Microbiological and Molecular Characteristics of Microorganisms of Importance in Dental Caries and Periodontal Disease: Research Contributions in Colombia*

Background: Dental caries is an infectious and multifactorial pathological process that destroys hard dental tissues, and Streptococcus mutans is found as the main microorganism associated with this disease. Periodontal disease is a multifactorial infectious disease, and, in its etiology, diverse microorganisms are involved, among which Porphyromonas gingivalis is of vital importance due to its virulence factors and role in the development of periodontal pathology. Purpose and Method: This integrative review article built on the basis of previously published research experiences of the authors of this document and contrasted with other studies worldwide, aimed to describe the microbiological and molecular characteristics of S. mutans and P. gingivalis, microorganisms, respectively, of great importance in dental caries and periodontal disease. Important studies were found with the use of the following keywords or medical subject headings (MeSH) in Pubmed, Scopus and Google Scholar: “S. mutans” and “P. gingivalis” and "phenotypic identification" and "genotypic characterization”. Results: From S. mutans, different biotypes were identified according to the methodology used, wide antimicrobial susceptibility and genotypic variability by AP-PCR that depends on the nesting of the primers on the DNA. Of P. gingivalis, high susceptibility to antimicrobials and broad genotypic discrimination were found by the AFLP technique. Conclusion: the deep phenotypic and genotypic knowledge of S. mutans and P. gingivalis, bacteria of great importance in dental caries and periodontal disease, together with the classical epidemiological study could be very important in the application of better prevention and control strategies that impact the oral public health in these two diseases.


INTRODUCTION
The balance of oral microbial diversity is of vital importance in the development of the functions of the different oral ecosystems, and in an adequate immune response to the invasion of pathogens that lead to diseases (1)(2)(3). Ecologically speaking, dental caries is the consequence of an imbalance in the oral environment that leads to the predominance of a flora previously considered normal in the oral cavity (1,2). Dental caries is an infectious, multifactorial, and communicable disease process that destroys hard dental tissues (4). The main microorganisms associated with caries production are, in order of frequency, Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Streptococcus gordonii (S. gordonii), and Lactobacillus and Actinomyces species (1,2,5). On the other hand, periodontal disease is a multifactorial infectious disease, and its etiology involves various microorganisms, among which Porphyromonas gingivalis (P. gingivalis) is of vital importance due to its virulence factors and role in the development of periodontal pathology.
Factors inherent to the host, smoking and environmental factors are important and determining in its evolution and severity (6)(7)(8)(9). Molecular biology techniques have greatly expanded knowledge on oral microbiology and the human oral cavity is currently considered to be the ecological niche with the greatest diversity in genera and species, known to date (10) and in the one that there are great possibilities of entering a huge bank of biodiversity, which is still in the process of knowledge and characterization (10).
In general terms, the recognition and characterization of oral microorganisms immersed in infectious processes requires the performance of four activities: the first, identification of microorganisms, begins with the culture of samples from the oral cavity, in culture media and conditions specific, followed by microscopic examination with Gram stain and standard biochemical tests performed on the isolated microorganism; the second activity involves the detection of phenotypic characteristics (enzymes, metabolism products, proteins, bacteriocins, etc.). The third activity involves the molecular epidemiological association or comparison of the isolates by genotypic characteristics, in order to control the infection or analyze the population (1,2). This activity has been enriched with molecular typing techniques for microbial DNA, which lead to the identification of clones (strains that have a high degree of genetic relationship) isolated from different sources and circumstances (1,2). Finally, in the fourth activity and thanks to the latest massive sequencing techniques, especially second-generation ones, and without depending on the limitations of microbiological cultures and standard identification procedures, a large number of microorganisms has been identified. with which the oral microbiota present in the different ecosystems has been built and structured in a better way.

MATERIALS AND METHODS
This integrative review article built on the basis of previously published research experiences of the authors of this document and contrasted with other studies worldwide, aimed to describe the microbiological and molecular characteristics of S. mutans and P. gingivalis, microorganisms, respectively, of great importance in dental caries and periodontal disease. To this end, important studies were searched and found with the use of the following keywords or medical subject headings (MeSH) in PubMed, Scopus and Google Scholar: "S. mutans" and "P. gingivalis" and "phenotypic identification" and "genotypic characterization." Regarding S. mutans, the microbiological characteristics, isolation and identification, biotyping, antimicrobial susceptibility and genotyping by AP-PCR are presented in sequence, and in relation to P. gingivalis, the microbiological characteristics, isolation and identification, antimicrobial susceptibility and genotyping are described. by AFLP.
