Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing

Background Few literatures pertain to the 16S ribosomal DNA （16S rDNA） analysis of bacteria contributing to primary and persistent endodontic lesions, with no information available for the Chinese population. As such, we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing, China using 16S rDNA gene sequencing techniques. Methods Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction. Pllymerase chain reaction （PCR） products were cloned and 100 clones from each generated library were randomly selected. Purified plasmid DNA with 16S rDNA gene inserts was sequenced, and the sequences were searched against GenBank databases using the BLASTN algorithm. Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping. Results More than 150 taxa were obtained. Primary endodontic infection was mainly associated with Burkholderia cepacia, Actinomyces, Aranicola spp. and Streptococcus sanguinis, whilst Burkholderia cepacia was predominant in the persistent endodontic infections. Conclusion There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beijing in comparison to other geographical or ethnic reports.

Novel mitochondrial 16S rRNA mutation, 3200T→C, associated with adult-onset type 2 diabetes

Objective To investigate the role of a potential diabetes related mitochondrial region, which includes two previously reported mutations, 3243AG and 3316GA, in Chinese patients with adult onset type 2 diabetes Methods A total of 277 patients and 241 normal subjects were recruited for the study Mitochondrial nt 3116-3353, which spans the 16S rRNA, tRNA leu(UUR) and the NADH dehydrogenase 1 gene, were detected using polymerase chain reaction (PCR), direct DNA sequencing, PCR restriction fragment length polymorphism and allele specific PCR Variants were analyzed by two tailed Fisher exact test The function of the variants in 16S rRNA were predicted for minimal free energy secondary structures by RNA folding software mfold version 3 Results Four homoplasmic nucleotide substitutions were observed, 3200TC, 3206CT, 3290TC and 3316GA Only the 3200TC mutation is present in the diabetic population and absent in the control population No statistically significant associations were found between the other three variants and type 2 diabetes The 3200TC and 3206CT nucleotide substitutions located in 16S rRNA are novel variants The 3200TC caused a great alteration in the minimal free energy secondary structure model while the 3206CT altered normal 16S rRNA structure little Conclusions The results suggest that the 3200TC mutation is linked to the development of type 2 diabetes, but that the other observed mutations are neutral In contrast to the Japanese studies, the 3316GA does not appear to be related to type 2 diabetes

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis(DGGE)was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high(10 mg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes(V3 to V5 region)were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes(Xuanwu,Mochou)have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes.Furthermore,the DGGE profiles of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further reamplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100%homology with known sequences(Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96%homology with known sequences(Acinetobacter,and Bacillus).The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples.