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Frequency of four different mutacin genes in Streptococcus mutans genotypes isolated from caries-free and caries-active individuals

Department of Oral Microbiology and Immunology, Piracicaba Dental School, State University of Campinas, Av. Limeira, 901 – Areião, Piracicaba, SP, Brazil

Correspondence Reginaldo B. Gonçalves Reginald{at}fop.unicamp.br

Received August 17, 2004
Accepted February 18, 2005

The ability of Streptococcus mutans to produce mutacins, combined with the production of other virulence factors such as lactic acid, may contribute to the pathogenesis of this bacterium. In the present study, the detection of genes encoding mutacin types I/III, II and IV was performed by PCR with specific primers to each type in a total of 63 S. mutans genotypes isolated from caries-active and caries-free individuals. In the caries-free group, PCR screening for mutacin IV revealed that 31.8 % of strains were positive for this mutacin. PCR for the other three mutacins tested (I/III and II) did not yield amplicons in any S. mutans strains in this group. The PCR with primers of mutacin IV showed 68.3 % positive genotypes in the caries-active group, on the other hand, the amplicons of mutacins I/III revealed 41.5 % positive strains that carried these genes. The chi square test showed significant differences in the number of positive strains to mutacin IV when comparing the caries-free and caries-active genotypes of S. mutans (P = 0.01). All tested S. mutans strains were negative by PCR for mutacin II. The low frequencies of detection of some mutacin genes suggest the existence of high diversity and polymorphism in the production of genetic determinants of mutacin-like substances. In addition, the production of a wide spectrum of mutacins can play an important biological role in colonization by S. mutans strains, mainly in the niche of high-complexity microbial communities.

	INTRODUCTION

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Mutans streptococci are generally accepted as one of the principal aetiological agents of dental caries (Loesche, 1986; Becker et al., 2002). The dental biofilm consists of a complex bacterial community, and the ability of specific strains of Streptococcus mutans to compete with other strains may be essential for colonization. Alaluusua et al. (1996) suggested that some strains of S. mutans might be able to colonize the host and induce dental caries better than other strains. Alternatively, dietary patterns of the host may be an important factor, since a high salivary mutans streptococci count does not necessarily exert a cariogenic challenge (van Palenstein Helderman et al., 1996).

Numerous factors affect the equilibrium among oral populations of micro-organisms, and several inhibitory substances have been identified, including mutacins (Fukushima et al., 1985; Caufield et al., 1985; Delisle, 1976). Mutacins are peptide or protein antibiotics that are mainly bactericidal for other bacteria of the same or closely related species, as well as for other Gram-positive micro-organisms, and are likely to confer an ecological advantage in diverse bacterial communities such as the dental biofilm (Parrot et al., 1990; Balakrishnan et al., 2002). Some studies have demonstrated that the mutacin activity of S. mutans could be related to the prevalence of this species in the dental biofilm, saliva and dental caries (Berkowitz and Jordan, 1975; Hillman et al., 1987).

Classification of mutacin-producer strains based on their bactericidal activity divides mutacins into four types, I, II, III and IV. The antimicrobial spectrum of mutacin IV is specifically against members of the mitis group of oral streptococci, while that of mutacins I, II and III is broader (Qi et al., 1999a, b, 2001).

The relationship between caries activity and the higher synthesis of some virulence factors by different strains of S. mutans has been demonstrated in the literature (Mattos-Graner et al., 2000). In a previous study, we observed a statistically significant positive association between the level of synthesis of water-insoluble glucan by S. mutans clinical isolates and the frequency of adherent cells in the presence of sucrose in caries-active subjects, but not in caries-free subjects (Napimoga et al., 2004). In addition, the strains of S. mutans isolated from caries-active individuals produced a wide spectrum of mutacins in comparison with those from caries-free individuals (Kamiya et al., 2005), suggesting that isolates from subjects with high caries activity were better at colonizing and accumulating on teeth, and, consequently, inducing caries.

In the present study, we analysed the relationships between the frequencies of detection of four different mutacins (mutacins I, II, III and IV) from S. mutans genotypes isolated from caries-free and caries-active individuals.

	METHODS

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Bacterial strains.

A total of 63 clinical isolates previously genotyped (Napimoga et al., 2004) and 3 reference strains, as positive controls, of S. mutans were used. The clinical S. mutans were previously isolated from individuals aged 18–29 years (mean ± SD, 23.5 ± 3.9). The clinical strains were identified by PCR and genotyped by arbitrarily primed PCR (Napimoga et al., 2004), and included 41 strains from eight caries-active individuals and 22 strains from eight caries-free individuals.

