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RESEARCH ARTICLE
Apple cider vinegar soaks do not alter the skin
bacterial microbiome in atopic dermatitis
Lydia A. LuuID
1
*, Richard H. Flowers
1
, Yingnan Gao
2
, Martin Wu
2
, Sofia Gasperino
3
, Ann
L. Kellams
4
, DeVon C. Preston
5
, Barrett J. Zlotoff
1
, Julia A. Wisniewski
5
, Steven
L. Zeichner
3
1Department of Dermatology, University of Virginia, Charlottesville, Virginia, United States of America,
2Department of Biology, University of Virginia, Charlottesville, Virginia, United States of America, 3Division
of Pediatric Infectious Diseases, Department of Pediatrics, Pendleton Pediatric Infectious Disease
Laboratory, Child Health Research Center, University of Virginia, Charlottesville, Virginia, United States of
America, 4Division of General Pediatrics, Department of Pediatrics, University of Virginia, Charlottesville,
Virginia, United States of America, 5Division of Allergy and Immunology, Department of Pediatrics and
Internal Medicine, University of Virginia, Charlottesville, Virginia, United States of America
*lal2ta@virginia.edu
Abstract
Introduction
Atopic dermatitis is a common skin disease characterized by altered cutaneous immunity in
which patients often exhibit lower skin microbiota diversity compared to healthy skin and are
prone to colonization by Staphylococcus aureus. Apple cider vinegar has been shown to
have antibacterial effects; however, its effects on the skin microbiome have not previously
been well-described.
Objectives
We aimed to examine the effects of topical dilute apple cider vinegar soaks on Staphylococ-
cus aureus abundance, skin bacterial microbiome composition, and skin bacterial micro-
biome diversity in atopic dermatitis participants compared to healthy skin.
Methods
Eleven subjects with atopic dermatitis and 11 healthy controls were enrolled in this random-
ized, non-blinded, single-institution, split-arm pilot study. Subjects soaked one forearm in
dilute apple cider vinegar (0.5% acetic acid) and the other forearm in tap water for 10 min-
utes daily. Skin bacteria samples were collected from subjects’ volar forearms before and
after 14 days of treatment. 16S sequencing was used to analyze Staphylococcus aureus
abundance and skin bacterial microbiome composition, and alpha diversity of microbiota
were determined using Shannon diversity index.
Results
There was no difference in skin bacterial microbiome in atopic dermatitis subjects after 2
weeks of daily water or apple cider vinegar treatments (p = 0.056 and p = 0.22, respectively),
or in mean abundance of S.aureus on apple cider vinegar-treated forearms (p = 0.60). At 2
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OPEN ACCESS
Citation: Luu LA, Flowers RH, Gao Y, Wu M,
Gasperino S, Kellams AL, et al. (2021) Apple cider
vinegar soaks do not alter the skin bacterial
microbiome in atopic dermatitis. PLoS ONE 16(6):
e0252272. https://doi.org/10.1371/journal.
pone.0252272
Editor: Thomas L. Dawson, Skin Research Institute
Singapore, SINGAPORE
Received: June 28, 2020
Accepted: May 13, 2021
Published: June 2, 2021
Copyright: ©2021 Luu 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: Sequences read were
submitted to the National Center for Biotechnology
Information, US National Library of Medicine, NIH,
Sequence Read Archive under accession number
PRJNA639330 and are freely available.
Funding: This study was funded by the University
of Virginia, UVA Child Health Research Center, the
Pendleton Pediatric Infectious Disease Laboratory,
and the Department of Dermatology. The funders
had no role in study design, data collection and
weeks, the skin bacterial microbiomes of healthy control subjects were not significantly dif-
ferent from the skin bacterial microbiome of atopic dermatitis subjects (p = 0.14, 0.21, 0.12,
and 0.05).
Conclusions
Our results suggest that daily soaks in 0.5% apple cider vinegar are not an effective method
of altering the skin bacterial microbiome in atopic dermatitis. Further studies are needed to
explore the effects of different concentrations of apple cider vinegar on skin microflora and
disease severity.
Trial number
UVA IRB-HSR #19906.
Introduction
Atopic dermatitis (AD), a common skin disease, affects up to 20% of children and 6% of adults
[1]. AD is associated with significant utilization of health care resources, as patients with AD
cost the health system $3,302 more annually in the United States than patients without AD [2].
