Denaturing Gradient Gel Electrophoresis Patterns Research Articles

Effect of Jhum Cycles on <i>Acidobacteria</i> and <i>Burkholderia</i> Community in the Rhizosphere of Mixed Crops of Different Jhum Fields

In the present study, culture-dependent and independent [Denaturing gradient gel electrophoresis (DGGE)] approach were used to analyze the effect of fallow cycle on bacterial populations associated with mixed crops selected from three different jhum fallow cycles (5-, 10- and 20- years) of Changki village, Nagaland. Two important bacterial phyla viz. Acidobacteria and Burkholderia were selected for DGGE analysis. According to the DGGE banding profile, Acidobacteria showed more richness compared Burkholderia. The presence and absence of few of the bands in the profile indicate the community shifts in the selected jhum fields. Shannon diversity index was higher in Acidobacteria than Burkholderia. Multivariate analysis showed that Acidobacteria community structure was similar among the crop plants of the same fallow cycle. Burkholderia community did not show clear separation among fallow cycles although crop of same fallow cycle tend to group together. ANOSIM and PERMANOVA showed significant differences in rhizosphere community pattern among fallow cycles and the crop plants of same fallow cycles indicated that both fallow cycle and crop plants are factors determining the Acidobacteria and Burkholderia community composition in jhum soils which was also confirmed by the culture-dependent analysis. Culturable bacterial counts were dependent on media used and significantly different at different fallow cycles. Bacterial communities (both culturable and DGGE patterns) were correlated with soil nutrients suggesting their importance in stability of jhum soil. The present study revealed that both fallow cycle and host plant may be the key factors in shaping many bacterial communities in jhum soils.

Microbial Communities of Hydrothermal Guaymas Basin Surficial Sediment Profiled at 2 Millimeter-Scale Resolution.

The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin.

Can the foregut nematode Haemonchus contortus and medicinal plants influence the fecal microbial community of the experimentally infected lambs?

The abomasal parasitic nematode Haemonchus contortus can influence the abomasal microbiome of the host. On the other hand, no information occurs on the influence of the parasite on the hindgut microbiome of the host. We evaluated the impact of Haemonchus contortus on the fecal microbial community of the experimentally infected lambs treated with a mixture of medicinal herbs to ameliorate the haemonchosis. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with a blend of medicinal herbs (Inf+Herb), uninfected control animals (Control), and uninfected animals supplemented with medicinal herbs (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herb blend (Herbmix) consisted of dry medicinal plants of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis, Melisa officinalis, Foeniculum vulgare, and Artemisia absinthium. Each animal was fed meadow hay and a commercial concentrate (600 + 350 g DM/d). Inf+Herb and C+Herb lambs were fed Herbmix (100 g DM/d and animal). Treatment lasted for 50 days. The fecal microbial fermentation parameters (short-chain fatty acids, ammonia, and pH) were evaluated at intervals of 0, 20, 32, and 50 days. The fecal eubacterial populations were evaluated by denaturing gradient gel electrophoresis (DGGE) at day 32 when H. contortus infection was the highest. No substantial effects of the H. contortus infection and the herbal treatment on fecal microbial fermentation parameters and fecal eubacterial populations were observed. Evaluation of DGGE patterns by Principal component analysis pointed to the tendency to branch the C+Herb group from the other experimental groups on Day 32. The results indicate that hindgut microbial activity was not disturbed by H. contortus infection and herbal treatment.

Use of the PCR-DGGE Method for the Analysis of the Bacterial Community Structure in Soil Treated With the Cephalosporin Antibiotic Cefuroxime and/or Inoculated With a Multidrug-Resistant Pseudomonas putida Strain MC1.

