Differences in gut microbiota profile between women with active lifestyle and sedentary women methods
Aim. Evidence-backed execution summary for Differences in gut microbiota profile between women with active lifestyle and sedentary women methods from Differences in gut microbiota profile between women with active lifestyle and sedentary women.
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human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Stool collection, DNA extraction and sequencing
reagent used in the protocol.
- Use
- Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was me...
Sequencing and bioinformatics
reagent used in the protocol.
- Use
- Microbiota composition analysis of fecal samples was performed by amplifying the hypervariable regions V3 and V4 of the 16s rRNA gene using the primer pair 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' [ ] to generate amplicons of 459 bp that were sequenced on a MiS...
Quantitative PCR analysis
reagent used in the protocol.
- Use
- Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 m...
Enzymatic fecal activity
reagent used in the protocol.
- Use
- Fecal enzymatic activity was determined using the API-ZYM system (Biomerieux, Marcy l'Etoile, France) according to the method described by Delgado et al. for fecal samples [ ] and the manufacturer´s instructions. The API-ZYM system is a semi-quantitative method that determines 19 enzyme activities: alkaline pho...
Fecal enzymatic activity
reagent used in the protocol.
- Use
- Bacterial enzymes in the intestine are involved in the metabolism of foods and drugs, and changes to the composition of intestinal bacterial populations may result in modifications to the bacterial enzyme profile that could impact host health status. Accordingly, enzymatic activities of fecal samples were compared b...
Physical activity and sedentary time determination
Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). This allowed us to verify in an objective manner the presorting of sedentary and active individuals as it determines body movements in all thr...
- Use
- Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). This allowed us to verify in an objective manner the presorting of sedentary and active individuals as it determines body movements in all thr...
Food frequency questionnaire
Dietary pattern characterization of the participants was carried out using a food frequency questionnaire (FFQ) with 97 food items. The FFQ was given to participants to complete the day they donated the fecal sample. Data from the FFQ were analyzed using Dietsource software 3.0 (Novartis, Barcelona, Spain) to obtain...
- Use
- Dietary pattern characterization of the participants was carried out using a food frequency questionnaire (FFQ) with 97 food items. The FFQ was given to participants to complete the day they donated the fecal sample. Data from the FFQ were analyzed using Dietsource software 3.0 (Novartis, Barcelona, Spain) to obtain...
Stool collection, DNA extraction and sequencing
Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was me...
- Use
- Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was me...
Sequencing and bioinformatics
Microbiota composition analysis of fecal samples was performed by amplifying the hypervariable regions V3 and V4 of the 16s rRNA gene using the primer pair 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' [ ] to generate amplicons of 459 bp that were sequenced on a MiS...
- Use
- Microbiota composition analysis of fecal samples was performed by amplifying the hypervariable regions V3 and V4 of the 16s rRNA gene using the primer pair 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' [ ] to generate amplicons of 459 bp that were sequenced on a MiS...
Quantitative PCR analysis
Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 m...
- Use
- Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 m...
Enzymatic fecal activity
Fecal enzymatic activity was determined using the API-ZYM system (Biomerieux, Marcy l'Etoile, France) according to the method described by Delgado et al. for fecal samples [ ] and the manufacturer´s instructions. The API-ZYM system is a semi-quantitative method that determines 19 enzyme activities: alkaline pho...
- Use
- Fecal enzymatic activity was determined using the API-ZYM system (Biomerieux, Marcy l'Etoile, France) according to the method described by Delgado et al. for fecal samples [ ] and the manufacturer´s instructions. The API-ZYM system is a semi-quantitative method that determines 19 enzyme activities: alkaline pho...
Statistical analysis
Statistical analysis was carried out using QIIME version 1.9.1, SPSS software 20.0 (SPSS, Chicago, IL) and the R statistical package 3.3.1. Variable normal distribution was assessed using the Shapiro-Wilk test. Comparisons of variables between sedentary and active subjects were analyzed by Student's t-test (two-tail...
- Use
- Statistical analysis was carried out using QIIME version 1.9.1, SPSS software 20.0 (SPSS, Chicago, IL) and the R statistical package 3.3.1. Variable normal distribution was assessed using the Shapiro-Wilk test. Comparisons of variables between sedentary and active subjects were analyzed by Student's t-test (two-tail...
Microbiota metagenomic analysis
The average number of reads per sample was 26,890. Rarefaction curves based on observed species, Chao1, and phylogenetic distance whole tree measures were virtually saturated, suggesting sufficient sequencing depth ( ). No significant differences between the active and sedentary groups were detected in alpha diversi...
- Use
- The average number of reads per sample was 26,890. Rarefaction curves based on observed species, Chao1, and phylogenetic distance whole tree measures were virtually saturated, suggesting sufficient sequencing depth ( ). No significant differences between the active and sedentary groups were detected in alpha diversi...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analysis was carried out using QIIME version 1.9.1, SPSS software 20.0 (SPSS, Chicago, IL) and the R statistical package 3.3.1. Variable normal distribution was assessed using the Shapiro-Wilk test. Comparisons of variables between sedentary and active subjects were analyzed by Student's t-test (two-tail...
