Donor and recipient group definition

Donors were healthy males and females between 22 and 40 years old, male and female, healthy individuals. Healthy individual is understood as a subject without visible skin problems and not immunocompromised. Subjects were evaluated visually by the researcher or dermatologist who ran the study and took samples. Additionally, written consent on the subject health was required before starting the study with each individual subject.

C. acnes strain isolation from the donors (PA mixtures)

A defined mixture of biologically active probiotic bacteria for topical administration was prepared as follows. A sample of skin microbiome was taken from a donor (forehead). The sample was then cultured in the laboratory, and a formulation was prepared.

Methods for analyzing the microbiome included DNA isolation, SLST amplification, and large-scale amplicon sequencing, as well as bioinformatics for the taxonomic assignment and quantification of diversity in microbial communities. Steps included are as follows:

1. Isolation of bacterial strains from a donor. Bacteria were collected using swabs. Swabs were moistened with water. 2. Growth in the laboratory. Bacteria were grown in reinforced clostridium agar (RCM) in anaerobic conditions at 37 °C. 3. Isolation and manipulation of the bacterial strains. The sample was enriched for 20 Cutibacterium strains and analyzed for positive genotypes with SLST primers. Strains H1, A1, and D1 were selected. 4. Formulation of a probiotic based on strains H1, A1, and D1. Colonies were picked and grown in liquid RCM medium, and spun down and resuspended in a saline solution with 0.5% peptone. 5. Application of 1mL of the probiotic suspension from step 4 to the recipient. The donor microbiome was applied using swabs, and the area was left to dry. 6. Genotyping of the modified recipient microbiome using an NGS-based genotyping approach discussed below.

Complete microbiome isolation from the donors (CM mixtures)

A mixture of biologically active probiotic bacteria for topical administration based on complete microbiomes was prepared as follows. A sample of skin microbiome was taken from a donor. The sample was then cultured in the laboratory, and a formulation was prepared. It is important to mention that by growing in vitro the microbiome, donor composition may become biased. Complete microbiome does not refer to comprehensive transfer from donor to acceptor but not-enriched donor sample preparation.

Methods for analyzing the microbiome included DNA isolation, 16S amplification, and large-scale amplicon sequencing, as well as bioinformatics for the taxonomic assignment and quantification of diversity in microbial communities. Steps included are as follows:

1. Isolation of bacterial strains from a donor. Bacteria were collected using swabs. Swabs were moistened with water. 2. Growth in the laboratory. Bacteria were grown in RCM agar in anaerobic conditions at 37 °C. 3. All colonies were collected from plates, grown in liquid RCM medium, and spun down and resuspended in a saline solution with 0.5% peptone. 4. Application of 1 mL of the probiotic from step 3 to the recipient. The donor microbiome was applied using swabs, and the area was left to dry. 5. Genotyping of the modified recipient microbiome using an NGS-based genotyping approach discussed below.

Skin microbiome donor preparation viability

PA and CM mixtures were grown in RCM as a liquid culture. After 2 days, the culture was spun down and washed first with PBS (phosphate-buffered saline, pH 7.4), and then with water. The pellet was resuspended to a final concentration of PBS and 0.5% peptone. Aliquots were stored either at room temperature or at 4 °C. In both cases, they were protected from sunlight. In regular intervals, about every 3–4 days, a dilution series of each sample was taken and the colony-forming unit (CFU) count was determined. The suspension was vortexed, and a serial dilution was prepared. To determine the CFU count, aliquots of the dilution were added on agar plates which are suitable to grow C. acnes. A volume of 10 μl of an appropriate dilution was introduced to RCM plates. The 10 μl were placed as a drop on top of the plate and run down. This method allows the placement of up to 4 drops on the plate. Each sample was determined in 4 technical replicates. After 3–4 days of anaerobic incubation, the colony numbers were counted (manually or using the software OpenCFU) and both the average and the standard deviation were determined. Thereby, a profile of the colony forming units was monitored over time. Combined samples (i.e., H1+D1; H1+D1+A1) were always mixed freshly before deployment https://www.future-science.com/doi/pdf/10.2144/97234bm22 [37].

Additionally, we performed some stability study of the probiotic solutions. Bacteria of the skin microbiome were stabilized in a neutral liquid matrix for several days at room temperature (i.e., saline solution or 0.5% peptone). It was demonstrated that Cutibacterium can survive weeks of storage at room temperature. Constant numbers of colony-forming units (CFUs) from a liquid matrix over a week were also recovered. To assess these numbers, methods that determine the CFU of liquids in a medium-throughput fashion were established as described below. It was shown that compositions were stable for at least 1.5 months.