AD is characterized by altered cutaneous immunity and skin barrier defects that increase sus-
ceptibility to bacterial infections [3,4]. AD patients exhibit lower skin microbiota diversity
compared to healthy skin [5], and over 90% of AD patients have colonization of lesional skin
with Staphylococcus aureus (S.aureus) [6,7], characteristics that have been targeted by AD
treatments such as topical steroids [8]. Low density S.aureus is also present in non-lesional
skin of AD patients and demonstrates resistance to common antimicrobials [9,10]. In con-
trast, S.aureus is absent from the healthy skin microbiome, except in moist higher pH intertri-
ginous zones and nares [11–13]. S.aureus-colonized patients have higher total serum IgE
levels and a higher food allergy prevalence [14]. AD subjects with IgE directed towards staphy-
loccocal enterotoxins also have a higher incidence of asthma [15,16].
Microbial dysbiosis and S.aureus colonization are driven by impairment of epidermal acid-
ification in AD [11,17–19]. Breakdown products of filaggrin, a key epidermal differentiation
complex protein deficient in AD skin, contribute to epidermal acidification and impair S.
aureus growth by forming pyrrolidone carboxylic acid and trans-urocanic acid (t-UCA) [20,
21]. The alkaline pH of AD skin likely arises from insufficient filaggrin-derived t-UCA; other
natural acidifiers may contribute to skin flora dysbiosis [11,22,23].
AD is difficult to treat and current treatments are not curative. Bath additives like dilute
sodium hypochlorite (bleach) are often recommended by dermatologists as adjuvant therapy
to reduce disease severity due to their potential anti-staphylococcal benefits [24]. However,
evidence supporting their effectiveness is sparse [13,25–27]. Given the acid mantle
impairment in atopic dermatitis, bleach is a counterintuitive approach from a pH standpoint
to manage S.aureus [11,22,23]. In addition, dilute bleach neither improves skin pH nor eradi-
cates S.aureus from AD skin [25,28,29]. Dilute bleach’s beneficial effects may be comparable
to water baths alone [29]. In ex-vivo studies, bleach concentrations of greater than 0.03%
sodium hypochlorite were required to eradicate S.aureus biofilms, but those levels are cyto-
toxic to human cells and should not be used clinically [30,31]. Evidence-based alternatives to
bleach that mitigate S.aureus are desirable.
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analysis, decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
There is increasing interest in complementary and alternative treatments for AD, especially
apple cider vinegar given its antimicrobial properties. Dilute vinegar (AA range 0.16% to
0.31%) inhibits ex-vivo growth and biofilm formation of various human skin pathogens,
including S.aureus [32]. ACV’s therapeutic potential for AD specifically is supported by
murine models that, after treatment with topical vinegar cream (pH 3.5), showed lower eczema
scores, increased stratum corneum hydration, and decreased transepidermal water loss, com-
pared mice treated with vehicle alone (pH 5.5) [33].
However, in spite of widespread recommendation of dilute ACV baths by dermatologists,
there is little high-quality data supporting its use for atopic dermatitis (34). One small case
series showed that vinegar baths with topical treatment improved AD disease severity [34]. In
contrast, a recent small study found that dilute ACV compresses did not reduce eczematous
skin S.aureus burden [35]. Similarly, in our pilot study of 11 AD patients and 11 healthy con-
trols, we showed that dilute ACV soaks did not improve skin barrier integrity as measured by
transepidermal water loss and skin pH, and caused skin irritation in a majority of subjects
[36]. In spite of theoretical and ex-vivo benefits, the effect of dilute ACV baths on S.aureus col-
onization and the skin microbiome are currently unknown [36].
In this study, we examined the effects of topical dilute ACV soaks on Staphylococcus aureus
abundance, skin bacterial microbiome composition, and skin bacterial microbiome diversity
in AD and healthy skin.
Methods
Study participants
Participants with AD and healthy controls, age 12 years, were recruited through advertise-
ment and from University of Virginia dermatology clinics [36]. Subjects were enrolled
between June 2017 and October 2017 (Fig 1A). All subjects met U.K. AD diagnostic criteria
[37]. Healthy controls had no current or prior history of AD. Subjects were excluded if they
used topical or systemic antimicrobials within 1 month of enrollment. All other treatments
used by subjects for AD, including topical steroids and immune modulators, were recorded.
Subjects were asked to continue these treatments as prescribed during study participation. All
participants or guardians signed written informed consent; minors provided verbal assent.
The University of Virginia Institutional Review Board approved the study.
Intervention
Patient characteristics were ascertained by questionnaire and medical record review. Disease
severity was determined using the Severity Scoring of Atopic Dermatitis Index (SCORAD)
[38]. Following a standardized education session, AD and control subjects followed identical
written protocols for soaking one forearm in dilute ACV (0.5% concentration acetic acid) and
the other in tap water for 10 minutes daily [36]. Participants were taught to prepare soaks
(below) and supervised soaking their forearms at the initial visit, then instructed to follow the
procedure every day for 2 weeks. The forearm selected for ACV treatment was determined by
a pseudo-random number generator before recruitment.
