Evaluation of different methods for the effective isolation of high-quality DNA from rodent faecal samples

Aims@#The inefficient lysis of recalcitrant bacterial cell wall and subsequent isolation of DNA from environmental samples can lead to a bias in the qualitative and quantitative assessment of bacteria present in the sample. Thus, the selection of an optimum DNA isolation method is the important first step for biosurveillance and metagenomic analyses. This study aims to determine the optimal DNA isolation method out of four commercial DNA isolation kits (A, B, C and D) and two conventional methods (E and F), for rodent faecal droppings. The key selection criterion is the general bacterial diversity contained in the isolated DNA, as evaluated by the Shannon-Weaver index based on the maximal number of PCR-amplicons of partial 16S rRNA gene, derived from each method. The amplicons were separated in accordance with their difference in nucleotide sequences via DGGE. @*Methodology and results@#Five faecal samples of wild rodents were collected from different sites and preserved in DNA/RNA shield reagent (Zymo Research). Each sample was extracted, and the DNA extracts were then subjected to amplification of the bacterial 16S rRNA and DGGE separation of the amplicons. Method E showed a higher yield of DNA (average 324.22 ng/µL) as compared to the other methods. However, the majority of the DNA extracts showed partial degradation. The DGGE profiles showed the highest number of amplicons were generated from DNA extracted from Method A and B with a total of 168 and 167 respectively. This is indicated by the Shannon-Weaver index which were 0.306 and 0.305, respectively. @*Conclusion, significance and impact of study@#Method A is the optimum DNA isolation method for rodent faecal samples as its isolated DNA contains the most diverse bacteria. The isolated DNA can then be used for PCR-biosurveillance or metagenomic sequencing and analyses.

Influence of different sources of contamination on the microbiological quality of lettuce in the Teresópolis region, RJ, Brazil / Influência de diferentes fontes de contaminação na qualidade microbiológica de alface na região de Teresópolis, RJ

ABSTRACT Irrigation water and cultivated soil have been identified as possible sources of contamination in several crops. In certain vegetables that are eaten raw, such as lettuce, this contamination can lead to public health problems. Aiming to evaluate the influence of these sources on the quality of lettuce grown in the Córrego Sujo Basin, Teresópolis, RJ, an important agricultural pole whose production services the metropolitan region of Rio de Janeiro, water from different sources (spring, weir and river) was collected in this region, as well as samples of soil and lettuce irrigated with these waters, to carry out conventional microbiological analyzes (counts of total heterotrophic bacteria and thermotolerant coliforms) and molecular analyzes (PCR-DGGE). The count of fecal coliforms in lettuce suggests that there is an influence of irrigation water and the cultivated soil on the contamination of these vegetables. The grouping of bacterial communities in the different samples obtained by the PCR-DGGE technique shows that irrigation water has a greater influence on the contamination of these vegetables in relation to the soil where they are grown. These results corroborate the need to monitor water bodies used for irrigation and demonstrate that the PCR-DGGE technique is of great value for the study of microbial communities and, when associated with specific primers, can help in the detection of pathogens in food.

RESUMO A água de irrigação e o solo de cultivo têm sido apontados como possíveis fontes de contaminação em diversas culturas. Em determinadas hortaliças consumidas cruas, como a alface, essa contaminação pode causar problemas de saúde pública. Objetivando avaliar a influência dessas fontes na qualidade das alfaces cultivadas na Bacia do Córrego Sujo, Teresópolis, RJ; importante polo agrícola com produção voltada à região metropolitana do Rio de Janeiro, coletou-se nesta região águas proveniente de diferentes fontes (nascente, açude e rio); solos e alfaces irrigados com essas águas, para realização de análises microbiológicas convencionais (contagens de bactérias heterotróficas totais e coliformes termotolerantes) e moleculares (PCR-DGGE). A contagem de coliformes fecais na alface sugere que existe influência da água de irrigação e do solo na contaminação desses vegetais. O agrupamento das comunidades bacterianas nas diferentes amostras obtido pela técnica de PCR-DGGE mostra que a água de irrigação tem influência maior na contaminação dessas hortaliças em relação ao solo onde são cultivadas. Esses resultados corroboram a necessidade de monitoramento de corpos d'água utilizados para irrigação e demonstram ser a técnica do PCR-DGGE de grande valia para o estudo das comunidades microbianas e, quando associada a iniciadores específicos podem ajudar na detecção de patógenos em alimentos.