The widespread use of cefuroxime (XM) has resulted in the increase in its concentration in hospital and domestic wastewaters. Due to the limited removal of antibiotics and antibiotic-resistant genes in conventional systems, the drugs enter the surface water and soils. Moreover, the introduction of XM and/or XM-resistant bacteria into soil may cause a significant modification of the biodiversity of soil bacterial communities. Therefore, the goal of this research was to assess the genetic diversity of a bacterial community in the cefuroxime (XM1 – 1 mg/kg and XM10 – 10 mg/kg) and/or antibiotic-resistant Pseudomonas putida strain MC1 (Ps – 1.6 × 107 cells/g)-treated soils as determined by the DGGE (denaturing gradient gel electrophoresis) method. The obtained data were also evaluated using a multivariate analysis and the resistance (RS)/resilience (RL) concept. Strain MC1 was isolated from raw sewage in the presence of XM and was resistant not only to this antibiotic but also to vancomycin, clindamycin and erythromycin. The DGGE patterns revealed that the XM10 and XM10+Ps treatments modified the composition of the bacterial community by the alteration of the DGGE profiles as well as a decline in the DGGE indices, in particular on days 30, 60, and 90. In turn, the XM1 and XM1+Ps or Ps treatments did not affect the values of richness and diversity of the soil bacteria members. A principal component analysis (PCA) also indicated that XM markedly changed the diversity of bacterial assemblages in the second part of the experiment. Moreover, there were differences in the RS/RL of the DGGE indices to the disturbances caused by XM and/or Ps. Considering the mean values of the RS index, the resistance was categorized in the following order: diversity (0.997) > evenness (0.993) > richness (0.970). The soil RL index was found to be negative, thus reflecting the progressing detrimental impact of XM on the genetic biodiversity of bacteria within the experiment. These results indicate that the introduction of XM at higher dosages into the soil environment may exert a potential risk for functioning of microorganism.

Breastfeeding increases microbial community resilience

ObjectiveSince the present group had already described the composition of the intestinal microbiota of Brazilian infants under low social economic level, the aim of the present study was to analyze the microbial community structure changes in this group of infants during their early life due to external factors. MethodsFecal samples were collected from 11 infants monthly during the first year of life. The infants were followed regarding clinical and diet information and characterized according to breastfeeding practices. DNA was extracted from fecal samples of each child and subjected to Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis analysis. ResultsThe results revealed a pattern of similarity between the time points for those who were on exclusive breastfeeding or predominant breastfeeding. Although there were changes in intensity and fluctuation of some bands, the Denaturing Gradient Gel Electrophoresis patterns in the one‐year microbial analysis were stable for breastfeeding children. There was uninterrupted ecological succession despite the influence of external factors, such as complementary feeding and antibiotic administration, suggesting microbiota resilience. This was not observed for those children who had mixed feeding and introduction of solid food before the 5th month of life. ConclusionThese results suggested an intestinal microbiota pattern resilient to external forces, due to the probiotic and prebiotic effects of exclusive breastfeeding, reinforcing the importance of exclusive breastfeeding until the 6th month of life.

Effector gene vap1 based DGGE fingerprinting to assess variation within and among Heterodera schachtii populations.

Abstract Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study inter-population variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast and – compared to sequencing approaches – inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.

Rapid detection of mutations in erm(41) and rrl associated with clarithromycin resistance in Mycobacterium abscessus complex by denaturing gradient gel electrophoresis

Clarithromycin resistance is increasing dramatically among Mycobacterium abscessus complex. The main resistance mechanisms are mutations in the erm(41) and rrl genes. Here we report PCR-based denaturing gradient gel electrophoresis (DGGE) as an alternative method for rapidly detection of mutations in erm(41) and rrl among M. abscessus isolates. Four primer sets targeting the full-length erm(41) gene and a 354bp fragment of the rrl gene were designed. A combination of 16 different DGGE patterns were observed for erm(41) gene, including 16 in M. abscessus subsp. abscessus and 1 in M. abscessus subsp. massiliense. Six DGGE patterns were obtained for rrl gene. Mutations in the erm(41) and rrl detected by DGGE were 100% identical to mutations detected by DNA sequencing. This is the first report to identify PCR-based DGGE as a practical, relatively inexpensive technique for rapidly detecting mutations in the erm(41) and rrl genes associated with clarithromycin resistance in M. abscessus complex.

Segment-specific responses of intestinal epithelium transcriptome to in-feed antibiotics in pigs.

