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RESEARCH ARTICLE
Scalp bacterial shift in Alopecia areata
Daniela Pinto
1,2,3
, Elisabetta Sorbellini
2,3
, Barbara Marzani
1,2,3
, Mariangela Rucco
3
,
Giammaria Giuliani
1,2
, Fabio RinaldiID
1,2,3
*
1Giuliani SpA, Milan, Italy, 2Human Advanced Microbiome Project-HMAP, Milan, Italy, 3International Hair
Research Foundation (IHRF), Milan, Italy
*fabio.rinaldi@studiorinaldi.com
Abstract
The role of microbial dysbiosis in scalp disease has been recently hypothesized. However,
little information is available with regards to the association between microbial population on
the scalp and hair diseases related to hair growth. Here we investigated bacterial communi-
ties in healthy and Alopecia areata (AA) subjects. The analysis of bacterial distribution at the
genus level highlighted an increase of Propionibacterium in AA subjects alongside a general
decrease of Staphylococcus. Analysis of log Relative abundance of main bacterial species
inhabiting the scalp showed a significant increase of Propionibacterium acnes in AA sub-
jects compared to control ones. AA scalp condition is also associated with a significant
decrease of Staphylococcus epidermidis relative abundance. No significant changes were
found for Staphylococcus aureus. Therefore, data from sequencing profiling of the bacterial
population strongly support a different microbial composition of the different area sur-
rounded hair follicle from the epidermis to hypodermis, highlighting differences between nor-
mal and AA affected the scalp. Our results highlight, for the first time, the presence of a
microbial shift on the scalp of patients suffering from AA and gives the basis for a larger and
more complete study of microbial population involvement in hair disorders.
Introduction
Alopecia areata (AA) is the second most common type of hair loss disorder for human beings.
It occurs in the form of a non-scarring alopecia which affects the scalp and, eventually, the
entire body [1]. An incidence higher than 2% has been reported for AA, with a lifetime risk of
1.7% both in men and women [2].
For subjects affected by AA, the catagen phase is either extremely short or doesn’t occur at
all, and in turn proceeds rapidly to telogen phase. From a clinical point of view, this led to sin-
gle or several annular or patchy bald lesions usually on the scalp [3,4]. These lesions can extend
to the entire scalp (Alopecia totalis) or to the entire pilar area of the body (Alopecia
universalis).
The management of AA still remains a challenge and is mainly aimed at containing it.
Among treatments currently available [5], in 2012, the British Association of Dermatologists
recommended two main treatments with a C grade of recommendation: i) topical and intrale-
sional corticosteroid (limited patchy hair loss); ii) immunotherapy (extensive patchy hair loss
and Alopecia totalis/universalis) [6].
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 1 / 11
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OPEN ACCESS
Citation: Pinto D, Sorbellini E, Marzani B, Rucco M,
Giuliani G, Rinaldi F (2019) Scalp bacterial shift in
Alopecia areata. PLoS ONE 14(4): e0215206.
https://doi.org/10.1371/journal.pone.0215206
Editor: Brenda A. Wilson, University of Illinois at
Urbana-Champaign, UNITED STATES
Received: July 6, 2018
Accepted: March 28, 2019
Published: April 11, 2019
Copyright: ©2019 Pinto et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: Financial assistance received from by
Giuliani SpA. The funder provided support in the
form of salaries for authors [DP, BM and FR], but
did not have any additional role in the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of
these authors are articulated in the ‘author
contributions’ section.
Competing interests: DP, BM are employed by
Giuliani S.p.A.; FR serves as consultant for Giuliani
S.p.A; GG is part of Board of Directors of Giuliani S.
Causes behind AA are not yet fully understood, and there have been debates dating back to
the beginning of the 1800s. Many associations have been proposed by researchers over the
years [7]. However, clinical evidence and association with other immune disorders [8] under-
line the role of immunity and inflammation in the early development of AA [9–11]. Interest-
ingly, authors [11] reported the efficacy of PRP (Platelet-rich plasma) on AA as a potent anti-
inflammatory agent by suppressing cytokine release and limiting local tissue inflammation
[11].
