Microbiome Signature and Diversity Profiling of Normal Skin of Human in Saudi Arabia

Abstract

Background: Studying human skin-associated bacterial communities is crucial to understanding human diseases, disease progression, and their role in maintaining human health. Objectives: This study aimed to identify normal (healthy) skin microbiome signatures of eight individuals living in Jeddah, Makkah Al-Mukarramah region, Saudi Arabia. Methods: The study involved the analysis of resident skin microbiome in inner elbow of the right arm after ethical approval is issued and an informed consent form is signed by participant individuals. Results: Phylogenetic tree indicated the existence of four phyla, e.g., Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria. Firmicutes were shown to be the highest in abundance, while Bacteroidetes were the lowest. At the genus level, Staphylococcus was the highest in abundance, while Enterococcus was the lowest. At the species level, Bacilluscereus was the highest in abundance, while Roseomonasmucosa was the lowest. The analysis for the highly abundant operational taxonomic units (OTUs) indicated a dramatic difference between sexes referring to either genera or species of which Staphylococcussp., Erwiniasp., Pseudomonassp., Sphingomonassp., Corynebacteriumsp., Propionibacterium acnes, Kocuria palustris are higher in males, while Bacillus cereus, Bacillus sp., Erwinia sp., Corynebacterium sp., Micrococcus sp., Pseudomonassp. are lower in males. Conclusions: The study succeeded in detecting the skin microbiome of individuals in Saudi Arabia.

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Jundishapur J Microbiol. 2021 February; 14(2):e113355.
Published online 2021 May 22.
doi: 10.5812/jjm.113355.
Research Article
Microbiome Signature and Diversity Profiling of Normal Skin of
Human in Saudi Arabia
Hanan AlQattan 1, *, Sherif Edris 2,3, 1 , Aala A.Abulfaraj 4, **, Raed ALbiheyri 1, Lojayn Tollbah 1,
Mohammed Alghamdi. 1, Ahmed Bahieldin 1, 2 , Sameer Zimmo 5and Rashad Al-Hindi 1
1Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
2Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt
3Princess Al-Jawhara Al-Brahim Centre of Excellence in Research of Hereditary Disorders (PACER-HD), Faculty of Medicine, King Abdulaziz University,Jeddah, Saudi Arabia
4Department of Biological Sciences, Science and Arts College, Rabigh Campus, King Abdulaziz University, Jeddah, Saudi Arabia
5Department of Dermatology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
*Corresponding author: Department of Biological Sciences, Faculty of Science, King Abdulaziz University,Jeddah, Saudi Arabia. Email: halgattan0001@stu.kau.edu.sa, Tel:
+966- 536935619
**Corresponding author: Department of Biological Sciences-Rabigh Campus, King Abdulaziz University, Jeddah 21589, Saudi Arabia. Email: aaabulfaraj@kau.edu.sa
Received 2021 March 19; Revised 2021 April 27; Accepted 2021 April 28.
Abstract
Background: Studying human skin-associated bacterial communities is crucial to understanding human diseases, disease progres-
sion, and their role in maintaining human health.
Objectives: This study aimed to identify normal (healthy) skin microbiome signatures of eight individuals living in Jeddah, Makkah
Al-Mukarramah region, Saudi Arabia.
Methods: The study involved the analysis of resident skin microbiome in inner elbow of the right arm after ethical approval is
issued and an informed consent form is signed by participant individuals.
Results: Phylogenetic tree indicated the existence of four phyla, e.g., Actinobacteria,Bacteroidetes,
Firmicutes,Proteobacteria.Firmicutes were shown to be the highest in abundance, while Bacteroidetes were the lowest. At the
genus level, Staphylococcus was the highest in abundance, while Enterococcus was the lowest. At the species level, Bacilluscereus
was the highest in abundance, while Roseomonasmucosa was the lowest. The analysis for the highly abundant operational taxo-
nomic units (OTUs) indicated a dramatic difference between sexes referring to either genera or species of which Staphylococcussp.,
Erwiniasp., Pseudomonassp., Sphingomonassp., Corynebacteriumsp., Propionibacterium acnes,Kocuria palustris are higher in males, while
Bacillus cereus,Bacillus sp., Erwinia sp., Corynebacterium sp., Micrococcus sp., Pseudomonassp. are lower in males.
