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

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 establish a systematic method for isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity.

METHODSSamples of the saliva, dental plaque and periapical granulation tissue were collected from 20 subjects with healthy oral condition and from 8 patients with different oral diseases. The bacteria in the samples were identified by morphological identification, VITEK automatic microorganism identification and 16s rRNA gene sequencing.

RESULTSVITEK automatic microorganism identification and 16s rRNA gene sequencing showed an agreement rate of 22.39% in identifying the bacteria in the samples. We identified altogether 63 bacterial genus (175 species), among which Streptococcus, Actinomyces and Staphylococcus were the most common bacterial genus, and Streptococcus anginosus, Actinomyces oris, Streptococcus mutans and Streptococcus mitis were the most common species. Streptococcus anginosus was commonly found in patients with chronic periapical periodontitis. Streptococcus intermedius and Staphylococcus aureus were common in patients with radiation caries, and in patients with rampant caries, Streptococcus mutans was found at considerably higher rate than other species.

CONCLUSIONAerobic and facultative anaerobic bacteria are commonly found in the oral cavity, and most of them are gram-positive. 16s rRNA gene sequencing is more accurate than VITEK automatic microorganism identification in identifying the bacteria.

Actinomyces ; isolation & purification ; Dental Caries ; Dental Plaque ; microbiology ; Humans ; Mouth ; microbiology ; RNA, Ribosomal, 16S ; genetics ; Saliva ; microbiology ; Staphylococcus aureus ; isolation & purification ; Streptococcus ; isolation & purification

OBJECTIVEBacterial cultures from respiratory aspirate or sputum have been the conventional diagnostic method for pneumonia, but the results of culture was often affected by early extensive use of antibiotics, sample collection and delivery. The objective of this study was to explore application of the combined detection of culture and polymerase chain reaction (PCR) assay in hospitalized children with pneumonia.

METHODSTotally 187 hospitalized children with pneumonia were enrolled. The age of the patients ranged from 1 month to 10 years, 124 were male, 63 female; 175 of the patients received antibiotics treatment before admission. Deep respiratory aspirate sample from patients was cultured by Streptococcus pneumoniae selective plate, Hemophilus influenzae selective plate and conventional plate. The aspirate samples were also amplified for DNA of 14 bacteria with target enriched multiplex polymerase chain reaction (Tem-PCR) and detected with Luminex xMAP technology platform.

RESULTSThe total positive rate by bacterial culture was 40.1% (75/187), of which 17.1% (24/187) were Hemophilus influenzae b, 8.6% (16/187) were Escherichia coli, 6.4% (12/187) were Klebsiella pneumoniae, 4.8% (9/187) were Staphylococcus aureus, 3.7% (7/187) were Streptococcus pneumoniae, 1.6% (3/187) were Pseudomonas aeruginosa, 1.1% (2/187) were Acinetobacter baumannii, and 1.1% (2/187) were Enterobacter cloacae. The total positive rate by combined detection of culture and Tem-PCR assay were 78.6% (147/187), of which 28.9% (54/187) were Hemophilus influenzae b, 19.3% (36/187) were Streptococcus pneumoniae, 8.6% (16/187) were Escherichia coli, 6.4% (12/187) were Klebsiella pneumoniae, 5.9% (11/187) were Staphylococcus aureus, 5.9% (11/187) were Acinetobacter baumannii, 2.7% (5/187) were Pseudomonas aeruginosa, and 1.1% (2/187) were Enterobacter cloacae.

CONCLUSIONThe Tem-PCR assay may increase the detection rate of Hemophilus influenzae b, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. The Combined detection may increase the positive rate of bacterial pathogens in hospitalized children with pneumonia, and the results might reflect the real patterns of bacterial etiology. The Tem-PCR needs further improvement for diagnosis of Escherichia coli and Klebsiella pneumoniae.

Child ; Child, Preschool ; Colony Count, Microbial ; Female ; Haemophilus influenzae ; genetics ; isolation & purification ; Humans ; Infant ; Male ; Pneumonia, Bacterial ; microbiology ; Polymerase Chain Reaction ; Streptococcus pneumoniae ; genetics ; isolation & purification

Anti-Bacterial Agents ; Child ; China ; epidemiology ; Drug Resistance, Bacterial ; genetics ; Erythromycin ; Humans ; Molecular Epidemiology ; Streptococcus pyogenes ; genetics ; isolation & purification ; pathogenicity ; Virulence Factors ; genetics

China ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Fast Foods ; microbiology ; Lactobacillus ; isolation & purification ; physiology ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S ; genetics ; Streptococcus ; isolation & purification ; physiology ; Yogurt ; microbiology