Application of Denaturing Gradient Gel Electrophoresis to the Analysis of Bacterial Communities Associated With Asymptomatic and Symptomatic Pericoronitis.

PURPOSE: Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities associated with asymptomatic and symptomatic pericoronitis. The aim of the study was to compare the fingerprinting patterns of these 2 clinical conditions. MATERIALS AND METHODS: The microbiota of mandibular third molar pockets associated with asymptomatic or symptomatic pericoronitis cases were collected and profiled by the polymerase chain reaction DGGE method. Banding patterns were compared by cluster analysis techniques. RESULTS: Thirteen symptomatic pericoronitis and 7 asymptomatic pericoronitis samples were collected. Comparative analysis of the 2 clinical conditions showed bands that were common to the symptomatic and asymptomatic cases, but most DGGE bands appeared to be unique to the clinical condition. No single band occurred in all profiles. The mean number of bands detected in the 16S rDNA community profiles was 23.8 ± 4.2 (range, 19 to 34) for samples from symptomatic cases and 24.1 ± 2.4 (range, 21 to 29) for those from asymptomatic cases. Cluster analysis and multidimensional scaling analysis of the DGGE banding pattern showed a distinction in the similarity of banding patterns according to the presence or absence of symptoms. CONCLUSIONS: These results suggest that the diversity of pericoronal pocket microbiota in asymptomatic pericoronitis cases differs markedly from that of symptomatic cases.

Changes in dental plaque microbial richness and oral behavioral habits during caries development in young Chinese children.

OBJECTIVE: To detect changes in the microbial richness of dental plaque and oral behaviors during caries development in young Chinese children. METHODS: Supragingival plaque samples and a survey of oral behaviors of 130 children aged 3 at baseline were analyzed at 6 months and 12 months. Total DNA was isolated from all samples and PCR-denaturing gradient gel electrophoresis analysis was conducted. RESULTS: In the follow-up, 44 children had caries or cavity fillings at 6 months, a further 28 children had caries or cavity fillings at 12 months. The other 58 children remained caries-free at 12 months. According to the changes in caries status at the 12-month follow-up, all participants were divided into three groups: caries-free, caries at 6 months and caries at 12 months. The changes in oral behaviors during the 12-month follow-up were not significantly different in the three groups. The frequency of eating sweets and eating sweets before sleeping was significantly different among the three groups at baseline. At baseline, the average detectable bands of caries in the 12-month caries group were similar to those of the caries-free group; both of them were higher than that of the 6-month caries group. At 6 months, the average detectable bands of the 12-month caries group were significantly lower than that of the caries-free group although the children of the 12-month caries group were caries-free at that time. CONCLUSIONS: For young Chinese children, the high frequency of eating sweets and eating sweets before sleeping are risk factors of caries onset, and the decrease in microbial richness could occur 6 months before the onset of caries.

HIV infection and microbial diversity in saliva.

Limited information is available about the effects of HIV and subsequent antiretroviral treatment on host-microbe interactions. This study aimed to determine the salivary microbial composition for 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that for 10 HIV-seronegative subjects. A conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV-positive subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, Streptococcus mutans, and Candida, in saliva than did HIV-negative subjects. The total cultivable microbial levels were significantly correlated with CD4+ T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. The human oral microbe identification microarray (HOMIM) assay, which compared the 16S rRNA genes, showed clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus were present in all 30 saliva samples. Only minor changes or no changes in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas, and Rothia were observed. Seven genera, Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium, were detected only in HIV-negative samples. The prevalences of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, Granulicatella, and Atopobium were increased after HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. The findings of this study suggest that HIV infection and HAART can have significant effects on salivary microbial colonization and composition.

PCR-DGGE analysis of the bacterial composition of a kaolin slurry showing altered rheology.

Kaolin is an important industrial raw material and a basis of a range of different products. Microbial spoilage is a detrimental process observed especially in kaolin slurries, leading to low quality products and economic loss. Although the alteration of kaolin slurries in ceramic industry was observed, the process and the microbial background have not been analyzed in details. This study provides the first data using a cultivation independent molecular biological approach (PCR-DGGE) regarding the bacterial composition of an altered kaolin slurry. The results show that potential exopolymer (EPS) producer bacteria (e.g. Acinetobacter, Pseudomonas) appear in the altered kaolin slurry, which may have an important role in the modification of kaolin slurries.

