Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Molecular analysis of the prevalent microbiota of human

male and female forehead skin compared to forearm skin
and the influence of make-up
T. Staudinger, A. Pipal and B. Redl
Division of Molecular Biology, Biocenter, Medical University Innsbruck, Innsbruck, Austria

Keywords
forearm skin, forehead skin, make-up, skin
bacteria, skin microbiota.
Correspondence
Bernhard Redl, Division of Molecular Biology,
Biocenter, Innsbruck Medical University,
A- Fritz Pregl Str. 3, 6020 Innsbruck, Austria.
E-mail: bernhard.redl@i-med.ac.at
The sequence data from this study have been
submitted to GenBank under accession no.
HQ16212–HQ16344.
2010 ⁄ 2116: received 22 November 2010,
revised 17 January 2011 and accepted 21
February 2011
doi:10.1111/j.1365-2672.2011.04991.x
Abstract
Aims: To compare the bacterial diversity of two different ecological regions
including human forehead, human forearm and to estimate the influence of
make-up.
Methods and Results: Twenty-two swab-scraped skin samples were analysed by
profiling bacterial 16S rRNA genes using PCR-based sequencing of randomly
selected clones. Of the 1056 clones analysed, 67 genera and 133 species-level
operational taxonomic units (SLOTUs) belonging to eight phyla were identi-
fied. A core set of bacterial taxa was found in all samples, including Actinobac-
teria, Firmicutes, and Proteobacteria, but pronounced intra- and interpersonal
variation in bacterial community composition was observed. Only 4Æ48% of the
genera and 1Æ50% of the SLOTUs were found in all 11 subjects. In contrast to
the highly diverse microbiota of the forearm skin, the forehead skin microbiota
represented a small-scale ecosystem with a few genera found in all individuals.
The use of make-up, including foundation and powder, significantly enlarged
the community diversity on the forehead skin.
Conclusions: Our study confirmed the presence of a highly diverse microbiota
of the human skin as described recently. In contrast to forearm skin, gender
does not seem to have much influence on the microbial community of the
forehead skin. However, the use of make-up was associated with a remarkable
increase in the bacterial diversity.
Significance and Impact of the Study: This study enhances our knowledge
about the highly complex microbiota of the human skin and demonstrates for
the first time the significant effect of make-up on the bacterial diversity of the
forehead skin.

follicles of the sebaceous glands were called resident flora.

Introduction
The human skin has a number of essential protective
functions in ensuring homeostasis of the whole body as it
forms a barrier against harmful effects of the environment
(Nemes and Steinert 1999; Madison 2003; Elias and Choi
2005; Proksch et al. 2008). It is colonized by a large
number of micro-organisms representing a complex
ecosystem. Originally, Price (Price 1938) classified the
microbiota of human skin into two groups. Those organ-
isms that grow on the surface and in the stratum corne-
um within the outmost layers of the epidermis and in the
They form a relatively stable population (Roth and James
1988; Wilson 2005), able to proliferate on the skin surface
and in the outer sweat glands (Montes and Wilborn
1969). A second group of micro-organisms was called
transient flora. They originate from external sources,
through the contact of humans with the environment.
These micro-organisms do not attach firmly to the skin
and their composition varies permanently. The resident
bacterial flora plays an important physiological role in
protecting the skin from being infected by external patho-
genic micro-organisms (Wilson 2005). Any change in the

