**2. Recommended methods**

*Bacterial Biofilms*

research.

**1.1 Biofilm, dental biofilm**

conditions of together growing bacteria.

all countries and all social communities.

**1.2 Dental biofilm bacterial composition**

biota with cancerogenesis [3, 4].

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

Bacterial biofilms are also characteristic of the growth of one type of bacteria,

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

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

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 micro-

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

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

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)

not able to decrease the number of dental diseases in the world [2].

diseases in humans and animals have been shown to be similar [7].

**180**

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

### **2.2 Taking of dental biofilm samples**

Samples of dental biofilms are easy to obtain, sampling is very simple, painless and noninvasive. Each human volunteer should confirm it with the signed agreement with taking samples, their next processing and provide the data in the anamnestic questionnaire concerning GDPR. In the case of domestic animals, dog or cat, owners have to agree with the possibility of sample taking and processing.

All things that are needed for the researcher are a sterile syringe needle and a sterile Eppendorf tube filled with sterile filtrated PBS commercial produced or according https://www.protocolsonline.com/recipes/phosphate-buffered-salinepbs/. Cultivation liquid medium can be use for this purpose too.

We provide Brain hearth infusion broth (Merck K GaA Darmstadt, Germany). In the case of lactic acid bacteria isolation, we use deMan, Rogosa and Sharpe MRS (CONDA S.A, Madrid Spain) broth. The blood agar (Tryptic soy agar (TSA)) with 5% ram's blood (BBL, Microbiology Systems, Cockeysville, USA) is often chosen as the first medium for the cultivation of bacteria in bacteriology. In case of selection of major streptococcal species it is good to use Mitis Salivarius Agar (Merck K GaA Darmstadt, Germany). The classical cultivation method is at 37.5°C during 24–48 hours under anaerobic or aerobic conditions, depending on target bacterial members of dental biofilm. We provide BD GasPak™ systems (Becton, Dickinson and Company) for anaerobic cultivation. The further selection of strains is according to the cultivation characteristics of selected colonies. Selected strains could be stored in the glycerol stock or Microbank system (Pro Lab Diagnostic). Each isolated strain has to be identified for further analysis. We provide MALDI-TOF mass spectrometry or Blast n analysis of 16S rRNA sequence for identification. The biochemical tests could help with the identification and reveal the characteristics of the tested strain.

### **2.3 Methods useful for identification of bacterial composition of dental biofilm**

For the study of the bacterial community and its composition, it is possible to use numerous methods. At first, it needs to be mentioned the classical microbiology. By classical bacteriology cultivation methods, we could select different types of cultivable bacteria in samples of dental biofilm. For this purpose, we could use different types of media, from liquid to solid, from basic to highly specific and selective media. Different conditions are also used in aerobic and anaerobic cultivation. The most numerous bacterial resident in the dental biofilm has better start line as low representative bacteria. On the other hand, the conditions in a cultivation medium could bring sometimes better conditions for the growth of former less presented bacteria in a tested sample. Due to this problem, it is hard to declare the ratio of different types of cultivable bacteria. Colonies forming units (CFU) method could reveal the approximate ratio of bacteria, but only the cultivable ones. Quantitative real-time PCR is a cultivation-independent perfect toll for declaring of the bacterial composition of cultivable, hard cultivable or uncultivable bacteria in tested sample, but it is limited due to numbers of selected bacterial groups. Amplicon sequencing is a sensitive method that is cultivation independent and good for declaring the composition of all bacterial members in the tested sample and it could quantify the ratio between bacterial groups [24, 25]. This method is cultivation free and principle is based on the amplification of total DNA isolated from the sample and next-generation sequencing (NGS) analysis. Big data obtained after sequencing are analyzed *in silico*.

If we combine the amplicon sequencing method with 16S rRNA identification of selected and isolated bacteria, we obtain perfect strategy and tools for confirmation of identified cultivable and uncultivable bacteria and also their semiquantitative ratio in our sample of dental biofilm.

**183**

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

It is necessary to know the numbers and ratios of bacteria in the sample because it can bring light to physiological or pathological parameters. On the other hand, it is hard to study this topic, due to the different bacterial composition of individual dental biofilms. Many isolated bacteria are autochthonous and host specific, and still found in a dental biofilm of the individuals. Based on these findings we can

Cultivation, isolation, identification, and storage of the strains are necessary steps for deep research of pathogens, potential pathogens, and potentially probiotic

This method is still necessary for valid research of potentially beneficial bacteria and their products in dental biofilm. For testing of potential candidates as probiotic bacteria from dental biofilm at first, we need to isolate and store it by microbiological cultivation techniques. The same goes for pathogenic bacteria. A very important step in bacteriology research is the identification of bacteria. Form of growth, Gram staining, catalase activity, biochemical parameters are helpful in the analysis of solitary bacterial colonies. These methods are in some cases imperfect for the exact identification of bacteria. In comparison with the methods mentioned above, the sequencing of genes coding 16S RNA or other PCR products and next Blast n analysis or MALDI-TOF mass spectrometry identification are more sensitive. Other growth characteristics as the possibility of growth inhibition of other bacteria are helpful in the selection of candidates with the production of bioactive substances, especially in the case of biosurfactants, bacteriocins, or bacteriocin-like

