**3. Possibility to produce bioactive substances detected in tested potential bacterial probiotic candidates by PCR (bacteriocins, biosurfactants, and exopolysaccharides)**

### **3.1 Recommended isolation of DNA**

The isolation of DNA from bacteria that are difficult to isolate, i.e. lactobacilli strains, is performed by the NucleoSpin® Tissue kit (Macherey-Nagel GmbH and Co. KG, Düren, Germany) using a lysis solution during overnight incubation at 95°C. The next steps of DNA isolation are according to the manufacturer's procedure. It is possible to use other kits for DNA isolation. It depends on researcher choice and routine practice in PCR laboratory. After isolation of DNA it is better to verify DNA quality and quantity. We use Nanodrop spectrophotometric (Wilmington, Delaware USA) analysis for this purpose.

For quick isolation of DNA it is also possible to use one bacterial colony and 100 μl DNAzol direct (Molecular research centre Inc. Cincinnati. USA), and heat it to 95°C during 15 min for isolation of DNA without measuring of DNA quantity, but storage of DNA samples for next analysis is time limited. For storage of DNA isolated by both methods we recommended −20°C. The isolation steps are according to the manufacturer and specific sample.

For PCR we could use Mastermix: One Taq 2X Master Mix (England Biolabs, Ipswich, Massachusetts, USA) and specific primers (**Tables 1–3**) in concentration of 33 μmol at volume 0.6 and 1–2 μl of DNA isolated with help of DNAzol direct.

### **3.2 Bacteriocins and methods for their detection**

A large number of lactic acid bacteria produce bacteriocins that kill other microorganisms. Lactobacilli bacteriocins have potential utility as pathogen inhibitors in humans [36]. Also, oral streptococci have their bacteriocins for example *Streptococcus mutans* have mutacin, and *Streptococcus salivarius* has salivaricin [37, 38]. There are a number of factors influencing the efficacy of bacteriocins *in vivo* and *in situ*, including the survival of the production strain, specific activity, and animal model and targeted pathogen. However, bacteriocins have a great deal of promise to manage various infections and may become an alternative to existing antibiotics. Bacteriocins will need to undergo the same rigorous, costly research and validation process as all other previously approved therapies used in therapy [26]. Recommended conditions for detection of genes coding bacteriocins of some oral potential beneficial bacteria by PCR are described in (**Table 1**).

The researcher could study probiotic or pathogenic bacteria depending of the particular relationship to diseases. For example, PCR condition for bacteriocin detection from *Lactobacillus* spp. is mentioned in the publication [46]. Detection of genes coding production of bacteriocins is only the start of the research. By this method, we could select potential candidates for further research. Inhibition potential can be detected by preferred sensitive bacterial strain for example like in case of *Streptococcus salivarius* salivaricin the sensitive strain is *Micrococcus luteus* [28]. After confirmation of bacteriocin gene presence in tested isolates, there is still much work to be done with purification, fractionation, and isolation of bacteriocins. Not

**185**

**Target gene** *Streptococcus salivarius*

Salivaricin *sal*

A

*Lactobacillus reuteri* glycerol

dehydrogenase

*gldC* (reuterin)

*Lactobacillus plantarum*

*Plantaricin*

*Lactobacillus plantarum*

*Plantaricin S*

*Streptococcus mutans*

*Mutacin*

*Bacillus subtilis*

*Subtilisin*

*Bacillus subtilis*

*Subtilosin*

**Table 1.**

*PCR conditions used for the detection of gene coding production of bacteriocins.*