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Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology 1381
Skin microbiota of human forehead compared to forearm
skin microbial balance can lead to harmful effects (Meloni
and Schito 1991; Brook 2000; Wilson 2005). Thus,
research on the microbiota has a long tradition. The use
of molecular approaches instead of culture-dependent
assays has helped to uncover the great diversity of skin
microbiota (Dekio et al. 2005; Gao et al. 2007; Fierer
et al. 2008; Grice et al. 2008, 2009). These studies revealed
a low level of interpersonal consensus with respect to bac-
terial community membership and structure, which is
approximately equal to intrapersonal variation. Also, a
highly dynamic microbiota composition was observed to
fluctuate greatly over time and gender variations have
been found (Gao et al. 2007; Fierer et al. 2008; Grice
et al. 2008, 2009).
In this work, we have investigated the diversity of skin
bacteria present on two different sites of the body, namely
the forehead and the forearm. An important feature of
the forehead skin is the presence of both sebaceous and
eccrine glands in high densities (Wilson 2005). This
results in enriched nutrient supplementation and in
elevated levels of antimicrobial peptides. The forehead is
described as a relatively oily and moist region and is one
of the most acidic regions of the skin. Because of its
exposed nature, the forehead is subject to large tempera-
ture variations. Furthermore, the use of cosmetics might
influence the number and species of bacteria present on
the forehead (Holland and Bojar 2002). Forearm skin is
described as a relatively dry environment, with a few
eccrine and apocrine glands, and only a low density of
sebaceous glands (Wilson 2005). However, the skin of the
forearm is in certain climate zones often covered with
clothes, which might affect the local environment, and
thus altering the growth conditions of the microbiota.
Materials and methods
Sample collection
Specimens from superficial skin were obtained from the
central forehead and the volar left forearm of 11 healthy
young subjects, five men and six women, with no history
of dermatological disorders or other chronic medical
disorders and with no current skin infections. Samples
were collected between late winter and early spring from
subject living in the area of Innsbruck, which has a classi-
cal alpine climate. The mean age of the subjects was
25Æ8 years (range 22–29 years). The skin characteristics of
all subjects are presented in Table S1. None of the sub-
jects had received any antibiotics for at least 1 month.
Female subjects F2, F4 and F5 used make-up (foundation
and powder) every day, whereas female subjects F1, F3
and F6 never used make-up but used skin cleansers and
skin cosmetics like moisturizers. None of the male
1382 Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology
T. Staudinger et al.
subjects used make-up. Subjects were instructed not to
wash with anything for an 8-h interval prior to sampling.
Samples were taken from 2 · 2 cm of skin, from the
central area of the forehead and volar left (flexor) fore-
arm, midway between wrist and elbow, by swabbing the
skin for 1 min with a sterile cotton swab that had been
soaked in sterile 0Æ15 mol l)1 NaCl with 0Æ1% Tween 20.
Samples from forehead and forearm were taken at the
same time. To minimize sample cross-contamination, a
fresh pair of sterile gloves was used for each individual
sampling process. Reagent controls consisted of sterile
cotton swabs moistened with a solution of sterile
0Æ15 mol l)1 NaCl and 0Æ1% Tween 20 and placed directly
in 1Æ5-ml microcentrifuge tubes. The study was approved
by the Ethics Committee of the Medical University Inns-
bruck (AN3616 ⁄ 4Æ13).
DNA extraction from swabs
DNA was extracted using the DNeasy Blood and Tissue
kit (Qiagen, Hilden, Germany). As Gram-positive bacteria
are more resistant to lysis than Gram-negative bacteria,
the manufacturer’s protocol for genomic DNA isolation
from Gram-positive bacteria was followed with modifica-
tions. The cotton tip of each swab was broken off directly
into a 1Æ5-ml tube to which 180 ll of lysis buffer has been
added. The tubes were capped and shaken by hand hori-
zontally for 30 s. The remaining steps were performed as
suggested by the manufacturer.
16S rRNA gene amplification, cloning procedure and
sequencing
PCR amplification was performed with primers specific
for conserved bacterial 16S rRNA sequences. PCR primers
F 8: 5¢-AGAGTTTGATYMTGGCTCAG-3¢ and R 1401:
5¢-CGGTGTGTACAAGGCCCG-3¢ amplified a bacterial
16S rRNA gene fragment from nucleotide positions
8–1401 (Escherichia coli numbering, GenBank J01859).
These primers span over 8 (V1–V8) of the nine
hypervariable regions of the bacterial 16S rRNA and were
chosen because most sequences in databases are available
for regions V1–V8 (Chakravorty et al. 2007). PCR ampli-
fication was performed using the following conditions
(final concentrations): 1· GoTaq reaction buffer (Pro-
mega, Madison, WI, USA), 0Æ2 mmol l)1 dNTPs, 50 pmol
of each primer and 2Æ5 U GoTaq Polymerase (Promega)
and 10 ng of DNA in a total volume of 50 ll. Twenty-
seven cycles of PCR amplification were performed. Each
of them entailed denaturation at 95C for 60 s, annealing
at 48C for 60 s and primer extension at 72C for 60 s.
PCR products were analysed on 1Æ2% agarose gels, stained
with ethidium bromide. The PCR fragments were gel
