[Dynamic and quantitative analysis of cariogenic bacteria and its proportion in the dental plaque of different caries-susceptible children].

PURPOSE: To investigate the ratio and change of bacteria in dental plaque in different caries-susceptible children. METHODS: Dental plaques were obtained from 60 children of 3-5 years old. Four targeted cariogenic bacteria were quantitatively detected by real-time PCR and the mean dmft score was calculated. The effectiveness was assessed and compared using SASS.17 software package. RESULTS: The decayed missing filled surfaces(dmfs) score and the percentages of cariogenic bacteria at 1 year increased significantly(P<0.01). There were significant differences in percentages of S.mutans and S.sobrinus in early childhood caries group and severe early childhood caries group between baseline and at 1 year (P<0.01). There was linear correlation between dmfs score and the sum of the percentages of S.mutans and S.sobrinus at baseline and 1 year. CONCLUSIONS: The ratios of cariogenic bacteria to total bacteria in plaque are closely associated with the prevalence of dental caries in children. The higher proportion of cariogenic bacteria, the higher susceptibility of subjects with early childhood caries. Supported by National Natural Science Foundation of China (81371158) and Special Research Fund for Health Care Industry (201002017).

[The influence of starvation on Candida albicans biofilm and persisters formation].

PURPOSE: To investigate the influence of starvation on Candida albicans (C. albicans) biofilm and persisters formation. METHODS: The growth curve was made by CFU counts methods in different culture times to find the exponential and stationary phases. C. albicans in exponential, stationary and starvation phases were developed respectively in 96-well microtiter plates to form 24 h biofilms model. Then the biofilms before and after amphotericin B (100 µg/ml) treatment were quantified to determine the number of live cells. Statistical analysis was performed using SPSS17.0 software package. The biofilm structure was observed by confocal scanning laser microscopy. RESULTS: Biofilm formation capacity of C. albicans in exponential, stationary and starvation phases reduced successively (P<0.05). The biofilms formed by starved C. albicans were predominately composed of yeast cells, compared with cells in exponential and stationary biofilms, which displayed filamentous growth. The ratios of persisters increased successively (P<0.05). CONCLUSIONS: The formation of C. albicans persisters can be influenced by environment events, and starvation can increase the percentage of persister cells.

[Dynamic monitoring and analysis of the persisters emerging in Candida albicans biofilm].

PURPOSE: To investigate the mechanism and approach related to persisters emerging in Candida albicans biofilm by studying its dynamic characteristics. METHODS: The Candida albicans biofilms model in vitro were formed by individual yeast cells and hyphal compartments respectively. The numbers of progenitive fungal cells and persisters at different stages were counted and analyzed with SPSS11.5 software package. The biofilm morphological changes were observed under confocal scanning laser microscopy. RESULTS: Different stages of biofilms which were formed by individual yeast cells and hyphal compartments had no significant difference both on the number of progenitive fungal cells and persisters. The number of progenitive fungal cells showed S-shaped growth curve until 12 hours. The persisters emerged within 0.5 hour and its number reached a steady state after 2 hours while the biofilm was in the period of microcolony formation under microscopy. CONCLUSIONS: The persisters emerging in Candida albicans biofilm are closely connected with the attachment to a surface(within 2 hours of the biofilm formation) and have little association with the maturity of the biofilm and its dimorphism.

[In vitro study of the secondary mesiobuccal canal of the maxillary second molar].

OBJECTIVE: To get some anatomical knowledge about the second mesiobuccal canal (MB2) of the maxillary second molar of Shandong region by studying the teeth in vitro. The anatomical knowledge may help us to improve the successful rate of root canal therapy of the maxillary molar. METHODS: 118 maxillary second molars were collected from the different region in Shandong province. Oblique square of pulp opening form was used, then root canals were detected and got through by C-type file. The rate of MB2 detection and getting through was recorded. By taken X-ray, the numbers, the morphologies and classifications of the root canal systems of these molars were recorded. Then the distance between mesiobuccal canal (MB) and MB2 was observed and measured by endodontic operating microscope and digital measurement. At last, we observed the apical foramen from the surfaces of these teeth, and try to analysis the differences between apical foramen and the end points of the roots. RESULTS: The rate of MB2 detection was 49.15%, among them 82.76% could be got through by files. 108 molars had three roots, among them, for the mesiobuccal root canal system, 46.30% were I root canal system, while II and III systems were 12.96% and 31.48%, respectively. The distance between MB and MB2 was 1.26 mm averaged. The distance between anatomical apical holes and the end points of roots was 1.13 mm averaged. CONCLUSION: MB2 can be detected in most of maxillary second molars in Shandong region. The dentists should try to detect MB2 in the root canal therapy, and should judge work length by clinical behavior and apical locator rather than by X-ray alone.