Diversity and abundance of denitrifiers during cow manure composting / Diversidad y abundancia de desnitrificadores durante el compostaje del estiércol de vaca

Diversity and abundance of the denitrifying genes nirK, nirS and nosZ were investigated in cow manure compost using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time quantitative PCR (qPCR), respectively. These three genes were detected in all the stages of the composting process. Phylogenetic analysis showed that the nirK gene was closely related to Rhizobiales, Burkholderiales, the nirS gene was closely related to Pseudomonadales and Burkholderiales, and the nosZ gene was closely related to Rhodospirillales, Rhizobiales, Pseudomonadales, and Alteromonadales. qPCR results showed that the abundance of these three genes (nirK, nirS and nosZ) reached the peak value in the late thermophilic stage of composting and abundance of the nirK gene was higher than that of the nosZ gene and the nirS gene. Redundancy analysis (RDA) showed that the diversity of the nirK and nirS genes was significantly correlated with ammonium (p < 0.05), the diversity of the nosZ gene was significantly correlated with pH (p < 0.05) and the abundance of the nirK nirS and nosZ genes was significantly correlated with temperature (p< 0.05).

La diversidad y la abundancia de los genes desnitrificadores nirK, nirS, nosZ en el compost de estiércol de vaca se investigaron por medio de la reacción en cadena de la polimerasa seguida de electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) y por PCR cuantitativa (qPCR) en tiempo real, respectivamente. Estos 3 genes fueron detectados durante todas las fases del compostaje. El análisis filogenético mostró estrecha relación del gen nirK con Rhizobiales y Burkholderiales, del gen nirS con Pseudomonadales y Burkholderiales y del gen nosZ con Rhodospirillales, Rhizobiales, Pseudomonadales y Alteromonadales. Los resultados de la qPCR mostraron que la abundancia de los genes nirK, nirSy nosZ alcanzó el valor máximo en la fase termofílica tardía del compostaje, y que la abundancia del gen nirK era más elevada que los de los genes nosZ y nirS. El análisis de redundancia (RDA) mostró que la diversidad de los genes nirK y nirS estaba significativamente correlacionada con la concentración de amonio (p<0,05), mientras que la del gen nosZ estaba significativamente correlacionada con el pH (p<0,05). También mostró que la abundancia de los genes nirK, nirS y nosZ estaba significativamente correlacionada con la temperatura (p<0,05).

Bifidobacterias como indicadoras de contaminación fecal en aguas tropicales

La contaminación fecal humana en el agua constituye un importante riesgo para la salud pública, sin embargo, los microorganismos indicadores comúnmente utilizados para detectar contaminación fecal no identifican su fuente específica. La detección de ciertas especies del género Bifidobacterium como B. adolescentis y B. dentium ha sido propuesta como un efectivo marcador de contaminación fecal humana, pero esto no ha sido evaluado en las condiciones ambientales tropicales. El objetivo de este trabajo fue determinar el perfil de bifidobacterias, en una muestra de agua de la Ciénaga de Mesolandia en el Caribe Colombiano y en 260 muestras fecales humanas y 94 de animales domésticos de un asentamiento humano periférico a la ciénaga. El ADN extraído de cada una de las muestras fue amplificado por PCR mediante el uso de cebadores específicos de género basados en la secuencia del gen 16S ARNr y separados por DGGE (Electroforesis en Gel con Gradiente Desnaturalizante). Las bandas obtenidas en DGGE, fueron extraídas del gel, re-amplificadas, secuenciadas y las secuencias comparadas con la base de datos del GenBank. El perfil de bifidobacterias en DGGE mostró la presencia de ocho especies de Bifidobacterias en la muestra de agua, las cuales también fueron identificadas en las heces humanas. B. adolescentis y B. dentium propuestas como marcadores de contaminación fecal humana, también fueron encontradas en animales domésticos. En este estudio bajo las condiciones ambientales y experimentales evaluadas no fue posible encontrar una especie de Bifidobacteria específica para ser utilizada como marcador de contaminación fecal humana en ambientes tropicales. Sin embargo, el método aplicado permitió una aproximación más cercana al origen de la contaminación fecal en relación con los métodos culturales tradicionales, ya que fue posible encontrar secuencias de ADN idénticas en el agua y en las muestras fecales.

Human fecal pollution in water constitute a serious risk to the public health, nevertheless the indicator microorganisms commonly used to detect the levels of fecal pollution does not identify the specific source. The detection of certain Bifidobacterium species as B. adolescentis and B. dentium have been proposed as an effective marker of human fecal contamination, but this has not yet been demonstrated in tropical environmental conditions. The aim of the present work was to determine the Bifidobacteria profile in one sample of water from the Mesolandia Swamp in the Colombian Caribbean and feces samples of 260 human and 94 domestic animals from a human settlement around the swamp. DNA from all the samples was amplified by PCR using specific specie primers based on the 16S rRNA gene sequence and separated by DGGE (Denaturing Gradient Gel Electrophoresis). DGGE bands were excised, reamplified, sequenced, and compared to GenBank database. Bifidobacterial DGGE profiles showed that eight species of Bifidobacterium that were found in the water sample, were also present in the human and animal feces. Bifidobacterium adolescentis and B. dentium described as potential human fecal pollution indicators were found in domestic animals. In this study under the environmental and experimental conditions evaluated was not possible to find a Bifidobacterium species as specific marker of human fecal contamination in tropical areas. However, the applied method in this study could be useful to detect fecal pollution in tropical waters allowing a nearest approximation to the origin of the fecal pollution compared with cultural traditional methods, since it was possible to find identical DNA sequences in the water and in the fecal samples.

Succession Law of Intestinal Flora after Death in SD Rats / 法医学杂志

OBJECTIVES@#To initially explore the sequential changes in the intestinal flora of corpse for the estimation of postmortem interval （PMI）.@*METHODS@#Rats were sacrificed by cervical dislocation, and samples were taken from their intestines using cotton swab to extract the DNA of intestinal flora. The 16S rRNA V3 universal primers were selected for PCR, and the PCR products were used for denatured gradient gel electrophoresis. The diversity and similarity analysis of intestinal flora were analyzed between groups, and the bands were cut from denaturing gradient gel electrophoresis. After purification, PCR and sequencing, the percentage of major bacteria in each group was obtained.@*RESULTS@#The flora diversity showed a reduced tendency from 1st to 30th day after death （ P<0.05）, while the intra-group similarity showed a downward trend （ P<0.05）. The number of bands and intra-group similarity coefficient （Cs） on the first day was higher than that of other groups （ P<0.05）. The intra-group Cs of the 25th and 30th day had a significant difference compared with the 5th day （ P<0.05）. At the genus level, the intestinal flora was mainly composed of Enterococcus sp. on the 1th and 5th day after death, Bacillus thuringienssis was the dominant species on the 10th, 15th and 20th day, and Enterococcus faecalis became the dominant species on the 25th and 30th day.@*CONCLUSIONS@#The composition and structure of intestinal flora change significantly in rats with the time after death, which indicates that the succession of intestinal flora is related to the postmortem interval.

16S rDNA-based molecular profiles of circulating bacteria in patients with severe acute pancreatitis / 肠外与肠内营养

Objective:This study aims to characterize the bacterial profile presenting in peripheral blood of severe acute pancreatitis (SAP) patients and investigate the potential role of circulating microorganisms in the development of systemic infection.Methods:A total of 30 patients with SAP were recruited in this study and divided into three groups:infected,sepsis and Septic shock (n =10 for each group).The peripheral blood was collected sterilely for extraction of DNA,which was subsequently amplified using the universe primers targeted the V6-V8 region of 16S ribosomal RNA genes.The amplicons were separated by denaturing gradient gel electrophoresis (DGGE),and then the gels were stained and photographed.The bands were cut out and sequenced to determine the closest bacterial relatives.Results:As shown in DGGE profile,multiple DNA bands (3 to 8 bands) were detected in peripheral blood from all (100％) of SAP patients complicated with septic shock.The microorganisms most frequently presenting in the blood of these cases included Escherichia coli,Bacillus coagulans,Pseudomonas putida,Pseudomonas aeruginosa,and Klebsiella pneumonia,with an incidence of 40 ％ or higher.In patients with sepsis,bacterial DNA consisting of 2 to 4 bands was observed in 90％ of the blood samples.The most common bacterial species was Pseudomonas putida (60％),followed by Shigella flexneri (40％),Staphylococcus aureus (30％) and Enterococcusfaecium (20％).By contrast,the positive rate of blood bacterial DNA was relatively lower in infected patients (70 ％).Of them,single bacterial species was commonly found in the blood samples.Conclusions:Our data showed that the bacterial profiles presenting in peripheral blood are distinct among SAP patients with different manifestations.Polymicrobial translocation could contribute to the development of systemic infection,offering novel insights into the pathogenesis of sepsis in SAE The findings are helpful for the prevention and treatment for bacterial infection and complications of SAP.

Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring

Background: Sulphur-oxidizing microorganisms are widely used in the biofiltration of total reduced sulphur compounds (odorous and neurotoxic) produced by industries such as the cellulose and petrochemical industries, which include high-temperature process steps. Some hyperthermophilic microorganisms have the capability to oxidize these compounds at high temperatures (N60°C), and archaea of this group, for example, Sulfolobus metallicus, are commonly used in biofiltration technology. Results: In this study, a hyperthermophilic sulphur-oxidizing strain of archaea was isolated from a hot spring (Chillán, Chile) and designated as M1. It was identified as archaea of the genus Sulfolobus (99% homology with S. solfataricus 16S rDNA). Biofilms of this culture grown on polyethylene rings showed an elemental sulphur oxidation rate of 95.15 ± 15.39 mg S l-1 d-1, higher than the rate exhibited by the biofilm of the sulphur-oxidizing archaea S. metallicus (56.8 ± 10.91 mg l-1 d-1). Conclusions: The results suggest that the culture M1 is useful for the biofiltration of total reduced sulphur gases at high temperatures and for other biotechnological applications.

Influence of gastric acid suppression on the structure and diversity of the jejunal microbiota in a rat model / 肠外与肠内营养

Objective:To investigate the dynamic changes of the luminal microbiota in the jejunum following administration of proton pump inhibitors (PPIs) in a rat model.Methods:Rats were randomized into six groups (n =6 each group).A group of rats were sacrificed just after anesthesia as normal control (0 d) and,other five groups were continuously administered with omeprazole (10 mg/kg twice daily,intraperitoneally) and were euthanized at 5,9,14,21,28 days following the treatment,respectively.Total DNA in the luminal contents of jejunum was extracted and was used for polymerase chain reaction (PCR) amplification with the primer set targeted the hypervariable V3 region of 16S ribosomal RNA genes.Subsequently,the amplicons were separated by denaturing gradient gel electrophoresis (DGGE).After the gels were stained and photographed,the bands were cut out and sequenced to determine the closest bacterial relatives with the BLAST.The DGGE profiles were analyzed to evaluate the shifts of the microbiota composition and diversity following treatments.Results:Changes of the jejunal microbiotas in rats were observed at 5 and 9 days post PPI administration,as characterized by outgrowth of Streptococcus pneumonia,Clostridium saccharolyticum and Lactococcus garvieae compared to those of the controls (0 d).With time extension of PPI treatment,the mictobiotas significantly shifted toward dysbiotic state,in which the opportunistic pathogens,including Ertterococcus faecalis and Clostridium difficile,were strikingly expanded,especially 21 days later.However,the commensals such as Lactobacillus reuteri and Weissella koreensis were markedly declined in PPI-treated animals compared with the controls.The similarity of the jejunal microbiotas between PPI-treated animals and controls was markedly reduced following PPI treatment,reaching (56.1 ± 16.7) ％ at 28 days.Conclusion:Our data demonstrate that the gastric acid suppression could induce shifts of the jejuna microbiota in a rat model.More importantly,long-term use (＞ 14 d) of PPI could lead to the dysbiosis of the jejunal microbiota,which might be related causally to increased susceptibility to enteric infection.

Changes of the fungal microbiota in inflamed intestinal mucosa of the patients with Crohn's disease / 肠外与肠内营养

Objective:The study aimed to characterize the difference of the fungal microbiota between the inflamed and non-inflamed intestinal mucosa in patients with Crohn's disease (CD) and explore the potential relation between their changes and the pathogenesis of CD.Methods:Seven patients with active CD were enrolled and the specimens was obtained from the inflamed and non-inflamed mucos a during operation.Tissue DNA was extracted and then amplified by nested polymerase chain reaction (PCR) with the premier sets NS 1/FR1 and EF390/GC-FR1.Denaturing gradient gel electrophoresis was subsequently conducted to profile the structure and composition of the fungal microbiota.Results:Compared to those of noninflamed region,the species richness and diversity of the fungal microbiota in inflamed region were significantly increased (P ＜ 0.05).The predominant fungal composition in inflamed region was significantly altered,mainly characterized by the increase of opportunistic pathogens including Candida albicans,Candida tropicalis,Gibberella moniliformis,Alternaria brassicicola and Cryptococcus neoformans relative to that of noninflamed region (P ＜ 0.05),compared with those of noninflamed region.Conversely,the proportion of the commensals,for example Saccharomyces cerevisiae,Saccharomy,ces castellii,Penicillum chrysogenum and Laccaria bicolor,were significantly decreased in inflamed region (P ＜ 0.05).Conclusion:The fungal microbiota of the inflamed intestinal mucosa is severely dysbiotic in CD patients.The colonization of some pathogenic fungi could participate and result in inflammatory damage.