ª 2011 The Authors
T. Staudinger et al.
eluted using Wizard SV Gel and PCR Clean-Up System
(Promega). Ten nanograms of DNA was ligated with the
pGEM-T vector (Promega) and transformed into E. coli
DH5a competent cells. The complete insert was cycle
sequenced using M13 primers for amplification and T7
and SP6 primers for sequencing on an ABI PRISM 3100
Genetic Analyser (Applied Biosystems, Carlsbad, CA).
Database and phylogenetic analysis
Analysis of closest relatives was performed by comparison
of the entire amplified rDNA sequence (without primer
sequences) with sequences available in the Ribosomal
Database Project (RDP) II (RDP Release 10Æ23) and
GenBank databases, by using the standard nucleotide-
nucleotide Blast program (Blastn-megablast; National
Center for Biotechnology Information, Bethesda, MD).
Forty-eight clones were analysed from each skin sample.
Sequences were examined for chimerism using Bellero-
phon at Greengenes (DeSantis et al. 2006).
Statistical analysis
The total number of species-level operational taxonomic
units (SLOTUs) (SLOTU cut-off was 98%) that may be
present in the sampled human skin and its associated
confidence intervals were calculated by using the non-
parametric richness estimator Chao1 (EstimateS ver.
5.0.1.) (Hughes et al. 2001).
Principal coordinate analysis (PCoA) was performed
within the UniFrac online suite of tools (http://bmf2.colo
rado.edu/unifrac/index.psp). For this purpose, a phylo-
genetic tree generated with mega ver. 4.0 (Tamura et al.
2007), along with environmental labels, was imported into
UniFrac (Lozupone et al. 2006). To facilitate the visuali-
zation of sample dissimilarity and diversity, the first two
orthogonal principal axes were obtained based on the
sample dissimilarity and were plotted to show the distri-
bution of samples in a two-dimensional space. The P test,
also available on the UniFrac suite of tools, assessed trees
for nonrandom distributions of lineages according to
environment. All P values reported were also corrected for
multiple comparisons (Martin 2002; Lozupone et al.
2006).
Results
Clone libraries, classification and distribution of clones
Samples from superficial skin of 11 healthy subjects were
collected, and a total of 1056 clones were analysed. Ini-
tially, 25 clones containing putative chimeric sequences
were removed from the phylogenetic analyses and were
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Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology
Skin microbiota of human forehead compared to forearm
replaced by new ones from the same master plate and the
same subject to obtain 48 clones for each skin sample.
These experiments revealed the presence of eight phyla,
67 genera and 133 SLOTUs, when using a SLOTU cut-off
of 98% (Table S2). Of the 1056 clones investigated, 1051
clones (99Æ53%) shared a nt sequence homology of ‡98%
with cultivated type strains and five clones (0Æ47%)
showed only significant homology to noncultured bacte-
ria. These include uncultured members of Acidobacteria
and members of the TM7 phylum. No clones representing
previously uncharacterized phylotypes were found.
Actinobacteria, Firmicutes, and Proteobacteria were the
predominant phyla, representing 1027 (97Æ25%) clones
and accounting for 36 (59Æ75% of the clones), 44
(25Æ09%) and 37 (12Æ41%) SLOTUs. These three phyla
were observed in all 11 subjects (Fig. 1a,b). The phylum
Bacteroidetes was observed in samples from eight subjects,
and the phylum Fusobacteria in samples from three sub-
jects. Acidobacteria were observed in samples from two
subjects, and each of the phyla Deinococcus-Thermus and
TM7 were observed in one subject (Fig. 1a,b).
Three genera including Propionibacterium (46Æ78% of
all clones), Staphylococcus (14Æ39%) and Corynebacterium
(5Æ40%) were observed in all 11 subjects analysed. How-
ever, of the 67 genera, 30 genera (44Æ78%) were identified
in only one subject, 11 (16Æ42%) in two subjects and 6
(8Æ96%) in three subjects; only 17 (25Æ37%) genera were
observed in ‡4 subjects, indicating substantial interper-
sonal variation at the genus level.
A total of 133 SLOTUs were found, reflecting the
microbiota of the forehead and of the volar left forearm
skin (Table S2). The number of SLOTUs in each subject
ranged from 16 to 42 (mean: 26Æ55 ± 7Æ57) (Fig. 2a,b).
The most numerous species identified belonged to the
genera Corynebacterium, Streptococcus and Staphylococcus.
Propionibacterium acnes was the most prevalent species
detected in all 11 subjects, accounting for 45Æ74% of all
clones analysed.
To estimate the SLOTU richness, the expected number
of SLOTUs was calculated using the Chao1 nonparametric
estimator. The results were visualized by collector’s curves
of observed and estimated SLOTU richness (Fig. S1a,b),
and the 95% confidential intervals were calculated to esti-
mate the precision of the richness estimation (Hughes
et al. 2001). We estimated that the microbiota of the
forehead samples contains c. 65 SLOTUs (95% CI, range
50–107). Consequently, the 42 SLOTUs observed in this
study represented 65Æ6% (95% CI, 46Æ7–68Æ0%) of the esti-
mated species or phylotypes. On the forearm skin, we
observed 128 SLOTUs in our study. However, the Chao1
score estimated that the microbiota of the forearm sam-
ples contains c. 170 SLOTUs (95% CI, range 149–211).
Based on this prediction, the present study identified
1383
Skin microbiota of human forehead compared to forearm T. Staudinger et al.