Conclusions: The study succeeded in detecting the skin microbiome of individuals in Saudi Arabia.
Keywords: Skin, Microbiome, 16S rRNA Gene, Swabbing, Next Generation Sequencing
1. Background
The human body is the home of more than one tril-
lion microbes with a diverse variety of commensal mi-
crobes that play an important role in the health of the
individual. These microbes inhabit diverse habitats such
as the gut, skin, vagina, oral, etc. The human skin is
the largest organ of the human body and plays an im-
portant role as the first line of defense against external
environmental changes and invading pathogens (1). The
skin is an ecosystem composed of microbial communities
that inhabita range of physiologically andtopographically
distinct niches, including sebaceous/nonsebaceous, hair-
bearing/glabrous, moist/dry, and creased/non-creased re-
gions (2,3). Human microbiome in healthy skin and the
overall well-being of the individual has been started to be
appreciated since years ago (4). Cataloging the healthy mi-
crobiome is a mandatory first step toward identification
and correction of the microbial configurations that are im-
plicated in diseases (5). The analysis of the human skin mi-
crobiome helps detect the cause behind the occurrence of
many complex diseases (6).
2. Objectives
The aim of this study was to identify normal skin mi-
crobiome signature of healthy Saudi individuals living in
Jeddah, Saudi Arabia through the analysis of 16S rRNA of
the resident skin microbiome.
Copyright © 2021, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
(http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly
cited.

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AlQattan H et al.
3. Methods
Eight healthy volunteers from Saudi Arabia (4 males
and 4 females), aged 20 to 37 years, were enrolled in the
current study. The inclusion criteria were no history of
dermatologic disorders or current skin infection (atopic
dermatitis, psoriasis, and stasis eczema), use of no skin
creams or moisturizer before sampling, no treatment with
chemotherapy or radiation, or subjects treated with antibi-
otics within the last three months. Samples were collected
by swabbing from inner elbow of the right arm for each
subject with no prior cleaning or treatment of skin surface
using iSWAB Microbiome Collection kit.
DNAs were extracted using the QIAamp® DNA Micro-
biome kit (Qiagen®51306; North Rhine-Westphalia, Ger-
many) according to the manufacturer’s instructions. PCR
of the V3-V4 regions of bacterial 16S rRNA using 338F
and 806R primers was done following standard proce-
dure (e.g., initial denaturation at 95°C for 5 min; 25 cy-
cles of denaturation at 95°C for 30s, annealing at 56°C
for 30s, and extension at 72°C for 40s, and final exten-
sion of 72°C for 10 min), while deep sequencing was done
at Beijing Genome Institute (BGI), China using Illumina
platform. Raw sequencing data were deposited in the Eu-
ropean Nucleotide Archive (ENA) and received no. PR-
JNA609106. These data were analyzed using the Quanti-
tative Insights Into Microbial Ecology 2 (QIIME2) package
v.2018.11; (https://qiime2.org). Subsequent bioinformatics
analysis was done following Abuljadayel et al. (7).
4. Results
4.1. Raw Data Statistics
Statistics of raw data for eight healthy skin micro-
biome are shown in Table 1, and data were described in
Appendix 1 and Appendix 2, while results of OTU annota-
tion are shown in Appendix 20. Alpha diversity was ap-
plied to analyze the complexity of species. Shannon and
Simpson indices (Alpha diversity measures) indicated no
significant differences between male and female groups
(Appendix 21 and Appendix 3). The results in Appendix 4
indicated that F1 and F3 subjects had the lowest richness
as referred to by Shannon index, while M4 and F4 showed
the highest (Appendix 4). As expected, the data of evenness
for Simpson index indicated opposite results (Appendix
4). Plot of principal coordinate analysis (PCoA) (shown
in Appendix 5) indicated separation between male and fe-
male samples. Rarefaction curves showed that the maxi-
mum permitted number of reads for further analysis was
~ 73,000 (Appendix 6).