The presence of genes coding bioactive substances could be easily detected by PCR, but better is to check the possibility of their production at first. For example, *Streptococcus salivarius* inhibition potential against *Micrococcus luteus* could help unfold the production of salivaricin [28]. *Streptococcus thermophilus* could induce cell lysis of *Pediococcus acidilactici* and reveal the potential of Thermophilin 110 production [29]. Generally, if the presence of bacteriocins is detected in tested potential probiotics by growth inhibition during cultivation with the target organism, the next step with the help of cultivation and proteomic methods is the overlaying gel test. It can detect the mass size of bacteriocins, i.e. in the case of *Lactobacillus plantarum* or *Streptococcus thermophilus* or other tested bacteria [29, 30]. Cultivation is also necessary to obtain a large volume of bacteriocin for further testing with the help of large volume fermentation. If bacteriocins are soluble and are produced in cultivation media, flow centrifuge is needed for their isolation. If bacteriocins are insoluble it is necessary to use ultracentrifuge in this step. The extraction of bacteriocins is an important step in isolation [31]. If we have pure bacteriocins for testing, cultivation is still needed. For example, salivaricin isolated from *Streptococcus salivarius* K12 is active against bacterial species involved in halitosis, by inhibition of *Micrococcus luteus* Il, *Streptococcus anginosus* T29, *Eubacterium saburreum* ATCC

Cultivation procedures are needed in case of studying of the capability of the other bioactive substances like biosurfactants or exopolysaccharides. These two products have antagonist effects. Exopolysaccharides enhance adherence and

It was detected that biosurfactant produced by *Lactobacillus reuteri* could very significantly down-regulate expression of *Streptococcus mutans* glucosyltransferase genes (*gtfB, gtfC*) and fructosyltransferase gene *ftf* [32]. These genes are very important for the production of exopolysaccharides which are responsible for

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

predict approximately similar conditions.

strains and research of their interaction.

inhibitory substances (BLIS) [26, 27].

33271 and *Micromonas micros* ATCC 33270 [28].

biosurfactants promote disruption of adherence.

**2.4 Classical cultivation necessary step in research**

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

It is necessary to know the numbers and ratios of bacteria in the sample because it can bring light to physiological or pathological parameters. On the other hand, it is hard to study this topic, due to the different bacterial composition of individual dental biofilms. Many isolated bacteria are autochthonous and host specific, and still found in a dental biofilm of the individuals. Based on these findings we can predict approximately similar conditions.

Cultivation, isolation, identification, and storage of the strains are necessary steps for deep research of pathogens, potential pathogens, and potentially probiotic strains and research of their interaction.

### **2.4 Classical cultivation necessary step in research**

This method is still necessary for valid research of potentially beneficial bacteria and their products in dental biofilm. For testing of potential candidates as probiotic bacteria from dental biofilm at first, we need to isolate and store it by microbiological cultivation techniques. The same goes for pathogenic bacteria. A very important step in bacteriology research is the identification of bacteria. Form of growth, Gram staining, catalase activity, biochemical parameters are helpful in the analysis of solitary bacterial colonies. These methods are in some cases imperfect for the exact identification of bacteria. In comparison with the methods mentioned above, the sequencing of genes coding 16S RNA or other PCR products and next Blast n analysis or MALDI-TOF mass spectrometry identification are more sensitive.

Other growth characteristics as the possibility of growth inhibition of other bacteria are helpful in the selection of candidates with the production of bioactive substances, especially in the case of biosurfactants, bacteriocins, or bacteriocin-like inhibitory substances (BLIS) [26, 27].

The presence of genes coding bioactive substances could be easily detected by PCR, but better is to check the possibility of their production at first. For example, *Streptococcus salivarius* inhibition potential against *Micrococcus luteus* could help unfold the production of salivaricin [28]. *Streptococcus thermophilus* could induce cell lysis of *Pediococcus acidilactici* and reveal the potential of Thermophilin 110 production [29]. Generally, if the presence of bacteriocins is detected in tested potential probiotics by growth inhibition during cultivation with the target organism, the next step with the help of cultivation and proteomic methods is the overlaying gel test. It can detect the mass size of bacteriocins, i.e. in the case of *Lactobacillus plantarum* or *Streptococcus thermophilus* or other tested bacteria [29, 30]. Cultivation is also necessary to obtain a large volume of bacteriocin for further testing with the help of large volume fermentation. If bacteriocins are soluble and are produced in cultivation media, flow centrifuge is needed for their isolation. If bacteriocins are insoluble it is necessary to use ultracentrifuge in this step. The extraction of bacteriocins is an important step in isolation [31]. If we have pure bacteriocins for testing, cultivation is still needed. For example, salivaricin isolated from *Streptococcus salivarius* K12 is active against bacterial species involved in halitosis, by inhibition of *Micrococcus luteus* Il, *Streptococcus anginosus* T29, *Eubacterium saburreum* ATCC 33271 and *Micromonas micros* ATCC 33270 [28].