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Materials and methods
Forty premenopausal women were recruited according to inclusion criteria: age 18-40 years and body mass index (BMI) 20-25 kg/m 2. Criteria for physical exercise were as follows: sedentary women who did not perform the minimal exercise established as healthy by WHO, ie 3 days of exercise per week for 30 minutes at a moderate intensity [ ]; and for active women, those who performed at least 3 hours of physical exercise per week. Exclusion criteria were any kind of pathology, previous gastrointestinal surgery, antibiotics intake during three months prior to the study, smoking, prebiotics, probiotics, vegetarian or vegan, nutritional or ergogenic complements, pregnancy or lactation. All subjects were Caucasian. The Ethics Committee for Clinical Research of the Ramón y Cajal Hospital (Madrid, Spain) approved the study. Written and informed consent was obtained from all pa...
Physical activity and sedentary time determination
Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). This allowed us to verify in an objective manner the presorting of sedentary and active individuals as it determines body movements in all three axes. Acceleration, energy expenditure, intensity of physical activity and body position was recorded. Results were analyzed with Actilife software (Actilife6, Actigraph). Recordings were performed for 7 days (5 weekdays and 2 weekend days). Participants were instructed to wear the accelerometer on the right wrist all day except when they had a shower or performed pool activities. Data were considered valid when they contained a record of at least five valid days including at least one weekend day, and at least 10 hours of activity were recorded. The time-sampling interv...
Stool collection, DNA extraction and sequencing
Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was measured using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA).
Quantitative PCR analysis
Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 1 minute at the established annealing temperature and 72°C for 45 seconds. Subsequently, melting curve analysis was performed, in which fluorescence was measured as the temperature increased from 50°C to 95°C. For bacterial quantification, standard curves were made using serial dilutions of a known DNA concentration corresponding to a known number of bacteria for each group or bacterial species. The calculation of the percentage for each bacterial species or group was performed considering the quantit...
Fecal water content
To obtain the fecal water content, 500 mg of each fecal sample was weighed and then lyophilized in a freeze-drier (Christ Alpha 1-2 LD, Osterode am Harz, Germany). Water content was expressed as the percentage of weight loss of stool samples.
Physical activity
When physical activity was compared between groups, there was a significant difference in the percentage of moderate physical activity in the active group (29.96±5.61%) versus the sedentary group (22.82±3.64%) (p = 0.039), while there were no significant differences in light physical activity measurements. The average expenditure was higher in the active group (145±31 kcal/h) than in the sedentary group (105±22 kcal/h), and consequently the total energy expenditure was also higher for active women (3432±911 kcals) than for sedentary women (2523±982 kcals) (p = 0.048). No significant differences were found for the remaining determined variables. Differences in sedentary behavior were also reflected in the variables "maximum time per sedentary break on working days" (SED = 691±96 min vs ACT = 515±91 min; p = 0.005), "average time...
Measurement outputs
What raw and processed outputs should exist?
Phyla and families in which the percentage of any of the 2 groups was >0.01% are shown. Data represent the mean±standard deviation. Differences were considered significant...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
PAM analysis favored partitioning into two clusters ( ), the Bacteroides and Prevotella enterotypes, whereas the Ruminococcus enterotype was not present in our samples ( ). Seve...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). Th...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Microbiota composition analysis of fecal samples was performed by amplifying the hypervariable regions V3 and V4 of the 16s rRNA gene using the primer pair 5'-TCGTCGGCAGCG...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Phyla and families in which the percentage of any of the 2 groups was >0.01% are shown.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Phyla and families in which the percentage of any of the 2 groups was >0.01% are shown. Data represent the mean±standard deviation. Differences were considered significant...; PAM analysis favored partitioning into two clusters ( ), the Bacteroides and Prevotella enterotypes, whereas the Ruminococcus enterotype was not present in our samples ( ). Seve...; Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). Th...; Microbiota composition analysis of fecal samples was performed by amplifying the hypervariable regions V3 and V4 of the 16s rRNA gene using the primer pair 5'-TCGTCGGCAGCG....
from paperStatistical comparison
Phyla and families in which the percentage of any of the 2 groups was >0.01% are shown. Data represent the mean±standard deviation. Differences were considered significant...; Statistical analysis was carried out using QIIME version 1.9.1, SPSS software 20.0 (SPSS, Chicago, IL) and the R statistical package 3.3.1. Variable normal distribution was asse...; Analysis of body composition parameters indicated significant differences in almost all adiposity and muscle parameters between sedentary and active women ( ).; Data on annual food consumption were collected with a FFQ and analyzed using Dietsource 3.0. Diets of both sedentary and active women conformed to the parameters of a balanced d...
from paperReporting output
Report representative outputs alongside summary comparisons for Phyla and families in which the percentage of any of the 2 groups was >0.01% are shown. Data represent the mean±standard deviation. Differences were considered significant..., PAM analysis favored partitioning into two clusters ( ), the Bacteroides and Prevotella enterotypes, whereas the Ruminococcus enterotype was not present in our samples ( ). Seve..., Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). Th..., Microbiota composition analysis of fecal samples was performed by amplifying the hypervariable regions V3 and V4 of the 16s rRNA gene using the primer pair 5'-TCGTCGGCAGCG....