4.2. Normal Skin Microbiome Signatures at the Phylum Up to
Species Levels.
A threshold of ≥10 reads was considered highly abun-
dant (Appendix 20) that was met for a number of 21 out
of the 28 OTUs (Appendix 22). These OTUs are described
in Appendix 23. Phylogenetic tree indicated the existence
of four phyla (Figure 1). They include Actinobacteria (six
genera), Bacteroidetes (one genus), Firmicutes (four gen-
era), and Proteobacteria (six genera). The results of Ap-
pendix 23 align with those of the heat maps at the differ-
ent taxa levels (Appendix 7-12). Venn diagram showed 17
OTUs common in both male and female groups (Appendix
13), while five OTUs were unique in male (Curtobacterium
spp.1, Roseomonas mucosa,Corynebacterium spp.4, Capno-
cytophaga spp.1, and Mogibacteriaceae, and six in female
(Agrobacteriumspp.1, Acinetobacter spp.1, Enterococcus spp.1,
Gardnerella spp.1, Lactobacillus spp.1 and Corynebacterium
spp.5). The latter results were not considered for further
analysis due to the low number of sequences for each OTU
(Appendix 13).
4.3. Abundance of Different Microbes Across Sex
Abundance of microbes (weighted Unifrac diversity
distances) of different subjects of male and female was
studied at the phylum (Appendix 14), class (Appendix15),
order (Appendix 16), family (Appendix 17), genus (Appendix
18) and species levels (Appendix 19). Weighted Unifrac di-
versity distances showed diversity in different microbiome
signatures. Four phyla, four classes, seven orders, 10 fami-
lies, 10 genera, and two species showed diversity in micro-
biomes of male and female (Appendix 14-19, respectively).
The four phyla included Actinobacteria,Bacteroidetes,Firmi-
cutes, and Proteobacteria (Appendix 14). The four classes
included Actinobacteria, Alphaproteobacteria, Bacilli, and
Gammaproteobacteria (Appendix 15).
The seven orders consisted of Actinomycetales,Bacil-
lales,Enterobacteriales,Lactobacillales,Pseudomonadales,
Rhizobiales, and Rhodospirillales (Appendix 16). The 10
families included Acetobacteraceae,Bacillaceae,Corynebac-
teriaceae,Enterobacteriaceae,Enterococcaceae,Microbacte-
riaceae,Moraxellaceae,Pseudomonadaceae,Rhizobiaceae,
and Staphylococcaceae (Appendix 17). The 10 genera in-
cluded Acinetobacter,Agrobacterium,Bacillus,Corynebac-
terium,Curtobacterium,Enterococcus,Erwinia,Pseudomonas,
Roseomonas, and Staphylococcus. (Appendix 18). The
two species included Bacilluscereus and Roseomonasmucosa
(Appendix 19). Highly abundant OTUs (15 OTUs with ≥10
reads) that appeared in all, or in at least three, subjects in-
dicated dramatic difference between sexes. The 15 OTUs
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AlQattan H et al.
Table1. Statistics of Data Generated from Deep Sequencing for Eight Saudi Individuals. M = Male, F = Female.
Sample ID Reads Length (Bp) RawData (Mbp) N Base (%) LowQuality (%) CleanData (Mbp) Data Utilization (%) Raw Reads Clean Reads Read Utilization (%)
M1 297:297 53.65 0.049 1.966 51.51 96.02 90,315 87,239 96.59
M2 298:297 53.71 0.047 1.713 51.74 96.34 90,266 87,448 96.88
M3 294:297 54.52 0.051 1.823 52.46 96.22 92,256 89,228 96.72
M4 299:297 53.89 0.071 2.011 51.62 95.78 90,425 87,122 96.35
F1 300:297 53.27 0.087 1.627 51.38 96.45 89,235 86,529 96.97
F2 296:297 54.33 0.041 1.911 52.26 96.19 91,616 88,602 96.71
F3 297:300 54.50 0.067 2.373 51.62 94.72 91,285 87,485 95.84
F4 300:297 53.68 0.076 2.044 51.46 95.87 89,911 86,656 96.38
Actinobacteria
Bacteroidetes
Firmicutes
Proteobacteria
Capnocytophage
Agrobacterium
Sphingomonas
Roseomonas
Pseudomonas
Acinetobacter
Erwinia
Bacilus
Enterococcus
Lactobacillus
Staphylococcus
Propionibacterium
Corynebacterium
Micrococcus
Gardnerella
Kocuria
Curtobacterium
Figure 1. Genus level phylogenetic tree of normal skin microbiome. Genera with the same color belong to the same phylum.