The isolates were previously evaluated for the production of mutacin-like substances (Kamiya et al., 2005) by a modification of the deferred antagonism method (Hamada & Ooshima, 1975). For mutacin-activity testing, the following 12 Streptococcus species were used as indicator strains: S. mutans CCT3440, S. mutans 32K, Streptococcus sobrinus ATCC 27607, S. sobrinus 6715, Streptococcus mitis A, S. mitis ATCC 903, Streptococcus salivarius ATCC 25975, S. salivarius 66.4, Streptococcus sanguinis CR311, S. sanguinis M5, S. sanguinis ATCC 10556 and Streptococcus oralis PB182. These strains were acquired from the respective collections of bacterial strains.

Extraction of chromosomal DNA.

The strains were grown planktonically in brain heart infusion broth (Difco). DNA from all strains was extracted using a simple DNA preparation in which the cells were washed and boiled for 10 min with TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) modified from Welsh & McClelland (1990) and Saarela et al. (1996), the debris pelleted and the supernatant used for detection of mutacin I/III, II and IV genes by PCR.

PCR screening of mutacin genes.

The detection of genes encoding mutacin types I, II, III and IV (Qi et al., 1999a, b, 2001) was performed by PCR using specific primers to each type. Only one pair of primers was used to detect genes encoding mutacin types I and III due to high homology between them (Qi et al., 1999b). Primers to the genes encoding mutacin types II and IV were designed based on sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov) (Table 1). The homologous genes to mutacin types I and III were detected by a pair of primers based on conserved amino acid sequences from mutacin 1140, mutacin NY266, epidermin and gallidermin (Qi et al., 1999b).

Amplification by PCR was performed in the GeneAmp PCR System 2400 (Perkin Elmer). The 50 µl PCR consisted of 1x PCR buffer containing 2.5 mM MgCl₂, 200 µM of each deoxynucleotide, 0.3 µM of each oligonucleotide primer, 1.25 U of Taq DNA polymerase (GIBCO) and 50 ng of template DNA. Besides the strains tested, positive and negative controls were used in each PCR reaction: purified genomic DNA from S. mutans UA159 was used as a positive control for mutacin type IV genes and two genotypes previously isolated from a mother/child pair (Klein et al., 2004) were used as positive controls to mutacin types I/III and II genes, respectively (unpublished results). Distilled water was used as a negative control.

The PCR conditions were optimized for the control strains. The PCR conditions included initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 52 °C for 1 min, extension at 72 °C for 2 min, and final extension at 72 °C for 7 min. The PCR products were analysed by electrophoresis in 1.0 % agarose gel using Tris/borate/EDTA buffer (pH 8.0). A 250 bp DNA ladder was included in each gel. The DNA was stained with 0.5 µg ml^–1 ethidium bromide and visualized under UV illumination.

Statistical analysis.

The chi-square test was applied to detect differences in the frequency of mutacin genes.

	RESULTS

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In the caries-free group, among the 22 S. mutans isolates analysed, four (18.2 %) showed inhibitory activity against mutans streptococci, 20 (90.9 %) showed inhibitory activity against mitis streptococci and five (22.7 %) against S. salivarius (Table 2). In the caries-active group, among 41 isolates, 13 (31.7 %) showed inhibitory activity against mutans streptococci, 33 (80.5 %) against mitis streptococci and seven (17.1 %) showed inhibitory activity against S. salivarius (Table 3). Two S. mutans strains from the caries-free and four from the caries-active group showed no inhibitory activity against any of the 12 indicator-strains.

In the caries-free group, PCR screening with primers of mutacin IV revealed that seven out of 22 (31.8 %) strains were positive for this mutacin. PCR for the other three mutacins tested (I/III and II) did not yield amplicons in any S. mutans strains in this group (Table 2).

The PCR with primers of mutacin IV showed that 28 out of 41 (68.3 %) strains were positive in the caries-active group; on the other hand, the amplicons of the mutacin I/III genes revealed that 17 out of 41 (41.5 %) strains carried these genes (Fig. 1). Significant differences were found in the number of positive strains that carried the mutacin IV gene when comparing the caries-free and caries-active genotypes of S. mutans (P = 0.01). PCR with mutacin II did not yield amplicons in any S. mutans strains (Table 3).

View larger version (89K): [in this window] [in a new window]   Fig. 1. PCR screening of four different mutacin genes in S. mutans. Lanes: 1, 250 bp marker ladder; 3 and 5, negative control (distilled water); 2, mutacin II (444 bp); 4, mutacin I/III (750/450 bp); 6, mutacin IV (1344 bp).

