OBJECTIVETo establish a simple and rapid method to detect Streptococcus mutans and streptococcus sobrinus simultaneously in human saliva.

METHODSChromosomal DNA from the bacteria was obtained by the extraction method with phenol-chloroform. A nested PCR method with two sets of primers specific for portions of the glucosyltransferase genes (gtfB of S. mutans and gtfI of S. sobrinus), was optimized to detect S. mutans and S. sobrinus from standard strains, clinical strains and directly in human saliva.

RESULTSThe first process of nested PCR was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 10(5) CFU cells of standard and clinical strains, or from 1 ml clinical saliva samples containing 10(5) CFU cells of either species. a second process of nested PCR, using the first PCR product as a template with new internal primers to detect 10(3) CFU of either streptococcal species in 1ml saliva samples.

CONCLUSIONNested PCR could detect S. mutans and S. sobrinus rapidly and simply in human saliva. This finding would be important to studies of elucidation the role of these two streptococcal species in the etiology of dental caries.

Humans ; Polymerase Chain Reaction ; Saliva ; microbiology ; Streptococcus mutans ; genetics ; isolation & purification ; Streptococcus sobrinus ; genetics ; isolation & purification

OBJECTIVETo analyze the community in dental plaque of elder people with root caries.

METHODSTotal DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification.

RESULTSThe dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114).

CONCLUSIONSThere were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

Age Factors ; Aged ; Capnocytophaga ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; Denaturing Gradient Gel Electrophoresis ; Dental Plaque ; microbiology ; Genotype ; Gram-Negative Bacteria ; genetics ; isolation & purification ; Gram-Positive Bacteria ; genetics ; isolation & purification ; Humans ; Middle Aged ; Neisseriaceae ; genetics ; isolation & purification ; Prevotella ; genetics ; isolation & purification ; Root Caries ; microbiology ; Selenomonas ; genetics ; isolation & purification ; Streptococcus mutans ; genetics ; isolation & purification ; Streptococcus oralis ; genetics ; isolation & purification ; Veillonella ; genetics ; isolation & purification

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects

PURPOSE: Erythromycin-resistant beta-hemolytic streptococci (BHS) has recently emerged and quickly spread between and within countries throughout the world. In this study, we evaluate the antimicrobial susceptibility patterns and erythromycin resistance mechanisms of BHS during 2003-2004. MATERIALS AND METHODS: The MICs of seven antimicrobials were determined for 204 clinical isolates of BHS from 2003 to 2004. Resistance mechanisms of erythromycin-resistant BHS were studied by the double disk test as well as by polymerase chain reaction (PCR). RESULTS: Compared with our previous study, resistance among Streptococcus pyogenes isolates to a variety of drugs decreased strikingly: from 25.7% to 4.8% in erythromycin; 15.8% to 0% in clindamycin; and 47.1% to 19.0% in tetracycline. The prevalent phenotypes and genotypes of macrolide-lincosamide-streptograminB (MLSB) resistance in Streptococcus pyogenes isolates have been changed from the constitutive MLSB phenotype carrying erm(B) to the M phenotype with mef(A) gene. In contrast with Streptococcus pyogenes, resistance rates to erythromycin (36.7%), clindamycin (43.1%), and tetracycline (95.4%) in Streptococcus agalactiae isolates did not show decreasing trends. Among the Streptococcus dysgalactiae subsp. equisimilis isolates (Lancefield group C, G), resistance rates to erythromycin, clindamycin, tetracycline and chloramphenicol were observed to be 9.4%, 3.1%, 68.8%, and 9.4%, respectively. CONCLUSION: Continual monitoring of antimicrobial resistance among large-colony-forming BHS is needed to provide the medical community with current data regarding the resistance mechanisms that are most common to their local or regional environments.
Anti-Bacterial Agents/*pharmacology ; Drug Resistance, Multiple, Bacterial ; Erythromycin/*pharmacology ; Genes, Bacterial ; Genotype ; Hospitals ; Humans ; Incidence ; Korea ; Microbial Sensitivity Tests ; Streptococcus/*drug effects/genetics/isolation & purification ; Streptococcus agalactiae/drug effects/genetics/isolation & purification ; Streptococcus pyogenes/drug effects/genetics/isolation & purification

The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pneumoniae ; genetics ; metabolism ; Transcription Factors ; biosynthesis ; genetics ; isolation & purification

China ; Genes, Bacterial ; Sequence Analysis ; Streptococcus suis ; classification ; genetics ; isolation & purification ; Virulence Factors ; genetics

OBJECTIVETo isolate and identify Streptococcus mutans (Sm) and Streptococcus sobrinus (Ss) in dental plaque of children with high dmft and no caries by selective medium, biochemical methods and arbitrarily primed-polymerase chain reaction (AP-PCR).

METHODSA total of 401 3-4-year-old children from seven kindergartens were recruited using cluster sampling and their dental caries status were examined. From 30% of children with the highest dmft score (dmft >/= 5), 20 children were chosen randomly as test group and 20 age and gender-matched caries-free children were selected as control. Plaque samples were collected from buccal surfaces of the molars and plated onto TYCSB plate. Sm and Ss were primarily identified by colony morphology and biochemical characteristics. Then chromosomal DNA of the strains was isolated and Sm or Ss were confirmed by AP-PCR.

RESULTSThe proportion positive for Sm and Ss in children with high dmft was 100% and 40% respectively while that in caries-free children was 75% and 5% by AP-PCR analysis. The differences were statistically significant between the two groups.

CONCLUSIONSThe proportions positive for Sm and Ss detected by AP-PCR method were significantly higher in children with high dmft than in caries-free children and it is a risk factor for high dmft in deciduous teeth harboring Sm and Ss.