Community structures of total bacterial DNA, cultivable bacteria and prototrophs in bulk soil and rhizospheres

Aims: It has been hypothesized that root exudates can be a nutritional factor influencing the bacterial community structure as well as the occurrence of prototrophs and auxotrophs in rhizospheres. The present study was performed to examine the community structures of total bacterial DNA, cultivable bacteria and prototrophs in 3 soil samples with different levels of abundance of root exudates. Methodology and results: Denaturing gradient gel electrophoresis (DGGE) was performed to examine the community structures of total bacterial DNA, cultivable bacteria and prototrophs in 3 soil samples including bulk soil, rhizosphere of a single plant species and rhizosphere of multiple plant species. For clustering analysis, a dendrogram generated from the DGGE patterns revealed the different bacterial community structures in these soil samples. Both rhizospheres claded together, separating from bulk soil. The DGGE patterns of cultivable bacteria showed particular fingerprints corresponding to kinds of media and soil samples. Nutrient agar (NA) medium, isolation medium for prototroph (IMP) and IMP supplemented with soil extracts were used for bacterial cultivations. Prototrophs were isolated and examined by random amplified polymorphic DNA (RAPD) and 16S rRNA gene sequence analysis. The genetic diversity of prototrophs in 3 soil samples was similar (approximately 5% to 10% similarities) and most of them (13 of 28 strains) were members of Pseudomonas with 97% to 100% identities. Conclusion, significance, and impact of study: The present study provides a strong evidence of the influence of root exudates and plant species on bacterial community structures.

Comparison of Total DNA Extraction Methods for Studying Endophyte Diversity in Mulberry / 广州中医药大学学报

Objective To compare the advantages and disadvantages of the four DNA extraction methods according to the endophytic diversity in the roots, stems, and leaves of mulberry analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) , and by taking the yield, purity and PCR amplification as indexes. Methods Four common methods, i.e., cetyltrimethyl ammonium bromide ( CTAB) , sterile phosphate buffered saline (SPBS) vibration, liquid nitrogen grinding (LNG) , and KIT methods, were used to extract the total DNA from different tissues of mulberry, and then were compared based on the diversity analysis results for endophyte by PCR-DGGE. Results From the roots and stems of mulberry, we got the highest concentration of DNA by LNG extraction method, and got the lowest concentration by SPBS extraction method. But for the leaves of mulberry, the results of the four extraction methods were completely opposite to those for the roots and stems. For different tissues of mulberry, the purity of DNA extracted by KIT method was the best. According to the endophytic bacteria diversity analyzed by 16S rDNA PCR-DGGE, the appropriate method for extraction of DNA was LNG or CTAB, but was not KIT. And according to the results of endophytic fungi diversity analyzed by ITS PCR-DGGE, the best extraction method was KIT, and the unsuited methods varied from the tissues of mulberry. Conclusion The optimum DNA extraction method for mulberry varies from the tissues of mulberry and endophtic bacteria.

Polymorphisms of SLC22A9 (hOAT7) in Korean Females with Osteoporosis

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SNP within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SNPs in the osteoporosis group (A645G and T1277C), three SNPs in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and 12.74 microM, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

Effects of dried whey on the intestinal bacterial community and probiotics in weaned laboratory rabbits / 中国比较医学杂志