Other comimon (common?) recognized offenders are hormonal imbalance, psychological
stress, genetic tendencies, other local skin disorders and also nutritional deficiencies [5]. More
recently, some authors reported evidence of the link between the gut microbiome and AA
[12,13] but little information is currently available as regards microbial communities on the
scalp [14,15]. Due to its unique features, the scalp is expected to harbor a specific microbiome,
which is expected to play a peculiar role in scalp conditions related to hair growth [16].
In this work, we present data on bacterial communities in healthy and AA subjects, on a
sample of Italian population. Our results highlight, for the first time, the presence of a signifi-
cative bacterial disequilibrium on the scalp of AA subjects compared to healthy population;
this disequilibrium also extends in the subepidermal compartments of the scalp.
Material and methods
Subjects recruitment
Fifteen healthy and AA subjects, respectively (20–60 years old; 40% male) were recruited from
a private Italian dermatological clinic (Milan, Italy).
All subjects have been enrolled under dermatological control. AA subjects have been previ-
ously evaluated about their disease history and by means of clinical examinations. Subjects
have been enrolled in control population after clinical examinations and in absence of any his-
tory of dermatological or scalp disorders.
All enrolled subjects had to meet the following criteria: i) no antibiotics in the 30 days lead-
ing up to the sampling; ii) no probiotics in the last 15 days; iii) the last shampoo was performed
48h before sampling; iv) no pregnancy or lactation; v) suffering from other dermatological dis-
eases; vi) no anti-tumor, immunosuppressant or radiation therapy in the last 3 months; vii) no
topical or hormonal therapy on the scalp in the last 3 months.
The study was approved by the Ethical Independent Committee for Clinical, not pharmaco-
logical investigation in Genoa (Italy) and in accordance with the ethical standards of the 1964
Declaration of Helsinki. All of the volunteers signed the informed consent.
Swab sample collection
The scalp surface has been sampled by means of swab procedure according to previously
reported methods [17,18] with minor modifications. Sterile cotton swabs were soaked for at
least 30s in ST solution (NaCl 0.15 M and 0.1% Tween 20) before sampling. A comb was used
to separate hair fibers and collect samples from a total area of 16cm
2
from a different area of
the scalp. After collection, the head of each swab was cut and stored in ST solution. Samples
from the same subjects were collected together and stored at 4˚C until DNA extraction. Sterile
cotton swabs placed in ST solution have been used as negative controls.
Biopsy samples collection
A total of 4 female subjects (two control and two AA, respectively) were also sampled for the
microbial community in the subepidermal compartments of the scalp. A 4-mm punch biopsy
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 2 / 11
p.A. This does not alter our adherence to PLOS
ONE policies on sharing data and materials.
specimen was collected from each subject. In AA subjects, the specimen was obtained from a
well-developed lesion. The sampled area was disinfected prior to the surgery to avoid contami-
nation from surface bacteria. Epidermis, dermis and hypodermis were aseptically separated
and stored in Allprotect medium (Qiagen) according to manufacturer conditions until DNA
extraction.
Bacterial DNA extraction
Bacterial DNA from scalp swabs was extracted by mean of QIAamp UCP Pathogen Mini Kit
(Qiagen, Milan, Italy) according to manufacturer protocol, with minor modifications [19].
The DNeasy Tissue kit (Qiagen, Milan, Italy) was used for DNA extraction from biopsy speci-
mens. Extracted DNA was finally suspended in DNAse free water and quantified by the
QIAexpert system (Qiagen, Milan, Italy) before qRT-PCR and sequencing.