Streptococci are Gram-positive cocci on Gram stain and negative behavior on the catalase test, which are grouped in pairs or chains. In the Streptococcus genus, the presence of organisms of the Streptococcus viridans (S. viridans) group stands out, as they are capable of producing 30-40% of cases of subacute bacterial endocarditis with an impact on highly significant bacteremia (11).
Streptococcus viridans are made up of the following groups: S. mutans group (S. mutans, S. sobrinus, S. cricetus, and S. rattus species), S. sanguinis group (S. sanguinis species, S. parasanguinis, S. gordonii, and S. crista), S. mitis group (S. mitis and S. oralis species), and S. salivarius group (S. salivarius and S. vestibularis species). These groups are differentiated by the production of acetoin, hydrolysis of esculin, production of arginine dehydrolase and urease, and the production of acid from mannitol and sorbitol (11).
S. mutans and S. sobrinus species of the S. mutans group are the main microorganisms associated with the initiation and pathogenesis of dental caries. In general, in the scientific community, there is consensus in pointing out S. mutans as the most important microorganism in dental caries, and therefore the isolation, phenotypic and molecular characterization, prevention and control strategies are mainly directed towards it. S. mutans, a Gram-positive coccus and negative catalase test, is named for the mutant forms in which it occurs, coccobacillus (oval shape) in an acid medium and coco (round shape) in an alkaline medium (11). In blood agar cultures, the colonies of this microorganism are alpha or gamma hemolytic (11). It is facultative anaerobic; however, its optimal growth occurs in anaerobiosis for 48-72 hours at 37 o C. Concomitantly with the synthesis of dextran from sucrose (a dietary disaccharide made up of glucose and fructose), colonies of this microorganism emit an aqueous exudate on the surface of the culture medium with sucrose, often abundant enough to that forms a puddle around the colony (11).
In culture media with sucrose, this microorganism can produce extracellular polysaccharides, acquiring an opaque, rough and white appearance, not adherent to the culture medium and occasionally surrounded by glucan polymers with a moist appearance. S. mutans produces extracellular polysaccharides from sucrose by the action of two enzymes, glucosyltransferase (GTF) and fructosyltransferase (FTF). GTF is able to synthesize glucan from glucose, and FTF, fructan from fructose. The insoluble glucans formed by this microorganism allow it to adhere to the smooth surfaces of the teeth and form the matrix of the biofilm. Specific and nonspecific adherence of S. mutans and other organisms to insoluble glucan bound to the tooth and subsequent acid formation leads to demineralization of tooth enamel and the initiation of carious lesions. The most common medium for the isolation of S. mutans is Mitis Salivarius Agar supplemented with bacitracin 0.2 U / ml and 20 % sucrose, which allows the selection of other streptococci (11).
Currently, studies are not only directed towards the search for S. mutans in saliva and dental plaque, but also towards the quantification of this microorganism (11)(12)(13)(14)(15). Different studies have shown a correlation between the counts of this microorganism in the oral cavity with the prevalence and incidence of caries (11)(12)(13)(14)(15). However, other studies have not found a correlation between the amount of S. mutans and the incidence of caries (11)(12)(13). Currently, the finding of a high count of S. mutans is a risk factor to consider in the prevention and control of dental caries.

S. mutans Isolation and Identification
For this purpose, samples of dental plaque and spontaneous or stimulated saliva are serially diluted The final count is expressed in colony forming units (CFU) per ml of saliva or gram of dental plaque (16). After bacterial count, colonies with characteristics of S. mutans are examined by Gram stain and subjected to biochemical tests. S. mutans has the following biochemical profile: positive fermentation of raffinose, mannitol, melobiose, trehalose, inulin, cellobiose, arbutin and amygdalin; positive hydrolysis of starch, negative hydrolysis of esculin in the presence of bile, and positive hydrolysis of esculin in the absence of bile; negative urease; negative hydrolysis of arginine and hippurate; and resistance to 2 U of bacitracin (16,17). In the study by Gamboa et al.  In total, 121 S. mutans strains were identified in the 86 children: 43 strains in the 30 children without dental caries and 78 in the 56 children with dental caries.

S. mutans Biotyping
At present, S. mutans biotyping is being carried out according to their enzymatic profile, with the api-ZYM system (bioMérieux, France) (17,19). This typing system has made it possible to establish differences in intra and interindividual S. mutans strains (20). The first evaluation of the api-ZYM system as a biotyping system was carried out in 1999 by De la Higuera et al. (17). In this study, 8 different phenotypes were identified in 160 clinical isolates of S. mutans only on the basis of the action of three enzymes of the microorganisms. Subsequently, the system was used to evaluate the enzymatic action on the 19 substrates included in the test (19)(20)(21)(22). The study by Lamby et al. (19) in children 3 to 6 years of age with incipient dental caries identified 17 phenotypes and the most frequent phenotype was 15 with 10 S. mutans strains. It is important to note that the greatest number of positive enzymatic reactions with substrates occur at an acidic pH (pH = 5.4), a situation that may be related to some of the specific characteristics of S. mutans, the ability to constantly produce acids at a pH low or acidic (acidity) and its property to tolerate and survive in acidic environments (acidophility) (11).
In the study by Gamboa et al. (21) S. mutans strains were grouped into 10 phenotypes, of which the most frequent phenotypes were 10 and 15, with 9 and 8 strains respectively, and it was possible to clearly establish inter-and intra-individual differences. In another study by Gamboa et al. (22) 119 S. mutans strains were grouped into 85 phenotypes: 33 phenotypes in strains isolated from children without cavities and 52 phenotypes in strains isolated from children with cavities. Two patients included in the study had 4 phenotypes in common (phenotypes 5, 6, 9 and 12). The most frequent phenotypes in children without caries were 6, 9, 5 and 3, and in patients with caries the most frequent phenotypes were 37,39, 6 and 9. In the study by Gamboa et al. (22) a large number of phenotypes represented by a single strain are highlighted. In another study (18), the grouping of 121 strains of S. mutans is reported in 38 biotypes: 24 in the caries group and 14 in the cariesfree group. The study draws attention to the difference found in the most frequent biotypes in children with and without dental caries. In the dental caries group the most frequent biotypes were XV, XI and XII, respectively, with 26, 12 and 11 strains and in the group without caries the most frequent biotypes were XXVI, XX and XXXVI, respectively, with 14. 8 and 7 strains.
Very likely the presence of unique profiles found in children with dental caries compared to children without dental caries may suggest a greater virulence capacity of S. mutans strains. In this sense, in the study by Napigoma et al. (23) the relationship between clonal diversity and some virulence factors of S. mutans isolated from individuals with and without dental caries was evaluated; the results showed 44 different profiles, with a maximum of 8 profiles in an individual.
They also found a large number of genotypes of S. mutans with greater ability to synthesize insoluble glucans, one of the important points of contribution in the genesis and development of dental caries (23). On the other hand, in the study by Krzysciak et al. (24) the usefulness of biotyping in the recognition of pathogenic determinants in S. mutans is discussed. They demonstrated that the biotype I prephenate dehydrogenase enzyme (typified with the STREPTOtest system) present in most of the S. mutans strains of children with dental caries had greater activity than that presented by S. mutans strains isolated from children without dental caries. Due to all the above and due to the usefulness of biotyping in the discrimination of S. mutans in children with and without dental caries, it is very necessary to continue with research to demonstrate the implications and relationships with the pathogenicity of S. mutans and its role in the genesis and development of dental caries and the indirect implications in maintaining balance in oral microbial ecology.
Regarding bacteriocins in S. mutans, the metabolic capacity of this microorganism to synthesize glucans from sucrose and produce bacteriocins is of great importance in the process of initiation and development of dental caries (25). Bacteriocins are antibiotic peptides or proteins, with strong bactericidal properties, produced by a wide variety of bacterial species. Hamada and Ooshima (26) demonstrated that many S. mutans strains are producers of bacteriocins that possess a wide range of activity against Gram-positive microorganisms and closely related species (26)(27)(28). The survival and proliferation of a microorganism can occur if it manages to eliminate or displace a competent organism in its ecological niche, where competition is very strong due to the diversity of species (29). It has been suggested that the function of bacteriocins is to allow the establishment and permanence of the bacterium that produces it in the niche that it colonizes. Most S. mutans strains produce bacteriocins, which are specifically called mutacins and which are those that exert antagonistic or inhibitory action on other microorganisms in the oral environment (29)(30)(31)(32). For many years, the search for S. mutans strains with antagonistic capacity and their application in replacement therapy or bacteriological control to displace virulent native strains of S. mutans has continued (29).
Different investigations indicate that the antagonistic capacity of S. mutans is due to its production of bacteriocins, which could confer a great ability to displace native strains of the same species in the oral cavity (16,19). In the study by Gamboa et al.

S. mutans Antimicrobial Susceptibility
In addition to dental caries and related pyogenic infections, S. mutans is also a very important infectious agent in endocarditis (33). The participation of this microorganism in oral and non-oral infections has generated interest in the knowledge of its susceptibility to antimicrobial agents. One of the most appropriate procedures to determine the antimicrobial susceptibility of S. mutans strains is the determination of the minimum inhibitory concentration (MIC) (34). The most widely used antimicrobial agents are: penicillin, amoxicillin, cefazolin, erythromycin, clindamycin, imipenem, and vancomycin. MIC is done using the agar dilution method. The protocol is briefly In the study by Gamboa et al. (20) the high sensitivity of S. mutans strains to penicillin, amoxicillin, cefazolin, erythromycin, clindamycin, imipenem and vancomycin is reported; 50 and 90 % of S. mutans strains were inhibited by all antibiotics at concentrations lower than 0.12 and 0.5 ug / ml, respectively. The lowest average value was that of penicillin. The MIC of all the strains were very similar to those observed by other authors (17,34). In another study (18) the antimicrobial susceptibility of S. mutans strains to penicillin, amoxicillin, cefazolin, erythromycin, clindamycin, imipenem, vancomycin and teicoplanin was evaluated. All strains were highly sensitive to the antimicrobials tested; 50 % and 90 % of the strains were inhibited, respectively, by concentrations lower than 0.12 and 1 ug / ml for all the antimicrobials evaluated. For penicillin, erythromycin, and imipenem, the lowest average MIC values were obtained, and all susceptibility patterns are in close agreement with those observed by other authors (14,(34)(35)(36). Although the treatment of dental caries is not done with antimicrobials, since they can lead to the nonspecific destruction of many microorganisms of the oral cavity, which leads to the alteration of the balance of the oral microbiota and a reinfection with S. mutans with the possibility of returning at pretreatment levels, it is important to make it clear that isolated Colombian S. mutans strains (18) remain sensitive and that in the event of systemic infections or endocarditis caused by S. mutans, a good number of antimicrobials are available that will have high inhibitory activity on this microorganism.

S. mutans Genotyping with Arbitrarily Primed PCR (AP-PCR)
One of the objectives of molecular typing is the identification of specific virulent clones within bacterial species and the study of the clones involved in an epidemiological event. Several molecular methods have allowed the study of oral streptococci (37,38), of which the analysis by restriction enzymes, ribotyping and typing with arbitrarily primed PCR (AP-PCR) have revealed considerable genetic heterogeneity between S. mutans strains (39,40). In recent years, AP-PCR has been widely applied in the genotypic characterization of different bacterial species, including oral pathogens (41,42). In recent years, a consensus has emerged on the use of the AP-PCR technique to detect different DNA profiles in clinical isolates of S. mutans (23,(43)(44)(45)(46). With AP-PCR, random DNA segments from the organism under study are amplified with simple primers of arbitrary sequence. The greatest advantage that AP-PCR offers for its development is that it is not necessary to know in advance the DNA sequence of the bacterial species to be amplified. It is very well established that there are different genotypes of S. mutans in children with dental caries and that genotyping with AP-PCR is useful in demonstrating the diversity of genotypes (23,(43)(44)(45)(46).
In the study by Gamboa  There are two strategies for using primers: one in which the primers OPA 02 and OPA 03 (23,27,46,49,50) are used and another in which the first OPA 05 is used (46,(51)(52)(53) . In order to determine the discriminatory potential of the primers, Machado et al.