Objective To investigate the effects of dried whey on the intestinal bacterial community and probiotics in weaned laboratory rabbits .Methods A single factor design was employed to investigate the effects of dried whey supplemented at levels of 0%, 2%, 5%and 10%, respectively, on 48 weaned (40-day-old) laboratory rabbits.At the day 30, eight rabbits in each group were taken and sacrificed after anesthesia .The total bacterial DNA from the ceacal content of each selected rabbit was drew to analyze the bacterial community and intestinal probiotics ( Bifidobacterium and Lactobacillius) population by PCR-DGGE and real-time fluorescence quantitative PCR, respectively.Results 1) The DGGE parameters of ceacal bacterial community were increased with the increasing dried whey supplemental levels .The number of DGGE band in 2%, 5%and 10%dried whey supplement groups (P0.05).Supplying dried whey has no significant effects on the homogeneity index (P>0.05).2) The population of Bifidobacterium and Lactobacillius in ceacal content had a trend of increase with the rising dried whey supplement levels .Compared with the 0% supplement group , the Lactobacillius population in the 2%, 5% and 10%supplement groups ( P <0.05 ) , the Bifidobacterium population in the 10% supplement group ( P <0.05 ) were significantly increased .Conclusions The results of our study indicate that: 1 ) Supplying dried whey in the feed of laboratory rabbit can effectively increase the diversity of ceacal bacterial community .2) Dried whey may effectively improve the intestinal probiotics population .

Denaturing Gradient Gel Electrophoresis for Detection of Bacterial Population and Diversity in Amniotic Fluids and Neonatal Gastric Fluids

PURPOSE: Intra-amniotic infection (IAI) is often polymicrobial, and the 16S rDNA PCR assay has a major limitation that its interpretation is difficult in the presence of multiple 16S rDNAs. Denaturing gradient gel electrophoresis (DGGE) can overcome this limitation by separating PCR products based on sequence. We performed the DGGE analysis to investigate bacterial prevalence and diversity in amniotic fluids from pregnant women with preterm births and gastric fluids from their newborns. METHODS: DNA was extracted from bacterial cells in amniotic fluid (AF) and gastric fluid (GF) and was amplified with universal 16S rDNA primers. For DGGE analysis, the PCR products were loaded onto polyacrylamide gels that were made with denaturing gradients. RESULTS: Bacterial 16S rDNA was detected by PCR from all AF and GF samples. The bacterial species in AF samples were the following: Lactobacillus reuteri (87.0%), uncultured Enterococcus species (65.2%), Ureaplasma urealyticum (13.0%), and Enterococcus faecalis (4.3%). The bacterial species in GF samples were the following: Lactobacillus reuteri (95.2%), uncultured Enterococcus species (42.9%), and Ureaplasma urealyticum (4.8%). Two or more species were identified from 69.6% of AF and 47.6% of GF samples. CONCLUSION: We suggest that DGGE analysis allows improved understanding of microbial diversity and community in AF and GF.

Bacterial analysis of combined periodontal-endodontic lesions using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) / 中华微生物学和免疫学杂志

Objective To compare the bacterial community profiles present in periodontium and root canals of the same tooth diagnosed as combined periodontal-endodontic lesions by using denaturing gradient gel electrophoresis (DGGE).Methods Samples were collected from 13 extracted teeth with advanced periodontitis,endodontic samples from root tip 1/3 root canal,and periodontal samples from the corresponding neighboring periodontium.Genomic DNA was collected for the following universal bacterial primersPCR.The PCR products were then loaded on the DGGE gels to gain separate bands.The typical DGGE bands were excised,PCR-cloned and sequenced.Results The number of bands,which was indicative of the number of bacterial species,was compared intra-group (periodontal and pulpal specimen from the same tooth).The difference was statistically significant (P＜0.01),but there was no positive correlation between them.The similarity (Dice Coefficient) between them was 13.1％-62.5％.Taxa identified through BLAST (≥98％ identity) were Campylobacter,Fusobacterium,Neisseria,et al in the periodontium,and Mogibacterium,Corynebacterium,Actinomyces,et al in the root canals.Conclusion Common bacteria existed between them,but not all of the periodontal bacteria would appear in neighboring root canal; and the bacteria in the root canal are not completely from neighboring periodontal tissue.The original bacteria in the root canals may resuscitate and enrich the bacterial community.In combined periodontal-endodontic lesions (periodontal source),it is probable that new species existed to be confirmed either in the periodontium or in the root canal.