[Distribution and genotypic subgroup of oral Saccharomyces albicans isolated from cancer patients receiving chemotherapy].

OBJECTIVE: The objective of this study is to study the distribution and molecular characteristics of the oral Saccharomyces albicans (S. albicans) in the cancer patients receiving chemotherapy. METHODS: 390 cancer patients receiving chemotherapy were sampled by oral mucosal swab. The Candida species were identified by CHROMagar Candida differential medium. All the S. albicans were genotypic grouped by PCR using primers reported to span a transposable intron region in the 25S rDNA gene. RESULTS: The frequency of oral Candida carriage of the cancer patients receiving chemotherapy was 53.85% (210/390). Most of them were Saccharomyces albicans, the frequency was 48.21% (188/390). The frequency of oral Candida glabrata carriage was 5.64% (22/390). Genotypic subgroup A, B, C of Saccharomyces albicans were determined, and genotypic group B was the predominant group 59.57% (112/188). CONCLUSION: Saccharomyces albicans, especially genotypic subgroup B, rather than subgroup A, is the prevalence subpopulation in the oral Candida obtained from cancer patients receiving chemotherapy.

[Roles of drug efflux pump genes in the mechanism of Saccharomyces albicans biofilm drug tolerance].

OBJECTIVE: Saccharomyces albicons is the main opportunistic pathogen which is also the common member of oral microflora, and the biofilms they formed have spontaneous drug tolerance compared with planktonic ones. Saccharomyces biofilm population can produce subpopulation of cells which can tolerant high concentration of antifungal drugs. They are called persisters. Many researches indicated that drug efflux gene such as CDR or MDR gene plays the very important roles in yeast drug resistance. The objective of this study is to illuminate the mechanism of Saccharomyces albicons biofilm drug tolerance related with drug efflux gene, especially to the persisters formation. METHODS: Test the minimal inhibitory concentration (MIC) and SMIC80 (sessile minimal inhibitory concentration 80%) of 24 h biofim of totally 7 defective strains of drug efflux pump gene with Amphotercin B. And treat the 24 h biofilm by fluconazole, micronazole, clotrimazole combined with CDR1 inhibitor Enniatin B respectively and antifungal drugs alone as control, then scrapped the biofilm, cultured on the YPD agar. By CFU counting, the numbers of biofilm persisters were determined. RESULTS: All the defective strains have the similar MIC and SMIC80 for 24 h biofilm with wide type strains. CDR1 inhibitor Enniatin B can help antifungal drugs especially micronazole and clotrimazole to eliminate the biofilm persisters. CONCLUSION: Saccharomyces albicans drug efflux gene may minor associated with 24 h biofilm drug tolerance. Drug efflux gene CDR1 may play an role in persisiters related biofilm drug tolerance.

Fluconazole susceptibility and genotypic heterogeneity of oral Candida albicans colonies from the patients with cancer receiving chemotherapy in China.

AIM: To identify heterogeneity of Candida albicans (C. albicans) isolated from the population with cancer in China by using identification medium, subculture molecular typing, and antifungal susceptibility test. METHODOLOGY: Oral cheek mucosal specimens from 52 cancer patients receiving chemotherapy were cultured on CHROMagar Candida plates for Candida identification. All the C. albicans colonies on the plates were subcultured and reconfirmed by API20C, then submitted to the antifungal drug susceptibility test with fluconazole and molecular typing using randomly amplified polymorphic DNA-PCR (RAPD) with primers RSD6 and RSD12. RESULTS: 54% (28/52) patients were oral yeast carriage in which C. albicans predominated. More than 7 C. albicans colonies were isolated from each of 12 patients (Group A), while less than 5 colonies were isolated from each of 16 patients (Group B). RSD6 and RSD12 were successful in eliciting 17 (A1-A17) and 2 (B1-B2) genotypes, respectively from among the 205 isolates. The two primers were combined to generate 21 genotypes. The C. albicans isolates obtained from the same patient and episode showed a diversity for fluconazole revealed by MIC50 and MIC90. CONCLUSION: The heterogeneity of the C. albicans colonies isolated from the same patients can be detected. C. albicans with varied fluconazole susceptibility and genotypic characteristics may coexist in the same oral Candida population.

