Human gut microbiota can be studied through the characterization of microorganisms present in feces. Metaproteomics has arisen as a good approach to investigate this vast community. However, the ...processing of fecal samples in order to obtain the largest number of proteins from gut microbiota to be subsequently analyzed by means of metaproteomics is a challenge. Here we describe a protocol to approach this task. It includes two main steps: the first step of humectation and dispersion of the feces, followed by the separation of microorganisms from other fecal components such as roughage and food debris, and the second step in which microbial cells are broken up and microbiota proteins recovered for MS analysis. Detailed procedures for sample preparation, protein extraction, trypsin digestion, and mass spectrometry analysis for gut microbiota samples are provided.
Fine‐scale estimation of trophic interactions is an important subject in the field of ecology. Diet analysis based on fecal DNA metabarcoding has been accepted as a noninvasive, accurate, and time‐ ...and cost‐effective tool to determine animal diets. Here, we summarize the trends of fecal metabarcoding studies as well as methodological characteristics using 155 original papers published from 2009 to March 2020. We calculated the frequencies of the methods and conditions used in each experimental procedure and bioinformatics approach. Mammals were the major target taxa for fecal metabarcoding. A few methods or conditions dominated each procedure: sampling, DNA extraction, PCR, sequencing, and bioinformatics, which might be specialized for metabarcoding of degraded fecal DNA. However, the disadvantages of common methods were noted in some studies, and further optimizations are required to obtain more accurate dietary data with high taxonomic resolution and quantitative performance. This review will help fecal metabarcode users, especially new scientists who are considering using fecal metabarcoding in their studies, understand the process and common methods of fecal metabarcoding. We also hope this review will facilitate further technical improvements in this method.
We summarize the trends of fecal metabarcoding studies as well as methodological characteristics. Mammals were the major target taxa. A few methods or conditions dominated each procedure, which might be specialized for metabarcoding of degraded fecal DNA. However, further optimizations are required to obtain more accurate dietary data with high taxonomic resolution and quantitative performance.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We report a hospitalized patient with COVID-19 whose fecal samples turned negative 22 days later than the respiratory samples. It highlights that the duration of virus release from patients is longer ...than previously expected. Current clinical examinations for treatment and discharge standard are limited to respiratory samples. However, we believe that nucleic acid testing of both respiratory and fecal samples is necessary for discharged patients. Further studies are needed to confirm the potential for fecal-oral transmission or fecal-respiratory transmission via aerosols.
Livestock plays a crucial role in ensuring food security and driving the global economy. However, viral infections can have far-reaching consequences beyond economic productivity, affecting the ...health of cattle, as well as posing risks to human health and other animals. Identifying viruses present in fecal samples, a primary route of pathogen transmission, is essential for developing effective prevention, control, and surveillance strategies. Viral metagenomic approaches offer a broader perspective and hold great potential for detecting previously unknown viruses or uncovering previously undescribed agents. Ubaté Province is Colombia's dairy capital and a key center for livestock production in the country. Therefore, the purpose of this study was to characterize viral communities in fecal samples from cattle in this region. A total of 42 samples were collected from three municipalities in Ubaté Province, located in central Colombia, using a convenient non-probabilistic sampling method. We utilized metagenomic sequencing with Oxford Nanopore Technologies (ONT), combined with diversity and phylogenetic analysis. The findings revealed a consistent and stable viral composition across the municipalities, primarily comprising members of the Picornaviridae family. At the species level, the most frequent viruses were Enterovirus E (EVE) and Bovine Astrovirus (BoAstV). Significantly, this study reported, for the first time in Colombia, the presence of viruses with veterinary importance occurring at notable frequencies: EVE (59%), Bovine Kobuvirus (BKV) (52%), and BoAstV (19%). Additionally, the study confirmed the existence of Circular replicase-encoding single-stranded (CRESS) Virus in animal feces. These sequences were phylogenetically grouped with samples obtained from Asia and Latin America, underscoring the importance of having adequate representation across the continent. The virome of bovine feces in Ubaté Province is characterized by the predominance of potentially pathogenic viruses such as BoAstV and EVE that have been reported with substantial frequency and quantities. Several of these viruses were identified in Colombia for the first time. This study showcases the utility of using metagenomic sequencing techniques in epidemiological surveillance. It also paves the way for further research on the influence of these agents on bovine health and their frecuency across the country.
Display omitted
•First report of Bovine Astrovirus, Bovine Kobuvirus (BKV), and Enterovirus E in Colombian cattle.•A consistent and stable viral composition across sites.•BKV sequence phylogenetically clustered with Brazilian sequences.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The investigation of wildlife gastrointestinal microbiomes by next-generation sequencing approaches is a growing field in microbial ecology and conservation. Such studies often face difficulties in ...sample preservation if neither freezing facilities nor liquid nitrogen (LQN) are readily available. Thus, in order to prevent microbial community changes because of bacterial growth after sampling, preservation buffers need to be applied to samples. However, the amount of microbial community variation attributable to the different preservation treatments and potentially affecting biological interpretation is hardly known. Here, we sampled feces of 11 sheep (
sp.) by using swabs and analyzed the effect of air-drying, an inexpensive self-made nucleic acid preservation buffer (NAP), DNA/RNA Shield™, and RNA
®, each together with freezing (for 10 days) or storing at room temperature (for 10 days) prior to 16S rRNA gene high-throughput sequencing to determine bacterial communities. Results revealed that the proportions of operational taxonomic units (OTUs) belonging to a bacterial phylum were affected by the preservation treatments, and that alpha diversities observed OTUs, Shannon index, and phylogenetic diversity (PD) were lower in all preservation treatments than in samples taken by forensic swabs and immediately frozen which is considered as the favored preservation treatment in the absence of any logistic constraints. Overall, NAP had better preservation qualities than RNA
® and DNA/RNA Shield™ making this self-made buffer a valuable solution in wildlife microbiome studies.