(a)
100%

80%

60%

40%

20%

0%

(b)
100%

80%

60%

40%

20%

0%
F1 F2 F3 F4 F5 F6 M1 M2 M3 M4 M5 F M Total
Total Total

Figure 1 Relative abundance of the most prevalent bacterial groups associated with each microenvironment depicted for each of the subjects
investigated. (a) Forehead skin, (b) forearm skin. Superscripts indicate phylum: 1, Actinobacteria; 2, Firmicutes; 3, Proteobacteria; 4, Bacteroidetes;
5, Acidobacteria; 6, Deinococcus-Thermus; 7, Fusobacteria. F, female; M, male. ( ) Propionibacteria spp.1; ( ) Corynebacteria spp.1; ( ) Other
Actinobacteria1; ( ) Bacillales2; ( ) Lactobacillales2; ( ) Clostridiales2; ( ) c-Proteobacteria3; ( ) a-Proteobacteria3; ( ) b-Proteobacteria3; ( )
Bacteriodales4; ( ) Flavobacteriales4; ( ) Acidobacteriales5; ( ) Deinococcales6; ( ) Fusobacteriales7; ( ) TM7 Phylum.

75Æ3% (95% CI, 65Æ9–78Æ5%) of the SLOTUs in this bacte-
rial ecosystem. This is in agreement with Gao et al. (Gao
et al. 2007), who calculated c. 246 SLOTUs (95% CI,
range 217–301) for this area. The Chao1 estimates for
both sampling sites stabilized to the end of the collector’s
curves indicating that the estimated richness might not
change with further sampling (Fig. S1a,b).
Forehead skin microbiota and the effect of gender
For the forehead samples, we observed four phyla (Fig. 1a),
with Actinobacteria being the most frequently isolated
microbial phylum in both men and women, representing
75Æ38% of all clones of the forehead skin (Table S2). Sub-
jects F4 (make-up using) and M3 contained an increased
proportion of Firmicutes sequences compared with the
other male and female subjects (Fig. 1a). Furthermore, the
phyla Firmicutes and Proteobacteria were detectable in both
sexes. The phylum Bacteroidetes could only be detected in
female subjects (Fig. 1a). From the 30 genera observed
on the forehead skin, Propionibacterium (73Æ11% of all
clones) and Staphylococcus (15Æ91% of all clones) were
most abundant. These two genera were detected in all 11
subjects and together they accounted for nearly 90% of all
clones. These findings indicate a small ecosystem on the
forehead skin (Fig. 1a), which was also evident on the
species level (Fig. 2a). Propionibacterium acnes was the
most predominant species on the forehead, accounting for
72Æ54% of all clones, but Staphylococcus epidermidis
(12Æ31%) was also frequently isolated from the forehead
(Table S3).
When comparing the samples from all subjects, the
forehead skin of women seems to harbour larger bacterial
diversity on the genus level (Fig. 1a). On this site, women
harboured on average 7Æ5 genera per subject and men
three genera per subject (Table S2). Also, on the species
level women showed an increased bacterial diversity com-
pared with male subjects (P £ 0Æ01) (Fig. 2a). However,
when excluding subjects using make-up (F2, F4 and F5)
in the comparison of the forehead microbiota of women