High throughput 16S amplicon generation, sequencing and analysis
DNA samples extracted from scalp swabs were amplified for the variable region V3-V4 using the
universal prokaryotic primers: 341 F CTGNCAGCMGCCGCGGTAA [20,21] and 806bR GGACTA
CNVGGGTWTCTAAT [22–24] utilizing a modified dual-indexed adapter-linked single step pro-
tocol. Library preparation and Illumina MiSeq V3-V4 sequencing were carried out at StarSEQ
GmbH, Mainz, Germany, according to the method of Caporaso et al. [25] and Kozich et al., [26]
with minor modifications. Amplicons were generated using a high fidelity polymerase (AccuS-
tart II PCR ToughMix, Quantabio, Beverly, MA). The amplicons were then normalized to equi-
molar concentrations using SequalPrep Plate Normalization Kit (ThermoFisher Scientific,
Monza, Italy) and the final concentration of the library was determined using a fluorometric kit
(Qubit, Life technologies, Carlsbard, CA, USA). Libraries were mixed with Illumina-generated
PhiX control libraries and denatured using fresh NaOH. Runs were performed using Real-Time
Analysis software (RTA) v. 1.16.18 and 1.17.22, MiSeq Control Software (MCS) v. 2.0.5 and
2.1.13, varying amounts of a PhiX genomic library control, and varying cluster densities. Four
sequencing runs were performed with RTA v. 1.18.54, MCS v. 2.6, a target of 25% PhiX, and
600–700 k/mm2 cluster densities according to Illumina specifications for sequencing of low
diversity libraries. We used 25% PhiX to balance the runs and use 600 bp V3 chemistry for
sequencing. Basecalls from Illumina High Throughput Sequencing (HTS) machines were con-
verted to fastQ files using bcl2fastq (Illumina) software, v2.20.0.42 and quality control carried
out by mean of, v0.11.5. bcl2fastq (Illumina) software, v2.20.0.422. Quality control of fastq reads
was carried out using FastQC v0.11.5. The quality trimming of primers and adaptors was carried
out using Cutadapt, v. 1.14 [27] and Sickle v. 1.33 [28] toolkits, respectively.
Paired-end reads were assembled using Pandaseq v. 2.11[29] using a threshold of 0.9 and a
minimum overlap region length of 50. Clustering was carried out using closed-reference OTU
picking and de novo OUT picking protocol of QIIME v1.9 [25] at 97% identity.
Greengenes database v13_8 was used as a reference for bacterial taxonomic assignment
[30]. Amplicon reads were also analyzed as regards alpha diversity by mean of Shannon index,
using QIIME v1.9.
Bacteria quantification by qRT-PCR
Relative abundance of bacterial DNA of main bacterial species on the scalp was assessed by
means of real-time quantitative PCR (RT qPCR). Microbial PCR assay kit (Qiagen, Milan, Italy)
with gene-specific primers and TaqMan MGB probe targeting Propionibacterium acnes,Staphy-
lococcus epidermidis and Staphylococcus aureus 16S rRNA gene, respectively, were used. Gen-
bank accession numbers of 16S rRNA gene sequences for P. acnes, S. aureus and S. epidermidis
Scalp bacterial shift in Alopecia areata
PLOS ONE | https://doi.org/10.1371/journal.pone.0215206 April 11, 2019 3 / 11
were ADJL01000005.1, ACOT01000039.1 and ACJC01000191.1, respectively. Samples were
mixed with 12.5μL of Microbial qPCR Mastermix, 1 μL of Microbial DNA qPCR Assay, 5ng of
genomic DNA sample and Microbial-DNA-free water up to a final volume of 25 μL.
Nine separate PCR reactions are prepared for each sample, including Positive PCR Control,
No Template Control, and Microbial DNA Positive Control, as well as the Microbial DNA
qPCR Assay. Pan-bacteria (Genebank accession number HQ640630.1) assays that detect a
broad range of bacterial species are included to serve as positive controls for the presence of
bacterial DNA. Assays for human GAPDH and HBB1 (Genebank accession numbers
NT_009759.16 and NT_009237.18, respectively) have been included to determine proper sam-
ple collection and used to assess the presence of human genomic DNA in the sample and,
eventually, subtracted from calculation. Thermal cycling conditions used were as follows; 95˚C
for 10 min, 40 cycles of 95˚C for 15 sec, 60˚C for 2 min. PCR reactions were performed in
duplicate using an MX3000p PCR machine (Stratagene, La Jolla, CA). Amplification-curve
plotting and calculation of cycle threshold (Ct) values were performed using MX3000p soft-
ware (v.3; Stratagene) and data were further processed by Excel. ΔΔCt method [31] was used
to calculate bacterial load of each swab sample. Obtained values have been used for calculation
of Bacterial Load-Fold Change (AA/Healthy subjects). Data is finally expressed as Log of the
relative abundance of each sample versus the control group.