referred to either genera or species of which Staphylococ-
cus (spp.1 and spp.2), Erwiniaspp.1, Pseudomonasspp.1, Sphin-
gomonas spp.1, Corynebacterium spp.2, Propionibacterium ac-
nes,Kocuriapalustris were higher in males (Figure 2), while
Bacilluscereus,Bacillus spp.1, Erwiniaspp.2, Corynebacterium
(spp.1 & spp.3), Micrococcusspp.1, Pseudomonasspp.2 were
lower in males (Figure 2).
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AlQattan H et al.
Microbe
Microbe
Abundance
AM
F
B
Staphylococcus spp.1
Staphylococcus spp.2
Erwinia spp.1
Pseudomonas spp.1
Sphingomonas spp.1
Corynebacterium spp.2
Propionibacterium Acnes
Kocuria Palustris
100000
100000
10000
1000
100
10
1
0.1
10000
1000
100
10
1
Bacillus Cereus
Bacillus spp.1
Erwinia spp.2
Corynebacterium spp.1
Corynebacterium spp.3
Micrococcus sPP.1
Pseudomonas spp.2
Abundance
M
F
Figure 2. High (a) and low (b) microbe abundance in male versus female skin microbiome of Saudi individuals. M = male, F = female.
5. Discussion
In the present study, healthy skin microbiome of Saudi
residents indicated the presence of as little as six, out of
28 OTUs that were detected at species level. They refer
to genera Bacillus (e.g., B. cereus), Roseomonas (e.g., P. mu-
cosa), Kocuria (e.g., K. palustris), Propionibacterium (e.g., P.
acnes), Pseudomonas (e.g., P. mendocina), and Corynebac-
terium (e.g., C.kroppenstedtii), respectively. These six gen-
era belong to phyla Firmicutes (e.g., Bacillus), Proteobacte-
ria (e.g., Roseomonas and Pseudomonas), and Actinobacte-
ria (e.g., Propionibacterium,Kocuria, and Corynebacterium).
There is one OTU (e.g., OTU26) that was detected only at
family level, e.g., Mogibacteriaceae (Appendix 23). This fam-
ily is part of phylum Firmicutes.
The other 21 OTUs refer to unassigned species of gen-
era Staphylococcus (Staphylococcus spp.1 and spp.2), Bacillus
(Bacillus spp.1), Enterococcus (Enterococcus spp.1), and Lacto-
bacillus (Lactobacillus spp.1) of phylum Firmicutes,Agrobac-
terium (Agrobacterium spp.1), Sphingomonas (Sphingomonas
spp.1), Erwinia (e.g., Erwinia spp.1 and spp.2), Pseudomonas
(Pseudomonas spp.1 and spp.2), and Acinetobacter (Acine-
tobacter spp.1) of phylum Proteobacteria,Curtobacterium
(Curtobacterium spp.1), Corynebacterium (Corynebacterium
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AlQattan H et al.
spp.1, spp.2, spp.3, spp.4 and spp.5), Micrococcus (Micrococ-
cus spp.1), Gardnerella (Gardnerella spp.1) of phylum Acti-
nobacteria, and Capnocytophaga (Capnocytophaga spp.1)
of phylum Bacteroidetes (Appendix 23).
