Objective To isolate, purify and identify clinical Streptococcus mutans (S.mutans) strains, construct their 412c-deficient mutant strains, and investigate on their proliferation and cariogenicity.
Methods Dental plaque samples were randomly collected from healthy adults without caries, were anaerobically cultivated on saliva or S.mutans bacitracin AGAR (MSA) medium and anaerobion were screened out. The isolated S.mutans strains were identified by colony morphology, Gram staining, biochemical test and polymerase chain reaction (PCR). The linearized recombinant plasmids were transformed into clinical S.mutans strains for homologous recombination to obtain 412c (hit) gene defect strains. The growth rate and acid resistance in the brain heart immersion (BHI) medium with different pH values. In BHI medium containing 3% sucrose, the acid production capacity of the two kinds of strains was compared.
Results The morphology of the dental enamel surface plaque of healthy adults without caries growing in MSA medium was dark blue hemispheric granular protrusions with rough surface and uneven edges. Using Gram staining, there were many long and short chained coccus-shaped bluish violet bacterium under an oil-microscopy. The isolated S.mutans was identified as "an equivalent S.mutans UA159" by biochemical test and PCR-Sanger sequencing of 412c gene segment. The hit-UP-pFW5-Down recombinant plasmids were transformed into the genetic competence of the isolated S. mutans strains. The 412c-deficient mutant strains were screened out. Similar to S.mutans ATCC 25175, the growth rate of the 412c-deficient mutant strains increased greatly compared with their parental S.mutans strains.
Conclusion The clinical isolate of S.mutans was "equivalent S.mutans UA159" strain, in which the 412c gene is growth-phase regulated, 412c-deficient S.mutans strains have significantly higher growth rates than its parental strain and have more cariogenic potential.
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