inferred from protocolStructured statistical methods
Phyla and families in which the percentage of any of the 2 groups was >0.01% are shown. Data represent the mean±standard deviation. Differences were considered significant...; Statistical analysis was carried out using QIIME version 1.9.1, SPSS software 20.0 (SPSS, Chicago, IL) and the R statistical package 3.3.1. Variable normal distribution was asse...; Analysis of body composition parameters indicated significant differences in almost all adiposity and muscle parameters between sedentary and active women ( ).; Data on annual food consumption were collected with a FFQ and analyzed using Dietsource 3.0. Diets of both sedentary and active women conformed to the parameters of a balanced d...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
Forty premenopausal women were recruited according to inclusion criteria: age 18-40 years and body mass index (BMI) 20-25 kg/m 2. Criteria for physical exercise were as follows: sedentary women who did not perform the minimal exercise established as healthy by WHO, ie 3 days of exercise per week for 30 minutes at a moderate intensity [ ]; and for active women, those who performed at least 3 hours of physical exercise per week. Exclusion criteria were any kind of pathology, previous gastrointestinal surgery, antibiotics intake during three months prior to the study, smoking, prebiotics, probiotics, vegetarian or vegan, nutritional or ergogenic complements, pregnancy or lactation. All subjects were Caucasian. The Ethics Committee for Clinical Research of the Ramón y Cajal Hospital (Madrid, Spain) approved the study. Written and informed consent was obtained from all participants. The study is registered in Clinicaltrials.gov with the accession number NCT02901912.
Levels of physical activity performed by both groups of women were measured with an ActiSleep V.3.4.2 accelerometer. (Actigraph, Manufacturing Technology Inc., Shalimar, FL). This allowed us to verify in an objective manner the presorting of sedentary and active individuals as it determines body movements in all three axes. Acceleration, energy expenditure, intensity of physical activity and body position was recorded. Results were analyzed with Actilife software (Actilife6, Actigraph). Recordings were performed for 7 days (5 weekdays and 2 weekend days). Participants were instructed to wear the accelerometer on the right wrist all day except when they had a shower or performed pool activities. Data were considered valid when they contained a record of at least five valid days including at least one weekend day, and at least 10 hours of activity were recorded. The time-sampling interval (epoch) was every minute during the 7 days. According to previous reports, a cut-off of <100 counts/minute was chosen to categorize sedentary time, which typically includes activities such as sitting or working quietly (eg, reading or typing on a computer) [ ]. A sedentary bout was defined as 1 o...
Participants were provided with a plastic device to collect stool samples, which were stored at -80°C until extraction. DNA was extracted using the commercial Power Soil DNA Kit (Mobio, CA) with a bead-beating homogenizer (Bullet Blender® Storm, Next Advance, NY). The concentration and purity of DNA was measured using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA).
Quantitative PCR (qPCR) analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (BioRad, Barcelona, Spain) using SYBR Green I chemistry (BioRad) in 20 µL reactions containing one microliter (1-10 ng) of DNA template and 200 nM of primers. Cycling parameters were 95 °C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 1 minute at the established annealing temperature and 72°C for 45 seconds. Subsequently, melting curve analysis was performed, in which fluorescence was measured as the temperature increased from 50°C to 95°C. For bacterial quantification, standard curves were made using serial dilutions of a known DNA concentration corresponding to a known number of bacteria for each group or bacterial species. The calculation of the percentage for each bacterial species or group was performed considering the quantity of bacteria obtained per gram of stool obtained with a universal primer pair as 100% [ ]. For Bifidobacterium longum and Roseburia hominis detection, primers were manually designed considering the conserved regions in the 16s rRNA gene and the hypervariable regions to avoid cross-reaction with phy...
To obtain the fecal water content, 500 mg of each fecal sample was weighed and then lyophilized in a freeze-drier (Christ Alpha 1-2 LD, Osterode am Harz, Germany). Water content was expressed as the percentage of weight loss of stool samples.
When physical activity was compared between groups, there was a significant difference in the percentage of moderate physical activity in the active group (29.96±5.61%) versus the sedentary group (22.82±3.64%) (p = 0.039), while there were no significant differences in light physical activity measurements. The average expenditure was higher in the active group (145±31 kcal/h) than in the sedentary group (105±22 kcal/h), and consequently the total energy expenditure was also higher for active women (3432±911 kcals) than for sedentary women (2523±982 kcals) (p = 0.048). No significant differences were found for the remaining determined variables. Differences in sedentary behavior were also reflected in the variables "maximum time per sedentary break on working days" (SED = 691±96 min vs ACT = 515±91 min; p = 0.005), "average time per sedentary break on working days" (SED = 81.15±14.71 min vs ACT = 58.82±15.89 min; p = 0.038), and in the variable "total time in sedentary bouts on working days" (SED = 311±21 min vs ACT = 367±35 min; p = 0.027).
Machine-readable layer
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