Typing of Streptococcus mutans strains isolated from caries free and susceptible subjects by multilocus enzyme electrophoresis.

This study was evaluated the clonal diversity of Streptococcus mutans in caries-free and caries-active subjects using MLEE. Strains from caries-free subjects were grouped in a single taxon. Unrooted dendrogram showed that different strains clustered in four different clades, also showed that more than one clonal type can be found in a same individual.

Streptococcus mutans, which is the primary cariogenic species, plays an important role in the generation of caries. This bacterium is widely investigated in order to determine its role in the cariogenic microbiota (Arakawa ; Baca ; Becker ; Kamiya ; Rosa ). S. mutans is widely disseminated not only in populations with moderate or high caries prevalence (Alaluusua ; Beighton ; Li ; Napimoga ) but also in populations having no or low caries experience (Carlson ; Li ; Matee ; Napimoga ).

For epidemiological purposes, establishment of some criteria that may separate two or more different genetic types, so-called clones, as distinct entities with their particularities is necessary. Intra-species genetic heterogenecity can be confirmed by DNA-based methods, such as restriction enzyme analysis (Caulfield ), ribotyping (Alaluusua ), arbitrary primed polymerase chain reaction (Gronroos and Alaluusua, 2000) and pulsed field gel electrophoresis (Jordan and LeBlane, 2002). Multilocus enzyme electrophoresis (MLEE) has been applied to study the epidemiology of several genera of pathogenic microorganism such as gram-positive, gram-negative bacteria (Baptist ; Caugant ; Rosa ; Schable ), mycoplasms (O’Brine ; Redmo ; Rosa ), filamentous fungi (Araujo ), yeasts (Naumov ; Rosa ), and protozoa (Meloni ). Gilmour proposed the use of MLEE to differentiate oral streptococci in some groups as mutans streptococci and sanguinis streptococci. However, the potential of MLEE for S. mutans specimen differentiation was evaluated in a few cases (Napimoga ; Redmo ; Rosa ). In this work, such potential was explored, as well as many electrophoresis/revelation systems were screened for the optimization of enzymatic bands detection. The aims of the present study were to evaluate the clonal diversity of S. mutans in caries-free and caries-active subjects and to compare some virulence traits between isolates of these two groups.

The study groups consisted of seven young adults with the mean age of 23 years without regard to sex. There was no history of antibiotic treatment at least 1 month prior to study. Caries-free individuals [DMF (Decayed, Missing, Filled) = 0] contained three subjects and the group of caries-susceptible individuals [DMF = 8 ± 2] contained four subjects.

Volunteers were informed not to brush their teeth during the preceding 12 h and not to drink or eat anything for 2 h before sampling. Pooled samples of dental plaque were taken with sterile dental curettes from three surfaces of the anterior and posterior teeth. The samples were immediately transferred to 3 mL of cold brain heart infusion broth (Difco Laboratories, Detroit, Mich.) and were vigorously shaken with a Vortex mixer to disperse the bacteria, then were transferred to a laboratory on ice and cultured immediately.

The serial dilutions of the samples were cultured on Mitis Salivarius Agar (Merck, Germany) supplemented with 2% sucrose (Synth) and 2.8 μg/mL of bacitracin (Sigma, USA). Plates were incubated at 37 °C for 48 h in an atmosphere of 10% CO2. After purification of S. mutans isolates, their identification was done according to Gram staining, Catalase and sugar fermentation tests also Rap ID STR kit (Remel, USA). Standard strain of S. mutans ATCC®35688 was also studied for comparison.

In order to obtain sufficient concentrations of proteins, 1011 cells of each isolate were grown in 100 mL Brain Heart Infusion (BHI) for 18 h at 37 °C with shaking (160 rpm) in a shaker incubator (Heidolph, Germany). Cells were collected by centrifugation (10,000 × g for 15 min) and washed three times in 40 mM potassium phosphate (pH 7.5) with 3 mM dithiothreitol, 10 mM L-cysteine HCl, 0.06 mM MnSO4 (Selander ). Cells were normally lysed by sonication (Hielscher, Germany) with microtip for 30 to 60 s, with ice-bath cooling. After lysis and centrifugation at 30,000 × g for 20 min, 0.5 mL of the several milliliters of lysate (supernatant) were transferred to three or four culture tubes and stored at −20 °C until used for electrophoresis. Protein concentrations were also determined by the Lowry method (Lowry ).