5.1. Race-specific Healthy Skin Microbiome Signatures
Consistent with Kim et al. (8), healthy skin micro-
biome in several populations at phylum level are Fir-
micutes,Actinobacteria, Proteobacteria, and Bacteroidetes.
However, our results demonstrated the occurrence of the
first three phyla, e.g., Firmicutes (342,875 reads), Actinobacte-
ria (58,322 reads), Proteobacteria (234,757 reads), while low
prevalence of Bacteroidetes (2 reads) in healthy skin micro-
biomes of individuals living in Saudi Arabia (Appendix 23).
Results of several other studies (9) align with these of the
present study in terms of abundance at the phylum level.
Silva et al. (10) also reported an increased level of Firmi-
cutes in healthy skin of different populations. These re-
sults align with those of the present study (Appendix 23).
Meisel et al. (11) showed that S. epidermidis and S. homi-
nis were prevalent for Staphylococcus. Our results align
with those of Kim et al. (8) referring to genus Propioni-
bacterium, while showed no existence of the two Staphy-
lococci species. Interestingly, our results indicated the oc-
currence of two new unassigned species of Staphylococ-
cus (Appendix 23). The two new species of Staphylococcus
might have the same action of the two-missing species in
microbiome signature of Saudi individuals.
5.2. Healthy Skin Microbiome and Gender
We suggest that differential abundance of microbes
due to gender represents extra environmental factors,
influencing such differences in microbiome signa-
ture. Previous studies indicated that Propionibacterium,
Corynebacterium, and Staphylococcus were more abundant
in males (12), while Enterobacteriales,Moraxellaceae,Lacto-
bacillaceae, and Pseudomonadaceae (according to Fierer et
al.) (13), and Lactobacillus,Enhydrobacter and Deinococcus
(According to Ling et al.) (12) were higher in females.
As women use cosmetics more frequently than men (in
accordance with Fierer et al.) (13), thereby altering the
microbial community structure and diversity of their
skin may definitely affect microbe richness and relative
abundance compared to men.
In the present study, relative abundances of assigned
species of genera Propionibacterium (e.g., Propionibac-
teriumacnes) and Kocuria (e.g., Kocuriapalustris) and
unassigned species of genera Staphylococcus,Erwinia,
Pseudomonas,Sphingomonas, and Corynebacterium are
higher in male microbiome, while relative abundances of
assigned (e.g., Bacillus cereus), and unassigned species of
genus Bacillus and unassigned species of genera Erwinia,
Corynebacterium,Micrococcus,Pseudomonas are lower in
male microbiome (Appendix 22 and Figure 2). The results
of Staphylococcus and Propionibacterium abundances in
the present study are in agreement with those of Ling et
al. (12) and Fierer et al. (13).
5.3. Conclusions
Overall, the study highlights skin microbiome signa-
ture of individuals in Saudi Arabia. This information will
be helpful when studying skin microbiomes of patients
with atopic dermatitis (AD), Psoriasis, or Acnevulgaris to-
ward the detection of biomarkers of the different diseases.
Supplementary Material
Supplementary material(s) is available here [To read
supplementary materials, please refer to the journal web-
site and open PDF/HTML].
Acknowledgments
Authors thank authorities at the Department of Biolog-
ical Sciences, Faculty of Science, King Abdulaziz University,
Jeddah, Saudi Arabia, for providing facilities and equip-
ment to do this research.
Footnotes
Authors’ Contribution: Conceptualization: HA, SE, AB,
SZ, RAH, Data collection: AH, LT, AA, RA, Methodology: AH,
SE, LT, RA, AM, SZ, Writing the manuscript: HA, AA, AB, RAH,
Review, editing, and correspondence: AA, AB.
Conflict of Interests: The authors declare no conflict of
interest.
Ethical Approval: Ethical approval to conduct skin micro-
biome analysis was obtained from the Ethics Committee
of King Abdulaziz University Hospital (KAUH), Saudi Arabia
(ref. no. 165-18).
Funding/Support: Not applicable.
Informed Consent: A consent form was prepared
and signed by volunteers or participants whose inclu-
sion/exclusion criteria were applied.
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