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1384 Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology
T. Staudinger et al. Skin microbiota of human forehead compared to forearm

(a) 18 (Fig. 2b). On the female forearm skin, an average of 25Æ2

16 SLOTUs per subject were found whereas an average of
14 18Æ4 SLOTUs were found on male forearm skin. The most
Number of Slotus

12 abundant species on the forearm was P. acnes, followed

10 by Staph. epidermidis, Rhizobium tropici and Micrococcus
8 luteus (Table S3).
6 Noteworthy, certain genera were exclusively found in
4 make-up-using women (Table S2). Examples include
2
Aerococcus (exclusively found in F5, both on forehead and
0
F1 F2 F3 F4 F5 F6 M1 M2 M3 M4 M5 forearm), Lautropia (forehead of F5; forearm of F4) and
Subjects Gordonia (forehead of F2 and F5; forearm of F2).

(b) 35
Effect of make-up on the forehead skin microbiota
30
Number of Slotus

25 Female subjects F2, F4 and F5, who used make-up every

20
15
10
5 to 5Æ66 genera per subject in the female subjects without
0
F1 F2 F3 F4 F5 F6 M1 M2 M3 M4 M5
Subjects
Figure 2 Distribution of species-level operational taxonomic unit
richness of bacterial communities present on the forehead (a) and
forearm skin (b). Subjects F2, F4 and F5 were make-up users.
and men, no significant differences (P = 0Æ26) in the bac-
terial diversity on the species level were detected, and also
the numbers of detected SLOTUs were similar (Fig. 2a).
Thus, gender does not seem to be a factor relevant for
microbial diversity on the forehead of the subjects investi-
gated.
Forearm skin microbiota A and the effect of gender
On the forearm skin, eight phyla were identified
(Table S2 and Fig. 1b). Actinobacteria, Firmicutes, Proteo-
bacteria, Bacteroidetes and Fusobacteria were found on
both men and women. The phylum Deinococcus-Thermus
was only detected in men, and an uncultured member of
the phylum Acidobacteria was only present in women.
The most frequently isolated genera on the forearm
were Propionibacterium (20Æ45%), Staphylococcus (12Æ88%)
and Corynebacterium (10Æ04%). In total, only 14 genera of
the 62 genera detected on the forearm were observed in
‡4 subjects, indicating substantial interpersonal diversity
at the genus level. A comparison of the microbiota diver-
sity of women and men showed clear differences on the
genus level (mean 18Æ5 genera per subject vs 14Æ4 genera
per subject). A similar result is also obtained when
comparing the number of SLOTUs found (P £ 0Æ01)
day, showed significantly higher microbial diversity
(P = 0Æ04) on their forehead skin than other female or
male subjects (P = 0Æ27) (Fig. 1a). These subjects
harboured an average of 10Æ33 genera ⁄ subject, compared
make-up and to 3Æ2 genera per subject in male subjects
(Table S2). Higher microbial diversity of the make-up
users is also evident from the number of SLOTUs found
with an average of 13Æ33 SLOTUs per subject present on
the forehead skin of make-up users compared to six
SLOTUs per subject in the female subjects without make-
up and to 4Æ2 SLOTUs per subject in male subjects
(Fig. 2a). Furthermore, the genera Selenomonas (in F5),
Aggregatibacter (in F5) and Aquicella (in F5) were exclu-
sively found in make-up-using women.
Thus, the use of make-up appears to be the most
important factor for causing differences in the bacterial
diversity on the forehead skin of the subjects investigated.
Skin nature does not appear to be a highly relevant
factor, because only female F4 had a more oily skin, but
all other women had normal or dry forehead skin
(Table S1).
Comparison of forehead and forearm microbiota
The comparison of the microbiota of forehead and fore-
arm skin clearly showed that the bacterial diversity of the
forearm was considerably greater than that of the
forehead (Fig. 1a,b). Even when including the data from
the make-up users, only 30 genera were observed on the
forehead compared to 62 genera on the forearm
(Table S2). Also on the species level the microbiota of the
forearm showed a much higher biodiversity than that of
the forehead. This is manifested by a mean of 23 SLOTUs
per subject compared to a mean of 7Æ18 SLOTUs per sub-
ject on forehead skin (Fig. 2a,b). However, the bacterial
communities from both sites showed some similarity in
qualitative terms. Of the 42 SLOTUs found on the

