Gut microbiome community analysis is used to understand many diseases like inflammatory bowel dis... more Gut microbiome community analysis is used to understand many diseases like inflammatory bowel disease, obesity, and diabetes. Sampling methods are an important consideration for human microbiome research, yet are not emphasized in many studies. In this study, we demonstrate that the preparation, handling, and storage of human faeces are critical processes that alter the outcomes of downstream DNA-based bacterial community analyses via qPCR. We found that stool subsampling resulted in large variability of gut microbiome data due to different microenvironments harbouring various taxa within an individual stool. However, we reduced intra-sample variability by homogenizing the entire stool sample in liquid nitrogen and subsampling from the resulting crushed powder prior to DNA extraction. We experimentally determined that the bacterial taxa varied with room temperature storage beyond 15 minutes and beyond three days storage in a domestic frost-free freezer. While freeze thawing only had an effect on bacterial taxa abundance beyond four cycles, the use of samples stored in RNAlater should be avoided as overall DNA yields were reduced as well as the detection of bacterial taxa. Overall we provide solutions for processing and storing human stool samples that reduce variability of microbiome data. We recommend that stool is frozen within 15 minutes of being defecated, stored in a domestic frost-free freezer for less than three days, and homogenized prior to DNA extraction. Adoption of these simple protocols will have a significant and positive impact on future human microbiome research.
Our previous studies revealed that offspring from rat dams fed fish oil (at 8% and 18% energy), d... more Our previous studies revealed that offspring from rat dams fed fish oil (at 8% and 18% energy), developed impaired intestinal barriers sensitizing the colon to exacerbated injury later in life. To discern the mechanism, we hypothesized that in utero exposure to fish oil, rich in n-3 polyunsaturated fatty acid (PUFA), caused abnormal intestinal reparative responses to mucosal injury through differences in intestinal microbiota and the presence of naïve immune cells. To identify such mechanisms, gut microbes and naïve immune cells were compared between rat pups born to dams fed either n-6 PUFA, n-3 PUFA or breeder chow. Maternal exposure to either of the PUFA rich diets altered the development of the intestinal microbiota with an overall reduction in microbial density. Using qPCR, we found that each type of PUFA differentially altered the major gut phyla; fish oil increased Bacteroidetes and safflower oil increased Firmicutes. Both PUFA diets reduced microbes known to dominate the inf...
Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious d... more Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious disease susceptibility remains unclear. Deciphering lipid-induced immunity is critical to understanding the balance required for protecting against pathogens while avoiding chronic inflammatory diseases. To understand how specific lipids alter susceptibility to enteric infection, we fed mice isocaloric, high-fat diets composed of corn oil (rich in n-6 polyunsaturated fatty acids [n-6 PUFAs]), olive oil (rich in monounsaturated fatty acids), or milk fat (rich in saturated fatty acids) with or without fish oil (rich in n-3 PUFAs). After 5 weeks of dietary intervention, mice were challenged with Citrobacter rodentium, and pathological responses were assessed. Olive oil diets resulted in little colonic pathology associated with intestinal alkaline phosphatase, a mucosal defense factor that detoxifies lipopolysaccharide. In contrast, while both corn oil and milk fat diets resulted in inflammation-induced colonic damage, only milk fat induced compensatory protective responses, including short chain fatty acid production. Fish oil combined with milk fat, unlike unsaturated lipid diets, had a protective effect associated with intestinal alkaline phosphatase activity. Overall, these results reveal that dietary lipid type, independent of the total number of calories associated with the dietary lipid, influences the susceptibility to enteric damage and the benefits of fish oil during infection.
Gut microbiome community analysis is used to understand many diseases like inflammatory bowel dis... more Gut microbiome community analysis is used to understand many diseases like inflammatory bowel disease, obesity, and diabetes. Sampling methods are an important consideration for human microbiome research, yet are not emphasized in many studies. In this study, we demonstrate that the preparation, handling, and storage of human faeces are critical processes that alter the outcomes of downstream DNA-based bacterial community analyses via qPCR. We found that stool subsampling resulted in large variability of gut microbiome data due to different microenvironments harbouring various taxa within an individual stool. However, we reduced intra-sample variability by homogenizing the entire stool sample in liquid nitrogen and subsampling from the resulting crushed powder prior to DNA extraction. We experimentally determined that the bacterial taxa varied with room temperature storage beyond 15 minutes and beyond three days storage in a domestic frost-free freezer. While freeze thawing only had an effect on bacterial taxa abundance beyond four cycles, the use of samples stored in RNAlater should be avoided as overall DNA yields were reduced as well as the detection of bacterial taxa. Overall we provide solutions for processing and storing human stool samples that reduce variability of microbiome data. We recommend that stool is frozen within 15 minutes of being defecated, stored in a domestic frost-free freezer for less than three days, and homogenized prior to DNA extraction. Adoption of these simple protocols will have a significant and positive impact on future human microbiome research.
Our previous studies revealed that offspring from rat dams fed fish oil (at 8% and 18% energy), d... more Our previous studies revealed that offspring from rat dams fed fish oil (at 8% and 18% energy), developed impaired intestinal barriers sensitizing the colon to exacerbated injury later in life. To discern the mechanism, we hypothesized that in utero exposure to fish oil, rich in n-3 polyunsaturated fatty acid (PUFA), caused abnormal intestinal reparative responses to mucosal injury through differences in intestinal microbiota and the presence of naïve immune cells. To identify such mechanisms, gut microbes and naïve immune cells were compared between rat pups born to dams fed either n-6 PUFA, n-3 PUFA or breeder chow. Maternal exposure to either of the PUFA rich diets altered the development of the intestinal microbiota with an overall reduction in microbial density. Using qPCR, we found that each type of PUFA differentially altered the major gut phyla; fish oil increased Bacteroidetes and safflower oil increased Firmicutes. Both PUFA diets reduced microbes known to dominate the inf...
Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious d... more Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious disease susceptibility remains unclear. Deciphering lipid-induced immunity is critical to understanding the balance required for protecting against pathogens while avoiding chronic inflammatory diseases. To understand how specific lipids alter susceptibility to enteric infection, we fed mice isocaloric, high-fat diets composed of corn oil (rich in n-6 polyunsaturated fatty acids [n-6 PUFAs]), olive oil (rich in monounsaturated fatty acids), or milk fat (rich in saturated fatty acids) with or without fish oil (rich in n-3 PUFAs). After 5 weeks of dietary intervention, mice were challenged with Citrobacter rodentium, and pathological responses were assessed. Olive oil diets resulted in little colonic pathology associated with intestinal alkaline phosphatase, a mucosal defense factor that detoxifies lipopolysaccharide. In contrast, while both corn oil and milk fat diets resulted in inflammation-induced colonic damage, only milk fat induced compensatory protective responses, including short chain fatty acid production. Fish oil combined with milk fat, unlike unsaturated lipid diets, had a protective effect associated with intestinal alkaline phosphatase activity. Overall, these results reveal that dietary lipid type, independent of the total number of calories associated with the dietary lipid, influences the susceptibility to enteric damage and the benefits of fish oil during infection.
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