Dataset Information

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species.

ABSTRACT: Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host-microbial interactions. Fecal DNA analysis can provide information about the host's microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers' instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples.

SUBMITTER: Eriksson P

PROVIDER: S-EPMC5678435 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species.

Eriksson Per P Mourkas Evangelos E González-Acuna Daniel D Olsen Björn B Ellström Patrik P

Infection ecology & epidemiology 20171026 1

<b>Introduction</b>: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host-microbial interactions. Fecal DNA analysis can provide information about the host's microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This stu ...[more]

PMID: 29152162

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Project description:BackgroundHost-associated microbes comprise an integral part of animal digestive systems and these interactions have a long evolutionary history. It has been hypothesized that the gastrointestinal microbiome of humans and other non-human primates may have played significant roles in host evolution by facilitating a range of dietary adaptations. We have undertaken a comparative sequencing survey of the gastrointestinal microbiomes of several non-human primate species, with the goal of better understanding how these microbiomes relate to the evolution of non-human primate diversity. Here we present a comparative analysis of gastrointestinal microbial communities from three different species of Old World wild monkeys.Methodology/principal findingsWe analyzed fecal samples from three different wild non-human primate species (black-and-white colobus [Colubus guereza], red colobus [Piliocolobus tephrosceles], and red-tailed guenon [Cercopithecus ascanius]). Three samples from each species were subjected to small subunit rRNA tag pyrosequencing. Firmicutes comprised the vast majority of the phyla in each sample. Other phyla represented were Bacterioidetes, Proteobacteria, Spirochaetes, Actinobacteria, Verrucomicrobia, Lentisphaerae, Tenericutes, Planctomycetes, Fibrobacateres, and TM7. Bray-Curtis similarity analysis of these microbiomes indicated that microbial community composition within the same primate species are more similar to each other than to those of different primate species. Comparison of fecal microbiota from non-human primates with microbiota of human stool samples obtained in previous studies revealed that the gut microbiota of these primates are distinct and reflect host phylogeny.Conclusion/significanceOur analysis provides evidence that the fecal microbiomes of wild primates co-vary with their hosts, and that this is manifested in higher intraspecies similarity among wild primate species, perhaps reflecting species specificity of the microbiome in addition to dietary influences. These results contribute to the limited body of primate microbiome studies and provide a framework for comparative microbiome analysis between human and non-human primates as well as a comparative evolutionary understanding of the human microbiome.

Project description:The gut microbiota contributes to host health by maintaining homeostasis and improving digestive efficiency. Therefore, identifying gut microbes will shed light on the annual life cycle of animals and in particular those that are threatened or endangered. Nonetheless, the gut microbial composition of the majority of bird species is still unknown. Here, for the first time, 16S rRNA gene sequencing was used to characterize and compare the community composition and diversity of gut microbiotas from six species of birds raised at the Wildlife Conservation Center in Baotou, China: relict gull (Larus relictus; n = 3), muscovy duck (Cairina moschata; n = 3), ruddy shelduck (Tadorna ferruginea; n = 3), demoiselle crane (Anthropoides virgo; n = 4), whooper swan (Cygnus cygnus; n = 3), and black swan (Cygnus atratus; n = 5). A total of 26,616 operational taxonomic units from 21 samples were classified into 32 phyla and 507 genera. Chao1, Shannon diversity, observed species, and Simpson index analysis revealed differences in the community richness and diversity between the different species. Proteobacteria was the dominant bacterial phylum in whooper swan and relict gull, whereas Firmicutes was the dominant bacterial phylum in the other species. At the genus level, 11 dominant genera were detected (Lactobacillus, Psychrobacter, Enterococcus, Carnobacterium, Weissella, Burkholderia, Escherichia/Shigella, Leuconostoc, Buttiauxella, Desemzia, and Staphylococcus). Principal component and cluster analyses revealed that, while the microbial community composition of different individuals of the same species clustered together, the gut microbial composition varied between the bird species. Furthermore, the most abundant bacterial species differed between bird species. Because many avian gut microbes are derived from the diet, the eating habits and natural living environment of birds may be important contributing factors to the observed differences. Short-term changes to the diet and living environment have little effect on the composition of the avian gut microbiota. This study provides a theoretical basis for bird protection, including disease prevention and control.

Project description:IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS.

Dataset Information

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species.

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Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species.

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