Child, Preschool ; Dental Caries ; microbiology ; Dental Plaque ; microbiology ; Female ; Humans ; Male ; Streptococcus mutans ; genetics ; isolation & purification ; Streptococcus sobrinus ; genetics ; isolation & purification

OBJECTIVETo investigate the relationship between genotypic diversity of Streptococcus mutans (Sm) and deciduous teeth caries in Xinjiang Uygur autonomous region.

METHODSIsolates of Sm were obtained from 17 caries-free and 17 caries-active Uygur preschool children aged from 3 to 5 years. A total of 143 isolates were subcultured, biochemically characterised and identified by polymerase chain reaction (PCR) as Sm and Streptococcus sobrinus (Ss). Then the Sm isolates were genotyped by arbitrarily primed-PCR (AP-PCR).

RESULTSA total of 81 Sm isolates from caries-active subjects and 62 isolates from caries-free subjects were identified by PCR and forty distinct genotypes identified from 143 clinical isolates. In seventeen caries-active group, 9 children had only 1 genotype, 5 children had 2 genotypes, 3 children had 3 genotypes. In seventeen caries-free group, 14 children had only 1 genotype, 3 children had more than 2 genotypes. The Spearman correlation test showed a strong association between genotypic diversity and caries experience (r = 0.342, P < 0.05).

CONCLUSIONSIsolates of Sm in caries-active Uygur preschool children show apparent more genetic diversity than those in caries-free children. The genotypes of isolates might be related to differences in caries susceptibility.

Child, Preschool ; China ; ethnology ; Dental Caries ; ethnology ; microbiology ; Genetic Variation ; Genotype ; Humans ; Polymerase Chain Reaction ; Streptococcus mutans ; genetics ; isolation & purification ; Streptococcus sobrinus ; isolation & purification

OBJECTIVETo evaluate the genotypic diversity of Streptococcus sobrinus (Ss) between children suffering with severe early childhood caries (S-ECC) and caries-free children by arbitrarily primed-polymerase chain reaction (AP-PCR).

METHODSA total of 178 children aged from 42 to 54 months were recruited from 14 urban kindergartens. The S-ECC group contained 87 children with more than 5 decayed teeth, and the control group was composed of 91 caries-free children. Stimulated whole saliva was collected by chewing paraffin. All mutans streptococcus isolates were subcultured, biochemically characterised and identified by PCR as Streptococcus mutans (Sm) and Ss. Then the Ss isolates were genotyped by AP-PCR.

RESULTSThe frequency of Ss detection was 18% in S-ECC children, which was significantly higher than 3% in caries-free children (P < 0.01). Twenty-two distinct genotypes of Ss were identified from 53 clinical isolates. In S-ECC group, one to three genotypes of Ss were detected in each saliva sample. Only one genotype of Ss was detected in all the caries-free children. One genotype of Ss were shared by three S-ECC children. The genotypes of isolates in S-ECC group were relate to decayed-missing-filled teeth (r = 0.50, P < 0. 05).

CONCLUSIONSThe rate of Ss detection was significantly higher in S-ECC children than in caries-free children. Isolates of Ss displayed genetic polymorphism. The multi-genotypes of Ss was related to differences in caries susceptibility. Strains of Ss with same genotype were present in unrelated subjects.

Child, Preschool ; Dental Caries ; microbiology ; Female ; Genotype ; Humans ; Male ; Polymorphism, Genetic ; Streptococcus sobrinus ; genetics ; isolation & purification

OBJECTIVETo compare the gene sequence and protein structure of lactate dehydrogenase (LDH) in Streptococcus oligofermentans with those of other bacteria with different acid generating capacities in oral cavity and to analyze the differences of their LDH.

METHODSLDH gene sequence of Streptococcus oligofermentans was measured by Institute of Microbiology, Chinese Academy of Sciences. LDH gene sequences of four Streptococcus and Lactobacillus casei in the NCBI Genbank was identified and compared among the six bacteria's LDH gene sequences and amino acid sequences by BLAST software. ExPASy database was used to predict the physical-chemical characteristics, secondary structure, trans-membrane regions, and spatial structure of Streptococcus oligofermentans LDH protein, which was compared with those of other bacteria.

RESULTSThe full-length of the LDH gene sequences of Streptococcus oligofermentans was 987 base pairs. The highest similarity was 89% with that of the Streptococcus sanguis, and 81% similarity with Streptococcus mutans, and 70% similarity with Lactobacillus casei. LDH amino acid sequence of Streptococcus oligofermentans was similar to Streptococcus sanguinis, with the highest similitude of 96%, with a similitude of 81% to Streptococcus mutans, but differed greatly from that of Lactobacillus casei, with a similitude of only 66%. Streptococcus oligofermentans LDH protein's physical-chemical characteristics, trans-membrane region's numbers, proportions of secondary structure, structural domain's location resembled those of Streptococcus mutans, Streptococcus sanguinis and Lactobacillus casei. Spatial structure differences between the LDH of Streptococcus oligofermentans and that of Streptococcus mutans and Lactobacillus casei were distinct.

CONCLUSIONSStreptococcus oligofermentans LDH's gene sequence, amino acid sequence, and spatial structure all vary from those of Streptococcus mutans and Lactobacillus casei, and these differeces may be a inherent reason that lead to the changes of its LDH's biological functions and incapacity of producing lactic acid.

Amino Acid Sequence ; Base Sequence ; L-Lactate Dehydrogenase ; genetics ; isolation & purification ; metabolism ; Lactic Acid ; metabolism ; Molecular Weight ; Protein Structure, Secondary ; Species Specificity ; Streptococcus ; enzymology ; genetics ; Streptococcus mutans ; enzymology ; genetics ; Streptococcus sanguis ; enzymology ; genetics