Statistical analysis
Data is expressed as log Relative abundance (RA) ±SEM for qRT-PCR analysis. Results were
checked for normal distribution using D’Agostino & Pearson normality test before further
analyses. Statistically significant differences on bacterial community between healthy and AA
group were determined using Wilcoxon test (p 0.05). All the comparisons were performed
pairwise for each group. Analyses were performed with GraphPad Prism 7.0 (GraphPad Soft-
ware, Inc., San Diego, CA). P–values equal to or less than 0.05 were considered significant.
Results
Microbiota profiling of the scalp in AA subjects
The human scalp’s bacterial composition of Control (n = 15) and AA (N = 15) subjects have
been analyzed by IlluminaSeq (Fig 1). We obtaining about 585,219 and 544,578 high quality
reads for the total V3-V4 sequences from control and AA subjects, respectively. About 56.3%
of sequences from the control group were assigned to Actinobacteria phylum and 35.2% to Fir-
micutes. As regards, AA group Actinobacteria were around 57.4% and Firmicutes decreased to
29.2%. The analysis of bacterial distribution at the genus level, interestingly, highlighted an
increase of Propionibacterium from 45.6% to 55.1% in AA subjects. Alongside data showed a
general decrease of Staphylococcus from 32.6% to 27.4% (Fig 1A). Therefore, the percentage of
other less abundant bacteria genus was similar (around 5%) both in control and AA subjects.
Alpha-diversity (Shannon diversity index) was significantly higher (p 0.001) in AA subjects
than in the control group (Fig 1B).
Microbial shift of the scalp surface in AA subjects
As previously reported by other authors [14,15], P.acnes,S.epidermidis and S.aureus are the
three major microbial species found on the scalp.
Relative abundance of predominant bacteria on scalps both of control and AA subjects has
been analyzed by mean of RT q-PCR. Primers and TaqMan MGB probe specific for 16S region
of P.acnes,S.epidermidis and S.aureus were used.
Scalp bacterial shift in Alopecia areata
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Pan bacteria specific targets designed to detect the broadest possible collection of bacteria
involved in human biology were used as control. Student’s test analysis of log Relative abun-
dance comparing control and AA subjects showed a significant (p<0.01) increase of P.acnes
(from 1.6 to 1.8 log RA) in AA subjects compared to control ones (Fig 2A). AA scalp condition
is also associated with a significant (p<0.05) decrease of S.epidermidis relative abundance
(from 1.4 to 1.01 log RA) (Fig 2B) while no significant changes were found for S.aureus
(Fig 2C).
Microbial shift due to AA is also clear as regards the proportion of bacterial populations
analyzed. The ratio P.acnes/ S.epidermidis is significantly higher (p<0.05) in AA subjects
(mean ratio = 2.1±0.3) compared to control subjects (mean ratio = 1.3±0.1) (Fig 2D). Addi-
tionally, the P.acnes/ S.aureus ratio was also significantly higher (p<0.01) in AA subjects
(mean ratio = 1.4±0.1 vs mean ratio = 1.2±0.1) (Fig 2E). No significative differences were
found in the ratio S.epidermidis / S.aureus (Fig 2F).
Fig 1. Bacterial profiling in control and AA subjects. (A)% of bacteria at genus level in the control and AA groups.
Results are presented as the percentage (%) of total sequences, (p0.05). (B) Shannon diversity index for bacterial
population observed in control and AA subjects (p0.05).
https://doi.org/10.1371/journal.pone.0215206.g001
Fig 2. Relative abundance of main bacterial species on the scalp of AA andcontrol subjects by RT qPCR. Box and
Whisker comparing the log relative abundance of P.acnes,S.epidermidis and S.aureus collected by swabbing the scalp.
