Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

<p>Abstract</p> <p>Background</p> <p>The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean^®Fecal DNA Isolation Kit, M; QIAamp^®DNA Stool Mini Kit, Q; FastDNA^®SPIN Kit, FSp; FastDNA^®SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.</p> <p>Method</p> <p>Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.</p> <p>Results</p> <p>Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.</p> <p>Conclusion</p> <p>We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.</p

Degradation of a benzene–toluene mixture by hydrocarbon-adapted bacterial communities

We examined the rate of degradation of a benzene–toluene mixture in aerobic microcosms prepared with samples of an aquifer that lies below a petrochemical plant (SIReN, UK). Five samples exposed to different concentrations of benzene (from 0.6 to 317 mg l−1) were used. Fast degradation (approx. 1–6 mg l−1 day−1) of both contaminants was observed in all groundwater samples and complete degradation was recorded by the seventh day except for one sample. We also identified the microbial community in each of the samples by culture-independent techniques. Two of the less impacted samples harbour the aerobic benzene degrader Pseudomonas fluorescens, while Acidovorax and Arthrobacter spp. were found in the most polluted sample and are consistent with the population observed in situ. Hydrogenophaga was found in the deepest sample while Rhodoferax spp. were recovered in an alkaline sample (pH 8.4) and may also be implicated in benzene degradation. Time series analysis shows that each of the samples has a different community but they remain stable over the degradation period. This study provides new information on a well not previously studied (no. 309s) and confirms that adapted communities have the ability to degrade hydrocarbon mixtures and could be used in further bioaugmentation approaches in contaminated sites

DNA-Based Diet Analysis for Any Predator

Background: Prey DNA from diet samples can be used as a dietary marker; yet current methods for prey detection require a priori diet knowledge and/or are designed ad hoc, limiting their scope. I present a general approach to detect diverse prey in the feces or gut contents of predators. Methodology/Principal Findings: In the example outlined, I take advantage of the restriction site for the endonuclease Pac I which is present in 16S mtDNA of most Odontoceti mammals, but absent from most other relevant non-mammalian chordates and invertebrates. Thus in DNA extracted from feces of these mammalian predators Pac I will cleave and exclude predator DNA from a small region targeted by novel universal primers, while most prey DNA remain intact allowing prey selective PCR. The method was optimized using scat samples from captive bottlenose dolphins (Tursiops truncatus) fed a diet of 6–10 prey species from three phlya. Up to five prey from two phyla were detected in a single scat and all but one minor prey item (2% of the overall diet) were detected across all samples. The same method was applied to scat samples from free-ranging bottlenose dolphins; up to seven prey taxa were detected in a single scat and 13 prey taxa from eight teleost families were identified in total. Conclusions/Significance: Data and further examples are provided to facilitate rapid transfer of this approach to any predator. This methodology should prove useful to zoologists using DNA-based diet techniques in a wide variety of study systems

Tidying Up International Nucleotide Sequence Databases: Ecological, Geographical and Sequence Quality Annotation of ITS Sequences of Mycorrhizal Fungi

Sequence analysis of the ribosomal RNA operon, particularly the internal transcribed spacer (ITS) region, provides a powerful tool for identification of mycorrhizal fungi. The sequence data deposited in the International Nucleotide Sequence Databases (INSD) are, however, unfiltered for quality and are often poorly annotated with metadata. To detect chimeric and low-quality sequences and assign the ectomycorrhizal fungi to phylogenetic lineages, fungal ITS sequences were downloaded from INSD, aligned within family-level groups, and examined through phylogenetic analyses and BLAST searches. By combining the fungal sequence database UNITE and the annotation and search tool PlutoF, we also added metadata from the literature to these accessions. Altogether 35,632 sequences belonged to mycorrhizal fungi or originated from ericoid and orchid mycorrhizal roots. Of these sequences, 677 were considered chimeric and 2,174 of low read quality. Information detailing country of collection, geographical coordinates, interacting taxon and isolation source were supplemented to cover 78.0%, 33.0%, 41.7% and 96.4% of the sequences, respectively. These annotated sequences are publicly available via UNITE (http://unite.ut.ee/) for downstream biogeographic, ecological and taxonomic analyses. In European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/), the annotated sequences have a special link-out to UNITE. We intend to expand the data annotation to additional genes and all taxonomic groups and functional guilds of fungi

Microbial Prevalence, Diversity and Abundance in Amniotic Fluid During Preterm Labor: A Molecular and Culture-Based Investigation

BACKGROUND: Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking. METHODS AND FINDINGS: In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r(2) = 0.42; P<0.002). CONCLUSIONS: The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship

Lack of Phylogeographic Structure in the Freshwater Cyanobacterium Microcystis aeruginosa Suggests Global Dispersal

Background : Free-living microorganisms have long been assumed to have ubiquitous distributions with little biogeographic signature because they typically exhibit high dispersal potential and large population sizes. However, molecular data provide contrasting results and it is far from clear to what extent dispersal limitation determines geographic structuring of microbial populations. We aimed to determine biogeographical patterns of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa. Being widely distributed on a global scale but patchily on a regional scale, this prokaryote is an ideal model organism to study microbial dispersal and biogeography. Methodology/Principal Findings : The phylogeography of M. aeruginosa was studied based on a dataset of 311 rDNA internal transcribed spacer (ITS) sequences sampled from six continents. Richness of ITS sequences was high (239 ITS types were detected). Genetic divergence among ITS types averaged 4% (maximum pairwise divergence was 13%). Preliminary analyses revealed nearly completely unresolved phylogenetic relationships and a lack of genetic structure among all sequences due to extensive homoplasy at multiple hypervariable sites. After correcting for this, still no clear phylogeographic structure was detected, and no pattern of isolation by distance was found on a global scale. Concomitantly, genetic differentiation among continents was marginal, whereas variation within continents was high and was mostly shared with all other continents. Similarly, no genetic structure across climate zones was detected. Conclusions/Significance : The high overall diversity and wide global distribution of common ITS types in combination with the lack of phylogeographic structure suggest that intercontinental dispersal of M. aeruginosa ITS types is not rare, and that this species might have a truly cosmopolitan distribution