Comparison of Microbial Diversity of Korean Commercial Makgeolli Showing High beta-Glucan Content and High Antihypertensive Activity, Respectively

We measured physiological functionalities, including antihypertensive angiotensin I-converting enzyme inhibitory activity and immun-stimulating beta-glucan content for sixty kinds of Makgeolli that is commercially available from the market. As a result, we selected R-12 commercial raw Makgeolli, with a high content of immuno-stimulating beta-glucan, and R-14 commercial raw Makgeolli, exhibiting high antihypertensive activity. Due to the similarities in their overall physicochemical properties and raw materials used for fermentation, we compared the microbial flora in order to investigate the reason for the differences in their functionalities. Nested PCR and denaturing gradient gel electrophoresis for yeasts and bacteria were performed for analysis of microbial diversity of two different kinds of Makgeolli (i.e., R-12, R-14), which showed immuno-stimulating beta-glucan content and exhibited a very high level of antihypertensive activity, respectively. Analysis of the 18S rDNA amplicon revealed a major presence of the yeast strain Pichia burtonii in every Makgeolli sample. Analysis of the 16S rDNA amplicon revealed a predominance of lactic acid bacteria, and the most frequent lactic acid bacteria were Lactobacillus ingluviei, L. fermentum, and L. harbinensis, and Lactobacillus sp. Among these, L. harbinensis was detected only in R-12 and L. ingluviei was found only in R-14. Different functionalities from the individual commercially available Makgeolli may be attributed to actions of different microbial flora during fermentation.

Characterization of leptospires using V3 region of 16S rDNA by denaturing gradient gel electrophoresis : A case study.

Serological and molecular characterization of Leptospiral isolates helps us to identify serovar, which is useful, for epidemiological study. Serological characterization is tedious and requires a panel of monoclonal antibodies and expertise to read the results. This study is a preliminary work to evaluate the usefulness of Denaturing Gradient Gel Electrophoresis (DGGE) to identify serovars of leptospira. The V3 region of most conserved 16S rDNA of five pathogenic leptospiral serovars and one saprophytic serovar was characterized. DGGE method was employed to separate the amplified V3 region based on the nucleotide sequence. On DGGE, amplified V3 region of leptospiral serovars, under study, showed bands at different positions indicating DGGE as the effective method of characterization in the future. DNA sequencing of V3 region of the three serovars showed great difference in nucleotide sequence supporting the results of DGGE.

Detection of two rare β -thalassemia mutations [-90 (C → T) and CD 26 (C → T)] among Indians.

BACKGROUND : β -Thalassemia (β -thal) is present in practically every caste group in Indians. Molecular characterization of β -thal in these groups has revealed an extremely heterogeneous picture. AIM : To identify all the mutations and to detect the novel mutations using a versatile mutation detection technique. MATERIALS AND METHODS : Denaturing gradient gel electrophoresis (DGGE) has been established to scan the entire β -globin gene to localize the mutation followed by DNA sequencing for characterization. The DNA samples from two families referred to us either for prenatal diagnosis or for DNA studies were studied. RESULTS : Atypical DGGE patterns in fragments B & A indicating the presence of the mutation, have been detected in both the families. DNA sequencing revealed two rare patterns fragments with patterns in fragments β -thal mutations [CD 26 (C→T) and -90 (C→T)]. CONCLUSION : DGGE is a useful mutation detection technique to identify β -thal mutations among the heterogeneous Indian population.

Comparative Analysis of Community Structure and Activity of Wastewater Treatment Biofilm Cultivated Under Different Conditions / 微生物学通报

The effect of phenol concentration on the structure and function of microbial communities,which were cultured in different conditions using coking wastewater biofilm as seeding,was investigated by Biolog and denaturing gradient gel electrophoresis(DGGE)methods.The less number of bands of cultivated sam-ples on the denaturing gradient gel electrophoresis fingerprint of 16S rRNA gene indicated reduction of di-versity after enrichment and cultivation.Some bands on the DGGE gel were significantly influenced by the phenol concentration in medium.The results of Biolog showed that the original biofilm sample had the highest substrate utility capacity as measured by average well color development(AWCD).But low concen-tration of phenol enriched sample S7 showed more diverse activity on the utility of Carboxylic acids.The principal component analysis(PCA)of Biolog data revealed that the metabolic patterns were similar when using the same phenol concentration,although the sample S7 much less similar to other cultivated samples.These results suggested that the enrichment and cultivation with phenol supplemented decreased the diver-sity and also changed the metabolic function of the microbial community.Lower phenol concentration in-creased the microbial community metabolic activity.The phenol degrading capacity of isolates from each samples indicated that the enrichment and cultivation condition had changed the type and property of cul-truable bacteria.Based on these results,we concluded that the different microorganisms will be isolated un-der different cultivation condition.