**1. Introduction**

Each form of life on earth needs to obtain water and some substances from the external environment for its growth. From viruses to whales, every form of life needs some substances. Differences are only in the mechanism of obtaining. Many types of organisms on earth for this purpose developed the digestive tract with the oral cavity during the evolution. The same mechanism is still on earth millions and millions of years. For example, dinosaurs had the same mechanism and during evolution developed dentition for good mechanical preparing of eaten food like humans today with some differences of course. We can deduct, that the dental problems in Jurassic age had the same cause as today if we are thinking about mechanical destruction. In the case of special dental diseases, like periodontitis or dental caries, the comparing is debatable. Maybe in Jurassic age were also some pathogens something like *Streptococcus mutans* nowadays, which were responsible for the destruction of dental enamel in Tyrannosaurus rex. Nowadays problems in dental diseases have the same causative mechanism. Many of them are caused by the effect of dental biofilm bacteria.

### **1.1 Biofilm, dental biofilm**

A biofilm comprises any syntrophic consortium of microorganisms in which cells stick to each other and often also to a surface. Biofilms are highly organized bacterial agglomeration, which diversity is depending on the external and internal conditions of together growing bacteria.

Bacterial biofilms are also characteristic of the growth of one type of bacteria, i.e. a biofilm of *Staphylococcus aureus* [1]. Biofilms may form on living or nonliving surfaces and can be founded in natural, industrial, and hospital conditions. In humans, a typical exam for biofilm is dental plaque. This microcosm was deeply characterized with the help of numerous basic or sophisticated methods of research. Microbiology procedures, microscopic techniques, genomic and proteomic methods bring new light on new findings in dental plaque (biofilm) research.

It is interesting, that the knowledge about dental biofilm from the discoveries of Anton van Leeuwenhoek (1632–1723) to today age is still not perfect because we are not able to decrease the number of dental diseases in the world [2].

Dental caries and periodontal diseases are the most common diseases in the world especially in areas with bad quality of dental medicine and in poor regions of the world. On the other side, it is also a disease, which is a wide range presented in all countries and all social communities.

Bacterial pathogens founded in dental enamel lesions are many times highly pathogenic and cause also systematic diseases like endocarditis, meningitis, pulmonary fibrosis, arthritis, and some findings predict the connection of dental microbiota with cancerogenesis [3, 4].

### **1.2 Dental biofilm bacterial composition**

Opinions on the number of bacteria living in the oral cavity vary. It has been estimated that about 500 species of bacteria inhabit the oral cavity in humans [5]. Molecular-based studies have shown that bacterial communities found in the oral cavity are highly complex with about 1000 species and have been shown to be the second most complex microbial community in the body after the colon [6]. Although the animal microbiocenosis of animals and humans has similar properties, there are also significant differences in relation to the microbial species and the relative proportions of these species in the oral cavity [7]. For example, rodents lack gender representatives *Peptostreptococcus, Bacteroides* (currently *Prevotella* and *Porphyromonas*), *Treponema*, *Vibrio* and *Leptotrichia* [8]. Oral microbiocenosis of dogs is believed to be more diverse than oral microbiocenosis in humans [9]. However, bacteria in dental biofilm that are responsible for periodontal infectious diseases in humans and animals have been shown to be similar [7].

The microbiota of the dental biofilm differs from the microbiota on the mucosal surfaces and the composition of the microbiota of the dental biofilm varies in different anatomical sites. Gingival crevice supplies nutrients to bacteria and has low redox potential; therefore, it is colonized predominantly by anaerobic species such as *Prevotella* spp., *Veillonella* spp. and *Fusobacterium* spp. In contrast, supragingival plaque consists mainly of Gram-positive facultatively anaerobic bacteria, especially *Streptococcus* spp. and *Actinomyces* spp. The composition of the oral microbiota is highly dependent on the clinical condition of the teeth and gingivae. Healthy oral plaque contains predominantly facultatively anaerobic Gram-positive species, while in periodontal diseases microbiota turns into obligate anaerobic Gram-negative species [10]. In the formation of dental biofilm, primarily Gram-positive cocci, especially

**181**

*Methods for Searching of Potential Beneficial Bacteria and Their Products in Dental Biofilm*