[Study on oral Candida albicans apoptosis in vitro].

PURPOSE: Candida albicans is one of the main opportunistic pathogen for human , the aim of this study is to investigate the phenomena of apoptosis in oral Candida albicans induced by acetic acid. METHODS: The Candida albicans of clinical strains were induced to apoptosis by using a weak acid acetic acid.The apoptosis was detected by flow cytometry and TEM. The data were processed for Chi-square test using SPSS11.5 software package. RESULTS: Oral Candida albicans had classic apoptosis when induced by proper concentration of acetic acid, and different concentrations of acetic acid had variable ability of inducing apoptosis of Candida albicans. CONCLUSIONS: Apoptosis can be detected in clinical strains of Candida albicans, the mechanism of apoptosis needs further research for the purpose of developing new antifungal drugs. Supported by National Natural Science Foundation of China(Grant No.30400498) and 2007 National College Student Innovative Planning Project.

[Dynamic study on saccharomyces albicans drug efflux pumps gene expression during the biofilm formation].

OBJECTIVE: To observe the drug resistance and drug efflux pumps gene mRNA of Saccharomyces albicans, including CDR1 gene and MDR1 gene, at different stage of biofilm formation in chemostat, furthermore to analysis the relationship between the drug efflux pump gene expression and the biofilm related drug resistance. METHODS: To form the mature biofilm in vitro in chemostat, then collect the biofilm strains at different development stages (2, 12, 24, 48 h) to semi-quantified mRNA amount of CDR1 gene and MDR1 gene by one step RT-PCR method. Using XTT reduction method to test the dynamic change of Saccharomyces albicans drug resistance in biofilm. RESULTS: Antifungal resistance of biofilm-grown cells increased conjunction with the biofilm maturation. Compared with earth stage of biofiom strains, the amount of CDR1 mRNA gene in mature biofilm strains increased, while MDR1 gene did not. CONCLUSION: There is positive correlation between drug resistance and biofilm maturation of Saccharomyces albicans. Biofilm related drug resistance appears to be partially associated with the upregulation of drug efflux pumps, although the variation is not shown coincidence. During the biofilm formation, CDR1 gene expression is actively up-regulated, but MDR1 gene expression is stable.

[Genetic diversity of F-ATPase subunits gene uncEBF amplified from Streptococcus mutans clinical isolates].

OBJECTIVE: The purpose of this research was to study the genetic diversity of F-ATPase subunit gene uncEBF derived from Streptococcus mutans (S. mutans) clinical isolates, furthermore to investigate the relationship between the genetic diversity of F-ATPase and S. mutans aciduric ability. METHODS: 38 S. mutans strains included 18 high acid tolerance strains and 20 low acid tolerance strains. Gene uncEBF of these isolates were amplified with specific primers from S. mutans genomic DNA, and the PCR products were analyzed by RFLP and sequenced. SPSS 11.0 statistic software assayed the results. RESULTS: It was testified that two genotypes A and B of PCR-RFLP were revealed when digested with Alu I and Dde I digested fragments of uncEBF displayed two different patterns C and D. Fisher exact two-tail test showed that the distributions of A and B genotype strains with different acidurance were different (P < 0.05), and the proportion of A genotype strains from high acidurance group was higher than that from low acidurance one. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I and Dde I recognized sites. CONCLUSION: This study indicated that uncEBF gene of S. mutans F-ATPase obviously exhibited genetic diversity.

[Genetic diversity and mRNA expression of F-ATPase subunit uncG gene of streptococcus mutans clinical isolates].

OBJECTIVE: To study the genetic diversity and the gene expression of membrane-bound proton-translocating ATPase (F-ATPase) subunit gene uncG derived from Streptococcus mutans (S. mutans) clinical isolates. METHODS: 38 S. mutans strains derived from caries-active and caries-free individuals including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncG was amplified with specific primers from S. mutans genomic DNA, then the PCR product was analyzed by RFLP and sequenced. The relative expression quantity of uncG gene against the housekeeping gene recA was determined by using RT-PCR method. A gel documentation system and QUANTITY ONES software were used to analyze the data results. RESULTS: It was testified that four genotypes A, B, C and D of PCR-RFLP were revealed when respectively digested with Alu I and Bsr I, but the distributions of the four genotype strains showed no difference (P > 0.05). The differences of uncG gene transcript quantities derived from different genotype or different aciduranc strains had no significance (P > 0.05). CONCLUSION: This study indicated that uncG gene of F-ATPase obviously displayed genetic diversity and existed polymorphism at mRNA expression level, while the Alu I-RFLP genotypes and the expression levels would not be responsive to different acid tolerance of S. mutans strains.