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Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology 1385
Skin microbiota of human forehead compared to forearm T. Staudinger et al.

forehead skin only five SLOTUs, including Acinetobacter were more similar to each other than they were to
haemolyticus, Aggregatibacter segnis, Aquicella siphonis, forearm skin samples (Fig. 3). An exception was M3,
Paracoccus sp., Selenomonas noxia were not present in the probable because of the high numbers of Staph. epidermi-
forearm samples (Table S2). Nevertheless, the overall mic- dis in this sample. Additionally, the forehead samples of
robiota differed substantially amongst the 11 subjects subject F1, F3 and F6 (women not using make-up) clus-
analysed (Fig. 1a,b). Propionibacterium acnes was the only tered closely to the forehead skin samples of the male
species present in all subjects on the forehead, and only subjects. The forehead samples of the make-up-using
four of the 42 SLOTUs were observed in more than ‡4 women (F2, F4 and F5) were more distant. Clustering of
subjects (Table S2). Conversely, 66Æ67% of all of the indi- the forearm samples of the 11 subjects revealed that they
vidual SLOTUs were identified only in a single individual. were not as closely related to each other as the forehead
The microbiota of the forearm revealed similar results. samples, clearly indicating greater diversity amongst the
Again P. acnes was the only SLOTU present in all 11 sub- forearm samples (Fig. 3).
jects (Table S2), 14 SLOTUs of the 128 SLOTUs were To determine whether individuals share a common skin
observed in more than ‡4 subjects and 54Æ69% of all of microbial diversity profile, the P test was used to assess
the individual SLOTUs were identified in a single subject intrapersonal and interpersonal variation (Table S4). Anal-
(Table S2). ysis of each pair of samples showed that no significance
This study also revealed that Gram-positive bacteria was found in skin samples from the same subject when
were much more abundant on the skin areas investigated both sites were considered. Furthermore, this analysis
(Table S5). About 95% of the bacteria found on forehead showed that if significance appeared between two samples
areas of both sexes were Gram-positive species. Gram- from different subjects, it did not follow any trend.
positive bacteria were also predominant on the forearm
areas (78Æ75 and 71Æ88%). The forearm areas of women
Discussion
showed slightly higher numbers of Gram-negative species
than male forearms (27Æ08% compared to 21Æ05%). Analysis of 1056 clones isolated from the superficial
To evaluate sample diversity and the relationship forehead and forearm skin revealed eight phyla, 67 genera
amongst samples PCoA was performed. This analysis sug- and 133 SLOTUs. Of these, the forehead microbiota
gested that the forehead skin samples from male subjects harboured four phyla, 30 genera and 42 SLOTUs, with an
estimated species coverage of c. 65Æ6%, and the forearm
PCA - P1 vs P2 microbiota harboured eight phyla, 62 genera and 128
0·5 SLOTUs, with estimated species coverage of c. 75Æ3%.
The most abundant phyla on the forehead as well
0·4
as on the forearm were Actinobacteria, Firmicutes and
P2 - Percent variation explained 18·91%

M4a Proteobacteria. These three phyla have also been reported

0·3
to be predominant on other skin areas (Dekio et al. 2005;
F2a
0·2 Gao et al. 2007; Fierer et al. 2008; Grice et al. 2008,
M1a
2009). The phylum Bacteroidetes could only be harboured
0·1 F3a F2f
on the forehead of female subjects. We cannot rule out
F5a
M1f
F6f F3f M2f the possibility, however, that increased sampling would
M4f F1f M5f
0·0 F6a show the presence of this phylum also in male subjects.
F5f
F4a
M5a On the forearm skin, the phylum Deinococcus-Thermus
–0·1 MBa was only detected in men, and an uncultured member of
M2a
M3f the phylum Acidobacteria was only present in women.
–0·2 F1a Again, we cannot rule out the possibility that with
F4f increased sampling these two latter phyla might be found
–0·3
in both genders.
–0·4 Although there appears to be a core set of phylotypes
–0·3 –0·2 –0·1 0·0 0·1 0·2 0·3 0·4 present on superficial human skin, the overall microbiota
P1 - Percent variation explained 48·61% differed substantially amongst the 11 subjects. Three
genera (Propionibacteria, Staphylococcus, and Coryne-
Figure 3 Principal coordinate analysis of species-level operational
taxonomic unit relatedness of 22 skin samples from 11 subjects. The
bacteria) were commonly found, but only 1Æ5 % of the
axes are labelled with the per cent of the variation explained by each SLOTUs and 4Æ48% of the genera were found in all 11
principal component. The circle represents all forehead skin samples, subjects. In contrast, 50Æ38% of all of the individual
which cluster closely together. F, female; M, male; f, face; a, arm. SLOTUs and 46Æ27% of the genera were identified only in