The ruminant gastrointestinal tract (GIT) microbiome plays a major role in the health, physiology and production traits of the host. In this work, we characterized the bacterial and fungal microbiota ...of the rumen, small intestine (SI), cecum and feces of 27 Nelore steers using next-generation sequencing and evaluated biochemical parameters within the GIT segments. We found that only the bacterial microbiota clustered according to each GIT segment. Bacterial diversity and richness as well as volatile fatty acid concentration was lowest in the SI. Taxonomic grouping of bacterial operational taxonomic units (OTUs) revealed that
(24.61 ± SD 6.58%) and
(20.87 ± SD 4.22%) were the two most abundant taxa across the GIT. For the fungi, the family
dominated in all GIT segments, with the genus
being the most abundant. Twenty-eight bacterial and six fungal OTUs were shared across all GIT segments in at least 50% of the steers. We also evaluated if the fecal-associated microbiota of steers showing negative and positive residual feed intake (n-RFI and p-RFI, respectively) was associated with their feed efficiency phenotype. Diversity indices for both bacterial and fungal fecal microbiota did not vary between the two feed efficiency groups. Differences in the fecal bacterial composition between high and low feed efficiency steers were primarily assigned to OTUs belonging to the families
and
and to the genus
. The fungal OTUs shared across the GIT did not vary between feed efficiency groups, but 7 and 3 OTUs were found only in steers with positive and negative RFI, respectively. These results provide further insights into the composition of the Nelore GIT microbiota, which could have implications for improving animal health and productivity. Our findings also reveal differences in fecal-associated bacterial OTUs between steers from different feed efficiency groups, suggesting that fecal sampling may represent a non-invasive strategy to link the bovine microbiota with productivity phenotypes.
Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial ...and human proteins which might be used later for an easier diagnosis.
Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used.
Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase.
Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases.
Display omitted
•Fecal metaproteome analyses separated healthy controls from CD patients, UC patients and GCA patients.•Increase of NETs and IgG and decrease of IgA and transcriptional regulatory protein RprY from B. fragilis while CD and UC.•Identification of potential marker metaproteins for CD, UC, IBS and GCA, such as human enzyme sucrose-isomaltase for CD.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is ...of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3-V4 and the other targeting the V4-V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results.
Venomous viperid snakes possess relatively large and fragile hollow fangs that are an integral part of the envenomation apparatus for predation. We hypothesized that fangs serve like disposable ...needles and predicted a high loss rate and, hence, high replacement rate in free-ranging snakes. Snakes also possess smaller rear teeth that aid in gripping and swallowing the prey. We reasoned that these teeth are less delicate than fangs and predicted that their loss would be at a slower rate than fangs. To test our predictions, we analyzed fecal samples of free-ranging Saharan sand vipers, Cerastes vipera, in the Northern Negev desert, Israel. Close to 25% of fecal samples contained fangs, averaging more than one fang per sample and, consequently, our first prediction was supported. We estimated that fangs are replaced each fourth predation, and that replacement rate under natural conditions is at a high rate of approximately every twenty days. Fecal samples contained rear teeth at the same proportion as fangs, which indicated that the rapid replacement of teeth was not limited only to fangs and, therefore, our second prediction was not supported. These findings reflect the importance of both front fangs and rear teeth in the hunting of prey in free-ranging C. vipera. This is the first quantitative report of fang and rear teeth loss in a free-ranging viperid which is based on their recovery in feces; and we believe that similar high rates of loss occur in other viperid species.
•Close to 25% of fecal samples from free-living Cerastes vipera contained fangs.•There were 1.4–2.0 fangs in each sample that contained fangs.•Most fecal samples contained one prey item which indicated one predation.•On the average, fangs are replaced every fourth predation.•Rear teeth are replaced at the same proportion as fangs.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Norovirus is one of the most common causes of gastroenteritis, a disease characterized by diarrhea, vomiting, and stomach pain. A rapid on-site identification of the virus from fecal samples of ...patients is a prerequisite for accurate medical management. Here, we demonstrate a rapid nucleic acid-based detection platform as an on-site biosensing tool that can concentrate viruses from fecal samples. Moreover, it can perform RNA extraction and identification, and signal amplification using G-quadruplex and hemin containing DNA probes (G-DNA probes) and graphene oxide (GO)-coated microbeads. Briefly, murine noroviruses are lysed without chemicals on the surface of the GO microbeads. Subsequently, the target RNA is hybridized with G-DNA probes, and the resultant RNA/G-DNA probe complex is separated from unbound G-DNA probes using GO beads and is mixed with the detection buffer (ABTS/H2O2). Presence of murine noroviruses causes a colorimetric change of the buffer from colorless to green. Thus, we integrated all processes required to detect murine noroviruses in stool samples in a simple foldable microfluidic chip. Moreover, it can detect 101 pfu of the virus in 30 min in a fecal sample.
•We developed the portable point-of-care biosensor to detect murine norovirus from the fecal sample in the facile foldable microchip.•Using graphene oxide (GO)-coated microbeads and G-quadruplex containing DNA probe, target RNA was successfully extracted from murine norovirus, isolated from other feces debris and unknown nucleic acid fragments, and detected with high sensitivity.•101 pfu of the virus in the fecal samples could be detected in 30 min by simple on-site microfluidic chip platform.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