Electrophoresis was carried out in 10% (w/v) poly-acrylamide resolving gels (1.5 M Tris-HCl pH 8.9, 10% acrylamide + bisacrylamide) and 2.5% stacking gels (0.5 M Tris-HCl pH 6.9, 3% acrylamide + bisacrylamide) by using 0.75 mm thick slab gels and Tris/HCl buffer (pH 6.8). The amount of 5 μL of Samples were diluted (total concentration of 50 g protein in diluted samples) in an equal volume of sample buffer (1 g sucrose, 500 μL β-mercaptoethanol, 0.002 g bromophenol blue, 1 mL of 1 M Tris/HCl). A marker of known molecular mass (Fermentas SM 0661 protein ladder) was also loaded (20 μL) along with the samples. The apparatus was connected with constant electric current (30 mA) till the bromophenol blue (BPB) reached the bottom of the plate. The gel was run in a cold room at 5 °C. After running (120 min, 30 mA), gels were revealed for enzyme active band detection of alcohol dehydrogenase (ADH), malic enzyme (ME), glucose dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G6PD), glucosyltransferase (GTF), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), superoxide dismutase (SOD), and glutamic-oxaloacetic transaminase (GOT).

For enzymatic band revelation, stain solution [100 mL Tris Glycine buffer 1X pH8, the specific substrate of each enzyme, coenzyme (100 mg NAD), Dye (50 mg NBT) and catalyst (20 mg PMS) and 40 mg MgCl2] was applied to the gel in an agar overlay. Gels were incubated at 37 °C in the dark until bands appear (45–60 min). The stain solution was then poured off and fixed in a 5% acetic acid solution.

After appearing enzymic bands, they were scored according to their respective relative mobility’s generating binary data (Schable ).

Genetic diversity for a locus is calculated as h = [1−∑xi2][n/(n−1)], where x is the frequency of ith allele at the locus, n is the number of isolates n/(n−1) is the correction for bias in small samples (Nei, 1978; Rosa ; Selander ).

A dendrogram was generated after the overall gel analysis using Euclidean Distance coefficient) calculated by the NTSYS 1.70 packages (Applied Biostatistics, Inc.). A tree of genetic distance was generated by the unweighted pair-group arithmetic average (UPGMA) clustering method (Rosa ; Sneath and Sokal, 1973).

In This Study, twelve S. mutans isolates were analyzed (Table 1). Six isolates were isolated from caries-susceptible subjects and, five other were isolated from caries-free persons.

Table 1

Electromorphotypes of S. mutans isolates of caries-free and caries susceptible individuals.

Subject	Isolate		Allele at enzyme locus*
			MDH	G6PD	GDH	ME	GTF	GOT	ADH	LDH	SOD
Caries-susceptible	1	C1	-	2	-	-	-	-	-	1	-
	1	C2	1	1, 2a	1	1	1	1	1	1	-
	2	D3	-	2	-	-	-	-	-	1	-
	3	F4, F5	-	2	-	-	-	-	-	-	-
	4	G7	-	2	-	-	-	-	-	-	-
Caries-free	5	A8	-	-	-	-	-	-	-	-	-
	6	B9, B11	-	-	-	-	-	-	-	-	-
	7	P12, P13	-	-	-	-	-	-	-	-	-
-	-	ATCC®35688	1	1, 2	1	1	1	1	1	1	-

Ac*according to the anodal migration.

Two alleles (allele 1 and allele 2) for G6PD were visualized while for others only one locus was seen.

All analyzed enzyme systems (ADH, GDH, G6PD, LDH, MDH, ME, GOT, GTF) showed no activity for any S. mutans isolates of caries-free subject group (Table 1). Glucose-6-phosphate dehydrogenase (G6PD) showed thin band zones that did not vary among all of the S. mutans isolates of caries susceptible group and ATCC®35688. Two alleles (allele 1 and allele2) for G6PD were visualized in C2 and ATCC®35688 strains while in other isolates only the second allele was observed and no locus of G6PD was seen in caries free isolates (Figure 1). For lactate dehydrogenase (LDH), the unexpected electrophoretic bands appeared only in C1, C2 and D3 (Table 1).

Figure 1

Multilocus enzyme electrophoresis analysis of S. mutans isolates of caries free & caries susceptible subjects.

All analyzed enzyme systems were active for S. mutans C2 isolated from caries susceptible subject group and ATCC®35688. For glucosyltransferase (GTF), a typical enzyme produced by S. mutans, the expected electrophoretic bands did not appear in all of the isolates and it is only appeared in C2 and ATCC®35688. The biofilm formation ability of these two isolates (C2 and ATCC®35688) was very high and they were classified as strongly adherent (data not shown).

