Fecal Calprotectin Is Increased in Stroke

Background: While there have been major advances in unveiling the mechanisms comprising the ischemic cascade of CNS, stroke continues to be a significant burden. There is a need to extend the focus toward peripheral changes, and the brain–gut axis has recently gained much attention. Our study aimed to evaluate gut inflammation and its association with blood variables in stroke using fecal calprotectin (FC). Methods: Fecal samples were obtained from 27 stroke patients and 27 control subjects. FC was quantitatively measured using a commercial ELISA. Laboratory data on the fecal sample collection were also collected, including CBC, ESR, glucose, creatinine, total protein, albumin, transaminases, and CRP. Results: There was a significant increase in FC levels in stroke patients compared to the controls. Furthermore, FC in stroke patients was negatively correlated with the Glasgow Coma Scale. Moreover, FC in stroke patients was positively correlated with CRP and negatively correlated with lymphocyte count and albumin. Conclusions: Our findings show that increased FC is associated with consciousness and systemic response in stroke and warrants further studies to elucidate the usefulness of FC in the management of stroke.

Predation of the Japanese keelback (Hebius vibakari Boie, 1826) by the Slender racer (Orientocoluber spinalis Peters, 1866)

Background The Slender racer (Orientocoluber spinalis Peters, 1866) has recently been reclassified to the new genus Orientocoluber from Hierophis. Ecological knowledge of this species is limited due to its highly mobile behavior. On 17 July 2020, we captured a female O. spinalis on Oeyeon Island, Boryeong-si, Republic of Korea, and collected its feces for a diet analysis. We observed snake scales from the collected feces and subsequently determined the prey species through morphological and molecular methods. Results We initially hypothesized that the extracted fecal sample scales belonged to H. vibakari, due to their thin keel and rhombus shape. We also amplified H. vibakari DNA from the extracted fecal sample using Illumina sequencing methods. Our morphological and molecular results suggest that O. spinalis predates H. vibakari on Oeyeon Island. Conclusion This is the first report of O. spinalis predating another snake species, ophiophagy, and implies that H. vibakari may be a crucial prey item for O. spinalis on Oeyeon Island.

Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

COVID-19 patients shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. In our benchmarking study, we recommend a standardized protocol for the preservation, extraction and detection of viral RNA from stool. This protocol includes a preservative, viral RNA extraction steps, and PCR-based quantification methods to maximize yield and detection of SARS-CoV-2 RNA. Our protocol takes advantage of commercially available reagents and equipment to maximize ease of access and consistency across studies. Additionally, we apply an attenuated bovine coronavirus vaccine as a spike-in control, and synthetic RNA standards to improve standardization and reliability of the assay. While we recommend both ddPCR and RT-qPCR-based assays, we acknowledge that ddPCR may be prohibitively expensive due to the necessity of specialized equipment and reagents. This protocol was developed with a focus on SARS-CoV-2 RNA, but may apply to other coronaviruses as well. We estimate that this protocol takes between 6 to 8 hours total to quantify the viral RNA load in a fecal sample.

Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics

Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug-microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at -80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

Assessment of Biolog EcoplateTM method for functional metabolic diversity of aerotolerant pig fecal microbiota

In the last decades, gut microbiota and its role in mammal host development and health have been increasingly investigated. Metabolites produced by gut microbiota can affect intestinal homeostasis and immune system maturity and activation, and in turn, they can influence the health and growth performance of livestock. Therefore, a better understanding of the functional metabolic capability of the gut microbiota would be appreciated by the scientific community. In this study, the BiologTM Ecoplates technology was applied for studying the metabolic potential of the aerotolerant microbial community of pig fecal samples, evaluating the interference of different storage conditions and cell concentrations. The length of time for which a fecal sample maintained detectable and unchanged microbial metabolic activity was also investigated. Two assays aimed to evaluate differences in the metabolic activities between fresh and snap-frozen fecal samples at different dilutions and at different lengths of times of preservation at −80°C were carried out. The biodiversity and the predicted functionality of the entire bacterial community through a targeted metagenomic approach were also explored. The results highlighted that snap freezing of fecal samples preserved the metabolic activity of the microbial community when compared to fresh feces. Sample storage at −80 °C did not significantly affect the metabolic activity of the microbial community, which was stable for 150 days. Furthermore, the highest metabolic activity was detected with 1:2 to 1:5 dilutions of the stock suspension. BiologTM Ecoplates technology is a rapid and useful method to explore microbial communities’ metabolism in animal fecal samples contributing to investigate host animal physiology. Key points • Freezing of samples can preserve the functional activity of the aerotolerant microbial community for 150 days. • The concentration of microbial cells strongly influences metabolic activity detection. • Sequencing coupled with the BiologTMEcoplates could be a strategy to evaluate the metabolic potential of the microbiota of the fecal sample. Graphical abstract

W27 IgA suppresses growth of Escherichia in an in vitro model of the human intestinal microbiota

W27 monoclonal immunoglobulin A (IgA) suppresses pathogenic Escherichia coli cell growth; however, its effect on the human intestine remains unclear. We aimed to determine how W27 IgA affects the human colonic microbiota using the in vitro microbiota model. This model was established using fecal samples collected from 12 healthy volunteers; after anaerobic cultivation, each model was found to retain the genera found in the original human fecal samples. After pre-incubating W27 IgA with the respective fecal sample under aerobic conditions, the mixture of W27 IgA (final concentration, 0.5 μg/mL) and each fecal sample was added to the in vitro microbiota model and cultured under anaerobic conditions. Next-generation sequencing of the bacterial 16S rRNA gene revealed that W27 IgA significantly decreased the relative abundance of bacteria related to the genus Escherichia in the model. Additionally, at a final concentration of 5 μg/mL, W27 IgA delayed growth in the pure culture of Escherichia coli isolated from human fecal samples. Our study thus revealed the suppressive effect of W27 IgA on the genus Escherichia at relatively low-concentrations and the usefulness of an in vitro microbiota model to evaluate the effect of IgA as a gut microbiota regulator.

Fecal sample collection methods and time of day impact microbiome composition and short chain fatty acid concentrations

Associations between the human gut microbiome and health outcomes continues to be of great interest, although fecal sample collection methods which impact microbiome studies are sometimes neglected. Here, we expand on previous work in sample optimization, to promote high quality microbiome data. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. We demonstrated that spot sampling of stool results in variable detection of some microbial members, and inconsistent levels of SCFA; therefore, sample homogenization prior to subsequent analysis or subsampling is recommended. We also identify a trend in microbial and metabolite composition that shifts over two consecutive stool collections less than 25 h apart. Lastly, we show significant differences in bacterial composition that result from collecting stool samples in OMNIgene·Gut tube (DNA Genotec) or Stool Nucleic Acid Collection and Preservation Tube (NORGEN) compared to immediate freezing. To assist with planning fecal sample collection and storage procedures for microbiome investigations with multiple analyses, we recommend participants to collect the first full bowel movement of the day and freeze the sample immediately after collection.