Preparation of apple cider vinegar and tap water soaks
White House Foods1Apple Cider Vinegar and 3-gallon soaking basins were provided to all
subjects. ACV was diluted to 0.5% AA by mixing 2.4 cups of ACV with 21.6 cups of tap water
in a 3-gallon soaking basin. A concentration of 0.5% AA was selected based on previous data
regarding safety, tolerability, and antimicrobial qualities [39]. A second basin was used to soak
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the opposite forearm in tap water simultaneously. Subjects were instructed to rinse basins with
tap water between uses.
Data collection
Samples for analysis of skin microbiota were collected at study visit 1 before and after the first
soak, and at study visit 2 after 14 days of daily soaking. For study visit 1, ACV and water soaks
were performed on opposite forearms simultaneously. Skin bacteria samples were collected
from subjects’ forearms using e-Swab
TM
Liquid Amies Collection and Transport System
(Copan e-Swab, Brescia, Italy). Swabs were rubbed across the volar forearm skin in a circular
motion for 15 seconds, then placed into the transport system. Skin bacteria samples from
study visit 1 before soaking and study visit 2 were analyzed.
Total DNA was isolated using modifications of previously described procedures, lysing
samples with a lysozyme/mutanolysin/lysostaphin cocktail, followed by proteinase K/SDS
treatment, and bead beating with a FastPrep-24 instrument [40,41]. DNA was further purified
with a Quick-DNA Fecal/Soil Microbe Kit (Zymo Research). DNA quality was assessed
spectrophotometrically and with gel electrophoresis. Negative extraction controls were pro-
cessed in parallel with each extraction.
16S amplicon sequencing was performed using V1-V3 primers on DNA extracted from
swabs at baseline and at 2 weeks. The V1-V3 hypervariable regions of 16S rRNA were ampli-
fied through PCR with broad range primers 27F and 534R [42] (PCR amplification: New
England Biolabs Phusion High-Fidelity PCR Master Mix with HF Buffer: M0531S; PCR prim-
ers: custom primers ordered from Integrated DNA Technologies; reaction purification/nor-
malization: Applied Biosystems SequalPrep™Normalization Plate Kit, 96-well: A1051001). 16S
rRNA amplicon libraries were pooled and sequenced using MiSeq Reagent Kit v3 (Illumina
MiSeq Reagent Kit v3 (600-cycle): MS-102-3003) and the Illumina MiSeq instrument. Micro-
bial amplicon variants present in each sample were identified and their relative abundances
were quantitated using DADA2 for quality filtering, chimeric sequence removal, identification
of unique amplicon variants, and taxonomic classification [43]. Sequences read were submit-
ted to the National Center for Biotechnology Information, US National Library of Medicine,
NIH, Sequence Read Archive under accession number PRJNA639330 and are freely available.
Statistical analysis
We calculated the Aitchison distance [44] using R package ‘microbiome’ to quantify differ-
ences between skin microbiota compositions [45]. We applied permutational multivariate
analysis of variance (PERMANOVA) using R package ‘vegan’ to partition variance of Aitchi-
son distance between fixed effects (i.e., status of AD, time after treatment and type of treat-
ment) and random effects. By examining R-squared and the p-values of each condition or
treatment, we determined if that condition or treatment results in significant microbiota com-
positional change. Statistical power of PERMANOVA was determined using R package
‘micropower’ [46]. Random forest analysis was applied using R package ‘randomForest’ to
identify genera that distinguish microbiota from the AD subjects and controls at baseline [47].
Shannon diversity index was used to quantify skin microbiota diversity through R package
‘microbiome’. We analyzed data on both intent-to-treat and as-treated bases. For intent-to-
Fig 1. Study design. A, Study schematic. B, Diagrammatic representation of skin bacterial microbiome comparisons between
forearms, (i) intention-to-treat and (ii) as-treated.
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treat analysis, all subjects were included in the analysis, regardless of treatment adherence. For
as-treated analysis, subjects who missed three or more days of treatment were excluded.
Results
Characteristics of AD and control subjects
Eleven subjects with AD (geometric mean (GM) age 20.6 years) and 11 healthy subjects (GM
age 28.8 years) were enrolled. Table 1 summarizes baseline characteristics of AD and healthy
subjects. The majority of AD subjects had mild-to-moderate disease (GM SCORAD 32.9
[23.8–45.4]). Emollients and topical corticosteroids were the most commonly used treatments.
Atopic dermatitis was present on volar forearms in 8/11 (73%) of AD subjects.
