543 Modern Skincare
baseline, there was an approximate 25% drop in sebum levels as early as day 3 in.the pilot
clinical study (Figure 6A).
As a part of their life cycle, C. acnes bacteria produce the molecule coproporphyrin III,
which will fluoresce a red-orange color under specific wavelengths of light.58 As such, it
has been utilized as a means of evaluating the population levels of C. acnes on the skin
of patients experiencing the effects of acne vulgaris.59 As shown in Figure 6B, there was
a visible decrease in C. acnes associated coproporphyrin III fluorescence from baseline to
day 7. The change in fluorescence intensity from baseline to day 7 was quantified using
ImageJ software (NIH), which indicated an approximate 20% reduction (data not shown).
To corroborate the fluorescence measurements from the coproporphyrin III examination,
16S rRNA sequencing was conducted on skin swabs collected from the study participants.
This sequencing method, first established by Dr. Carl Woese, allows for the differentiation
Figure 5. Three-dimensional blemished skin model. A) C. acnes targeted phage cocktail exhibits no tissue
toxicity when topically applied on a C. acnes-infused blemish-prone three-dimensional skin tissue model. The
phage cocktail was applied at three different dilution levels and allowed to incubate for 24 h. Histological
examination was conducted on those tissues as well as untreated tissue (negative control) and tissue exposed
to 0.1% SDS (cytotoxic positive control). B) The tissue described in (A) was further examined for a reduction
in C. acnes levels in response to the phage cocktail application. As shown, at all of the dilutions applied, the
phage cocktail diminished the C. acnes levels relative to tissue not exposed to the phage cocktail. C) The tissue
was also tested for changes in the levels of inflammatory factors normally associated with blemished skin.
The levels of IL-8 were significantly reduced in the blemished skin tissue model relative to untreated controls
(right side of graph, orange bars), and achieved levels as low as those observed in healthy skin tissue (left side
of graph, peach bars).

544 JOURNAL OF COSMETIC SCIENCE
between bacterial genera on the basis of genetically variable regions of the coding sequence
for the 16S ribosomal subunit.16,60 This technique is now commonly used in the clinical
setting to genetically characterize pathogenic bacteria in specimens collected from study
participants.61 As anticipated, the phage-infused finished formula produced an approximate
25% reduction in detected Cutibacterium genomes (Figure 6C), which is roughly consistent
with the diminished coproporphyrin III fluorescence measure. The 16S rRNA sequencing
also indicated little to no detectable fluctuation in the number of genomes detected for
a variety of different bacteria with the exception of S. epidermidis which showed a slight
increase at the start of the study with phage treatment. The slight boost in this bacterial
species at the single time point could be attributable to the reduced C. acnes levels as these
two bacteria sometimes occupy the same region of the skin. Moreover, the lack of any
significant impact on the populations of other bacteria collected in the swabs reinforces
the existing literature that phages are species-specific and suggests that phage offer a more
precise and selective approach to microbiome modulation.
DISCUSSION
Here, we sought to demonstrate that the more than 100-year-old concept of reducing
specific bacterial populations by employing the naturally occurring cognate phages of those
bacteria could be applied to modern skincare as microbiome modulatory ingredients. Indeed,
as the world is currently embroiled in a crisis of growing numbers of antibiotic resistant
bacteria, the scientific and healthcare community are exploring alternatives including older
strategies like phage therapy. At the beginning of the 21st century, the United States Food
and Drug Administration (US FDA) approved the use of phage cocktails as food sprays
to minimize the occurrence of outbreaks of bacterial food borne illnesses.62,63 In recent
Figure 6. Pilot clinical study with C. acnes phage cocktail. A 7-day pilot clinical study was conducted with
the phage cocktail, where a finished formulation containing the cocktail at 1% was applied twice-daily by
participants. The participants were examined at baseline and at the conclusion of the study for relative sebum
levels (A), C. acnes associated coproporphyrin III fluorescence levels (B), and the relative levels of skin bacteria
by 16S rRNA sequencing (C).