| Background: Dental caries is recognized as the common oral disease affecting physical and mental health even bring economic burden to the family.ECC is defined as “the presence of one or more decayed(non cavitated or cavitated lesions),missing(due to caries),or filled tooth surfaces in any primary tooth in a child under the age of six”.National oral epidemiological surveys at 2015 had shown that the prevalence of dental caries in 5-year old children was as high as 71.9%,which is up 5.9% from a decade ago.Streptococcus mutans(S.mutans)has been implicated as the main pathogenic bacteria of human caries.Its strong biofilm formation ability and unique acid-producing and acid-resistant ability accelerate the development of caries.Previous studies have shown that the virulence expression from various dental plaque is different.However,the virulence and genetic characteristics of site-specific dental plaque-derived S.mutans remains unclear.Objective: Analysis of virulence and genetic characteristics of site-specific dental plaque-derived S.mutans.Methods: In present study,c serotype of site-specific dental plaque-derived S.mutans was divided into three groups: caries-active site of ECC children(ECC-C group),caries-free site of ECC children(ECC-F group)and caries-free site of caries free children(CF group).The dental plaque from ECC children and caries free children were collected.The ECC children presented two sites as a cavitated lesion and a sound surface,while the caries free children presented one sound surface.The ECC children whose S.mutans could be isolated from both sites were selected into present study.Caries free children only provided dental plaque on the sound surface.1 Detection of the virulence phenotypes in S.mutans.There were 137 clinical strains of S.mutans isolated from 31 children with both cavitated and sound surfaces of ECC children and 28 clinical strains of S.mutans were isolated from 16 caries free children.S.mutans clinical isolates from 10 children with the top-10 dmfs scores were selected to form the caries-active group(n = 23)and caries-free group(n = 23)of children with ECC.All of the S.mutans clinical isolates(n = 28)from 16 caries free children were selected to form the caries free group.Virulence phenotype including growth pattern,biofilm formation and EPS formation were evaluated according to the turbidimetric method,crystal violet staining method and congo red staining method.The p H value change(△ p H)at the early stationary phase under planktonic status and the final p H of supernatant under biofilm status were detected by a p H meter.2 Detection of the relative expression levels of virulence genes in S.mutans.Among the 10 children with ECC,1 isolate of S.mutans from caries and sound surface sites of each children was selected randomly into caries-active(n = 10)and caries-free(n = 10)groups,while clinical isolates of S.mutans from 10 children which were selected randomly from 16 caries free children were made as the caries free group(n = 10).The relative expression levels of virulence genes(gtf B,gtf C,spa P,srt A,rel A,ffh,srn225147,srn821798)were detected by real-time quantitative PCR in S.mutans from above three groups.S.gordonii was used as the indicator strain to evaluate mutacin IV activity,S.mutans clinical isolates with high production of mutacin IV and free mutacin IV were selected to form High-IV(n = 10)and None-IV(n = 10)groups.The relative expression levels of mutacin IV gene(com D,nlm A,nlm B)and srn225147 were detected by real-time quantitative PCR.3 Multilocus sequence typing(MLST)technique in S.mutans.Among the 31 children with ECC,1 isolate of S.mutans from caries and sound surface sites of each children was selected randomly into caries-active(n = 31)and caries-free(n = 31)groups.Because only 9 caries free children had been isolated multiple S.mutans from the 16 caries free children,2 isolates of multiple S.mutans from each subject of 9 caries free children were selected randomly into caries-free group(n = 18).All of 80 isolates were subjected to whole genome sequencing,and the sequenced contig file were submitted to Pub MLST for finding reported STs and registering with new alleles and sequence types using Do MLST.Phylogenetic tree was constructed based on the sequence type and used to analysis genetic characteristics of site-specific dental plaque-derived S.mutans.Results: 1 Phenotypes of virulence in S.mutans.The similar growth pattern and p H value at the early stationary phase of S.mutans isolated from the three sites were found under planktonic status.No significant difference in the capacity of biofilm formation and △ p H was observed between the caries-active site and caries-free site of children with ECC(P = 0.947 for biofilm formation and P = 0.928 for p H value),while the capacity of biofilm formation,△ p H or extracellular polysaccharide was higher in S.mutans of ECC than that in the sound site of caries free children(P = 0.018 and P < 0.001 between caries-active site of ECC children and sound site of caries-free children;P = 0.017 and P = 0.046 between caries-active site of ECC children and sound site of caries-free children;P = 0.043 and P = 0.006 between caries-free site of ECC children and sound site of caries-free children).2 The relative expression levels of virulence genes in S.mutans.The relative expression levels of gtf B,spa P and srn225147 in the ECC-C group were higher than those in the CF group(P = 0.040 for gtf B;P = 0.011 for spa P;P <0.05 for srn225147).No significant difference in the relative expression levels of srn821798 was found among the three sites.The expression level of com D was decreased when the expression of srn225147 reached approximately 400-fold(P = 0.019),while the expression level of com D was increased when the expression of srn225147 reached approximately 1400-fold(P = 0.002).However,there was no significant difference in the production of mutacin IV under the approximately 400-fold or 1400-fold expression of srn225147. 3 Multilocus sequence typing(MLST)technique in S.mutans.There were 44 new alleles and 77 new sequence types identified from 80 isolates of S.mutans.A total of 94.44% children shared identical sequence type in children with ECC.All of the isolates in a single caries free child shared identical sequence type.Conclusions: The levels of biofilm and EPS formation,△ p H and virulence genes in S.mutans isolated from the ECC children were higher than those from the caries free children.The virulence of S.mutans in the sites of caries-active and caries-free of ECC children was similar.srn225147 had weak two-way regulation role in the com D gene and its real role in caries needs further exploration.S.mutans could spread through child-to-child transmission between children in the same kindergarten. |
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