P. gingivalis Microbiological Characteristics
Periodontal disease is a multifactorial oral infectious disease that affects a large number of people in the world population (6.7) and in Colombia this disease, evaluated by loss of clinical attachment affects a large number of the population (55). Periodontitis corresponds to a form of periodontal disease. Due to its insidious and asymptomatic behavior, its diagnosis is almost always made in advanced age and even in terminal stages of the disease (6)(7)(8)(9). This disease leads to progressive attachment and bone loss and is also characterized by the formation of pockets that can affect a variable number of teeth with different stages of progression (6)(7)(8). Factors inherent to the host, smoking and environmental factors are important and determining factors in its evolution and severity (6)(7)(8)(9). Various microorganisms are involved in its etiology, among which P. gingivalis is of vital importance due to its virulence factors and role in the development of periodontal pathology (6-9,56). P. gingivalis is a strictly anaerobic, Gram-negative bacillus, capable of developing brown-black colonies on anaerobic blood agar, and in the oral cavity it is found mainly immersed in the subgingival microflora (6)(7)(8). This microorganism meets the requirements to be considered a pathogen: it stimulates the host's immune response, evades defense mechanisms and destroys host tissues by secreting its own substances (6,(57)(58)(59). Different studies have shown that the frequency and distribution of periodontal microorganisms and in particular of P. gingivalis, in the subgingival microflora, is variable according to geographical region, race, diet, level of development and living conditions, among others (6,7,56-60).

P. gingivalis Isolation and Identification
For the correct isolation and identification of strict anaerobic microorganisms, it is very important to pay great attention to the selection, collection and transport of clinical samples (61)(62)(63)(64)(65). Regarding the isolation and identification of P. gingivalis from crevicular fluid samples, for the collection of samples in general, 2 to 5 sites with pocket depth ≥ 4mm and clinical insertion level ≥ 2 mm are selected. the supragingival biofilm with a sterile gauze, the area is isolated with sterile cotton and the paper cones are introduced into the periodontal bag for 1 minute. After this time, the cones are removed and placed in eppendorf tubes with 900 uL of thioglycollate broth (BBL TM Fluid, Becton Dickinson and Company) supplemented with hemin (5 ug / ml) and menadione (1 ug / ml) (62)(63)(64)(65), and are placed in jars with anaerobic sachets (Anaerogen, Oxoid) (no more than 4 hours) until reaching the laboratory. In the laboratory, the samples in the anaerobic jars are incubated for 4 hours at 37 ° C in order to achieve enrichment and consequently multiplication of the anaerobic microorganisms (64,65). After incubation, centrifugation (Eppendorf ® Centrifuge) is carried out at 4000 r.p.m for 10 minutes. 300 μl are removed from the centrifuged product and the remaining 600μl are stirred in Vortex (Maxi mix II Thermolyne ®) to achieve a homogeneous mixture of the sample. Subsequently, from the remaining thioglycollate broth, five serial dilutions (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ) are made in thioglycollate broth in order to isolate P. gingivalis. Regarding the clinical parameters, the values (mean ± standard deviation) of pocket depth found in patients with and without P. gingivalis were, respectively, 5.62 ± 1.4 mm and 5.77 ± 1.6 mm, and not statistically significant differences were found (P> 0.6514). According to probing depth in the 30 patients with chronic periodontitis and the presence of P. gingivalis, 13 patients (43.3%) were found in a pocket depth range of 4-5mm, 12 (40%) in a range of pocket depth between 5-7 mm, and 5 patients (16.7%) in a pocket depth range greater than 7 mm. Regarding the level of clinical attachment, it could be determined that in patients in whom P. gingivalis was detected, a value (mean ± standard deviation) of attachment level loss of 5.77 ± 3.1 was found and in patients in those for which P. gingivalis was not identified was 5.56 ± 2.6 mm. These differences were not statistically significant (P> 0.8687). Nor were statistically significant differences (P> 0.7511) found in terms of the severity of periodontitis between the group of patients with P. gingivalis According to the U-Mann Whitney statistical analysis, no statistically significant differences were found between these two groups (P> 0.5063). According to other studies, the frequency found by Everything seems to indicate that the variations found in the frequency of these microorganisms are due to differences in the taking, transport and processing of samples and isolation by bacteriological culture, as well as the use of molecular techniques, sociocultural and demographic aspects and particular living conditions ( 6.57.70-75). In the study by Sanai et al. (75) makes it clear that the transport of the samples may have led to the loss of viability of P. gingivalis, P.