Isolates #C1, #C2, #D3, #F4, #F5, and #G7 were isolated from caries susceptible subjects; Strain #S was S. mutans ATCC®35668; Slot E is the pure enzyme that was added as an electrophoretic control.

As it is shown in Table 1, isolate C1 also differed from C2 and they were isolated from the same individual (subject #1) and their enzymatic patterns suggest that they belong to different clones. Indeed, average genetic diversity between both clones was determined as 0.468. In this study, genetic diversities for the eight evaluated loci were, 0.59, 0.88, for G6PD and LDH respectively and 0.097 for other loci. The mean diversity for the loci was 0.228.

To determine the genetic distance among isolates as well as their clustering behavior, a non-rooted dendrogram was built (Figure 2). Such tree shows that different isolates could be clustered in four different taxa with genetic distances varying from 0.2398 to 0.9592.

Figure 2

Non-rooted dendrogram clustering isolates of S. mutans from caries-free (CF) and caries-susceptible (CS) patients in different taxa in function of their isozymic profiles.

All isolates from free caries subjects as listed in Table 1 belong to clade C as shown in Figure 2 (genetic distance d ≥ 0.4796).

Although in different taxa, different isolates of same individuals (patients #3, #6 and #7) presented no divergences (d = 0.0000). Patient #1, however, presented two isolates separated one to each other with a d = 0.9592, showing that more than one clonal type can be found in a same individual.

The differentiation ability of MLEE for cariogenic and non cariogenic S. mutans isolates was confirmed in this study. Isolates of caries susceptible group differed from isolates of caries free group.

According to the results, it is evident that caries-susceptible subjects bear more genotypes than caries-free. It is in agreement with earlier reports (Alaluusua ; Napimoga ). Redmo-Emanuelsson , in a study enrolling young adults (mean age of 25.2 years) found a maximum of seven genotypes in subjects who had previous caries experience and Napimoga found a maximum of eleven and eight genotypes in caries-active subjects using MLEE and AP-PCR respectively.

We obtained up to three genotypes in caries-susceptible subjects (4 subjects; 7 isolates) using MLEE. In contrast, the results of Kreulen showed a negative relationship between caries activity and genotype diversity. Redmo-Emanuelsson suggested that the larger number of genotypes found could be because of the larger number of isolates analyzed, which increases the possibility of detecting different genotypes.

In this study, all analyzed enzyme systems revealed no activity for any S. mutans isolates of caries free subject group (3 subject, 5 isolates) so, they belong to one clade. Perhaps, such enzymes are either produced in smaller quantities than the method can detect or they are not produced by those isolates. Alaluusua related that six caries-free children (3 to 7 strains per volunteer) only harboured one ribotype of S.mutans. According to these, it is probable that from plaque samples of caries free subjects the only primary strains were found and other strains, if they existed were below the detection level.

Getting activity for G6PD (in S. mutans isolates of caries susceptible group and ATCC®35688) and LDH (in C1, C2 and D3) in this study was in contrast with previous researches that showed no activity for the major part of dehydrogenases (ADH, GDH, G6PD, LDH, MDH, ME) in S. mutans strains (Napimoga ; Rosa ). These differences are probably due to the pH, type of buffer system used in preparation of enzyme extract or type and concentration of used gel. Occasionally, the relative mobilities of certain electromorphs are reversed in different buffer system (Selander ). According to Wunder and Bowen (2000), glucosyltransferases are enzymes that act at extracellular environment. This may, at least in part, explain why glucosyltransferase activity was not observed in all of the cases and it is only appeared in C2 and ATCC®35688.

A non-rooted dendrogram was shown that different isolates of this study could be clustered in four different taxa. All isolates from three caries-susceptible patients were grouped in a single taxon (taxon C). Patient #1, however, presented two isolates separated one to each other (d = 0.9592), showing that more than one clonal type can be found in a same individual. Rosa also could isolate two clusters of S. mutans isolates from a same individual, and their enzymatic patterns suggest that they belong to different clones. The isolates of caries free subjects, presented no divergences (d = 0.0000) although they were isolated from non-related individuals.

In parallel to the discrimination ability, MLEE has proven to be a useful tool for establishing genetic diversity, even in small or subdivided (Rosa ) populations. This method detects allelic frequencies prompter than other methodologies, such as RAPD and RFLP. These facts support the premise that MLEE may be used in surveys in which intra-species determination of S. mutans is required. In conclusion, the results here presented support the proposition of MLEE employment for clonal differentiation of Streptococcus mutans. Three clonal types and one clonal type were observed in caries-susceptible and caries-free subjects, respectively.