*Streptococcus sanguis* and *Streptococcus mitis*, are involved in primary colonization, which colonizes the teeth for the first 4 hours after professional cleansing [11]. Other early colonizers include *Actinomyces* spp., *Capnocytophaga* spp., *Eikenella* spp., *Haemophilus* spp., *Prevotella* spp., *Propionibacterium* spp., and *Veillonella* spp. [12]. One of the major bacteria that serve as a bridge between early and late oral biofilm colonizers is *Fusobacterium nucleatum* [13]. Although it is an anaerobic bacterium, it could tolerate oxygen in the biofilm. This ability allows *F*. *nucleatum* to promote the growth of other strictly anaerobic bacteria such as *Porphyromonas gingivalis* [14]. Later colonizers are *Lactobacillus* spp., *Porphyromonas* spp., *Actinobacillus* spp., *Prevotella* spp., *Eubacterium* spp., *Selenomonas* spp., *Tannerella* spp., *Aggregatibacter* and *Treponema* spp. [15, 16]. Which type of bacteria, pathogens or potential beneficial members of dental microbiota will be chosen for research depends on the researcher. Currently, preparations containing probiotic strains such as: *Lactobacillus reuteri* (BioGaia Prodentis), *Bacillus coagulans* (Life Extension Advanced Oral Hygiene) and *Streptococcus salivarius* K12 (Bactoral) are available on the market [17–20]. *Bacillus subtilis* in form of tablet [VITALREXTM (VL)] is also used in the treatment of periodontal diseases [21]. Depending on the findings of the beneficial effect of living bacteria, there is also the possibility to use only their metabolic products for research

**2.1 Selection criteria useful for studying of dental biofilm and sample obtaining**

In the oral cavity area, it is possible to study apart from dental biofilm also other biofilms, i.e. buccal, lingual, prosthesis, filled live or death teeth, soft tissue biofilms, etc. Our preferred place for obtaining of dental biofilm samples are sites of tooth surfaces close to the salivary duct orifices, because proteins produced in saliva could help to form biofilm and calculus. In humans, it is the lingual surface of the lower front teeth and decreases towards the third molar teeth. On the upper jaw, the supragingival calculus is often formed on the buccal surfaces of the first molars [22]. Also, in veterinary patients, supragingival calculus usually accumulates more rapidly and in larger amounts on the buccal surfaces of the upper jaws [23]. Places for sampling are variable depending on the anatomical proportion of hosts that are used for research as volunteers. Except for humans, it is possible to study dental biofilm also on domesticated or wild animals. Important criteria in the case of human biofilm are smoke, veganism, celiac disease, age, health condition, therapy with medication and so on. Each external and internal factor could change the composition of biofilm and each human has individual microbiota in the mouth. It is better when the group of volunteers has similar dental care (a type of toothpaste used) and similar food consumption habits. The selection of volunteers should be based on the targeted microbiota from the dental biofilm e.g. autochthonous or allochthonous or obtaining of pathogenic bacteria from target pathological lesions in the oral cavity, e.g. caries, etc. Autochthonous microbiota is isolated from volunteers who starve overnight after carefully brushing their teeth. The dental biofilm sample has to be obtained immediately after waking up. Volunteers could not eat, drink or brush their teeth before sampling. The composition of autochthonous or allochthonous microbiota depends on sampling time. If sampling takes place during the day, samples also contain allochthonous microbiota. Better condition for obtaining samples of autochthonous microbiota is from

*DOI: http://dx.doi.org/10.5772/intechopen.88024*

aimed at preventing and treating dental diseases.

volunteers, which several days do not brush the their teeth.

**2. Recommended methods**

*Methods for Searching of Potential Beneficial Bacteria and Their Products in Dental Biofilm DOI: http://dx.doi.org/10.5772/intechopen.88024*

*Streptococcus sanguis* and *Streptococcus mitis*, are involved in primary colonization, which colonizes the teeth for the first 4 hours after professional cleansing [11]. Other early colonizers include *Actinomyces* spp., *Capnocytophaga* spp., *Eikenella* spp., *Haemophilus* spp., *Prevotella* spp., *Propionibacterium* spp., and *Veillonella* spp. [12]. One of the major bacteria that serve as a bridge between early and late oral biofilm colonizers is *Fusobacterium nucleatum* [13]. Although it is an anaerobic bacterium, it could tolerate oxygen in the biofilm. This ability allows *F*. *nucleatum* to promote the growth of other strictly anaerobic bacteria such as *Porphyromonas gingivalis* [14]. Later colonizers are *Lactobacillus* spp., *Porphyromonas* spp., *Actinobacillus* spp., *Prevotella* spp., *Eubacterium* spp., *Selenomonas* spp., *Tannerella* spp., *Aggregatibacter* and *Treponema* spp. [15, 16]. Which type of bacteria, pathogens or potential beneficial members of dental microbiota will be chosen for research depends on the researcher. Currently, preparations containing probiotic strains such as: *Lactobacillus reuteri* (BioGaia Prodentis), *Bacillus coagulans* (Life Extension Advanced Oral Hygiene) and *Streptococcus salivarius* K12 (Bactoral) are available on the market [17–20]. *Bacillus subtilis* in form of tablet [VITALREXTM (VL)] is also used in the treatment of periodontal diseases [21]. Depending on the findings of the beneficial effect of living bacteria, there is also the possibility to use only their metabolic products for research aimed at preventing and treating dental diseases.