(A) Log Relative abundance of P.acnes in Control and AA subjects. (B) Log Relative abundance of S.epidermidis in
Control and AA subjects. (C) Log Relative abundance of S. aureus in Control and AA subjects. Ratios P. acnes/ S.
epidermidis (D), P. acnes/ S. aureus (E) and S. epidermidis / S. aureus (F) in Control and AA subjects. Values are
presented as mean +/- SEM, in duplicate. Box-and-Whiskers plot showing median with 25th to 75th percentile. The
center line of each box represents the median; data falling outside the whiskers range are plotted as outliers of the data.
https://doi.org/10.1371/journal.pone.0215206.g002
Scalp bacterial shift in Alopecia areata
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AA alteration of bacterial distribution in the subepidermal compartments
of the scalp
Two bioptic samples were collected respectively from control and AA subjects and divided in
the main subepidermal compartments. Extracted genomic DNAs were analyzed by Illumina-
Seq and analyzed for bacterial distribution.
Similar proportions of Firmicutes (24.6% vs 27.6%) and Proteobacteria (16.2% vs 16.9%)
were reported in epidermis of both control and AA subjects (Fig 3) while a higher proportion
of Actinobacteria (33.3% vs 22.4%) and Bacteroidetes (20.1% vs 9.9%) were found in AA sub-
jects compared to control (Fig 3). Bacterial community in dermis shifted to a lower proportion
of Actinobacteria (6.1% vs 11.3%) in AA subjects while Proteobacteria (14.9% vs 8.1%) and
Bacteroidetes (14.2% vs 4.0%) increased compared to control (Fig 3). Also hypodermis showed
a peculiar bacterial distribution which results, also in this case affected by scalp condition. AA
subjects showed a significative higher proportion of Proteobacteria,Bacteroidetes and espe-
cially Firmicutes than control subjects (Fig 3). In general less variability was observed for bacte-
rial communities in AA subjects and this may reflect in a compromised healthiness of the
scalp.
Most interesting, the analysis at species level of bioptic samples highlighted the presence of
Prevotella copri in both AA samples, in all analyzed compartments.
Akkermansia muciniphila was also found (less than 1.5% of total population) in AA sub-
compartments of the scalp, in particular in the hypodermis.
Discussion
In this study, we reported, for the first time, the relationship between microbial shift on the
scalp and hair growth disorder, in particular, Alopecia areata. We conducted analysis by mean
of qRT-PCR and 16S sequencing.
A diversified and abundant microbial community host the skin [32] and this symbiotic rela-
tionship results, most of the time, as beneficial for both the host and microbial community
[33–35]. Bacteria mainly belong to Corynebacteriaceae, Propionibacteriae, and
Fig 3. Bacterial profiling of scalp biopsy samples from control and AA subjects. % of bacteria at phylum level in the
control and AA groups in the epidermis, dermis and hypodermis. Results are presented as the percentage (%) of total
sequences.
https://doi.org/10.1371/journal.pone.0215206.g003
Scalp bacterial shift in Alopecia areata
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Staphylococcaceae [36–39] and are differently distributed according to the physiochemical
properties of each skin site they host [39,40]. Many scientific published evidence reported the
strict correlation between microbial disequilibrium and skin conditions [41–45]. Little is still
reported with regards to the microbiome inhabiting the scalp and hair growth disorders
[14,15,46]. Clavaud and collaborators [15] and, more recently, Xu et al. [14] reported, the
implication of microorganisms in the development of dandruff. Characterization of scalp bac-
terial species involved in hair disorders such as Alopecia androgenetica, Alopecia areata, and
Lichen Planopilaris has been poorly investigated and, only recently, the piece bit of evidence
has been reported [16].
We focused our attention on bacterial population of the scalp of healthy and AA subjects
looking at main bacterial species on the scalp [15] (P.acnes,S.aureus, and S.epidermidis) and
at their reciprocal balancing. We quantified their relative abundance by means of accurate
gene-specific primers and probe targeting 16S region, by RT qPCR. Our results are concurrent
with Wang’s work [46] highlighting the reciprocal inhibition exerted by bacteria, each other,
on the scalp (Propionibacterium vs Staphylococcus and vice-versa). AA subjects showed an
increase in P.acnes and a decrease of Staphylococcus, especially S.epidermidis, suggesting the
role of Propionibacterium/Staphylococcus balancing in AA. A role of P.acnes with hair casts
and Alopecia has previously been hypothesized by Wang and collaborators [46] even though
not deeply investigated. P.acnes is able to synthesize many enzymes involved in the metabo-
lism of porphyrins that, once activated, may contribute to oxidation and follicular inflamma-
tion. Therefore, a speculation about the role of the hypoxic condition of the follicular region
may be speculated in AA and this may encourage P.acnes overgrowth. A role of hypoxia has
been reported in the progression of other skin condition such as psoriasis [47] and atopic der-
matitis [48]. The presence of A.muciniphila, a strictly anaerobic bacteria, around the hair folli-
cle in analyzed AA subjects may be suggestive of a hypoxic ecosystem in which this bacteria
can find favorable growth conditions.