**Primers** SalAUS 5′GTAGAAAATATTTACTACATACT3′

SalADS 5′GTTAAAGTATTCGTAAAACTGATG3′

GD1f 5′GTTCAGTCCGCCGCATATC3′

GD1r 5′GCCGCTCTTCGTGGATTTC3′

plaF 5′-GGCATAGTTAAAATTCCCCCC-3′

plaR 5′-CAGGTTGCCGCAAAAAAAG-3′

plnF 5′-GCCTTACCAGCGTAATGCCC-3′

plnR 5′-CTGGTGATGCAATCGTTAGTTT-3′

F 5′-AGTTTCAATAGTTACTGTTGC-3′

R 5′-GCCAAACGGAGTTGATCTCGT-3′

spaSFwd

5′CAAAGTTCGATGATTTCGATTTGGATGT3′

spaSRev 5′GCAGTTACAAGTTAGTGTTTGAAGGAA3′

sboAFwd 5′CGCGCAAGTAGTCGATTTCTAACA3′

sboARev R 5′CGCGCAAGTAGTCGATTTCTAACA3′

**PCR protocol** 95°C, 13 min, 30× (95°C, 30 sec, 55°C, 1 min, 72°C, 1 min)

72°C, 5 min

94°C, 5 min, 34× (94°C, 1 min, 58°C, 30 sec, 72°C, 50 sec)

72°C, 7 min

94°C, 5 min, 30× (94°C 45 sec, 53.2°C, 45 sec, 72°C, 45 sec)

428 bp 475 bp 750/450 bp

722 bp

[45]

[44]

[43]

[42]

72°C, 5 min

94°C, 5 min, 30× (94°C, 45 sec, 62.3°C, 30 sec, 68°C,

2 min sec) 68°C, 5 min

94°C, 5 min, 34× (94°C, 1 min, 58°C, 30 sec, 72°C, 50 sec)

72°C, 7 min

94°C, 5 min, 34× (94°C, 30 sec, 55°C, 30 sec, 65°C, 60 sec)

65°C, 7 min

94°C, 5 min, 34× (94°C, 30 sec, 50°C, 30 sec, 65°C, 60 sec)

565 bp

[45]

65°C, 7 min

**Product size**

338 bp 562 bp

[41]

[38–40]

**Source**

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

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


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

**Table 1.**

*PCR conditions used for the detection of gene coding production of bacteriocins.*

*Bacterial Biofilms*

**and exopolysaccharides)**

**3.1 Recommended isolation of DNA**

(Wilmington, Delaware USA) analysis for this purpose.

ing to the manufacturer and specific sample.

**3.2 Bacteriocins and methods for their detection**

potential beneficial bacteria by PCR are described in (**Table 1**).

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.

**3. Possibility to produce bioactive substances detected in tested potential bacterial probiotic candidates by PCR (bacteriocins, biosurfactants,** 

The isolation of DNA from bacteria that are difficult to isolate, i.e. lactobacilli strains, is performed by the NucleoSpin® Tissue kit (Macherey-Nagel GmbH and Co. KG, Düren, Germany) using a lysis solution during overnight incubation at 95°C. The next steps of DNA isolation are according to the manufacturer's procedure. It is possible to use other kits for DNA isolation. It depends on researcher choice and routine practice in PCR laboratory. After isolation of DNA it is better to verify DNA quality and quantity. We use Nanodrop spectrophotometric

For quick isolation of DNA it is also possible to use one bacterial colony and 100 μl DNAzol direct (Molecular research centre Inc. Cincinnati. USA), and heat it to 95°C during 15 min for isolation of DNA without measuring of DNA quantity, but storage of DNA samples for next analysis is time limited. For storage of DNA isolated by both methods we recommended −20°C. The isolation steps are accord-

For PCR we could use Mastermix: One Taq 2X Master Mix (England Biolabs, Ipswich, Massachusetts, USA) and specific primers (**Tables 1–3**) in concentration of 33 μmol at volume 0.6 and 1–2 μl of DNA isolated with help of DNAzol direct.

A large number of lactic acid bacteria produce bacteriocins that kill other microorganisms. Lactobacilli bacteriocins have potential utility as pathogen inhibitors in humans [36]. Also, oral streptococci have their bacteriocins for example *Streptococcus mutans* have mutacin, and *Streptococcus salivarius* has salivaricin [37, 38]. There are a number of factors influencing the efficacy of bacteriocins *in vivo* and *in situ*, including the survival of the production strain, specific activity, and animal model and targeted pathogen. However, bacteriocins have a great deal of promise to manage various infections and may become an alternative to existing antibiotics. Bacteriocins will need to undergo the same rigorous, costly research and validation process as all other previously approved therapies used in therapy [26]. Recommended conditions for detection of genes coding bacteriocins of some oral

The researcher could study probiotic or pathogenic bacteria depending of the particular relationship to diseases. For example, PCR condition for bacteriocin detection from *Lactobacillus* spp. is mentioned in the publication [46]. Detection of genes coding production of bacteriocins is only the start of the research. By this method, we could select potential candidates for further research. Inhibition potential can be detected by preferred sensitive bacterial strain for example like in case of *Streptococcus salivarius* salivaricin the sensitive strain is *Micrococcus luteus* [28]. After confirmation of bacteriocin gene presence in tested isolates, there is still much work to be done with purification, fractionation, and isolation of bacteriocins. Not

**184**


### **Table 2.**

*PCR conditions used for the detection of gene coding production of biosurfactants.*

all bacteria which present genes for bacteriocins are also capable inhibit pathogens. Some inhibition effects are caused by bacteriocins like inhibitory substances or by others active molecules which are waiting to discovered.

### **3.3 Biosurfactants and methods for their detection**

Biosurfactants are naturally produced molecules that demonstrate potentially useful properties such as the ability to reduce surface tensions between different phases [47]. The release of biosurfactants by adhering microorganisms as a defense mechanism against other colonizing strains on the same substratum surface has been described previously for probiotic bacteria in the urogenital tract, the intestines, and the oropharynx, but not for microorganisms in the oral cavity [48]. The antimicrobial properties observed in dialyzed biosurfactants produced by the tested lactobacilli open possibilities for their use against microorganisms responsible for oral diseases [49]. Biosurfactants (BS) obtained from *Lactobacillus* spp. exhibit antibiofilm and antiadhesive activity against a broad spectrum of microbes [50]. For example, they are active against biofilm formation of *Candida albicans* [51] or *Staphylococcus aureus* [52]. Biosurfactants produced by the *Bacillus subtilis* SPB1 strain (HQ392822) revealed a wide spectrum of actions including antimicrobial activity towards multidrug-resistant microorganisms [53, 54]. For the detection of biosurfactants production, i.e. in the case of *Bacillus subtilis*, it is recommended to use PCR with the help of specific primers listed in **Table 2**.

**187**

**Target gene** *Str. mutans.* Glucosyltransferase gene

*Str. salivarius.* Glucosyltransferase gene

*Str. oralis.* Glucosyltransferase gene (*gtf)*

*Lactobacillus* spp. Glucosyltransferase

gene (*gtf)*

**Table 3.**

*PCR conditions used for the detection of gene coding production of exopolysaccharides.*

(*gtf)*

(*gtf)*

**Primers** MKD-F

5′GGCACCACAACATTGGGAAGCTCAGTT3′

MKD-R 5′GGAATGGCCGCTAAGTCAACAGGAT3′

MKK-F 5′GTGTTGCCACATCTTCACTCGCTTCG3′

MKK-R

5′CGTTGATGTGCTTGAAAGGGCACCATT3′

*gtfR* MKR-F 5′TCCCGGTCAGCAAACTCCAGCC3′

*gtfR* MKR-R 5′GCAACCTTTGGATTTGCAAC3′

DexreuV 5′GTGAAGGTAACTATGTTG3′

DexreuR 5′ATCCGCATTAAAGAATGG3′

**PCR protocol** 95°C, 13 min, 30× (95°C, 30 sec, 67°C, 1 min, 72°C,

1 min) 72°C, 5 min

95°C, 13 min, 30× (95°C, 30 sec, 66°C, 1 min, 72°C,

544 bp

[60]

1 min) 72°C, 5 min

95°C, 13 min 30× (95°C, 30 sec, 66°C, 1 min, 72°C, 1 min)

374 bp 600 bp

[62]

[60]

72°C, 5 min

94°C, 5 min, 31× (94°C, 1 min, 47°C, 1 min, 72°C, 1 min)

72°C, 10 min

**Product size**

433 bp

[60, 61]

**Source**

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

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



*PCR conditions used for the detection of gene coding production of exopolysaccharides.*

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

*Bacterial Biofilms*

*Bacillus subtilis* surfactin sfp

*Bacillus subtilis*  surfactin *srfAA*

*Bacillus subtilis* fengycin *fenB*

*Bacillus subtilis* fengycin *fenD*

*Bacillus subtilis* iturín *ituD*

*Bacillus subtilis* iturín *ituC*

**Table 2.**

**186**

all bacteria which present genes for bacteriocins are also capable inhibit pathogens. Some inhibition effects are caused by bacteriocins like inhibitory substances or by

**Target gene Primers PCR protocol Product size Source**

95°C, 3 min, 30× (95°C, 30 sec, 50°C, 30 sec, 72°C, 45 sec) 72°C, 10 min

95°C, 3 min, 30× (95°C, 30 sec, 60°C, 30 sec, 72°C, 30 sec) 72°C, 10 min

95°C, 3 min, 30× (95°C, 30 sec, 57°C, 30 sec, 72°C, 45 sec) 72°C, 10 min

95°C, 3 min, 30× (95°C, 30 sec, 60°C, 30 sec, 72°C, 1 min) 72°C, 5 min

95°C, 3 min, 30× (95°C, 30 sec, 57°C, 30 sec, 72°C, 32 sec) 72°C, 10 min

95°C, 3 min, 30× (95°C, 30 sec, 58°C, 30 sec, 72°C, 30 sec) 72°C, 10 min

675 [55]

201 [55]

201 [55]

670 [55]

482 [55]

423 [55]

sfp F 5′ATGAAGATTTACGGAATTTA3′ sfp R 5′TTATAAAAGCTCTTCGTACG3′

*srfAA* F 5′TCGGGACAGGAAGACATCAT3′ *srfAA* R 5′CCACTCAAACGGATAATCCTGA3′

*fenB* F 5′CCTGGAGAAAGAATATACCGTACCY3′ *fenB* R 5′GCTGGTTCAGTT KGATCACAT3′

*fenD* R 5′GCTGGTTCAGTT KGATCACAT3′ *fenD* F 5′GGCCCGTTCTCTAAATCCAT3′

*ituD* F 5′TTGAAYGTCAGYGCSCCTTT3′ *ituD* R 5′TGCGMAAATAATGGSGTCGT3′

*ituC* F 5′GGCTGCTGCAGATGCTTTAT3′ *ituC* R 5′TCGCAGATAATCGCAGTGAG3′

*PCR conditions used for the detection of gene coding production of biosurfactants.*

Biosurfactants are naturally produced molecules that demonstrate potentially useful properties such as the ability to reduce surface tensions between different phases [47]. The release of biosurfactants by adhering microorganisms as a defense mechanism against other colonizing strains on the same substratum surface has been described previously for probiotic bacteria in the urogenital tract, the intestines, and the oropharynx, but not for microorganisms in the oral cavity [48]. The antimicrobial properties observed in dialyzed biosurfactants produced by the tested lactobacilli open possibilities for their use against microorganisms responsible for oral diseases [49]. Biosurfactants (BS) obtained from *Lactobacillus* spp. exhibit antibiofilm and antiadhesive activity against a broad spectrum of microbes [50]. For example, they are active against biofilm formation of *Candida albicans* [51] or *Staphylococcus aureus* [52]. Biosurfactants produced by the *Bacillus subtilis* SPB1 strain (HQ392822) revealed a wide spectrum of actions including antimicrobial activity towards multidrug-resistant microorganisms [53, 54]. For the detection of biosurfactants production, i.e. in the case of *Bacillus subtilis*, it is recommended to

others active molecules which are waiting to discovered.

use PCR with the help of specific primers listed in **Table 2**.

**3.3 Biosurfactants and methods for their detection**

Other species producing biosurfactants and condition for their detection are able in research papers for example: *Lactobacillus paracasei* produced biosurfactants with anti-adhesive properties [56]. *Streptococcus mitis* biosurfactants plays a protective role in the oral cavity and protects against colonization of saliva-coated surfaces by cariogenic *Streptococcus mutans* [48]. Based on *Bacillus subtilis* SPB1 lipopeptides production researcher predict their possibility used in toothpaste formulation [53]. Biosurfactants are promising bioactive molecules for oral-related health applications [47].

### **3.4 Exopolysaccharides and methods for their detection**

Lactic acid bacteria are the most frequently mentioned in studies of exopolysaccharides (EPS) in oral microbiota [57]. Except for lactobacilli, which are participated in the later stages of dental biofilm formation, streptococci are one of the first bacteria capable of producing EPS. Streptococci are able to assert themselves and adhere to the hard tissues of the oral cavity immediately after washing the teeth. This property of adherence is predetermined and is encoded in genes that are also responsible for the production of glucosyltransferases. Glucosyltransferases (Gtfs) are produced by several types of lactic acid bacteria [58]. Gtfs are generally characterized as Gtf-S (glucosyltransferase-soluble) or Gtf-I (glucosyltransferaseinsoluble) enzymes, depending on whether the glucan they produce is water soluble or insoluble [59]. For detection of exopolysaccharides production in oral lactic acid bacterial members is useful PCR with help of specific primers see in **Table 3**.

## **4. Testing of growth inhibition activity against pathogens**

Testing of bacterial isolates as potential beneficial candidates or their products is necessary step in new discoveries. We are able declarate production of bioactive substance by very easy PCR reactions, as mentioned above in part 3. Activity of these substances is easy to declare by simply *in vitro* tests. At first for activity it is possible to use spot on or disc diffusion test. Same mechanism of declaration is for live bacteria isolates as for isolated bioactive substances.

If we found bacteria with interesting effect in spot or disc diffusion test it predict selection criteria of former characterized bacteria for next research.

### **4.1 The disc diffusion method for** *Lactobacillus reuteri* **for testing of growth inhibition activity against pathogens**

We recommend the disc diffusion test for the detection of the inhibitory properties of beneficial microorganisms. Selected lactobacilli strains were grown on MRS agar (CONDA S.A, Madrid Spain) for 48 hours. anaerobically (Gas Pak Plus, BBL, Microbiology Systems, Cockeysville, USA) at 37°C. Then, a standardized suspension with an optical density of 1 McFarland by dissolving several solitary colonies in 5 ml of physiological saline was prepared. Sterile clean discs (6 mm diameter, BBL, Cockeysville, USA) were placed on Petri dishes (Ø 90 mm) with 20 ml of PYG agar (HiMedia Laboratories GmbH Einhausen, Germany). The sterile paper discs were inoculated with 5 μl of standardized suspensions of lactobacilli.

As a negative control, one Petri dish with PYG agar is served with a clean paper discs soaked with sterile MRS broth.

The plates with discs were incubated for 48 hours. anaerobically (Gas Pak Plus, BBL, Microbiology Systems, Cockeysville, USA) at 37°C. The discs were removed with a sterile syringe needle or tweezer after incubation. Subsequently, 3 ml of 0.7% PYG agar was inoculated with 0.3 ml of the indicator pathogenic strain and put into

**189**

**5. Conclusion**

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

plates with lactobacilli. Pathogenic strains were incubated for 18 hours in PYG broth at 37°C. The plates with YPG medium inoculated with pathogen were incubated for 24 hours aerobically at 37°C. After incubation, the diameter of the inhibition zones was measured. The results were recorded in the table as the arithmetic means of the