Despite widespread use of antibiotics for treatment of human diseases and promotion of growth of agricultural animals, our understanding of their effects on the host is still very limited. We used a model in which pigs were fed with or without a cocktail of antibiotics and found, based on the denaturing gradient gel electrophoresis (DGGE) patterns, that the fecal bacteria from the treatment and control animals were distinct. Furthermore, the total bacterial population in the feces tended to be decreased by the antibiotic treatment (P = 0.07), and the counts of Lactobacillus and Clostridium XIVa were significantly reduced (P < 0.05). To explore the effects of antibiotics on host intestinal epithelium, we assessed gene expression profiles of the jejunum and ileum and their response to antibiotic administration. The results indicate that in-feed antibiotics increased expression of genes involved in immune functions in both the jejunum and ileum, some of which were clustered in the coexpression network. Gene ontology terms of metabolic processes were altered predominantly in the jejunum but not in the ileum. Notably, antibiotics diminished intestinal segment-specific transcriptional changes, especially for genes associated with metabolic functions. This study reveals segment-specific responses of host intestinal epithelium to in-feed antibiotics, which can be a valuable resource for deciphering antibiotic-microbiota-host interactions.

Breastfeeding increases microbial community resilience

ObjectiveSince the present group had already described the composition of the intestinal microbiota of Brazilian infants under low social economic level, the aim of the present study was to analyze the microbial community structure changes in this group of infants during their early life due to external factors. MethodsFecal samples were collected from 11 infants monthly during the first year of life. The infants were followed regarding clinical and diet information and characterized according to breastfeeding practices. DNA was extracted from fecal samples of each child and subjected to Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis. ResultsThe results revealed a pattern of similarity between the time points for those who were on exclusive breastfeeding or predominant breastfeeding. Although there were changes in intensity and fluctuation of some bands, the Denaturing Gradient Gel Electrophoresis patterns in the one-year microbial analysis were stable for breastfeeding children. There was uninterrupted ecological succession despite the influence of external factors, such as complementary feeding and antibiotic administration, suggesting microbiota resilience. This was not observed for those children who had mixed feeding and introduction of solid food before the 5th month of life. ConclusionThese results suggested an intestinal microbiota pattern resilient to external forces, due to the probiotic and prebiotic effects of exclusive breastfeeding, reinforcing the importance of exclusive breastfeeding until the 6th month of life.

Efficiency of temporary storage of geothermal waters in a lake system: Monitoring the changes of water quality and bacterial community structures.

Disposal of used geothermal waters in Hungary often means temporary storage in reservoir lakes to reduce temperature and improve water quality. In this study, the physical and chemical properties and changes in the bacterial community structure of a reservoir lake system in southeast region of Hungary were monitored and compared through 2 years, respectively. The values of biological oxygen demand, concentrations of ammonium ion, total inorganic nitrogen, total phosphorous, and total phenol decreased, whereas oxygen saturation, total organic nitrogen, pH, and conductivity increased during the storage period. Bacterial community structure of water and sediment samples was compared by denaturing gradient gel electrophoresis (DGGE) following the amplification of the 16S rRNA gene. According to the DGGE patterns, greater seasonal than spatial differences of bacterial communities were revealed in both water and sediment of the lakes. Representatives of the genera Arthrospira and Anabaenopsis (cyanobacteria) were identified as permanent and dominant members of the bacterial communities.

Unique clade of alphaproteobacterial endosymbionts induces complete cytoplasmic incompatibility in the coconut beetle

Maternally inherited bacterial endosymbionts in arthropods manipulate host reproduction to increase the fitness of infected females. Cytoplasmic incompatibility (CI) is one such manipulation, in which uninfected females produce few or no offspring when they mate with infected males. To date, two bacterial endosymbionts, Wolbachia and Cardinium, have been reported as CI inducers. Only Wolbachia induces complete CI, which causes 100% offspring mortality in incompatible crosses. Here we report a third CI inducer that belongs to a unique clade of Alphaproteobacteria detected within the coconut beetle, Brontispa longissima This beetle comprises two cryptic species, the Asian clade and the Pacific clade, which show incompatibility in hybrid crosses. Different bacterial endosymbionts, a unique clade of Alphaproteobacteria in the Pacific clade and Wolbachia in the Asian clade, induced bidirectional CI between hosts. The former induced complete CI (100% mortality), whereas the latter induced partial CI (70% mortality). Illumina MiSeq sequencing and denaturing gradient gel electrophoresis patterns showed that the predominant bacterium detected in the Pacific clade of B. longissima was this unique clade of Alphaproteobacteria alone, indicating that this endosymbiont was responsible for the complete CI. Sex distortion did not occur in any of the tested crosses. The 1,160 bp of 16S rRNA gene sequence obtained for this endosymbiont had only 89.3% identity with that of Wolbachia, indicating that it can be recognized as a distinct species. We discuss the potential use of this bacterium as a biological control agent.