Data from IlluniaSeq profiling also suggested a higher diversity of bacterial species inhabit-
ing the scalp of AA subjects. These results are in line with previous work [15] on other scalp
conditions. On the basis of the present and previous results, a link with a higher susceptibility
of an unhealthy scalp to be colonized by microorganisms could be postulated but further anal-
ysis are needed to understanding the reason behind this high variety.
Beyond the superficial relationship between the microorganism with skin, microbes can
also communicate with cells of the subepidermal compartments [49] and are involved also in
deep immunological response [50–54]. As reported by Nakatsuji et al., [49] high interpersonal
variability was observed as regards epidermal and subepidermal microbial population. In this
study, data from sequencing profiling of the bacterial population strongly support a different
microbial composition of different area surrounded hair follicle from the epidermis to hypo-
dermis, highlighting differences between normal and AA affected scalp. We can hypothesize
the role of this different microbial composition in AA symptoms and manifestations.
Microbial changing at different subepidermal compartment may be linked to an autoim-
mune component of the pathology as to skin barrier skin disruption, as previously shown for
other skin disorders [55].
Most interesting, the analysis at species level of bioptic samples highlighted the peculiar
presence of P.copri and A.muciniphila in both AA samples, in all analyzed compartments.
These findings are very intriguing. The finding of Prevotella copri as one of the most abundant
bacteria in subepidermal compartments of AA scalp may be linked to the autoimmune compo-
nent of this hair condition. For example, P.copri has been found as relevant in the pathogene-
sis of rheumatoid arthritis [56], another chronic inflammatory autoimmune disorder that can
affect other parts of the body including the skin. Therefore the identification of A.muciniphila
Scalp bacterial shift in Alopecia areata
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in the subepidermal compartments of the scalp of AA subjects could open to new therapeutic
approaches in the management of AA. The link between A.muciniphila and skin disease has
been yet discussed as it has been considered a gut signature of psoriasis [57].
The present work reported data from an initial pilot study. Future studies should be aimed
at better investigate both the role of microbial community shifts and hypoxia in hair scalp dis-
eases. Also the study of additional factors such as inclusion of samples from non-lesional sites
in AA and non-AA subjects and from other baldness disease besides AA and the role should
be considered.
Conclusions
Our study highlighted, for the first time, the presence of a microbial shift on the scalp of
patients suffering from AA and gives the basis for a larger and more complete study of micro-
bial population involvement in hair disorders. Therefore, the reported findings as the availabil-
ity of sophisticated and quick methods to evaluate the microbial composition of the scalp open
to new therapeutic approaches in the management of hair disorders.
Larger studies are still needed for a more precise identification of bacterial community on
the scalp as for the analysis of fungal component in AA subjects but the results of the present
work permit to asses, for the first time, the involvement of microbial changing in hair disorder,
in particular AA, also in the subepidermal compartments of the scalp.
Author Contributions
Conceptualization: Elisabetta Sorbellini, Barbara Marzani, Giammaria Giuliani, Fabio
Rinaldi.
Data curation: Daniela Pinto, Mariangela Rucco.
Formal analysis: Daniela Pinto.
Investigation: Daniela Pinto, Elisabetta Sorbellini, Mariangela Rucco, Fabio Rinaldi.
Methodology: Daniela Pinto, Fabio Rinaldi.
Supervision: Elisabetta Sorbellini, Fabio Rinaldi.
Writing – original draft: Daniela Pinto, Elisabetta Sorbellini, Barbara Marzani, Fabio Rinaldi.
Writing – review & editing: Daniela Pinto, Elisabetta Sorbellini, Barbara Marzani, Fabio
Rinaldi.
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