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1386 Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology
T. Staudinger et al.
a single individual. These data indicate that the superficial
human skin microbiota is highly diversified with a low
level of interpersonal consensus. Similar findings were
reported in recent studies (Gao et al. 2007; Fierer et al.
2008; Grice et al. 2008, 2009). A pairwise comparison of
all subjects revealed that significance did not follow any
trend, suggesting that occurrence of significance was more
random than directed amongst different subjects. The low
interpersonal consensus is also evident when comparing
our results to the study of Gao et al. (2007), because of
the 67 genera found in our study only 36 genera were
also found in the previous study.
The most frequently isolated organism on the forehead
as well as on the forearm was P. acnes, which was the
only species present in all skin samples of the ten
subjects. Moreover, the forehead microbiota was domi-
nated by P. acnes accounting for 72Æ54% of all clones. In
contrast, P. acnes represented only 18Æ94% of the clones
isolated from the forearm skin. Thus, we conclude that a
rich population of Propionibacteria is a stable characteris-
tic of the skin of the forehead. So far, no other exposed
microenvironment of the human body has been found to
be similarly dominated by a single bacterial species (Evans
1975). Propionibacterium, and P. acnes in particular, is the
most dominant organism in normal human skin, as
shown in prior culture-based studies and in molecular
analyses (Fredricks 2001; Gao et al. 2007, 2008). Although
it has been suggested that P. acnes is associated with seri-
ous skin disorders, such as acne vulgaris and rosacea
(Leyden 2001), it is likely that it might protect the skin
against more aggressive microbes (Eady and Ingham
1995; Leyden 2001; Bek-Thomsen et al. 2008). It is not
yet clear what factors dictate the level of the Propionibac-
teria population, but it seems probable that the critical
substances will be found in sebum (Evans 1975).
It is interesting to note that subject F4 appears to be a
major outlier with respect to community composition.
On the forehead skin of subject F4, P. acnes was found to
be underrepresented. Instead, an increased proportion of
Firmicutes sequences was detected. These sequences
appear to be derived from Staphylococcus species, suggest-
ing that subject F4 was colonized with Staphylococcus, as
found by cultivation studies in c. 5–10% of healthy adults
(Nagase et al. 2002). As subject F4 was using make-up,
this different bacterial composition might be attributed to
an influence of make-up.
Of the ten most common species on the forearm skin
found by Gao et al. (2007), six (including P. acnes,
Corynebacterium tuberculostearicum, Corynebacterium
mucifaciens, Streptococcus mitis, Staph. epidermidis and
Corynebacterium amycolatum) were also found in our
study. However, in our study only P. acnes, Staph.
epidermidis and Strep. mitis were present in high num-
ª 2011 The Authors
Journal of Applied Microbiology 110, 1381–1389 ª 2011 The Society for Applied Microbiology
Skin microbiota of human forehead compared to forearm
bers. Acinetobacter junii and Deinococcus AJ549111 were
not found in our study, but we detected other closely
related Acinetobacter and Deinococcus species (Table S2).
Enhydrobacter aerosaccus, identified in a study by Gao
et al. (Gao et al. 2007), was not detected in subjects of
the present study.
We also detected some potential pathogens on the fore-
head as well as on the forearm skin, such as Staphylococ-
cus aureus, Neisseria meningitis, Streptococcus pneumonia
and Haemophilus influenzae. These organisms are consid-
ered as transient colonizers of the human skin producing
various kinds of skin-damaging toxins and are suspected
to play a role in serious skin diseases (Roth and James
1988; Wilson 2005). They are ubiquitously present in the
environment, e.g. in soil, tap water and crops. Therefore,
these species are able to cope with harsh environmental
conditions. Consequently, they are well equipped to sur-
vive the rapidly changing conditions of the surface of the
skin and it is possible that the skin microbiota may be a
reservoir of such opportunistic pathogens derived from
the environment (Dekio et al. 2005).
It was reported previously that Gram-positive bacteria
are more abundant on superficial human skin than
Gram-negative bacteria (Roth and James 1988; Wilson
2005; Gao et al. 2007). This was confirmed by our study.
On the other hand, from culture-based studies it has also
been suggested that the skin of male individuals, being