Denaturing gradient gel electrophoresis profiles of bacteria from the saliva of twenty four different individuals form clusters that showed no relationship to the yeasts present

ObjectivesThe aim was to investigate the relationship between groups of bacteria identified by cluster analysis of the DGGE fingerprints and the amounts and diversity of yeast present. MethodsBacterial and yeast populations in saliva samples from 24 adults were analysed using denaturing gradient gel electrophoresis (DGGE) of the bacteria present and by yeast culture. ResultsEubacterial DGGE banding patterns showed considerable variation between individuals. Seventy one different amplicon bands were detected, the band number per saliva sample ranged from 21 to 39 (mean±SD=29.3±4.9). Cluster and principal component analysis of the bacterial DGGE patterns yielded three major clusters containing 20 of the samples. Seventeen of the 24 (71%) saliva samples were yeast positive with concentrations up to 103cfu/mL. Candida albicans was the predominant species in saliva samples although six other yeast species, including Candida dubliniensis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida rugosa and Saccharomyces cerevisiae, were identified. The presence, concentration, and species of yeast in samples showed no clear relationship to the bacterial clusters. ConclusionDespite indications of in vitro bacteria-yeast interactions, there was a lack of association between the presence, identity and diversity of yeasts and the bacterial DGGE fingerprint clusters in saliva. This suggests significant ecological individual-specificity of these associations in highly complex in vivo oral biofilm systems under normal oral conditions.

Metagenomic and Culture-Dependent Analysis of the Bacterial Diversity of a Hot Spring Reservoir as a Function of the Seasonal Variation

A natural thermal habitat, Tulasi Shyam, was studied for the culture-dependent and culture-independent diversity of the thermophilic bacteria. Soil and water samples of winter, summer and monsoon seasons were analyzed to assess the impact of the seasonal variation on the microbial diversity. The soil was fertile and contained high humic substances. The temperature of the water was 50 °C, which did not significantly vary with seasons. Various methods were assessed for the extraction of the metagenomic DNA to obtain quality DNA in good quantity. The 16S rRNA genes were amplified using the metagenomic DNA as template and the products were analyzed further by the denaturing gradient gel electrophoresis (DGGE) for evaluating the bacterial diversity. The metagenomic diversity of the hot spring reservoir-1 as compared to reservoir-2 was less influenced by the seasonal variations. The DGGE patterns of the soil samples suggested significant effect of the seasonal changes. The metagenomic diversity was distinct when compared with the culture-dependent microbial diversity. The diversity was further analyzed by the principal component analysis (PCA) and diversity indices. The apparent shift in the diversity appears to be governed by the prevailing environmental conditions.

Insights into the fecal microbiota of captive Iberian lynx (Lynx pardinus).

The Iberian lynx (Lynx pardinus) is an endangered species restricted to several areas of Spain and Portugal. Its low genetic diversity likely provokes immune depression and high susceptibility to infectious diseases. The intestinal microbiota is closely related to host health and nutrition. In order to contribute to the knowledge of the Iberian lynx intestinal microbiota, fecal microbiota of captive specimens from two breeding centers ("La Olivilla" and "El Acebuche"), located in Southern Spain, were studied by Denaturing Gradient Gel Electrophoresis (DGGE). Results grouped microbiota in two main clusters (I and III) which included DGGE patterns of 19 out of 36 specimens, cluster I being the most frequent in "La Olivilla" (50%) and cluster III in "El Acebuche" (55.55 %) specimens. Bacteroidetes, Firmicutes and Proteobacteria phyla were identified. Segregation of clusters I and III was attributed to different microorganism presence (Pseudomonas koreensis, Pseudomonas migulae, Carnobacterium sp., Arthrobacter, Robinsoniella peorensis and Ornithinibacillus sp.) and ability to use different carbon sources. Biolog EcoPlates® results indicate high functional diversity of fecal microbiota, it being higher in cluster III. The great impact of intestinal microbiota on host health supports the importance of its microbial composition understanding. This study is the first report of captive Iberian lynx fecal microbiota composition. [Int Microbiol 20(1): 31-41 (2017)].

