Project description:In studies of the human microbiome, faecal samples are frequently used as a non-invasive proxy for the study of the intestinal microbiota. To obtain reliable insights, the need for bacterial DNA of high quality and integrity following appropriate faecal sample collection and preservation steps is paramount. In a study of dietary mineral balance in the context of type 2 diabetes (T2D), faecal samples were collected from healthy and T2D individuals throughout a 13-day residential trial. These samples were freeze-dried, then stored mostly at -20°C from the trial date in 2000/2001 until the current research in 2014. Given the relative antiquity of these samples (~14 years), we sought to evaluate DNA quality and comparability to freshly collected human faecal samples. Following the extraction of bacterial DNA, gel electrophoresis indicated that our DNA extracts were more sheared than extracts made from freshly collected faecal samples, but still of sufficiently high molecular weight to support amplicon-based studies. Likewise, spectrophotometric assessment of extracts revealed that they were of high quality and quantity. A subset of bacterial 16S rRNA gene amplicons were sequenced using Illumina MiSeq and compared against publicly available sequence data representing a similar cohort analysed by the American Gut Project (AGP). Notably, our bacterial community profiles were highly consistent with those from the AGP data. Our results suggest that when faecal specimens are stored appropriately, the microbial profiles are preserved and robust to extended storage periods.
Project description:Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to -80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants' homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to -80 °C. Refrigeration at 4 °C, storage at ambient temperature, and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to -80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.
Project description:A novel Gram-stain-positive, coccus-shaped, obligately anaerobic bacterium was isolated from a faecal sample obtained from an individual in a traditional community located off the southern coast of Peru. Comparative 16S rRNA gene sequence analysis showed the novel bacterium belonged to the genus Peptoniphilus but showed no particular relationship with any species, demonstrating less than 91?% 16S rRNA gene sequence similarity with all members of the genus. The major cellular fatty acids of the novel isolate were determined to be C10?:?0, C14?:?0, C16?:?0, C18?:?1?9c and C18?:?2?6,9c/anteiso-C18?:?0. The DNA G+C content was 34.4?mol%. End-products of metabolism from peptone-yeast-glucose broth (PYG) were determined to be acetate and butyrate. Based on the phenotypic, chemotaxonomic and phylogenetic results, the organism represents a novel species of the genus Peptoniphilus, for which the name Peptoniphilus catoniae sp. nov. is proposed. The type strain is M6.X2DT (?=?DSM 29874T?=?CCUG 66798T).
Project description:The gut microbiome has a fundamental role in human health and disease. However, studying the complex structure and function of the gut microbiome using next generation sequencing is challenging and prone to reproducibility problems. Here, we obtained cross-sectional colon biopsies and faecal samples from nine participants in our COLSCREEN study and sequenced them in high coverage using Illumina pair-end shotgun (for faecal samples) and IonTorrent 16S (for paired feces and colon biopsies) technologies. The metagenomes consisted of between 47 and 92 million reads per sample and the targeted sequencing covered more than 300 k reads per sample across seven hypervariable regions of the 16S gene. Our data is freely available and coupled with code for the presented metagenomic analysis using up-to-date bioinformatics algorithms. These results will add up to the informed insights into designing comprehensive microbiome analysis and also provide data for further testing for unambiguous gut microbiome analysis.
Project description:Vancomycin-resistant Enterococcus faecalis, E. faecium and E. durans isolates with the genotype vanA were detected in 7 of 118 faecal samples (5.9%) of natural gilthead seabream recovered off the coast of Portugal, and one vancomycin-resistant isolate/sample was further characterized. The genes erm(B), tet(L), tet(M), aac(6')-aph(2"), aph(3')-IIIa and/or ant(6)-Ia were identified in most of the 7 vancomycin-resistant enterococci. Sequence types ST273, ST313 and ST76 were detected in three E. faecium isolates and ST6 in two E. faecalis isolates. VanA-containing enterococci are suggested to be disseminated in fish in marine ecosystems close to areas of human activity.
Project description:African apes are endemically infected with numerous Plasmodium spp. including close relatives of human Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Although these ape parasites are not believed to pose a zoonotic threat, their ability to colonise humans has not been fully explored. In particular, it remains unknown whether ape parasites are able to initiate exo-erythrocytic replication in human hepatocytes following the bite of an infective mosquito. Since animal studies have shown that liver stage infection can result in the excretion of parasite nucleic acids into the bile, we screened faecal samples from 504 rural Cameroonians for Plasmodium DNA. Using pan-Laverania as well as P. malariae- and P. vivax-specific primer sets, we amplified human P. falciparum (n?=?14), P. malariae (n?=?1), and P. ovale wallikeri (n?=?1) mitochondrial sequences from faecal DNA of 15 individuals. However, despite using an intensified PCR screening approach we failed to detect ape Laverania, ape P. vivax or ape P. malariae parasites in these same subjects. One faecal sample from a hunter-gatherer contained a sequence closely related to the porcupine parasite Plasmodium atheruri. Since this same faecal sample also contained porcupine mitochondrial DNA, but a matching blood sample was Plasmodium-negative, it is likely that this hunter-gatherer consumed Plasmodium-infected bushmeat. Faecal Plasmodium detection was not secondary to intestinal bleeding and/or infection with gastrointestinal parasites, but indicative of blood parasitaemia. Quantitative PCR identified 26-fold more parasite DNA in the blood of faecal Plasmodium-positive than faecal Plasmodium-negative individuals (P?=?0.01). However, among blood-positive individuals only 10% - 20% had detectable Plasmodium sequences in their stool. Thus, faecal screening of rural Cameroonians failed to uncover abortive ape Plasmodium infections, but detected infection with human parasites, albeit with reduced sensitivity compared with blood analysis.
Project description:Clinical trial faecal collections present challenges through geographical spread and inexperienced participants. Collection techniques have been developed and tested to overcome these challenges, but previous studies investigating these techniques have demonstrated a highly variable capacity for sample preservation. Furthermore, these studies typically only examine either preservation of genetic content or metabolites, not both. This study investigated the Stool Nucleic Acid Collection and Preservation Tube (Norgen BioTek Corp) for the preservation of both microbial DNA and microbial organic acid metabolites in human faecal samples when compared to frozen samples. Twenty six healthy adult participants were instructed to collect a bowel movement, subsample into collection tubes and immediately transfer the remaining bulk to -20 °C storage. Resulting organic acid concentrations remained comparable across methods when the preservation tubes were used correctly. The 16S rRNA gene sequencing data revealed twenty significantly different bacterial genera between the two collection methods. Ten Gram-negative genera were more abundant in the collection tubes, and ten Gram-positive genera were more abundant in the fresh frozen samples. This study has illustrated that faecal collection methods bias the microbial community profile according to Gram status and this should be considered when designing studies that collect and store human faecal samples.