In both groups, there were some strains that produced mutacin and showed in vitro inhibitory activity against at least one indicator strain but did not yield amplicons to mutacin genes. On the other hand, in the caries-active group, some genotypes that showed amplicons to mutacin IV and to mutacins I/III did not reveal inhibitory activity against any of the indicator strains tested (Table 3).

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Mutacins have been implicated as virulence factors in dental caries (Hillman et al., 1987; Novak et al., 1994). In this study, we investigated the detection frequency of mutacin genes in S. mutans strains isolated from caries-free and caries-active individuals. We found that S. mutans strains recovered from caries-active individuals showed a higher frequency of detection of mutacin IV than the S. mutans strains recovered from caries-free individuals. In addition, only S. mutans in the caries-active group showed amplicons corresponding to mutacin I/III genes.

The oral biofilm is subjected to variable environmental stress, including the availability of nutrients, acidic pH (Carlsson, 1989) and mutacin activity (Qi et al., 2001). Clinically, mutacins have been considered important for the establishment and equilibrium of bacteria in dental biofilms: the mutacin-producing strains might colonize more easily and suppress non-producing or susceptible strains (Hillman et al., 1987).

Longo et al. (2003) analysed 19 strains isolated from children and found only one strain that showed an amplicon homologous to the gene for mutacin II. Qi et al. (2001) searched clinical isolates of S. mutans for the presence of mutacin IV genes by PCR and found >50 % positive results. According to Qi et al. (2001), mutacin IV is produced by planktonic cells while mutacin I is produced by biofilm-like cells. Different mutacins may serve different purposes during the process of colonization by S. mutans. For instance, production of mutacin IV by planktonic cells in saliva may help S. mutans kill the primary colonizers on the tooth surface to make room for its own population. Supporting this hypothesis, the antimicrobial spectrum of mutacin IV is specifically against members of the mitis group of oral streptococci (Qi et al., 2001). Nevertheless, our study suggests that given the increasing complexity of the oral microbiota, as found in caries-active individuals (Napimoga et al., 2004), the S. mutans strains producing a wide spectrum of mutacins, including mutacins I, II and III, could become prevalent in most oral sites.

Longo et al. (2003) related no association between mutacin inhibitory spectrum and infecting levels of mutans streptococci or caries incidence in the host, suggesting that the mutacin production may not be relevant to the ability of the strain to colonize the host and induce disease. In a previous study we showed distinct mutacin production profiles between S. mutans isolated from caries-active and S. mutans isolates from caries-free individuals, which can be related to the different colonization profiles described in these individuals (Kamiya et al., 2005).

In the caries-active individuals the sites from which S. mutans were recovered were more diverse, probably because production of organic acids and mutacins within the biofilm resulted in a more complex community compared to caries-free individuals (Paddick et al., 2003). Probably due to this complexity, S. mutans genotypes recovered from caries-active individuals presented higher frequencies of mutacin IV and a wide spectrum of mutacins, such as I/III, and presented greater mutacin activity in vitro compared to S. mutans recovered from caries-free individuals (Kamiya et al., 2005).

We also found some mutacin-producing strains in both groups that showed in vitro inhibitory activity against at least one indicator strain but did not yield amplicons to mutacin genes. These data suggest a high genetic diversity at the mutacin locus or absence of the structural genes encoding these genes. One possible explanation is that the polymorphism at the mutacins locus may have compromised primer annealing in the PCR, which suggests that there are different mutacin-coding genes that have a similar phenotype.

On the other hand, in the caries-active group, some genotypes showed amplicons to mutacin IV and to mutacin I/III but did not reveal inhibitory activity against any of the indicator strains. One possible explanation is the modification of only one amino acid, which has already been shown to alter or prevent the activity of certain mutacins (Mulders et al., 1991; Rollema et al., 1995; Chan et al., 1996). The inhibition assay previously performed (Kamiya et al., 2005) could also result from the production of more than one inhibitory substance, showing that the understanding of the genetic determinants of several mutacins still needs to be improved.

This study evaluated the frequency of mutacins I, II, III and IV in S. mutans strains recovered from caries-active and caries-free individuals, and compared them with the phenotypic profiles of these substances in vitro. Our results suggest high diversity and polymorphism in the production of genetic determinants of mutacin-like substances. In addition, the production of a wide spectrum of mutacins can play an important biological role in colonization by S. mutans strains, mainly in the niche of high-complexity microbial communities.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This research was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP # 01/11787-4), the Fundo de Amparo ao Ensino e à Pesquisa (FAEP # 1158-01; # 1505-02) and the Conselho Nacional de Pesquisa (CNPq # 140949-04).

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES