Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA.
medRxiv : the preprint server for health sciences
COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.
View details for DOI 10.1101/2021.04.10.21255250
View details for PubMedID 33880485
Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA.
2021; 12 (1): 5753
Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.
View details for DOI 10.1038/s41467-021-25576-6
View details for PubMedID 34599164
Gut microbiota impairs insulin clearance in obese mice.
OBJECTIVE: Hyperinsulinemia can be both a cause and consequence of obesity and insulin resistance. Hyperinsulinemia can result from increased insulin secretion and/or reduced insulin clearance. While many studies have focused on mechanisms triggering insulin secretion during obesity, the triggers for changes in insulin clearance during obesity are less defined. Here, we investigated the role of the microbiota in regulating insulin clearance during diet-induced obesity.METHODS: Blood glucose and insulin clearance were tested in conventional male mice treated with antibiotics and in germ-free mice colonized with microbes from mice that were fed control (chow) diet or an obesogenic high fat diet (HFD). The composition of the fecal microbiota was analyzed using 16S rRNA sequencing.RESULTS: Short-term HFD feeding and aging did not alter insulin clearance in mice. Oral antibiotics mitigated impaired blood insulin clearance in mice fed HFD for 12 weeks or longer. Germ-free mice colonized with microbes from HFD-fed donor mice had impaired insulin, but not C-peptide, clearance. Microbe-transmissible insulin clearance impairment was only observed in germ free mice after more than 6 weeks post-colonization upon HFD feeding. Five bacterial taxa predicted >90% of the variance in insulin clearance. Mechanistically, impaired insulin clearance was associated with lower levels of hepatic Ceacam-1, but increased activity of liver and skeletal muscle insulin degrading enzyme (IDE).CONCLUSIONS: Gut microbes regulate insulin clearance during diet-induced obesity. A small cluster of microbes, or their metabolites, may be targeted for mitigating defects in insulin clearance and hyperinsulinemia during the progression of obesity and type 2 Diabetes.
View details for DOI 10.1016/j.molmet.2020.101067
View details for PubMedID 32860984
NOD2 in hepatocytes engages a liver-gut axis to protect against steatosis, fibrosis, and gut dysbiosis during fatty liver disease in mice.
American journal of physiology. Endocrinology and metabolism
Obesity promotes non-alcoholic fatty liver disease (NAFLD). The intestinal microbiota contributes to NAFLD progression through a gut to liver pathway that promotes inflammation and fibrosis. Gut microbiota-derived factors can travel to the liver and activate immune responses in liver-resident cells to promote inflammation and NAFLD. Little is known about bacterial sensors or immune responses that can protect against NAFLD. We tested if the bacterial cell wall sensor nucleotide-binding oligomerization domain-containing (NOD)2 protects against diet-induced NAFLD in mice. Whole-body deletion of NOD2 exacerbated liver steatosis and fibrosis in mice fed a NAFLD-promoting diet. Mice with a hepatocyte-specific deletion of NOD2 (Nod2-/-HKO) also had higher liver steatosis and fibrosis compared to littermate wild type mice (WTloxp) fed a NAFLD-promoting diet. Hepatocyte-specific NOD2 deletion altered the composition of the gut microbiome. Nod2-/-HKO mice had increased relative abundance of Clostridiales and lower Erysipelotrichaceae among other changes in cecal bacteria compared to littermate WTloxp mice. Hepatocyte-specific NOD2 deletion altered a transcriptional program of liver inflammation, metabolism, and fibrosis. Nod2-/-HKO mice had higher levels of transcripts involved in lipid and cholesterol metabolism. Nod2-/-HKO mice had higher transcript levels of transforming growth factor beta and collagen isoforms, which coincided with higher levels of liver collagen compared to WTloxp mice. These data show that bacterial cell wall sensing within hepatocytes can engage retrograde crosstalk from the liver to the gut, where liver immunity communicates with the gut to influence the intestinal host-microbe relationship during diet-induced NAFLD, and NOD2 within the hepatocyte confers protection from liver steatosis and fibrosis.
View details for DOI 10.1152/ajpendo.00181.2020
View details for PubMedID 32516028
The gastrointestinal microbiota controls cancer cell intrinsic mechanisms to promote the progression of acute lymphoblastic leukemia.
AMER ASSOC CANCER RESEARCH. 2020: 58–59
View details for Web of Science ID 000526057000081
Strain-resolved microbiome sequencing reveals mobile elements that drive bacterial competition on a clinical timescale.
2020; 12 (1): 50
Populations of closely related microbial strains can be simultaneously present in bacterial communities such as the human gut microbiome. We recently developed a de novo genome assembly approach that uses read cloud sequencing to provide more complete microbial genome drafts, enabling precise differentiation and tracking of strain-level dynamics across metagenomic samples. In this case study, we present a proof-of-concept using read cloud sequencing to describe bacterial strain diversity in the gut microbiome of one hematopoietic cell transplantation patient over a 2-month time course and highlight temporal strain variation of gut microbes during therapy. The treatment was accompanied by diet changes and administration of multiple immunosuppressants and antimicrobials.We conducted short-read and read cloud metagenomic sequencing of DNA extracted from four longitudinal stool samples collected during the course of treatment of one hematopoietic cell transplantation (HCT) patient. After applying read cloud metagenomic assembly to discover strain-level sequence variants in these complex microbiome samples, we performed metatranscriptomic analysis to investigate differential expression of antibiotic resistance genes. Finally, we validated predictions from the genomic and metatranscriptomic findings through in vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples.During the 56-day longitudinal time course that was studied, the patient's microbiome was profoundly disrupted and eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allowed us to identify differences in substrain populations over time. Based on this, we predicted that particular mobile element integrations likely resulted in increased antibiotic resistance, which we further supported using in vitro antibiotic susceptibility testing.We find read cloud assembly to be useful in identifying key structural genomic strain variants within a metagenomic sample. These strains have fluctuating relative abundance over relatively short time periods in human microbiomes. We also find specific structural genomic variations that are associated with increased antibiotic resistance over the course of clinical treatment.
View details for DOI 10.1186/s13073-020-00747-0
View details for PubMedID 32471482
Adipose Tissue Inflammation Is Directly Linked to Obesity-Induced Insulin Resistance, while Gut Dysbiosis and Mitochondrial Dysfunction Are Not Required.
Function (Oxford, England)
2020; 1 (2): zqaa013
Obesity is associated with adipose tissue hypertrophy, systemic inflammation, mitochondrial dysfunction, and intestinal dysbiosis. Rodent models of high-fat diet (HFD)-feeding or genetic deletion of multifunctional proteins involved in immunity and metabolism are often used to probe the etiology of obesity; however, these models make it difficult to divorce the effects of obesity, diet composition, or immunity on endocrine regulation of blood glucose. We, therefore, investigated the importance of adipose inflammation, mitochondrial dysfunction, and gut dysbiosis for obesity-induced insulin resistance using a spontaneously obese mouse model. We examined metabolic changes in skeletal muscle, adipose tissue, liver, the intestinal microbiome, and whole-body glucose control in spontaneously hyperphagic C57Bl/6J mice compared to lean littermates. A separate subset of lean and obese mice was subject to 8 weeks of obesogenic HFD feeding, or to pair feeding of a standard rodent diet. Hyperphagia, obesity, adipose inflammation, and insulin resistance were present in obese mice despite consuming a standard rodent diet, and these effects were blunted with caloric restriction. However, hyperphagic obese mice had normal mitochondrial respiratory function in all tissues tested and no discernable intestinal dysbiosis relative to lean littermates. In contrast, feeding mice an obesogenic HFD altered the composition of the gut microbiome, impaired skeletal muscle mitochondrial bioenergetics, and promoted poor glucose control. These data show that adipose inflammation and redox stress occurred in all models of obesity, but gut dysbiosis and mitochondrial respiratory dysfunction are not always required for obesity-induced insulin resistance. Rather, changes in the intestinal microbiome and mitochondrial bioenergetics may reflect physiological consequences of HFD feeding.
View details for DOI 10.1093/function/zqaa013
View details for PubMedID 34278304
Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens.
To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2-5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6-9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.
View details for DOI 10.1038/s41564-019-0595-2
View details for PubMedID 31659298
Large-Scale Analyses of Human Microbiomes Reveal Thousands of Small, Novel Genes.
Small proteins are traditionally overlooked due to computational and experimental difficulties in detecting them. To systematically identify small proteins, we carried out a comparative genomics study on 1,773 human-associated metagenomes from four different body sites. We describe >4,000 conserved protein families, the majority of which are novel; 30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain and almost half are not represented in reference genomes. We identify putative housekeeping, mammalian-specific, defense-related, and protein families that are likely to be horizontally transferred. We provide evidence of transcription and translation for a subset of these families. Our study suggests that small proteins are highly abundant and those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.
View details for DOI 10.1016/j.cell.2019.07.016
View details for PubMedID 31402174
Long term but not short term exposure to obesity related microbiota promotes host insulin resistance.
2018; 9 (1): 4681
The intestinal microbiota and insulin sensitivity are rapidly altered after ingestion of obesogenic diets. We find that changes in the composition of the fecal microbiota precede changes in glucose tolerance when mice are fed obesogenic, low fiber, high fat diets (HFDs). Antibiotics alter glycemia during the first week of certain HFDs, but antibiotics show a more robust improvement in glycemic control in mice with protracted obesity caused by long-term feeding of multiple HFDs. Microbiota transmissible dysglycemia and glucose intolerance only occur when germ-free mice are exposed to obesity-related microbes for more than 45 days. We find that sufficient host exposure time to microbiota derived from HFD-fed mice allows microbial factors to contribute to insulin resistance, independently from increased adiposity in mice. Our results are consistent with intestinal microbiota contributing to chronic insulin resistance and dysglycemia during prolonged obesity, despite rapid diet-induced changes in the taxonomic composition of the fecal microbiota.
View details for PubMedID 30409977