Gut content microbiota of introduced bigheaded carps (Hypophthalmichthys spp.) inhabiting the largest shallow lake in Central Europe

Studying the microbiota in the alimentary tract of bigheaded carps (Hypophthalmichthys spp.) gained special interest recently, as these types of investigations on non-native fish species may lead to a better understanding of their ecological role and feeding habits in an invaded habitat. For microbiological examinations, bigheaded carp gut contents and water column samples from Lake Balaton (Hungary) were collected from spring to autumn in 2013. Denaturing Gradient Gel Electrophoresis (DGGE) and pyrosequencing of the 16S rRNA gene were performed to reveal the composition. According to the DGGE patterns, bacterial communities of water samples separated clearly from that of the intestines. Moreover, the bacterial communities in the foreguts and hindguts were also strikingly dissimilar. Based on pyrosequencing, both foregut and hindgut samples were predominated by the fermentative genus Cetobacterium (Fusobacteria). The presence of some phytoplankton taxa and the high relative abundance of cellulose-degrading bacteria in the guts suggest that intestinal microbes may have an important role in digesting algae and making them utilizable for bigheaded carps that lack cellulase enzyme. In turn, the complete absence of typical heterotrophic freshwater bacteria in all studied sections of the intestines indicated that bacterioplankton probably has a negligible role in the nutrition of bigheaded carps.

Evolution of Ciona intestinalis Tumor necrosis factor alpha (CiTNFα): Polymorphism, tissues expression, and 3D modeling

Although the Tumor necrosis factor gene superfamily seems to be very conserved in vertebrates, phylogeny, tissue expression, genomic and gene organization, protein domains and polymorphism analyses showed that a strong change has happened mostly in invertebrates in which protochordates were a constraint during the immune-molecules history and evolution.RT PCR was used to investigate differential gene expression in different tissues. The expression shown was greater in the pharynx.Single-nucleotide polymorphism has been investigated in Ciona intestinalis Tumor necrosis factor alpha (CiTNFα) mRNA isolated from the pharynx of 30 ascidians collected from Licata, Sicily (Italy), by denaturing gradient gel electrophoresis (DGGE). For this analysis, CiTNFα nucleotide sequence was separated into two fragments, TNF-1 and -2, respectively, of 630 and 540 bp. We defined 23 individual DGGE patterns (named 1 to 10 for TNF-1 and 1 to 13 for TNF-2). Five patterns for TNF-1 accounted for <10% of the individuals, whereas the pattern 13 of TNF-2 accounted for >20% of the individuals. All the patterns were verified by direct sequencing. Single base-pair mutations were observed mainly within COOH-terminus, leading to 30 nucleotide sequence variants and 30 different coding sequences segregating in two main different clusters. Although most of the base mutations were silent, four propeptide variants were detected and six amino acid replacements occurred within COOH-terminus. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure on COOH-terminus domain. Lastly we displayed the in silico 3D structure analysis including the CiTNFα variable region.

Changes in the water quality and bacterial community composition of an alkaline and saline oxbow lake used for temporary reservoir of geothermal waters.

Geothermal waters exploited in the southeastern region of Hungary are alkali-hydrogen-carbonate type, and beside the high amount of dissolved salt, they contain a variety of aromatic, heteroaromatic, and polyaromatic hydrocarbons. The majority of these geothermal waters used for heating are directed into surface waters following a temporary storage in reservoir lakes. The aim of this study was to gain information about the temporal and spatial changes of the water quality as well as the bacterial community composition of an alkaline and saline oxbow lake operated as reservoir of used geothermal water. On the basis of the water physical and chemical measurements as well as the denaturing gradient gel electrophoresis (DGGE) patterns of the bacterial communities, temporal changes were more pronounced than spatial differences. During the storage periods, the inflow, reservoir water, and sediment samples were characterized with different bacterial community structures in both studied years. The 16S ribosomal RNA (rRNA) gene sequences of the bacterial strains and molecular clones confirmed the differences among the studied habitats. Thermophilic bacteria were most abundant in the geothermal inflow, whereas the water of the reservoir was dominated by cyanobacteria and various anoxygenic phototrophic prokaryotes. In addition, members of several facultative anaerobic denitrifying, obligate anaerobic sulfate-reducing and syntrophic bacterial species capable of decomposition of different organic compounds including phenols were revealed from the water and sediment of the reservoir. Most of these alkaliphilic and/or halophilic species may participate in the local nitrogen and sulfur cycles and contribute to the bloom of phototrophs manifesting in a characteristic pink-reddish discoloration of the water of the reservoir.

