Longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis.

From birth onwards, the gastrointestinal (GI) tract of infants progressively acquires a complex range of micro-organisms. It is thought that by 2 years of age the GI microbial population has stabilized. Within the developmental period of the infant GI microbiota, weaning is considered to be most critical, as the infant switches from a milk-based diet (breast and/or formula) to a variety of food components. Longitudinal analysis of the biological succession of the infant GI/faecal microbiota is lacking. In this study, faecal samples were obtained regularly from 14 infants from 1 month to 18 months of age. Seven of the infants (including a set of twins) were exclusively breast-fed and seven were exclusively formula-fed prior to weaning, with 175 and 154 faecal samples, respectively, obtained from each group. Diversity and dynamics of the infant faecal microbiota were analysed by using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. Overall, the data demonstrated large inter- and intra-individual differences in the faecal microbiological profiles during the study period. However, the infant faecal microbiota merged with time towards a climax community within and between feeding groups. Data from the twins showed the highest degree of similarity both quantitatively and qualitatively. Inter-individual variation was evident within the infant faecal microbiota and its development, even within exclusively formula-fed infants receiving the same diet. These data can be of help to future clinical trials (e.g. targeted weaning products) to organize protocols and obtain a more accurate outline of the changes and dynamics of the infant GI microbiota.

Use of Pyrosequencing and Denaturing Gradient Gel Electrophoresis to Examine the Effects of Probiotics and Essential Oil Blends on Digestive Microflora in Broilers Under Mixed Eimeria Infection

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements.

Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Technical note: Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) to assess bacterial diversity in the rumen of sheep

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intenstity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans.

Rumen microbial diversity under influence of a polyclonal antibody preparation against lactate-producing and proteolytic bacteria in cows fed different energy sources

Nine ruminally cannulated cows fed different energy sources were used to evaluate an avianderived polyclonal antibody preparation against specific ruminal bacteria and monensin on microbial community diversity. The experimental design was three Latin squares 3 x 3 distinguished by the main energy source in the diet [dry-ground corn grain, high moisture corn silage or citrus pulp]. Inside each Latin square, animals received one of the feed additives per period [control, monensin or polyclonal antibody preparation]. Each period lasted 21 days where 20 were used for treatments adaptation and the last one for sampling collection. Microbial diversity was evaluated by protozoa counts and denaturing gradient gel electrophoresis. Polyclonal antibodies plus citrus pulp (CiPu) addition in the diet resulted in an increase of relative counting of Isotricha protozoa that indicates a possible effect on this ruminal ciliate population. In general lines, in the present experiment, it was not possible to assign that there was a pattern in the structures of amplification of Bacteria and Archaea communities of the ruminal content. Oral passive immunization is a technology that arises as an effective alternative for feed additive production. Further research is still necessary to better understand its mechanisms of action.

Rumen microbial diversity under influence of a polyclonal antibody preparation against lactate-producing and proteolytic bacteria in cows fed different energy sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

Detection of competing DNA structures by thermal gradient gel electrophoresis: from self-association to triple helix formation by (G,A)-containing oligonucleotides

Sequence-specific recognition of DNA can be achieved by triple helix-forming oligonucleotides that bind to the major groove of double-helical DNA. These oligonucleotides have been used as sequence-specific DNA ligands for various purposes, including sequence-specific gene regulation in the so-called ‘antigene strategy’. In particular, (G,A)-containing oligonucleotides can form stable triple helices under physiological conditions. However, triplex formation may be in competition with self-association of these oligonucleotides. For biological applications it would be interesting to identify the conditions under which one structure is favoured as compared to the other(s). Here we have directly studied competition between formation of a parallel (G,A) homoduplex and that of a triple helix by a 13 nt (G,A)-containing oligonucleotide. Temperature gradient gel electrophoresis allows simultaneous detection of competition between the two structures, because of their different temperature dependencies and gel electrophoretic mobilities, and characterisation of this competition.

Microeukaryoticdiversity in the extreme environments of the Iberian PyriteBelt: a comparison between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning

FEMS Microbiology Ecology, Vol. 57, nº 1

Molecular weight estimation of esterase isoenzymes in closely related Drosophila Species (Diptera: Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Endophytic bacteria in long-term in vitro cultivated axenic pineapple microplants revealed by PCR-DGGE

In vitro propagated plants are believed to be free of microbes. However, after 5 years of in vitro culture of pineapple plants, without evidence of microbial contamination, the use of culture-independent molecular approach [classifying heterogeneous nucleic acids amplified via universal and specific 16S rRNA gene by polymerase chain reaction (PCR)], and further analysis by denaturing gradient gel electrophoresis (DGGE) revealed endophytic bacteria in roots, young and mature leaves of such plants. The amplification of 16S rRNA gene (Bacteria domain) with the exclusion of the plant chloroplast DNA interference, confirmed the presence of bacterial DNA, from endophytic microorganisms within microplant tissues. PCR-DGGE analysis revealed clear differences on bacterial communities depending on plant organ. Group-specific DGGE analyses also indicated differences in the structures of Actinobacteria, Alphaproteobacteria and Betaproteobacteria communities in each part of plants. The results suggest the occurrence of a succession of bacterial communities colonizing actively the microplants organs. This study is the first report that brings together evidences that pineapple microplants, previously considered axenic, harbor an endophytic bacterial community encompassing members of Actinobacteria, Alphaproteobacteria and Betaproteobacteria group which is responsive to differences in organs due to plant development.

Endophytic Colonization of Potato (Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte, Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.

Occurrence and Structure of Arbuscular Mycorrhizal Fungal Communities in Cassava after Cultivation of Cover Crops as Observed by the “PCR-DGGE” Technique

ABSTRACT Cassava (Manihot esculenta Crantz) is a highly mycotrophic crop, and prior soil cover may affect the density of arbuscular mycorrhizal fungi (AMFs), as well as the composition of the AMFs community in the soil. The aim of this study was to evaluate the occurrence and the structure of AMFs communities in cassava grown after different cover crops, and the effect of the cover crop on mineral nutrition and cassava yield under an organic farming system. The occurrence and structure of the AMFs community was evaluated through polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A randomized block experimental design was used with four replications. Six different cover crop management systems before cassava were evaluated: black oats, vetch, oilseed radish, intercropped oats + vetch, intercropped oats + vetch + oilseed radish, plus a control (fallow) treatment mowed every 15 days. Oats as a single crop or oats intercropped with vetch or with oilseed radish increased AMFs inoculum potential in soil with a low number of propagules, thus benefiting mycorrhizal colonization of cassava root. The treatments did not affect the structure of AMFs communities in the soil since the AMFs communities were similar in cassava roots in succession to different cover crops. AMFs colonization was high despite high P availability in the soil. The cassava crop yield was above the regional average, and P levels in the leaves were adequate, regardless of which cover crop treatments were used. One cover crop cycle prior to the cassava crop was not enough to observe a significant response in variables, P in plant tissue, crop yield, and occurrence and structure of AMFs communities in the soil. In the cassava roots in succession, the plant developmental stage affected the groupings of the structure of the AMF community.

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem™ and GenePhor™, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem™ and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.