[A study of genetic diversity in lactate dehydrogenase of Streptococcus mutans from clinical isolates].

OBJECTIVES: Lactate dehydrogenase (LDH) is shown to be an important virulence factor resulting in acid production of Streptococcus mutans (S. mutans), on which the cariogenic potential of S. mutans depends. Differences in cariogenic abilities of S. mutans isolates may be determined by genetic heterogeneity from virulent factors. The relationship between LDH activity or genetic diversity and cariogenicity of S. mutans (serotype c) isolates was studied in this research. METHODS: The genome DNA of S. mutans isolates were isolated and LDH gene (ldh) were amplified with specific primers. These isolates came from 34 caries-active individuals and 36 caries-free ones, in which 24 strains showed the high LDH activity and 21 strains showed the low LDH activity. Then genetic diversity of PCR products were analyzed or assessed by restriction fragment-length polymorphism (RFLP). Some of amplified LDH genes from different group were sequenced and assayed. RESULTS: It is testified that two genotypes A and B of ldh-RFLP were revealed when LDH genes were digested with Mse I, but Hph I, Mnl I, Dde I, Nla III and Alu I digesting fragments of Idh gene did not show different pattern. Furthermore, Fisher Exact one-Tail Test showed that the proportion of genotype B among strains from caries-free individuals was higher than that from caries-active ones (P = 0.033), while the distribution of genotypes with different LDH activity was different between two groups (two-Tail Test P = 0.017). The sequencing DNA testified that the specific base mutation would lead to multiple kind of genotype resulted. CONCLUSION: This study indicated that LDH gene of S. mutans clinical strains from different individuals is conservative, while there still is the gene mutation in. The ldh genetic diversity may be related to the low caries sensitivity, and closely correlated with the differences in LDH enzyme activity of S. mutans strains.

[Genetic diversity of F-ATPase subunits gene uncA amplified from Streptococcus mutans clinical isolates].

OBJECTIVE: To study the genetic diversity of F-ATPase alpha subunit gene uncA derived from Streptococcus mutans (S. mutans) clinical isolates and to investigate the relationship between the genetic diversity of acidurance factor and S. mutans aciduric ability, also and the cariogenicity. METHODS: Sixty-four S. mutans strains derived from 34 caries-active individuals and 30 caries-free individuals, including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncA was amplified with specific primers from S. mutans genomic DNA, then the PCR products were analyzed by RFLP and sequenced. RESULTS: Two genotypes A and B of PCR-RFLP were revealed when digested with Hph I. Mbo II also produced two different pattern C and D. The distributions of A and B genotype strains with different caries-sensitivity groups were different (P < 0.05), and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. The distributions of C and D genotype strains with different acidurance strains were different (P < 0.05), and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance group. These amplified uncA genes from different group were sequenced and there existed variation of Hph I and Mbo II recognized sites. CONCLUSIONS: This study indicates that uncA gene of S. mutans F-ATPase obviously displayed genetic diversity. The different Hph I-RFLP and Mbo II-RFLP genotypes could be related to the cariogenicity and acid tolerance of S. mutans strains.

[The distribution of Candida spp. in the oral cavities of normal children].

OBJECTIVE: To study the distribution of Candida spp. mainly Candida albicans in the oral cavities of health children. METHODS: Four groups children of different ages, A1: newborn babies, A2: 3.2 years old (average) children,A3, 7.2 years old,A4:12.7 years old and B control group 20.4 years old,mucosal swab sampling with centrification,CHROMagar Candida identified culture medium for culture and identification, and different methods for isolating Candida albicans for A3 group. RESULTS: The isolation rates of Candida spp were A1 7.5%,A2 70%,A3 56.36%,A4 49.12%,B 27.5%,the proportion of Candida albicans also diversely,and the method of PCR was more sensitive than the one of culture. CONCLUSION: Candida spp can be isolated from the normal oral cavities in different aged children, most of them were Candida albicans,both the isolation rates of Candida spp and the proportion of Candida albicans were different.