutes, bacilli (Streptococci and Lactobacilli), Actinobacteria (especially Actinomyces), Spirochaetes, Proteobacteria and various other 6

anaerobes, such as bacteroidetes (Asikainen & Karched, 2008). Streptococci form a major component of the oral flora. Gram negative anaerobic bacteria populate periodontal samples. This includes Actinobacilli actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. Endodontal samples have been found to contain Gram negative species, Porphyromonas endodontalis, P. Gingivalis, and many Prevotella species. In dental caries the species found are predominantly Gram positive facultatively anaerobic bacteria. Among these, Streptococci mutans is the most studied along with other streptococci found in the mouth (Asikainen & Karched, 2008). Corby, et. al. (2005) performed a study to identify bacteria associated with dental caries in children. Their study found that there was an overabundance of Actinomyces sp., S. mutans and Lactobacillus spp. Bacterial composition not only differs in each ecosystem found in the oral cavity, but also amongst individuals. Factors affecting the host's oral environments are the host's diet, oral hygiene, whole body health status, presence or absence of teeth, medication and genetic composition. Bacterial colonizing the mouth can vary depending on age. At birth the oral cavity is sterile until it becomes colonized as a result of the environment and their first feeding. The oral cavity is mainly composed of soft tissues. An infant's oral cavity is a moist environment due to the secretions from the salivary glands and the lack of hard tissue. As a result Streptococcus salivarius is the principal bacterial species in an infant's oral cavity (Tanzer, Livingston, & Thompson, 2001). Many Staphylococci spp. are found in the oral cavity, but Staphylococcus aureus is often found in the oral cavity of young children but not adults (Samaranayake, 2002). Actinomyces gerencseriae, Bifidobacteria, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum are found to be associated with caries in childhood (Becker, et. al., 2002). Actinobacilli spp. is notably found in the gingival pockets in juvenile gingivitis. During puberty bacteroides and spirochetes colonize (Samaranayake, 2002). 7

Dental diseases are multibacterial infections caused by the resident bacteria in the oral cavity. This means that the pathogens that cause the disease are found in healthy individuals although in lower quantities in the healthy subjects than in those with dental disease. If the host bacteria are allowed to colonize into large numbers within the oral cavity they can become opportunistic and the interactions between bacteria can cause disease and infection to develop. Because dental diseases are caused by many bacteria (Suzuki, Yoshida, Nakano 2005), mouthwash is expected to be able to reduce the growth of a wide range of bacteria in both children and adults. 1.2 Oral Health, dental plaque and dental caries Most oral bacteria survive by adhering to the structures in the mouth. The adherence molecules contribute to the formation of bacterial communities, known as dental plaque, which adhere to the tooth surface. Fig 1.2 shows the structure of a healthy tooth. Fig 1.2 The structure of a healthy human tooth (Tortora, Funke, & Case, 2007) 8

There are many areas won the tooth where bacteria can attach and accumulate. The tooth has a unique exterior surface compared to other surface in the body. They have a hard surface that doesn't shed its outer layer. As a result, bacteria can attach to the tooth surface and form a biofilm, or plaque. Patterson (1996) describes plaque as a dense adhesive microbial mass that colonizes teeth and is linked to caries and human oral disease. This is true as resident bacteria, which are also pathogens, are responsible for the formation of plaque. Streptococci mutans are a major contributor. Plaque can build up on the tooth, especially in the gingival crevice where the bacteria are protected from shearing actions of chewing, or flushing action of saliva produced in the mouth. The bacteria colonize on tooth surfaces by binding to salivary glycoproteins, like mucins, that form a thin protein film known as the enamel pellicle (Tortora, Funke, & Case, 2007). The bacteria, having established itself on the tooth surface, produce adhesion molecules and colonize, forming plaque. The bacteria within the plaque produce acid which causes the loss of tooth mineral from the tooth surface. The bacteria utilise fermentable carbohydrates that are consumed by the host through the ingestion of food. Saliva, an indigenous fluid, coats the entire oral cavity. It is composed of inorganic ions such as sodium, potassium, calcium and bicarbonate along with proteins. Usually, the saliva replenishes the mineral lost preventing the breakdown of the tooth enamel and the bicarbonate can neutralise the acid, therefore it can actively regulate the growth of oral bacteria (Samaranayake, 2002). However, if food containing high levels of fermentable carbohydrates is continuously consumed, the bacteria will constantly produce acid, even after the food has been swallowed. This acid will lower the ph within the mouth and the conditions become favourable to bacteria, such as S. mutans and Lactobacilli (Loesche, 1996). Not only does this sustain and increase plaque but these bacteria have the ability to store polysaccharides (S. mutans in particular) which they will 9

utilise to produce and secrete acid. A high level of plaque build up will also reduce its permeability to saliva, therefore the acid cannot be neutralized and the tooth surface will remain inaccessible to the saliva, further preventing remineralisation (Tortora, Funke, & Case, 2007). The loss of the tooth mineral will become higher than mineral replenished resulting in an overall loss in tooth mineral. Therefore decay is when the solubilisation of the tooth mineral becomes irreversible due to the high amount of acid. Fig 1.3 shows the progression of dental plaque to decay (or dental caries). Fig 1.3 Stages of tooth decay. (Tortora, Funke, & Case, 2007) The decayed lesion can reach the tooth pulp resulting in the formation of an abscess in the tissues surrounding the root. This allows for the accumulation of more anaerobic bacteria. A limited number of bacterial species contribute to the development of dental caries or other oral diseases. Streptococci mutans have been found to be the main cause due to its ability to ferment a wide range of carbohydrates (Coykendall, 1989; Loesche, 1996) and its ability to adhere to the tooth surface through the production of adhesion molecules. Lactobacilli have been found to be associated with the progression of dental caries (Loesche, 1996). 10

1.3 Streptococci mutans Streptococci mutans is a gram positive, facultative anaerobic bacterium which is found to be a major component of the oral flora (Coykendall, 1989). They are a group of seven species which are phenotypically similar, two of which are common in humans (Patterson, 1996). Coykendall (1989) described the bacteria as 'a docile tenant of the mouth and gut, capable of causing diseases and death among hosts'. This is because it is a primary cause of dental caries (Tortora, Funke, & Case, 2007). S. mutans is thought to be one of the main initiators of dental plaque because of its ability to ferment nearly all sugars. Amongst the sugars the S. mutans utilises are glucose, lactose, raffinose, mannitol, inulin and sorbitol. This allows the bacteria to produce an extracellular polysaccharide, glucan, and an intracellular starch-glycogen polysaccharide (Coykendall, 1989). A form of glucan produced is dextran. This is produced when the S. mutans metabolises sucrose into glucose and fructose. The glucose is converted into the unique, adhesive glycocalyx called Dextran seen in Fig 1.4. Fig 1.4 (a) S. mutans growing in a glucose broth; (b) S. mutans growing in sucrose broth. Pink arrows point to the S. mutans cells amongst the dextran (Tortora, Funke, & Case, 2007) This is done by a specialised enzyme, glucosyltransferase. When grown in a broth with added sucrose the production of the glucan, dextran, causes the cells to adhere to eachother (Wu-Yuan, 11

Tai, & Slade, 1978). Hence the initiation of plaque is caused by the attachment of the S. mutans and other bacteria to the surface of the teeth via these adhesive molecules. The dextran allows the S. mutans along with other bacteria to bind to the teeth forming the base of a biofilm (plaque) (Wu-Yuan, Tai, & Slade, 1978). The dextran forms a coating, producing an anaerobic environment, which will attract other bacteria. For this reason, most S. mutans are found harboured in dental plaque on the tooth surfaces (Coykendall, 1989). The residual fructose and other sugars are fermented into lactic acid. The acids erode the teeth enamel, initiating caries and this makes the S. mutans cariogenic. The increase in caries production is not solely due to the S. mutans but also because the glucan allows other acid producing bacteria to adhere to the tooth surface, therefore the S. mutans is classified as the most cariogenic and virulent bacteria (Coykendall, 1989; Patterson, 1996; Loesche, 1996) due to its ability to synthesize acid and glucan from fermentable carbohydrates. 1.4 Mouthwash and Oral Hygiene Although dental caries can be removed from the teeth, they leave the tooth vulnerable and more susceptible to bacterial growth. Restorative procedures, such as fillings, act to protect the weakness in the tooth, however over time bacteria can populate underneath and cause further damage. Periodontal diseases, where the gums detach from the teeth, are a result of poor oral hygiene. It is due to the build up of bacteria within plaque and this initiates an inflammatory response. The inflammatory response leads to loss of the periodontal tissues, allowing the formation of periodontal pockets, bleeding gums and the loosening and loss of teeth. Streptococci mutans and other bacteria are not only the cause of oral disease but can also cause endocarditis. The adhesive quality of the bacteria can allow it to stick to the walls of damaged heart valves, initiating subacute bacterial endocarditis and therefore oral hygiene is important in maintaining whole body health. 12

Brushing and flossing are important in maintaining good oral hygiene however these methods do cannot remove bacteria from all areas. Rinsing the mouth, appropriately, with a solution containing antimicrobial agents can act as another method to enhance oral hygiene. It has the ability to remove the bacteria in areas that are missed in brushing and flossing, and includes the soft tissue, under poor quality restorative work, such as fillings or crowns, in addition to the teeth. Dental caries are of multibacterial etiology and mouthwash must be able to accommodate for the different types of bacteria. Boutaga, et. al. (2007) conducted a study which involved analyzing and quantifying bacteria within mouthwash samples. The study found high frequencies of bacteria- A. actinomycetemcomitans, P. gingivalis and T. forsythensis. This proves that mouthwash does have the ability to remove multiple bacteria. The use of current antimicrobial agents can eradicate bacteria, such as S. mutans, significantly reducing infections. An ideal mouthwash should not only have the ability to remove bacteria, but also to inhibit bacterial growth, working to prevent colonization of pathogenic bacteria. In order to do this mouthwash are designed containing a variety of ingredients to counteract bacterial growth. This is known as the active ingredient. This ingredient usually kills bacteria, preventing regrowth for a limited time. Various brands of mouthwash use similar active ingredients. These include sodium fluoride and other antiseptics. These can be found in different concentrations depending on the mouthwash brand. 1.5 Active ingredients in mouthwash Common active ingredients include a variety of antiseptics. Sodium fluoride is one of the most common ingredients seen in mouthwash. It is known to be able to prevent or reduce cavities by up to 50% in the young (Loesche, 1996). Fluoride primarily works by either inhibiting the demineralization of the tooth, by enhancing the remineralization, producing a layer on the tooth surface that is highly resilient to acid attack, or lastly, by inhibiting the bacterial enzymes 13

(Featherstone, 1999). Fluoride is found in the water supplies, dentifrices and materials used by dentists. Featherstone (1999) found that a low, but slightly higher than normal, level of fluoride in the saliva and plaque helps to prevent and reverse caries by inhibition of the demineralization process and enhancing the remineralization. Fluorides have antibacterial effects, interfering with the metabolic processes of bacteria; however it has low antifungal effects. Flisfisch, et. al. (2007) comprised a study looking at the effects of fluorides on Candida albicans, a common cause of oral infection in the elderly. The study showed that sodium fluoride had little antifungal effects on the C. albicans. Pandit, et. al., (2010) investigated the effects of sodium fluoride on S. mutans biofilms. They found that sodium fluoride can inhibit the bacterial virulence factors and effect the biofilm composition, allowing it to successfully inhibit dental plaque accumulation. These results support the study from 2007, conducted by Griffin, et. al. Hexetidine, found in Oraldene mouthwash, is known to have antiseptic and antifungal activity. It reduces the adherence of fungal species (C. albicans) by inhibiting its morphogenesis (Jones, et. al., 1997). It acts againsts Gram positive and negative species, fungi and parasites. Chlorhexidine gluconate is an antiseptic and disinfectant agent that is extremely effective on both Gram positive and negative bacteria, although less effect on Gram neagtive microbes (Source: World Health organisation). Emilson C. (1994) found chlorhexidine to be an effective antimicrobial in preventing dental caries. The mode of action for this antiseptic is through reduction in the S. mutans bacterial count. However it could not prevent bacterial regrowth. Chlorine dioxide is also an antiseptic like the other chemicals; however it also reduces malodour in the mouth. This is because of its ability to oxidise volatile sulphur compounds, such as hydrogen sulphide, that cause the malodour. The sulphur compounds are oxidized into non-odorous 14

compounds. If used over a long period of time, the chlorine dioxide will also reduce plaque; tongue coating accumulation and Fusobacterium spp in the saliva (Shinada, et al., 2010). Dentyl Active contains an antiseptic known from reducing dental plaque and gingivitis, cetyl- pyridinium chloride (CPC). Garcia, et. al. (2010) conducted a study that proved this. They analysed the plaque inhibitory effects of CPC. The study found that CPC does reduce plaque formation; however the study did not specify which bacteria the CPC had effected. Alcohol is predominantly used as a solvent in mouthwash, however around 10 percent acts as a preservative, antiseptic and caustic agent, (McCullough & Farah, 2008). There is an ongoing debate as to whether mouthwash containing alcohol can cause oral cancer. McCullough and Farah believe that there is sufficient evidence to suggest that mouthwash increases the risk of developing oral cancer and that alcohol containing mouthwash also increase this risk. However, this is dependent on the concentration and duration of exposure to the alcohol. Alcohol in the oral cavity eliminates the lipid component of the epithelial cells that form a barrier in the oral cavity. Short term exposure to alcohol can increase the permeability of the ventral tongue mucosa. This may be due to the lipid molecules in the epithelium undergoing conformational changes, resulting in the opening f intercellular epithelial routes. This can allow other carcinogens, such as tobacco, to penetrate across the oral mucosa (McCullough & Farah, 2008). The alcohol predominantly found in mouthwash is ethanol. Ethanol can be metabolised, by the enzyme alcohol dehydrogenase, into acetaldehyde which is the primary metabolite. Acetaldehyde has been shown to have mutagenic properties making it cancerous. This metabolism can occur in the oral cavity due to oral tissue expression enzymes. This allows the acetaldehyde to build up. Particular species of streptococci contain alcohol dehydrogenase. Individuals with poor oral hygiene will have an increased number of these enzyme expressing bacteria, resulting in an increased risk of developing oral cancer (McCullough & Farah, 2008). 15

Hydrogen peroxide can also be used as an antiseptic in mouthwash. A study by Wennström and Lindhe (1979) demonstrated that hydrogen peroxide not only prevents the colonization of filaments but also of spirochaetes in plaque development. The mechanism of action of hydrogen peroxide is through hindering the colonization and multiplication of anaerobic bacteria (Wennström & Lindhe, 1979). Other antiseptics include thymol, methyl salicalate, benzylkonium chloride and domiphen bromide. 1.6 Investigation into the effects of mouthwash and bactericidal properties of mouthwash Oral diseases are multifactorial therefore many methods should be implemented to maintain oral hygiene. It is important to maintain a high standard throughout life, from infancy to adulthood. Studies have shown that The majority of research conducted on the bactericidal properties of mouthwash predominantly focuses on the active ingredient within mouthwash. The experiments use the chemical itself as opposed to the mouthwash as a whole. This causes the laboratory results, in vitro, to have reduced relevance in the clinical aspects, or in vivo, conditions. The research conducted in vivo mainly involves mouth rinses being used as a sole form of oral hygiene. This has its advantages in that the result will give a full representation of the actions of the mouthwash, however, assuming the environment and diet of the subjects are controlled, this will not fully represent the effect of mouthwash as the composition of the oral flora will differ. 1.7 Investigation aims The aim of this project is to compare the bactericidal properties of mouthwash on Streptococci mutans. This project will not only identify which mouthwash is most effective at inhibiting the 16

growth of the bacteria but also identify which mouthwash is most capable at preventing bacterial growth. 1.8 Designing the Method for Investigation For this experiment a selection of mouthwash with different ingredients will be compared using their antimicrobial quality. The method of choice for the investigation is the antibiotic resistance test. This method is a simple, but effective test that is still being used today. It allows for easy comparison between the results from the mouthwash. The bactericidal property of the mouthwash will be determined depending on the resistance of the bacteria to the mouthwash. The bacterium of choice for this investigation is Streptococci mutans. This bacterial species is easy to cultivate in the laboratory. It also plays an important role in oral diseases, and therefore we can assume that it is a common target amongst mouth rinses. 17