Cultivation procedures are needed in case of studying of the capability of the other bioactive substances like biosurfactants or exopolysaccharides. These two products have antagonist effects. Exopolysaccharides enhance adherence and biosurfactants promote disruption of adherence.

It was detected that biosurfactant produced by *Lactobacillus reuteri* could very significantly down-regulate expression of *Streptococcus mutans* glucosyltransferase genes (*gtfB, gtfC*) and fructosyltransferase gene *ftf* [32]. These genes are very important for the production of exopolysaccharides which are responsible for

*Bacterial Biofilms*

**2.2 Taking of dental biofilm samples**

Samples of dental biofilms are easy to obtain, sampling is very simple, painless and noninvasive. Each human volunteer should confirm it with the signed agreement with taking samples, their next processing and provide the data in the anamnestic questionnaire concerning GDPR. In the case of domestic animals, dog or cat,

All things that are needed for the researcher are a sterile syringe needle and a sterile Eppendorf tube filled with sterile filtrated PBS commercial produced or according https://www.protocolsonline.com/recipes/phosphate-buffered-saline-

We provide Brain hearth infusion broth (Merck K GaA Darmstadt, Germany). In the case of lactic acid bacteria isolation, we use deMan, Rogosa and Sharpe MRS (CONDA S.A, Madrid Spain) broth. The blood agar (Tryptic soy agar (TSA)) with 5% ram's blood (BBL, Microbiology Systems, Cockeysville, USA) is often chosen as the first medium for the cultivation of bacteria in bacteriology. In case of selection of major streptococcal species it is good to use Mitis Salivarius Agar (Merck K GaA Darmstadt, Germany). The classical cultivation method is at 37.5°C during 24–48 hours under anaerobic or aerobic conditions, depending on target bacterial members of dental biofilm. We provide BD GasPak™ systems (Becton, Dickinson and Company) for anaerobic cultivation. The further selection of strains is according to the cultivation characteristics of selected colonies. Selected strains could be stored in the glycerol stock or Microbank system (Pro Lab Diagnostic). Each isolated strain has to be identified for further analysis. We provide MALDI-TOF mass spectrometry or Blast n analysis of 16S rRNA sequence for identification. The biochemical tests could help with the identification and reveal the characteristics of the tested strain.

**2.3 Methods useful for identification of bacterial composition of dental biofilm**

For the study of the bacterial community and its composition, it is possible to use numerous methods. At first, it needs to be mentioned the classical microbiology. By classical bacteriology cultivation methods, we could select different types of cultivable bacteria in samples of dental biofilm. For this purpose, we could use different types of media, from liquid to solid, from basic to highly specific and selective media. Different conditions are also used in aerobic and anaerobic cultivation. The most numerous bacterial resident in the dental biofilm has better start line as low representative bacteria. On the other hand, the conditions in a cultivation medium could bring sometimes better conditions for the growth of former less presented bacteria in a tested sample. Due to this problem, it is hard to declare the ratio of different types of cultivable bacteria. Colonies forming units (CFU) method could reveal the approximate ratio of bacteria, but only the cultivable ones. Quantitative real-time PCR is a cultivation-independent perfect toll for declaring of the bacterial composition of cultivable, hard cultivable or uncultivable bacteria in tested sample, but it is limited due to numbers of selected bacterial groups. Amplicon sequencing is a sensitive method that is cultivation independent and good for declaring the composition of all bacterial members in the tested sample and it could quantify the ratio between bacterial groups [24, 25]. This method is cultivation free and principle is based on the amplification of total DNA isolated from the sample and next-generation sequencing (NGS) analysis.

If we combine the amplicon sequencing method with 16S rRNA identification of selected and isolated bacteria, we obtain perfect strategy and tools for confirmation of identified cultivable and uncultivable bacteria and also their semiquantitative

owners have to agree with the possibility of sample taking and processing.

pbs/. Cultivation liquid medium can be use for this purpose too.

Big data obtained after sequencing are analyzed *in silico*.

ratio in our sample of dental biofilm.

**182**

adherence of oral streptococci [33, 34]. In the case of *Streptococcus mutans* glucosyltransferase genes are responsible for cariogenic activity [35]. These genes are also useful for the differentiation of streptococcal candidates which are often difficult to differentiate because they have high homologous sequences in the 16S rRNA gene.