Comparative analysis of microbial DNA extraction protocols for groundwater samples.

A comparative analysis of four different DNA extraction protocols was performed to determine the best choice for groundwater microbial diversity studies using temperature gradient gel electrophoresis (TGGE) analysis. The methods used were a chelex-based method, a modified salting out procedure (MSOP), and the commercial kits Epicentre and FastDNA. Both commercial kits exhibited the greatest reproducibility in their methods; however, their band patterns were very different. The protocol that showed the highest diversity was the chelex-based method, and the one that showed the lowest diversity was the FastDNA kit.

Nitinol as a suitable anode material for electricity generation in microbial fuel cells.

Nitinols (Nickel-titanium alloys) have a good electrical conductivity and biocompatibility with human tissue and bacteria and, therefore, can be effectively used as an anode material in bioelectrochemical systems. This paper aimed to use nitinols (at different Ni/Ti ratios) as an anode material for microbial fuel cells (MFCs) in order to achieve higher power density. The maximum power densities of the MFCs using NiTi-1, NiTi-2, and NiTi-3 electrodes were 555 mW/m2, 811 mW/m2, and 652 mW/m2, respectively. More bacterial adhesion was observed on the NiTi-2 electrode. Electrochemical impedance spectroscopy (EIS) results showed low charge transfer resistance at MFCs fabricated with NiTi. The biofilm observations indicate that bacterial attachment is better with NiTi-2 as compared with that on NiTi-1 and NiTi-3. The resulting mesopore and macropore rich structure significantly promote microbial colonization, enabling formation of compact electroactive biofilms with additional benefit from the excellent biocompatibility and chemical stability of NiTi-2. Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) results indicated that five groups of bacteria were the dominant phyla in the MFCs: environmental samples, b-proteobacteria, g-proteobacteria, d-proteobacteria, and CFB group bacteria. The high biocompatibility, electrical conductivity and stability of nitinols make them a more attractive anode material for MFCs.

Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

BACKGROUND: The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. METHODS: Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. RESULTS: Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P <0.001). The average Cs of the RC and PP samples was 93.81% ± 10.26%. Overall, 60 genera/species were identified by sequencing. Of these, the predominant genera in RCs were Porphyromonas sp. (13.9%), Filifactor sp. (12.5%), and Parvimonas sp. (11.1%), similar to the genera obtained from PP samples. In total, 43 genera/species were common to the RC and PP samples. The most prevalent bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. CONCLUSIONS: The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

Polyacrylamide temperature gradient gel electrophoresis.

Temperature Gradient Gel Electrophoresis (TGGE) is a form of electrophoresis in which temperature gradient is used to denature molecules as they move through either acrylamide or agarose gel. TGGE can be applied to analyze DNA, RNA, protein-DNA complexes, and, less commonly, proteins. Separation of double-stranded DNA molecules during TGGE relies on temperature-dependent melting of the DNA duplex into two single-stranded DNA molecules. Therefore, the mobility of DNA reflects not only the size of the molecule but also its nucleotide composition, thereby allowing separation of DNA molecules of similar size with different sequences. Depending on the relative orientation of electric field and temperature gradient, TGGE can be performed in either a parallel or a perpendicular mode. The former is used to analyze multiple samples in the same gel, whereas the later allows detailed analysis of a single sample. This chapter is focused on analysis of DNA by polyacrylamide TGGE using the perpendicular mode.

Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P < 0.01), there was no positive correlation; similarity (Dice coefficient) was 13.1% to 62.5%. Some bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

Dynamics of oral microbial community profiling during severe early childhood caries development monitored by PCR-DGGE.

OBJECTIVE: To monitor the longitudinal changes in oral microbial diversity of children with severe early childhood caries (S-ECC) compared to caries free (CF) controls. METHODS: Dental plaque samples of 12 children in each group at 8, 14, 20, 26 and 32 months of age were analysed. Total microbial genomic DNA was isolated from each sample, and PCR-denaturing gradient gel electrophoresis (DGGE) analyses were carried out. RESULTS: The number of bands was significantly higher in the CF group (18.17±4.91 bands) than in the S-ECC group (14.54±5.56 bands) at 32 months of age (P<0.05). A total of 21 genera were identified in all subjects, and there were no significant differences between the two groups at genus level. DGGE profiles showed that most of the clusters were constructed from one individual over time in the both groups. CONCLUSIONS: The onset of S-ECC is accompanied by a decrease in microbial diversity. The overall composition of the microbiota is highly similar within an individual over time.

Tris-acetate polyacrylamide gradient gels for the simultaneous electrophoretic analysis of proteins of very high and low molecular mass.

Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and that it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-acetate-PAGE system a very helpful tool for protein analysis.

[Research on population structure and distribution characteristic of indigenous microorganism in post-polymer-flooding oil reservoir].

Denaturing gradient gel electrophoresis (DGGE) method and principal component analysis (PCA) method were used to analyze the structures of microorganism population in injection wells and production wells of a post-polymer-flooding oil reservoir in Daqing oil field. The results showed that the dominant species in injection wellhead were aerobic bacteria Pseudomonas and Acinenobacter. Facultative anaerobic bacteria Enterbacter was the dominant bacteria in near area of injection wells. Bacteria detected in production wells included Thauera, Clostridia, Pseudomonas, Petrobacter and some uncultured bacteria. Methanosaeta turned out to be the only archaea detected in injection wells, which was an aceticlastic methane-producing archaeon. Archaea detected in production wells consisted of Methanomicrobium, Methanospirillum and Methanobacterium. In general, aerobic bacteria, facultative anaerobe, and strictly anaerobic bacteria distributed successively from injection wells to production wells in this block. The dominant populations of archaea were different between injection wells and production wells, while were influenced by different environments and microbial metabolism products.

Effect of external resistance on bacterial diversity and metabolism in cellulose-fed microbial fuel cells.

External resistance affects the performance of microbial fuel cells (MFCs) by controlling the flow of electrons from the anode to the cathode. The purpose of this study was to determine the effect of external resistance on bacterial diversity and metabolism in MFCs. Four external resistances (20, 249, 480, and 1000 Ω) were tested by operating parallel MFCs independently at constant circuit loads for 10 weeks. A maximum power density of 66 mW m(-2) was achieved by the 20 Ω MFCs, while the MFCs with 249, 480, and 1000 Ω external resistances produced 57.5, 27, and 47 mW m(-2), respectively. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes showed clear differences between the planktonic and anode-attached populations at various external resistances. Concentrations of short chain fatty acids were higher in MFCs with larger circuit loads, suggesting that fermentative metabolism dominated over anaerobic respiration using the anode as the final electron acceptor.

Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

OBJECTIVE: To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. METHODS: Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. RESULTS: Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. CONCLUSION: The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

Denaturing gradient gel electrophoresis analysis with different primers of subgingival bacterial communities under mechanical debridement.

DGGE of 16S rDNA is one of the most frequently used methods to study microbial communities. In this study, the DGGE profiles of different 16S rDNA regions of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella nigrescens were investigated. The results suggested that V3-V5 and V6-V8 fragments may be suitable for community analysis of subgingival bacteria. Further analysis of subgingival samples with V3-V5 and V6-V8 regions as target fragments suggested that, in chronic periodontitis, re-colonization by periodontal bacteria with a population very similar to the baseline may occur by 6 weeks after mechanical debridement.

Avaliação da comunidade microbiana, com ênfase para a espécie Enterococus faecalis, em dentes decíduos com necrose pulpar através de técnicas fenotípicas e moleculares / Evaluation of microbiota with emphasis on species of Enterococcus faecalis, in primary teeth root canals with pulp necrosis, using conventional and molecular microbiological techniques