**4.2 The disc diffusion method for** *Streptococcus salivarius* **for testing of growth** 

The disc diffusion test with *Micrococcus luteus* was used for the preliminary testing of *Streptococcus salivarius* inhibition [39]. This test analyses the activity of the BLIS produced in agar and determines the activity spectrum of Sal9 producers. Briefly, the tested strain was inoculated across the surface of the Blood agar medium (BBL, Microbiology Systems, Cockeysville, USA) in a glass Petri dish (Ø 90 mm) as a 1 cm-wide streak. After incubation, the strain growth was stopped by its exposure to chloroform vapor for 30 min. The plate was then aired for 15 min before 24 hours inoculating cultures as the indicator strains across the original tested strain. The plate was incubated for 24 hours and examined for the zones of the indicator strain growth inhibition. The inhibition activity against the selected standard indicators was recorded in code form by inoculating the indicators in three triplets. The inhibition of the first member of a triplet was given a score of 4, the second a score of 2, and the third a score of 1. The absence of the inhibitory action against an indicator was scored as 0. The code was recorded as a sequence of three numbers representing the sum of each triplet. All tests were performed in duplicate, and further testing was undertaken until the consistency of the inhibition patterns was obtained [63].

It is necessary to know the composition of the dental biofilm of healthy individuals and the bacterial composition in pathological conditions to identify species responsible for disease initiation and progression. Identification of species and their characterization is essential for the selection of pathogenic, potentially pathogenic and potentially probiotic species. Blast n analysis of 16S RNA or MALDI-TOF mass spectrometry identification is perfect tools for identification of bacterial species. The ability to modulate the microbiocenosis of the dental biofilm by bacteria living together in the biofilm should be studied. The some bacteria are capable of producing bioactive substances whose presence we can quickly and easily declare with help of PCR. Sequencing and comparing of genes coding bioactive substances can uncover differences between tested bacteria isolates. Presence of these genes and prove the ability to inhibit the growth of other bacterial species are important steps in selection of potentially probiotic candidates. These bacteria are of great interest for further study and may be useful in the development of new antibacterial agents. Bioactive substances can be extracted by physical methods (centrifugation, separation and fractionation), by chemical methods (purification) and detected by modern analytical method (HPLC) or proteomic methods (MALDI-TOF MS). Next important step is declaration of activity pure extracted substance. Bioactive substances of bacterial origin can be used in dental preparations and serve as prevention or supplementary therapy of periodontal diseases. During recent years there has occurred a shift towards ecological and microbial community based approach to the therapy of oral cavity diseases. With the increasing resistance to antibiotics, the use of probiotics appears as a prospective alternative treatment or preventative measure in the control of periodontal diseases. From the clinical point

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

three measurements ± standard deviation.

**inhibition activity against pathogens**

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

plates with lactobacilli. Pathogenic strains were incubated for 18 hours in PYG broth at 37°C. The plates with YPG medium inoculated with pathogen were incubated for 24 hours aerobically at 37°C. After incubation, the diameter of the inhibition zones was measured. The results were recorded in the table as the arithmetic means of the three measurements ± standard deviation.

## **4.2 The disc diffusion method for** *Streptococcus salivarius* **for testing of growth inhibition activity against pathogens**

The disc diffusion test with *Micrococcus luteus* was used for the preliminary testing of *Streptococcus salivarius* inhibition [39]. This test analyses the activity of the BLIS produced in agar and determines the activity spectrum of Sal9 producers. Briefly, the tested strain was inoculated across the surface of the Blood agar medium (BBL, Microbiology Systems, Cockeysville, USA) in a glass Petri dish (Ø 90 mm) as a 1 cm-wide streak. After incubation, the strain growth was stopped by its exposure to chloroform vapor for 30 min. The plate was then aired for 15 min before 24 hours inoculating cultures as the indicator strains across the original tested strain. The plate was incubated for 24 hours and examined for the zones of the indicator strain growth inhibition. The inhibition activity against the selected standard indicators was recorded in code form by inoculating the indicators in three triplets. The inhibition of the first member of a triplet was given a score of 4, the second a score of 2, and the third a score of 1. The absence of the inhibitory action against an indicator was scored as 0. The code was recorded as a sequence of three numbers representing the sum of each triplet. All tests were performed in duplicate, and further testing was undertaken until the consistency of the inhibition patterns was obtained [63].