Donor microbiome solution application to the recipient

Microbiome donor solution was applied once a day for 3 days using swabs onto a delimited area of the chest of the recipient (Fig. 1a). Prior to application, the area was cleaned and disinfected. Sampling for genotyping was carried out before new donor samples were applied.

Treatment areas measure 100 cm2 so that sampling can be performed in adjacent sides and prevent potential measurement artifacts associated with using 3S-Biokit repeatedly at the exact same point.

Different areas of sampling defined in the study may have slightly different properties (i.e., variation of pilosebaceous unit) and experience different environmental features (access to wash, clothes contact, etc.). Due to the lack of previous reports of this study, we prioritized perhaps a noisier but less biased sampling scheme. A rotatory scheme may prevent the observation of results that may be very unique to one specific site and provide more generalizable results.

Engraftment

In this study, we used natural strains that cannot be distinguished between donor and acceptor. We determined the engraftment computing the differences in the proportions of strains present in the acceptor and in the applied solution. We defined engraftment as the distance between the composition of the site and the applied solution. The distance was computed using Pearson’s correlation.

Strain genotyping

An NGS-based genotyping approach was used for identifying different strains:

1. The microbiome was collected using strips daily. Strip kit 3S-Biokit from C+K electronic was used. 2. The sample was incubated at high temperature to isolate the DNA. The QuickExtract™ kit from Epicentre, Chicago, IL, was used with some modifications. A volume of 80 μL of 0.05 M NaOH was added to the suspension solution. The incubation was conducted for 45 min at 60 °C, followed by a 5-min incubation at 95 °C. After incubation, 920 μL of 1 M Tris-HCl (pH 7.0) was added. A volume of 0.5 μL was used for PCR. 3. PCR was conducted on the sample using 16S primers, and SLST primers characterize the population. Samples were amplified using KAPA polymerase (Initial denaturation for 5 min at 95 °C followed by 35 cycles of 98 °C for 20 s, 62 °C for 25 s, and 72 °C 30 s; and a final elongation for 1 min at 72 °C).

Primers used for 16S amplification were as follows:

5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3’ and

5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3’

The Illumina overhang adapter sequence is in bold, and the 16S V3 and V4 region priming sequence described in Klindworth et al. [38] is in italics.

Primers used for SLST amplification were as follows:

5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-TTGCTCGCAACTGCAAGCA-3’ and

5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-CCGGCTGGCAAATGAGGCAT -3’

The Illumina overhang adapter sequence is in bold, and the sequence used for SLST targeting is in italics. We used a shorter SLST amplicon version which can be fully sequenced by Miseq PE300 Illumina sequencing:

4. Library preparation. The library was constructed using two rounds of PCR. The 10 first round used 16S primers and SLST primers which included sequences compatible with Illumina sequencing. The second round was used to barcode the different samples for sequencing in a single Illumina flow cell. 5. Illumina MiSeq sequencing was conducted. We reserved a depth of ~ 10,000 reads for each sample. 6. Samples were analyzed using a two computational pipeline for 16S and SLST. The 16S pipeline was performed as described in Willis et al. [30]. SLST typing pipeline consisted on quality filtering; SLST amplicon was mapped to SLST database [27] using BWA mapping software; BAM file processing and visualization were conducted with R statistical language.

Significance of engraftment

We performed Wilcoxon-Mann tests between measured engraftment before treatment (day 1) and after treatment (days 2, 3, 4, 5, 8, 10, 12 ,17, 24, 38, and 72). Multiple testing was adjusted using the BH method.

Normalization and filtering of 16S and SLST data

The 16S rRNA gene counts and the SLST counts for the samples in this study were stored and analyzed using the R package Phyloseq (version 1.16.2) [36]. The counts were normalized per sample by dividing each value by the sum of all counts for a given sample and multiplying by 100, leaving the relative abundance of each genus/strain within that sample, with all values between 0 and 100.

Clustering and dermatotype analyses

The Jensen-Shannon divergence (JSD) was used to produce a distance matrix between the genera/strains of all samples and then partitioning around medoids (PAM) clustering to group samples with similar overall abundances. We used the Calinski-Harabasz (CH) index to determine the optimal number of clusters, and we further verified this by calculating the average silhouette width of the samples, which is a measure of the separation of samples within one cluster from those of another cluster. The functions for these calculations come from the R packages cluster (version 2.0.4) [38] and clusterSim (version 0.44-2) [38]. A Principal Coordinate Analysis (PcoA) was used to visualize the clustering of the samples within their respective dermatotypes with the R package ade4 (version 1.7-4) [39].