Microbiome study reveals potential relationship between bacterial diversity in a gym and human health
Test items and sampling methods
Rhythmic Gymnastics Group (GR)
Thirty female RG students from a university in western China were used in this study (age: 20.70 years; height: 165.20 cm; weight: 52.96 kg; training time: 7.16 years ).
Rhythmic gymnast flippers (n = 30 people), a rhythmic gymnastics mat (13 × 13 m2), 3 bars (length: 4 m/bar), rhythmic gymnastics hoops (n = 30) and balls (n = 30).
A small amount of sterile water was poured into a sterile medical bag to moisten the gauze. The tester wore disposable gloves (replaced for each sampled subject), wiped both of the subject’s palms 4–5 times with gauze after exercise, and then immediately put the gauze in a sterile plastic bag.
The gym mat was sampled after training. The mat was divided into 9 rectangles (diagonal: 30 cm × 30 cm) with wide adhesive tape. Three pieces of gauze were used to sample each rectangle. A gloved tester wiped each rectangle with damp gauze 4-5 times. The gauze was placed in a sterile plastic bag. To compensate for the insufficient biological samples obtained by wiping the carpet, some of the fibers on the surface of the carpet were cut as a sample at any three locations within each sample rectangle area.
The hoop, ball and bar were sampled after the gymnasts practiced. A gloved sampler wiped the surface of the hoops and balls 2–3 times with damp gauze; each bar was wrapped with two pieces of wet gauze and wiped 2-3 times. After all the samples were collected, they were mixed into 5 pooled samples.
Example of RG code
Mat (aa), bar (ab), hoop (ac), ball (ad), palm (ae).
Artistic Gymnastics Group (AG)
Thirty-seven GA students from a university in western China were included in the study, including 12 boys and 25 girls (age: 20.81 years; height: 165.97 cm; weight: 55.88 kg; duration of training: 4.57 years).
Artistic gymnast flippers (37 people), a gymnastics mat (14 × 14 m2), 3 sets of gymnastic wall bars, 10 bars per set (length: 77 cm/bar), 3 pairs of horizontal bars (length: 23.7 cm/bar) and 4 pairs of parallel bars (length: 35 cm/ Rod).
Same as sampling method for rhythmic gymnasts.
A gym mat was sampled after training. The mat was divided into 9 rectangles (diagonal: 47cm × 47cm) with wide tape. Three pieces of gauze were used to sample each rectangle. A gloved tester wiped each rectangle with damp gauze 4-5 times. The gauze was placed in a sterile plastic bag. To compensate for the insufficient biological samples obtained by wiping the carpet, some of the fibers on the surface of the carpet were cut as a sample at any three locations within each sample rectangle area.
Samples were taken from the wall bars, parallel bars and horizontal bars after training. A gloved sampler whose gloves were changed for each piece of equipment sampled wrapped each wall bar with two pieces of damp gauze and wiped the surface 2-3 times. The same method as for the espaliers was used to sample the parallel bars and the horizontal bars. After all samples were collected, they were mixed into 5 pooled sample bags.
AG Code Example
Mat (ba), wall bars (bb), horizontal bar (bc), parallel bars (bd), palm tree (be).
DNA extraction, amplification and sequencing
DNA extraction and PCR amplification
Total DNA was extracted using a FastDNA Spin Kit for Soil (MP, USA) according to the manufacturer’s instructions. DNA concentration and purity were detected using a NanoDrop2000, in which the nucleic acid was selected after placing the 2 μL DNA sample, and the quality of the extraction DNA was detected by 1% agarose gel electrophoresis. The PCR experiment used primers 338F (5′-ACTCCTACGGGAGGCAGCAGCAG-3′) and 806R (5′-GGACTach VGGGTWTCTAat-3′) for PCR amplification of the v3–V4 variable region. The amplification procedure was as follows: predenaturation for 3 min at 95°C, 27 cycles (95 cycle denaturation for 30 s, 55 annealing for 30 s, 72 for 30 s extended) and a maximum extension of 10 min (PCR : ABI GeneAmp 9700). The amplification system consisted of 20 μL, 4 μL of 5* FastPfu buffer, 2 μL of 2.5 mm dNTPs, 0.8 μL of primer (5 μM), and 0.4 μL of FastPfu polymerase. Briefly, 10 ng of template DNA. PCR products were recovered using a 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA ), eluted with 10× TE buffer and detected by 2% agarose electrophoresis. Quantification was performed using QuantiFluor uantiPromega, USA). 2*300 PE libraries were constructed from the amplified fragments purified according to standard operating procedures of the Illumina MiSeq platform (Illumina, San Diego, USA).
Data quality control
quick (https://github.com/OpenGene/fastpversion 0.20.0) was used for quality control of the original sequencing sequence, and FLASH (http://www.cbcb.umd.edu/software/flash, Version 1.2.7) splicing software was used. For grouping OTU sequences, the UPARSE software (http://drive5.com/uparse/, version 7.1) was used with 97% similarity, and chimeras were eliminated. Species classification annotation was performed using the RDP classifier (http://rdp.cme.msu.edu/Version 2.2), and the comparison threshold was set at 70% for the Silva 16S rRNA (V138) database.
Functional prediction. FAPROTAX is a functional prediction software based on 16S sequencing. It integrates several prokaryotic functional databases from published culturable literature. The database contains 7600 functional information for more than 4600 species, including 80 functional groups, including potential pathogenesis, methanogenesis, fermentation, etc. In use, the annotated OTU table in the Greengenes or Silva database is read, and the annotated OTU information is compared with the species information in the database through the Python program, and the results of the prediction of various functions are produced18. BugBase is another tool to predict the phenotypic function of the 16S microbiome. Based on OTU tables and mapping files, BugBase uses existing databases, annotations and frameworks to compare and measure a large amount of information, providing users with microbiome-level phenotypic prediction at the organism level.19. Including pathogenic potential, biofilm formation, Gram positive and Gram negative, etc. BugBase also provides grouped statistics and visualization. BugBase can be used as a web application (http://bugbase.cs.umn.edu) to use, can also be downloaded for free (https://github.com/knights-lab/BugBase).
Bar chart of microbial composition and relative abundance of samples, Venn and KRONA annotation, FaproTax and Bugbase function prediction analysis as well as LefSe analysis (http://huttenhower.sph.harvard.edulefse/) were made using BMKCloud (http://www.biocloud.net/). Specifically, for analysis of species with significant differences (known as biomarker analysis) between groups, LefSe was used to estimate the impact of the abundance of each component (species) on the differential effect. The LDA score for significant differences was set at 3.0 and p
The ethics committee of Chengdu Sports University approved the study protocol. The study was conducted in accordance with current guidelines and regulations.
Informed consent was obtained from all individual participants included in the study.