Relationship Between Frailty and Gut Microbiota in Older Adults: An Educational Program for Healthy Aging

Fecal samples will be collected by the participants themselves at home. For the determination of gut microbiota, the following steps will be carried out:

DNA Extraction: DNA will be extracted using the QIAamp DNA Stool Mini Kit (QIAGEN, Barcelona, Spain).

Sequencing: Polymerase Chain Reaction (PCR) will be used to amplify the extracted DNA with primers targeting the hypervariable V3 and V4 regions of the bacterial 16S rRNA gene, using the following primers: forward 16S Amplicon PCR primer: 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, and reverse 16S Amplicon PCR primer: 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.

Bioinformatic Analysis: Illumina bcl2fastq2 v2.20 software will be used to demultiplex the raw sequences, and the raw data will be imported into the open-source QIIME 2 2020.8 software.

Functional Profile Determination: PICRUSt2 will be used to predict potential functional profiles for the sequenced samples.

Through the Fried questionnaire, which includes five domains: i) unintentional weight loss, ii) self-reported exhaustion and fatigue, iii) low weekly physical activity measured using the short version of the Minnesota Leisure Time Physical Activity Questionnaire, iv) gait speed over 4.57 meters, and v) low grip strength measured with a hand-held dynamometer.

Using the Short Physical Performance Battery (SPPB), balance was assessed (with the feet together, semi-tandem, and tandem tests), gait speed was measured (with the 4-meter walking test), and lower body strength was evaluated (with the five-times chair stand test).

Functional dependence was assessed using the Barthel Index, which is a reliable and validated tool designed to evaluate an individual's ability to perform ten Activities of Daily Living. These activities include feeding, transferring from a chair to the bed, bathing, dressing, grooming, bowel control, bladder control, using the toilet, walking, and climbing stairs. Additionally, the Lawton and Brody scale was applied, which is a reliable and validated tool used to assess Instrumental Activities of Daily Living, covering eight domains: use of the telephone, transportation, medication management, financial transactions, meal preparation, shopping, household maintenance, and laundry.

Measured using wrist-worn accelerometers (GENEActiv, ActivInsights Ltd., Kimbolton, UK), which participants wore for at least six consecutive days. The accelerometers were set to 60 Hz, and raw data were downloaded using GENEActiv software version 3.3. All raw data files were analyzed on the servers of the University of Málaga using the open-source R package GGIR, version 2.7-0. This R package auto-calibrated the data according to local gravity and calculated the Euclidean Norm Minus One (ENMO) for accelerations to minimize sensor calibration error.

Using the Pittsburgh Sleep Quality Index, which has been validated in this population, the questionnaire includes 19 items assessing seven distinct dimensions of sleep. These dimensions include subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, use of sleep medication, and daytime dysfunction.

The EuroQoL 5-Dimension 5-Level (EQ-5D-5L) was used to assess health-related quality of life in the older participants. It consists of five dimensions (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression), with participants rating each dimension on a five-point scale from 1 (no problems) to 5 (extreme problems). These ratings were combined into a five-digit number representing their health state. After generating 3,125 possible health states, a formula was used to convert them into a single health index ranging from 0 to 1, with 1 representing the maximum achievable score. In addition, the questionnaire included a Visual Analog Scale (VAS) to assess self-reported health levels. The VAS consisted of a scale from 0 (representing the worst imaginable health state) to 100 (representing the best imaginable health state), and participants were asked to indicate the score that best represented their health on the day of the assessment.

Through the 15-item Yesavage Geriatric Depression Scale (GDS-15), a validated assessment tool for depression in older adults. The GDS-15 uses a binary response format (yes or no) and evaluates only the cognitive symptoms of a major depressive episode experienced during the past 15 days, excluding somatic depressive symptoms such as sleep difficulties, changes in appetite, lack of concentration, and fatigue.

Through the Duke-UNC Functional Social Support Questionnaire (Duke-UNC-11), which has been validated in the Spanish population.

The cognitive status of the participants was assessed using the Portable Short Portable Mental Status Questionnaire (SPMSQ), which has demonstrated good validity and reliability in the Spanish population. This test is widely used due to its brevity and ease of administration, making it a rapid screening tool. It consists of 10 questions that evaluate short- and long-term memory, orientation, and the ability to perform serial mathematical tasks.

Through the Mini-Nutritional Assessment (MNA). This evaluation includes questions related to various aspects such as lifestyle, medication, mobility, number of meals, food and fluid intake, independence in eating, and self-perception of health and nutrition.