more humid because of enhanced sweat production, is
more frequently colonized by Gram-negative bacteria than
that of women, as high moisture is necessary for survival
of these bacteria (Roth and James 1988). This observation
could not be confirmed by our study. We found that the
skin of women was more frequently colonized by
Gram-negative species than that of men, which is in accor-
dance with previous molecular genetic analysis (Gao et al.
2007). As Gram-negatives were more abundant on the
forearm, it might be suggested that occlusion of the skin
by dressing could have a profound effect on the micro-
environment of the forearm skin area by reducing the
evaporate water loss. Thus, increasing the water content of
the occluded region may lead to a higher humidity suitable
for the growth of Gram-negative bacteria (Wilson 2005).
In contrast, the cutaneous microbiota of the forehead
is always exposed to the external environment and there-
fore subject to UV, temperature and enhanced mechanical
stress. Probably Gram-negative bacteria are more sensitive
to these conditions because of their cell wall structure
(Roth and James 1988; Wilson 2005). This might explain
our finding of their less abundant presence on the
forehead skin.
Analysis of the distribution of SLOTUs between
forehead and forearm revealed that only five of the 42
SLOTUs found in the forehead samples were not shared
1387
Skin microbiota of human forehead compared to forearm
with the forearm samples, but quantitatively there was
more discrepancy between the two regions. The forehead
skin harboured only a few species (mean: 7Æ18 SLOTUs
per subject). Whereas the forearm microbiota showed a
more complex community structure (mean: 23 SLOTUs
per subject), suggesting that the forearm skin is a highly
diverse ecosystem. This difference in bacterial diversity
between the forehead samples and the forearm samples
was statistically significant (P = 0Æ03). Together these
results demonstrate that the phylotype richness of the
forearm was higher than that of the forehead skin area.
These results were surprising, as the forehead was
expected to have higher levels of diversity than the fore-
arm because of the more frequent contact with potential
invaders from the environment. On the other hand, a
higher concentration of antimicrobial substances on the
forehead could inhibit the growth of some species and
the dominant presence of P. acnes could protect this site
from other species by competing for nutrients, niches and
receptors (Gao et al. 2008).
Comparing all forehead samples from men and women
revealed significant diversity amongst them. However,
when samples from make-up-using women (F2, F4 and
F5) were excluded, these differences between male and
female samples were not longer observed. Thus, the use
of make-up appears to strongly interfere with the compo-
sition of the bacterial communities. It is important to
note, however, that further experiments using higher
numbers of samples are needed to substantiate this con-
clusion. For instance, the observed differences might also
be a consequence of a bacterial contamination of the
make-up. In fact, especially the presence of Aquicella,
which is usually found in thermal water, strongly indi-
cates a contamination of make-up. Use of skin cleansers
and skin moisturizers does not appear to influence the
skin in a way as make-up does. Female subjects F1, F3
and F6 in our study never used make-up but used skin
cleansers and skin cosmetics like moisturizers in their
daily life. These subjects did not show increased numbers
of phylotypes on the forehead skin.
Acknowledgements
The research was partially funded by a COMET project
from the Austrian Research Promotion Agency (FFG).
The authors thank Rajam Csordas and Alexandra Lusser
for critical reading and correction of the manuscript.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Collector’s curves of observed and estimated
SLOTU richness of pooled forehead (a) and forearm (b)
skin samples from the 11 subjects investigated.
Table S1 Skin characteristics of the individuals investi-
gated, (F) female, (M) male.
Table S2 Representation of all 1056 clones investigated,
(F) female, (M) male.
Table S3 Most common species represented in forehead
and forearm samples.
Table S4 Pairwise P-values of P-test analysis in bacterial
population distribution of the 22 samples from the 11
subjects, (F) female, (M) male, (f) face, (a) arm.
Table S5 Representation of all Gram-positive and
Gram-negative phyla detected on the superficial human
skin of the forehead and of the forearm in 11 subjects.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
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