Reaction-denaturing Gradient Gel Electrophoresis Method Research Articles

Analysis of bacterial community in rumen fluid of cattle supplemented with different protein and energy sources

Context Source and composition of feed influence rumen microbial community, which determines efficiency of feed digestion and thus productivity in ruminants. Therefore, changes in the structure, function and diversity of the rumen microbial populations in response to changes in diet provide an understanding in the rumen fermentation process. Aims The present study, consisting of two experiments, was conducted to determine the effects of supplementing different protein and energy sources on the rumen bacterial community in cattle. Methods The dietary treatments of the first experiment, which evaluated the effect of protein sources, were as follows: (i) Hymenachne acutigluma grass, rice straw and rice bran (1.5 kg/head.day; C1), (ii) C1 plus 120 g urea/head.day (C1 + U), (iii) C1 plus 720 g soybean/head.day (C1 + SM), and (iv) C1 plus 720 g of blood and feather meal (in 1:1 ratio)/head.day (C1 + BFM). The treatments in the second experiment were (i) Hymenachne acutigluma grass, rice straw and concentrate (1.5 kg/head.day; C2), (ii) C2 plus 250 g fish oil/head.day (C2 + FO) and (iii) C2 + 250 g soybean oil/head.day (C2 + SO). At the end of the 90-day feeding trial, rumen fluids were extracted for microbial DNA isolation to identify the microbe species by the polymerase chain reaction–denaturing gradient gel electrophoresis method and sequencing of the 16S rRNA region. Key results The sequences of some DNA bands were closely related to the bacteria strains of the Prevotella, Cytophaga, Capnocytophaga, Cyanobacterium, Catonella, Faecalibacterium, Lachnospiraceae, Ruminococcaceae, Propionivibrio, Galbibacter, Moorellaglycerin, Escherichia coli and Klebsiella alba groups, with similarity levels ranging from 73% to 96%. In addition, the Prevotella species was found in both the protein and the energy supplement trials, and irrespective of diet supplements, the Firmicutes and Bacteroidetes were the prominent groups in the rumen. Conclusions Firmicutes and Bacteroidetes are the two dominant groups of rumen microflora, and Bacteroidia and Clostridia classes together with the Prevotella genus are predominant in the rumen irrespective of protein and energy sources. Implications Our findings provided evidence on the effect of diet on the interaction of rumen microbial community and have important implications in establishing optimal diets for cattle.

Effect of different short-term tillage management on nitrogen-fixing bacteria community in a double-cropping paddy field of southern China.

Soil nitrogen (N)-fixing bacteria community plays an important role in the N cycling process in soil, but there is still limited information about how the soil microbes that drive this process to respond to combined application of tillage and crop residue management under the double-cropping rice (Oryza sativa L.) paddy field in southern of China. Therefore, the effects of 6-years short-term tillage treatment on soil N-fixing bacteria community under the double-cropping rice paddy field in southern China were studied by using the polymerase chain reaction-denaturing gradient gel electrophoresis method. The field experimentincluded four tillage treatments: conventional tillage with crop residue incorporation (CT), rotary tillage with crop residue incorporation (RT), no-tillage with crop residue retention (NT), rotary tillage with crop residue removed as control (RTO). The results showed that the diversity index and richness index of cbbLR and nifH genes with CT, RT, and NT treatments were increased, compared with RTO treatment. Compared with RTO treatment, the abundance of cbbLR gene with CT, RT, and NT treatments were increased by 6.54, 4.73, and 2.78 times, respectively. Meanwhile, the abundance of nifH gene with CT, RT, and NT treatments were 5.32, 3.71, and 2.45 times higher than that of RTO treatment. The results also indicated that soil autotrophic Azotobacter and nitrogenase activity with CT and RT treatments were significantly higher (p < .05) than that of RTO treatment. There was an obvious difference in characteristic of soil N-fixing bacteria community between the application of crop residue and without crop residue input treatments. In summary, the results indicated that the abundance of N-fixing bacteria community in the double-cropping rice paddy field increased with conventional tillage and rotary tillage practice.

