SKIN MICROBIOME 77
one genus (Corynebacterium), genus/species quantification using quantitative (real time) PCR (qPCR) were done. All qPCRs were performed with LightCycler 480 II (Roche) thermocycler using LightCycler®
Table 1: Inflammatory and non-inflammatory lesions variation between D0 and D8. Parameters
Multiplex DNA Master
(Roche, ref 07339577001) core qPCR kit and fluorescent TaqMan probe chemistry for species specific qPCR, and LightCycler® 480 SYBR Green I Master (Roche, ref 04707516001) for genus specific qPCR. For each qPCR, standards made of known amount of corresponding genomic DNA extracted from pure strains (105 to 10 genome equivalent) were integrated to qPCR assay.
Cutibacterium acnes Variable C. acnes region described by Scholtz et al.11
was amplified and
sequenced on Illumina Miseq using paired- end technology using mofidified primers. All sequences processing was performed using QIIME2 suite (
https://qiime2.org/) using default settings. After quality filtering, reads pairs were merged by overlapping and clustered and chimeric sequences were removed. SLST (Single Locus sequence Type) were assigned to sequences against SLST reference database (
http://medbac.dk/slst/pacnes).
Clinical evaluations They were conducted by the same investigator on Day 0 and Day 28 and included the scoring of lesions (blackheads, microcysts, papules and pustules) on each hemi-face (except nasal pyramid, the vermillion border, the crease of the chin and the rim of the scalp), and the reporting of local tolerance issues, acne signs and symptoms. The variations (D28-D0) in the number of lesions are calculated for each kind of lesions. Local tolerance issues and self-assessment of the products were also evaluated.
Statistical analysis Raw data were used to determine taxa abundances and α-diversity indices after removing OTU with less than 2 counts. Filtered and normalised data were used to compare populations between treatments and days (β-diversity) and to compare taxa abundances between same samples’ groups. Abundances for five taxonomic levels (Phylum to Genus) were calculated by summing sequences numbers assigned to same taxa for the considered level. Shannon diversity indice was calculated using Past 3.20 software. Indices increase with diversity. Concerning the genomic quantitative study of major genus/species of skin microbiome, statistics were performed on normalised data. Normalisation was done for each sample by dividing observed taxon abundance by the sum of all taxa abundances. Statistical comparison of taxa
April 2020
Blackheads Papules
Global non-inflammatory lesions Global inflammatory lesions
abundances was performed uniquely for taxa identified in at least half of tested samples. Missing values (i.e. samples where no species or genus was detected) were replaced by a random value sampled around the minimum value +/- 50%. Taxa abundances were compared using either parametric paired T-test or non-parametric Wilcoxon and Sign rank tests. Normality of data distribution was verified using Shapiro-Wilk test.
SLST types and phylotypes abundances and diversities were calculated for each sample and compared to evaluate treatment effect between groups made of volunteers treated with active formula and volunteers treated with placebo formula and treatment effect overtime using non- parametric Wilcoxon Test. For all hypothesis testing, p-value threshold was set at 0.05 to determine whether observed differences were statistically significant.
Results The RT active ingredient contains polyphenols (10% per dry matter), organic acids (2% per dry matter), rhomomyrtone (5 ppm) and piceatannol (500 ppm). As shown in Figure 1, major phyla were Actinobacteria (avg. 56%), followed by Firmicutes (avg. 32%), Cutibacterium being the most abundant genus (avg. 40%), followed by Staphylococcus (avg. 18%), Corynebacterium (avg. 12%) and Streptococcus (avg. 7%) both at D0 and D28, with no significance differences between placebo and RT extract treatment. When diversity index was compared between treatment using non-parametric Wilcoxon test, diversity expressed by Shannon index appeared to be statistically higher (p-<0.05) after 28 days of treatment with RT active ingredient when compared with D0 as shown in Figure 2. At D0, Cutibacterium acnes was the major species in term of mean abundance, followed by Staphylococcus epidermidis. Staphylococcus hominis was also present with a mean abundance close to that of S. epidermidis. At D28, a significant increase in Corynebacterium genus (p< 0.008) was noted only for the Placebo treatement. Cutibacterium granulosum prevalence was decreased from 53% to 29% after the RT active ingredient treatment. In most samples and all groups, A1 was the most abundant SLST type, and IA1 the
Variation -3.5±1.1 -1.6±0.4 -4.1±1.4 -2.2±0.6
p value 0.007 0.001 0.009 0.001
most abundant Phylotype, followed by Phylotypes II, IB, IA2, IC and III. This repartition was the same whatever the treatment except for RT treatment at D28, which for the 2 latter were inversed (Fig 2). Diversity expressed by Simpson index appears to be statistically higher after 28 days of treatment with active cosmetic when compared with D0 (Data not shown). RT active ingredient induced significant
decrease in blackheads, papules, general non-inflammatory lesions, and global inflammatory lesions (Table 1). This was not the case for the Placebo. 83% of subjects noted that RT extract treatment reduced imperfections (36% after the placebo treatment, 77% observed that it eliminated sebum excess (53% for the placebo treatment), and 71% that it tightened the pores (30% for the placebo).
Discussion
The aim of this exploratory study was to determine the microbiota on the surface of skin with acne lesions and to evaluate changes in the microbiota profile and C. acnes phylotype biodiversity after 28 days of twice daily application of RT extract or placebo. This study reveals that prior to the application of RT extract, the skin surface microbiota in acne subjects was dominated by Actinobacteria followed by Firmicutes and Proteobacteria. At genus level, Cutibacterium (Actinobacteria) was the most abundant genus followed by Staphylococcus (Firmicutes) and Corynebacterium (Actinobacteria), with no changes after RT active ingredient treatment. This finding is consistent with literature for healthy face skin, comparable repartition being also reported for patients suffering from acne.12 At D28, Corynebacterium significantly
increased after Placebo treatment, this genus being identified as dominant mediators of skin immunity and inflammation, and recognised as pathogens, particularly among immunocompromised hosts.13
Of the five
bacterial species evaluated, Cutibacterium acnes was the major species in terms of mean abundance, followed by Staphylococcus epidermidis, both these taxa being detected in all tested samples at D0. Staphylococcus hominis, a skin commensal bacteria and opportunistic pathogen, was detected in almost all samples (84%), its abundance not being affected by the
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