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(GCP) is a requirement. Thus a well-described study population, freedom from bias (using randomisation, placebo, controls, etc) and consideration of any confounding interactions are attributes of good clinical trials. In the cosmetic field, skin microbiome studies consist in manipulating the human skin microbiota in order to harness its potential positive effects upon the skin. One can divide studies into: i) antimicrobial manipulation aiming at a significant reduction of the numbers of all skin microorganism; ii) manipulation aiming at an increase or a restauration of the number of selected, potentially beneficial microorganisms using prebiotics or probiotics; and iii) verification that the application of one product (e.g. detergents) will not modify the equilibrium of a healthy skin microbiota. For skin microbiome studies, determining the composition of microbial communities involves analysing the DNA recovered from skin samples. Historically culture-based microbiological techniques were used but they had the limitations that they were focused on a single or on a few bacteria and that some microorganisms escaped detection. Now there are two methods widely used, both based on the analysis of DNA sequences: the first one is the 16S amplicon sequencing and the second is the Whole Genome Shotgun (WGS) metagenomic sequencing. The increasing use of these culture-independent methods has been driven by advances in sequencing and bioinformatics analysis tools. These methods investigate the taxonomic diversity as well as the functional metagenomics which attempts to describe which biological activities can be done by the microorganisms
from the studied community. In the 1990s, Carl Woese developed the 16S rRNA genes sequencing technique to identify bacterial genera and species. The principle of this method is based on the fact that the 16S rRNA gene is present in all bacterial species and contains both highly conserved regions and highly variable sequences which can be used as a ‘molecular fingerprint’. After swabbing of the skin to collect bacterial samples and isolation of DNA, the genetic material is amplified by Polymerase Chain Reaction (PCR) using primers covering hypervariable regions of the 16S rRNA gene. Since nine hypervariable regions can be used to identify species or genera of bacteria, the careful choice of the primer for 16S amplicon generation is critical. The V4 hypervariable region has been traditionally selected but recent work has shown that the V3-V4 region of the 16S rRNA gene captures well the bacterial community diversity and that the V1-V3 region is recommended for skin microbiome studies. Amplicon sequencing is not without limitations. One problem of the 16S community analysis is that the method does not distinguish between living, dormant and dead members of the microbial community. WGS metagenomic sequencing is an alternative approach to the study of uncultured microbiota. Shotgun metagenomic sequencing also starts with DNA extraction from all the cells of the community sampled. But, instead of targeting a specific locus for amplification, all DNA is cut into tiny fragments that are independently sequenced. Some of these DNA sequences will be sampled from taxonomically informative genomic loci (e.g. 16S) and others will be sampled from coding
sequences that provide insight into the biological functions encoded in the genome. With WGS, a more accurate determination of the composition of the skin microbiome can be achieved compared to the 16S amplicon sequencing, but its cost is higher and the bioinformatics of WGS sequencing is more complex. Data generated by metagenomics experiments are enormous and noisy and their analysis would not have been feasible without the fantastic progress of bioinformatics. Large public databases of DNA sequences are available as well as specific tools to identify microorganisms, to analyse the functions of the microbial communities, etc.
Methodological choices are known to
affect observed results in human microbiome studies. Such factors include the relevant choice of subject inclusion and exclusion criteria (antibiotics use, diet supplements including probiotics and fibre/prebiotics supplements, skin moisturising level, ethnicity, age, gender), how skin is sampled, genetic material extraction approach, storage solutions and conditions, sequencing platform, regions sequences and bioinformatics data processing and analysis. The first step of all skin microbiome studies consists in the acquisition of a sample which must contain, if possible exhaustively, all resident microorganisms from the cutaneous surface. Sampling the skin gives low biomass and there is a real necessity to eliminate potential contaminants. Swabs are the usual way to collect samples. With swabs, it is feasible to sample skin before and immediately after an intentional perturbation because swab- based sampling is not expected to drastically
September 2018
PERSONAL CARE EUROPE
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