intermedia, and P. nigrescens. Differences in the sensitivities (recovery and identification of the bacteria in culture) obtained by culture could also be due to situations that generate changes in the subgingival microflora, among which are: poor hygiene habits and attitudes, chronic underlying diseases, smoking, alcoholism and previous antimicrobial therapies (7,57,58,72,76). In the study by Urban et al. (77), the detection of periodontopathogenic bacteria is made by the traditional anaerobic culture method and by commercial PCR. The PCR test detected almost the same number of positive samples for P. gingivalis as those detected by culture (77), with only two discrepant results and reached a concordance of 94%. According to these last results, it can be deduced that commercial PCR can be recommended for its speed (2-3 hours), sensitivity and specificity in the routine of an oral microbiological diagnostic laboratory. However, the culture method despite being tedious, slow and requires expertise, allows to evaluate antimicrobial susceptibility and carry out other studies that require the bacteria in live form to be used in typing or in virulence and pathogenicity studies. When choosing any of these methodologies, laboratories must assess their needs, the impact they may have on the diagnosis, and the limitations of the two methodologies.
From the results seen with the culture and the PCR, everything seems to indicate that it is possible that the joint use of the two methodologies is required due to the individual contributions of each technique (74,77,78).

P. gingivalis Antimicrobial Susceptibility
If antimicrobial therapy is necessary in patients with periodontal disease, to control P. gingivalis or another strict anaerobic periodontopathogen, its antibiotic resistance or susceptibility profile must be known (58,79,80). In this sense, various susceptibility patterns have been found in this microorganism (70,75,(81)(82)(83)(84). Through in vitro antimicrobial susceptibility tests, the profiles and changes in the behavior of microorganisms can be determined against the different therapeutic alternatives in periodontal treatment, in order to contribute to the development of appropriate antibiotic management policies and consequently delay the emergence of antimicrobial resistance (58,70,84,85). In the microbiological process, after the isolation and identification of P. gingivalis, the antibiotic susceptibility (MIC) for different antimicrobials is evaluated using the M.I.C.
In the case of the M.I.C. Evaluator proceeds to make a fresh isolation of the P. gingivalis strains in Wilkins Chalgren anaerobic agar (Oxoid) supplemented with hemin and menadione at 1% (v / v) and lamb blood at 5% (v / v). All isolates are left in incubation at 37 ° C for 5 days in an anaerobic atmosphere, in order to obtain fresh colonies for the subsequent mounting of supplemented with hemin and menadione at 1% (v / v) and lamb blood at 5% (v / v), the different MIC system antibiotic strips Evaluator and test media are incubated at 37 ° C for 5 days in an anaerobic atmosphere. Finally, the MIC is read, according to the recommendations of the manufacturer of the system used and according to the cut-off points for the evaluated antibiotics, it is defined whether the strains are sensitive or resistant.
In the study carried out by Gamboa et al. (69) (83). In this sense, these situations that lead to the generation of bacterial resistance should be avoided every day (78,79). In the study by Gamboa et al. (69) the resistance presented by 10% to tetracycline is striking. The most common mechanism of resistance to this antimicrobial is through the synthesis of efflux pump proteins, which in Gram-negative microorganisms are encoded by tet genes (82). Sanai et al. (75) in 2002 determined the presence of the tetracycline resistance gene (tet-Q) in 3 of 5 isolates (60%) of P. gingivalis isolated from children and that seem to belong to the same clone of origin. It is important to consider that antimicrobial resistant bacteria residing in the oral cavity can be an important source of transmission of antimicrobial resistance genes to other pathogenic bacteria (75).

P. gingivalis Genotyping with Amplified Fragment Length Polymorphism (AFLP)
The genetic diversity of P. gingivalis in the population suffering from periodontal disease is high and it is possible to find an association between the clonal type of P. gingivalis and the type or of P. gingivalis by AFLP. The entire procedure (5 steps) followed for the study by AFLP is described below: • Step 1. Digestion of bacterial DNA. All 65 P. gingivalis isolates and the reference strain P.