Infecção endodôntica em dentes decíduos tem sido pouca avaliada, apesar da influência destes sobre a dentição permanente. No presente estudo foi avaliada a microbiota, com ênfase na espécie de Enterococcus faecalis, de canais radiculares de dentes decíduos com diagnóstico de necrose pulpar, utilizando-se técnicas microbiológicas convencionais e moleculares. Para tanto, um estudo do tipo seccional, clínico e laboratorial foi desenvolvido, sendo a coleta do material endodôntico realizada na clínica de Odontopediatria da Faculdade de Odontologia da UFRJ. Para o estudo, 244 crianças saudáveis foram examinadas no período de um ano, e destas, 43 se enquadravam nos critérios de inclusão. O material foi coletado do canal radicular utilizando-se cones estéreis de papel, dos quais dois foram inoculados em caldo seletivo-indicador Enterococcosel e os outros dois em TSB-DMSO sob congelamento a -20º C. A identificação de E. faecalis foi realizada a partir do cultivo inicial no caldo e subcultivo em agar sangue para observação de colônias bacterianas características. Testes bioquímicos e enzimáticos e a Reação em Cadeia da Polimerase (PCR) para o gene do rRNA 16S também foram utilizados para identificação da espécie. A técnica de Eletroforese em Gel com Gradiente Desnaturante (DGGE) foi utilizada para avaliação do perfil da comunidade microbiana presentes nos espécimes clínicos. Os resultados mostraram que dos 43 espécimes clínicos obtidos, 18 foram excluídos devido à contaminação no controle. Entre os 25 casos estudados, 10 foram positivos no caldo de enterococcosel, sendo cinco (20%) positivos para a espécie E. faecalis nos testes fenotípicos e na PCR. Outros cinco espécimes foram positivos no caldo, mas as amostras bacterianas não apresentaram bioquímica compatível com a espécie E. faecalis, e foram então submetidas ao seqüenciamento de um fragmento do gene do rRNA 16S. Foram identificados Lactobacillus plantarum (4 amostras) e Lactobacillus rhamnosus (1 amostra). Não houve relação dos dados clínicos com a presença de E. faecalis (p > 0,05). A técnica de DGGE mostrou uma comunidade polimicrobiana nos 25 espécimes analisados e, considerando-se o número de bandas no gel, um número ≥ 20 foi relacionado com pacientes com idade ≤ 4 anos e  20 bandas com pacientes com mais de 4 anos de idade (p  0,05). Relação significativa também foi observada entre idade  4 anos e cárie em dente posterior, assim como entre idade  4 anos e cárie em dente anterior. Trauma como causa de infecção endodôntica foi significativa entre crianças com 4 anos de idade. Os resultados demostram a presença de E. faecalis em infecções endodônticas com necrose pulpar em dentição decídua, confirmando sua presença na cavidade oral destes pacientes. Adicionalmente, a técnica de DGGE mostrou uma comunidade polimicrobiana, indicando associação entre idade do paciente e doença cárie.

Endodontic infections in primary teeth have been poorly evaluated despite their influence on permanent dentition. In the present study we assessed the microbiota, with emphasis on species of Enterococcus faecalis, in primary teeth root canals with pulp necrosis, using conventional and molecular microbiological techniques. Thus, a cross-sectional study of clinical and laboratory data was developed. The material was collected at the endodontic clinic of Pediatric Dentistry, Faculty of Dentistry, UFRJ. For the study, 244 healthy children were examined during one year, and of these, 43 met the inclusion criteria. Material was collected from root canals using sterile paper cones. Four paper cones were used for each canal: two were inoculated in the selective broth indicator Enterococcosel and anothers two placed in TSB-DMSO and frozen at -20 ° C until required. Identification of E. faecalis was initially made using culture in broth and subculture on blood agar for observational characteristics of bacterial colonies. Biochemical and enzymatic analysis as well as Polymerase Chain Reaction (PCR) for the 16S rRNA gene were also used for species identification. The Gradient Gel Electrophoresis Agents denaturants (DGGE) technique was used to assess the profile of the microbial communities present in the clinical specimens. Eighteen (18) of the 43 clinical specimens obtained were excluded due to contamination. Among the 25 cases studied, 10 were positive for the species E. faecalis in Enterococcosel broth, five (20%) were positive in the phenotypic tests and PCR. Another five specimens were positive in the broth medium, but the bacterial samples showed no biochemical species compatible with E. faecalis, and so were subjected to sequencing of a fragment of the 16S rRNA gene. Lactobacillus plantarum were identified (4 samples) and Lactobacillus rhamnosus (1 sample). There was no statistically significant correlation of clinical data with the presence of E. faecalis (p> 0.05). The DGGE analysis showed a community polymicrobial. About 20 bands or more were associated with patients ≤ 4 years old and that lesser 20 bands were associated with patients over 4 years old (p  0,05). A significant relationship was also found between patients > 4 years old and caries in posterior teeth, as well as between patients  4 years and anterior tooth caries. Trauma as the cause of endodontic infection was significant among children  4 years old. The results show the presence of E. faecalis in endodontic infections with pulp necrosis in the primary dentition, confirming its presence in the oral cavity of patients. Additionally, DGGE showed a polymicrobial community in 25 samples obtained, suggesting an association between patient age and tooth decay.