A comparison of microbial profiles of different regions of the equine hindgut

The microbial profiles of the luminal content of five hindgut segments of one healthy horse were compared with rectal sample to elucidate the effect of anatomical region on bacterial and archaeal community structure and to evaluate the use of faeces as a representative model of large intestine. The qualitative and quantitative changes of the microbial community composition of caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and faeces were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR using universal primers amplifying the V3 region of 16S rDNA. DGGE fingerprints revealed extensive bacterial as well as archaeal diversity in all studied samples and reflected shifts in the community structure among the caecum, the different segments of the colon and the faeces. Archaeal DGGE pattern of the caecum differed from all the other parts of the hindgut. Microbial profile similarities were found between the left and the right dorsal colon and between the left ventral colon and the faeces. The excised DGGE bands were related to uncultured bacteria and methanogens, the dominant archaeal bands of caecum and faeces were related to Methanocorpusculum sp. Diversity indices indicated the higher diversity for bacteria than for archaea and the dominance of some methanogenic species. The real-time PCR revealed the differences in the microbial quantitative composition of each segment, showing the highest number of total bacteria and archaea in the right ventral colon. The analyses of bacterial and archaeal composition along the one equine hindgut indicate that the faecal sample is similar to that of the left ventral colon, but does not represent the microbial community of the caecum and other parts of the colon.

The Effect of Probiotic Treatment on Patients Infected with the H7N9 Influenza Virus.

BackgroundA novel avian-origin influenza A (H7N9) virus emerged and spread among humans in Eastern China in 2013. Prophylactic treatment with antibiotics and probiotics for secondary infection is as important as antiviral treatment. This study aims to assess the ability of probiotic treatment to restore internal homeostasis under antibiotic pressure and to reduce/ameliorate the risk of secondary infections resulting from infection with the H7N9 virus.MethodsThis is a retrospective study in archival samples. Between April 1 and May 10, 2013, 113 stool, sputum, and blood specimens were collected and analyzed by denaturing gradient gel electrophoresis (DGGE) to determine the composition of the patient microbiomes. Microbial diversity was calculated using Gel-Pro analyzer and Past software. Cluster analysis of DGGE pattern profiles was employed to create a phylogenetic tree for each patient, and multidimensional scaling (MDS) and principal component analysis (PCA) were performed to visualize relationships between individual lanes.ResultsFive patients had secondary infections, including Klebsiella pneumonia, Acinetobacter baumanii and Candida albicans infection. The DGGE profiles of fecal samples obtained at different time points from the same individual were clearly different, particularly for patients with secondary infections. Shannon’s diversity index and evenness index were lower in all infected groups compared to the control group. After B. subtilis and E. faecium or C. butyricum administration, the fecal bacterial profiles of patients who had not been treated with antibiotics displayed a trend of increasing diversity and evenness. C. butyricum failed to reduce/ameliorate secondary infection in H7N9-infected patients, but administration of B. subtilis and E. faecium appeared to reduce/ameliorate secondary infection in one patient.ConclusionH7N9 infection might decrease intestinal microbial diversity and species richness in humans. C. butyricum failed to reduce/ameliorate secondary infection in H7N9-infected patients. B. subtilis and E. faecium may also play a role in reducing/ameliorating secondary infection in these patients.

Effects of digestate on soil chemical and microbiological properties: A comparative study with compost and vermicompost

Anaerobic digestion has become increasingly popular as an alternative for recycling wastes from different origins. Consequently, biogas residues, most of them with unknown chemical and biological composition, accrue in large quantities and their application into soil has become a widespread agricultural practise. The aim of this study was to evaluate the effects of digestate application on the chemical and microbiological properties of an arable soil in comparison with untreated manure, compost and vermicompost. Once in the soil matrix either the addition of compost or digestate led to an increased nitrification rate, relative to unamended and manure-treated soil, after 15 and 60 days of incubation. Faecal coliform and E. coli colony forming units (CFUs) were not detected in any of the amended soils after 60 days. The highest number of Clostridium perfringens CFUs was recorded in manure-amended soil at the beginning of the experiment and after 15 days; whilst after 60 days the lowest CFU number was registered in digestate-treated soil. Denaturing gradient gel electrophoresis patterns also showed that besides the treatment the date of sampling could have contributed to modifications in the soil ammonia-oxidising bacteria community, thereby indicating that the soil itself may influence the community diversity more strongly than the treatments.