gingivalis ATCC were processed to lyse the bacteria, obtain and quantify DNA. In order to digest the DNA, 300 ng of genomic DNA were taken, 5 ul of NB4 buffer and 0.5 ul of BSA were added, and then 1 unit of MSEI (New England) and 5 units of ECORI (New England) were added. Subsequently, DEPC water (Invitrogen) was completed at 50 ul of final volume, incubated at 37 ºC for 1 hour, to finally leave at 65 ºC for 20 minutes. Digestion was verified by taking 4 ul of the digestion and run on 1% agarose gel%. Regarding results, the dendrogram analysis of the products generated by digestion with MSE1C-ECOR1A allowed clustering into two larger clusters A and B. In cluster A, 28 isolates were grouped and subclusters A1 and A2 were presented. In subcluster A1, there were two subgroups called A1a and A1b; Subgroups 1 (13 strains) and 2 (1 strain) were derived from subgroup A1a.
In subcluster A2, two subgroups called A2a and A2b were found. Subgroups 1 and 2 were derived from subgroup A2a, subgroup 1 was affiliated with 6 strains and subgroup 2 was affiliated with only 1 strain. Two subgroups called 1 and 2 were derived from subgroup A2b, and in each of these 1 strain was affiliated. In cluster B 38 isolates were grouped and subclusters B1 and B2 were presented. In subcluster B1, two subgroups called B1a and B1b were found; from subgroup B1a, subgroups 1 (15 strains) and 2 (a single strain) were derived. In subcluster B2 there were two subgroups called B2a and B2b; Subgroups 1 (6 strains) and 2 (14 strains) were derived from subgroup B2a. On the other hand, the dendrogram analysis of the products generated by digestion with MSE1C-ECOR1T also allowed the grouping of bacteria into two larger clusters A and B. In cluster A, 56 isolates were grouped and subclusters A1 and A2 were presented; Subgroups 1 and 2 were derived from subgroup A1a. 15 strains were affiliated with subgroup 1 and three strains affiliated with subgroup 2. Subgroups 1 (13 strains) and 2 (24 strains) were derived from subgroup A1b. Only one strain affiliated to subcluster A2. In cluster B 10 isolates were grouped and subclusters B1 and B2 were presented. In subcluster B1, two subgroups called B1a and B1b were found; from subgroup B1a, subgroups 1 and 2 were derived. Subgroup 1 was affiliated with 1 strain and subgroup 2 was also affiliated with a single strain. Subgroups 1 (3 strains) and 2 (1 strain) were derived from subgroup B1b. Only one strain affiliated to subcluster B2. Regarding the discriminatory capacity of the two digestion systems, MSE1C-ECOR1A and MSE1C-ECOR1T, both allowed the grouping of P. gingivalis strains into two large clusters, A and B. However, the MSE1C-ECOR1A allowed greater discrimination in both groups, since it distributed 28 strains in cluster A and 38 strains in cluster B. In contrast, the MSE1C-ECOR1T digestion system allowed greater discrimination of the strains within subcluster A1.
In conclusion, the work carried out by Gamboa et al. (article in preparation) allowed to typify the 65 isolates by AFLP, in two larger clusters (A and B) and in several smaller clusters, and obtain a high discrimination between the isolates, which consequently indicates the importance of the AFLP technique as a useful tool. in molecular epidemiology.

CONCLUSIONS
In this article, fundamental aspects of the microbiological and molecular characterization of S. mutans and P. gingivalis, microorganisms of great importance in dental caries and periodontal disease, were discussed, based on the research experiences of the authors of this review compared to other important findings from the world literature. Complete isolation strategies from clinical samples, dilutions, culture media, and biochemical identification protocols were described for both microorganisms. Regarding S. mutans, different biotypes of importance and associated with pathogenicity were found according to the methodology used, wide antimicrobial susceptibility of the isolates and genotypic variability by AP-PCR that depends on the random nesting of the simple primers on the DNA of the bacteria studied. The AP-PCR genotyping technique, because it is fast and reproducible, is also of great value in distinguishing species of S. mutans and S. sobrinus and other oral streptococci. From P. gingivalis, clinical isolates were highly susceptible to antimicrobials, including metronidazole and tetracycline, and high genotypic discrimination between isolates by the AFLP technique, which indicates the importance and usefulness of this in molecular epidemiology. The deep phenotypic and genotypic knowledge of S. mutans and P.
gingivalis, together with the classical epidemiological study, could be very important in the application of better prevention and control strategies that impact oral public health in dental caries and periodontal disease.