Genetic association of CTLA4 gene with polycystic ovary syndrome in the Chinese Han population.

The autoimmune and gene etiology are implicated in the pathogenesis of polycystic ovary syndrome (PCOS). The cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is important for negative regulation of T-cell activation, and CTLA4 gene has been identified as a risk factor for some autoimmune diseases. However, none studies have been performed about the association between PCOS and the CTLA4 gene before. Here, we aimed to investigate the association of CTLA4 with PCOS in the Chinese Han population though a case-control association analysis of 606 individuals. The tagging variants rs733618 and rs231775 in the CTLA4 gene were detected using polymerase chain reaction-denaturing gradient gel electrophoresis method. Further analysis found the rs733618 was significantly different between case and control groups in either genotypic or allelic distribution (P = .01 and .009, respectively) while rs231775 not. Moreover, rs733618 was significantly associated with higher body mass index in the dominant model (P = .003) and with higher waist/hip ratio in the recessive model (P = .02). Interestingly, rs733618 was only found to have significant association with homeostatic model assessment for insulin resistance (HOMA-IR) in both dominant and recessive model (P = .009 and .0065, respectively). This is the first study to investigate the association of CTLA4 gene with PCOS. The CTLA4 gene is suggested to correlated with PCOS, and influence PCOS through regulating obesity and the HOMA-IR in a novel way.

Dynamic changes of bacterial communities and nitrite character during northeastern Chinese sauerkraut fermentation.

Northeastern Chinese sauerkraut is a well-known traditional fermented vegetable in China. Incomplete identification of the microorganisms' (bacteria in spontaneous fermentation) diversity and accumulation of nitrite make it difficult to normalize the fermentation process and product qualities of northeastern Chinese sauerkraut. Conventional culturing and polymerase chain reaction-denaturing gradient gel electrophoresis methods were combined to describe microbial structure and diversity. Lactobacillus, Leuconostoc, Enterobacter, Accumulibacter, Thermotoga, Pseudomonas, Clostridium, Rahnella and Citrobacter were predominant microorganisms in different fermentation periods. The pH value and nitrite concentration presentedacertainrelevancetothe amount of lacticacidbacteria. Lactobacillus and Leuconostoc had the ability to decrease nitrite by inhibiting nitrate-reducing bacteria such as Enterobacter. Therefore, Northeastern Chinese sauerkraut should not be eaten until 4weeks of fermentation for the safety and quality of fermented foods. Northeastern Chinese sauerkraut is rich in lacticacidbacteria, which demonstrate its ability as an excellent probiotic for applications in functional foods.

Association of diverse bacterial communities in human bile samples with biliary tract disorders: a survey using culture and polymerase chain reaction-denaturing gradient gel electrophoresis methods.

Bacterial infection is considered a predisposing factor for disorders of the biliary tract. This study aimed to determine the diversity of bacterial communities in bile samples and their involvement in the occurrence of biliary tract diseases. A total of 102 bile samples were collected during endoscopic retrograde cholangiopancreatography (ERCP). Characterization of bacteria was done using culture and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods. Antimicrobial susceptibility of the isolates was determined based on the Clinical and Laboratory Standards Institute (CLSI) guidelines and identity of the nucleotide sequences of differentiated bands from the DGGE gels was determined based on GenBank data. In total, 41.2% (42/102) of the patients showed bacterial infection in their bile samples. This infection was detected in 21% (4/19), 45.4% (5/11), 53.5% (15/28), and 54.5% (24/44) of patients with common bile duct stone, microlithiasis, malignancy, and gallbladder stone, respectively. Escherichia coli showed a significant association with gallstones. Polymicrobial infection was detected in 48% of the patients. While results of the culture method established coexistence of biofilm-forming bacteria (Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Enterococcus spp., and Acinetobacter spp.) in different combinations, the presence of Capnocytophaga spp., Lactococcus spp., Bacillus spp., Staphylococcus haemolyticus, Enterobacter or Citrobacter spp., Morganella spp., Salmonella spp., and Helicobacter pylori was also characterized in these samples by the PCR-DGGE method. Multidrug resistance phenotypes (87.5%) and resistance to third- and fourth-generation cephalosporins and quinolones were common in these strains, which could evolve through their selection by bile components. Ability for biofilm formation seems to be a need for polymicrobial infection in this organ.

The Role of Epsilonproteobacteria in Children With Gastroenteritis

A polymerase chain reaction-denaturing gradient gel electrophoresis method was used to examine 50 stool samples from children in Belgium with gastroenteritis for an extensive range of Epsilonproteobacteria species. During the 3-month study period, Campylobacter concisus was the most common species. Our observations suggest that C. concisus displays similar microbiologic and clinical features as Campylobacter jejuni.

Landfill leachate pollutant removal performance of a novel biofilter packed with mixture medium

Landfill leachate pollutants were treated in a biofilter filled with a mixture of aged refuse and slag, and the performance was compared with those reactors filled solely with either medium. Cultural counting method showed that bioreactor filled with slag had the highest amount of nitrifying bacteria, while polymerase chain reaction–denaturing gradient gel electrophoresis method showed that reactor filled with both media had the highest bacterial community diversity. Particle size distributions measurement showed that slag contained less fine particles than aged refuse, which provided better permeability. The reactor containing both media exhibited a high efficiency in removal of pollutants, and a higher resistance to shock loading and low temperature compared with single-medium reactors. It also overcame both the poor permeability of aged refuse filling and the low bacteria diversity of slag. The study shows that a mixture of aged refuse and slag as a new biofilter medium for leachate pollutant removal is technically viable.

Characterization of cyclohexane and hexane degradation by Rhodococcus sp. EC1

Cyclohexane is a recalcitrant compound that is more difficult to degrade than even n-alkanes or monoaromatic hydrocarbons. In this study, a cyclohexane-degrading consortium was obtained from oil-contaminated soil by an enrichment culture method. Based on a 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis method, this consortium was identified as comprising Alpha-proteobacteria, Actinobacteria, and Gamma-proteobacteria. One of these organisms, Rhodococcus sp. EC1, was isolated and shown to have excellent cyclohexane-degrading ability. The maximum specific cyclohexane degradation rate ( V max) for EC1 was 246 μmol g-DCW −1 (dry cell weight) h −1. The optimum conditions of cyclohexane degradation were 25–35 °C and pH 6–8. In addition to its cyclohexane degradation abilities, EC1 was also able to strongly degrade hexane, with a maximum specific hexane degradation rate of 361 μmol g-DCW −1 h −1. Experiments using 14 C-hexane revealed that EC1 mineralized 40% of hexane into CO 2 and converted 53% into biomass. Moreover, EC1 could use other hydrocarbons, including methanol, ethanol, acetone, methyl tert-butyl ether, pyrene, diesel, lubricant oil, benzene, toluene, ethylbenzene, m-xylene, p-xylene and o-xylene. These findings collectively suggest that EC1 may be a useful biological resource for removal of cyclohexane, hexane, and other recalcitrant hydrocarbons.

Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis.

Affected members of most kindreds with Albright hereditary osteodystrophy have a partial deficiency of functional Gs, the guanine nucleotide-binding protein that stimulates adenylyl cyclase. By use of the polymerase chain reaction to amplify genomic fragments with the attachment of a high-melting G + C-rich region (GC clamp) and analysis of these fragments by denaturing gradient gel electrophoresis, heterozygous mutations in the Gs alpha-subunit gene were found in two kindreds. These included a G----C substitution at the donor splice junction of intron 10 and a coding frameshift created by a single base deletion within exon 10. The findings illustrate the heterogeneity of genetic defects in Albright hereditary osteodystrophy and the usefulness of the polymerase chain reaction-denaturing gradient gel electrophoresis method to search rapidly for mutations in a large candidate gene.