Effect of 14 daily 10-minute ACV soaks on skin microbiota
At baseline, the bacterial microbiomes in AD participants and control subjects were signifi-
cantly different (p = 0.011) (Table 2, Figs 1B and 2A). Disease status explained 6.2% of the
variations in skin bacterial microbiome composition. Random forest analysis of baseline sam-
ples at the genus level revealed 8 genera that distinguished AD subjects and healthy controls:
Halomonas,Delftia,Massilia,Cutibacterium,Shewanella,Leuconostoc,Sphingomonas, and
Staphylococcus. These genera had an average abundance greater than 0.1% in at least one
Table 1. Characteristics of study participants at enrollment.
Atopic Dermatitis N = 11 Control Subjects N = 11 p-value
Age (years)
1
20.6 [16.2–26.2] 28.8 [22.6–36.8] 0.15
Gender
2
Male 36% 45% 1.00
Female 64% 55%
Ethnicity
2
Caucasian 46% 36% 1.00
African American 18% 0%
Asian 36% 46%
Other 0% 18%
Birth history
2
Vaginal delivery 73% 91% 0.59
Caesarian delivery 27% 9%
Medical history
2
Allergies
3
100% 36% 0.0039
Family history
2
Allergies
3
100% 64% 0.09
SCORAD
1
33 [23.8–45.4] 0 [0–0] N/A
AD severity
4
Mild 36% N/A <0.01
Moderate 46%
Severe 18%
AD medications
2
Emollients 100% 0% <0.01
Topical steroids 91% 0%
Systemics 18% 0%
Skin barrier
1
TEWL 11.1 [8.6–14.3] 7.1 [6.0–8.4] 0.0064
pH 4.88 [4.67–5.1] 4.86 [4.6–5.13] 1.00
Presence of dermatitis
2
ACV-treated forearm 73% 0% <0.01
Water-treated forearm 73% 0%
1
Geometric mean [95% confidence interval]
2
Percentage of subjects (prevalence)
3
Positive history of allergies included asthma, food, or environmental allergies
4
AD severity was mild (if SCORAD <25), moderate (if SCORAD 25–50), or severe (if SCORAD >50).
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Table 2. Pairwise PERMANOVA of skin microbiota composition.
Intention-to-treat Baseline Day 14
Healthy control AD subject Healthy control AD subject
Water-treated
forearm
ACV-treated
forearm
Water-treated
forearm
ACV-treated
forearm
Water-
treated
forearm
ACV-treated
forearm
Water-treated
forearm
ACV-treated
forearm
Baseline Healthy
control
Water-
treated
forearm
R
2
= 0.0371 R
2
= 0.0544 R
2
= 0.0545
P = 0.4675 P = 0.0350 P = 0.0609
β= 0.662 β= 1.000 β= 0.991
ACV-
treated
forearm
R
2
= 0.0596 R
2
= 0.0615
P = 0.0200 P = 0.0110
β= 1.000 β= 0.999
AD
subject
Water-
treated
forearm
R
2
= 0.0314
P = 0.3846
β= 0.746
ACV-
treated
forearm
Day 14 Healthy
control
Water-
treated
forearm
R
2
= 0.0384 R
2
= 0.0418 R
2
= 0.0507 R
2
= 0.0497 R
2
= 0.0335 R
2
= 0.0525 R
2
= 0.0529
P = 0.6623 P = 0.0969 P = 0.1499 P = 0.2298 P = 0.5055 P = 0.1818 P = 0.1538
β= 0.571 β= 0.890 β= 0.999 β= 0.992 β= 0.647 β= 0.995 β= 0.998
ACV-
treated
forearm
R
2
= 0.0387 R
2
= 0.0390 R
2
= 0.0528 R
2
= 0.0526 R
2
= 0.0518 R
2
= 0.0551
P = 0.7283 P = 0.4685 P = 0.0879 P = 0.1089 P = 0.2348 P = 0.1059
β= 0.526 β= 0.669 β= 1.000 β= 0.998 β= 0.999 β= 1.000
AD
subject
Water-
treated
forearm
R
2
= 0.0534 R
2
= 0.0563 R
2
= 0.0384 R
2
= 0.0401 R
2
= 0.0339
P = 0.1179 P = 0.0599 P = 0.2947 P = 0.3506 P = 0.3227
β= 0.998 β= 1.000 β= 0.570 β= 0.606 β= 0.602
ACV-
treated
forearm
R
2
= 0.0570 R
2
= 0.0627 R
2
= 0.0371 R
2
= 0.0378
P = 0.0340 P = 0.0130 P = 0.7822 P = 0.6014
β= 1.000 β= 1.000 β= 0.323 β= 0.557
As-treated Baseline Day 14
Healthy control AD subject Healthy control AD subject
Water-
treated
forearm
ACV-treated
forearm
Water-
treated
forearm
ACV-treated
forearm
Water-
treated
forearm
ACV-treated
forearm
Water-
treated
forearm
ACV-treated
forearm
Baseline Healthy
control
Water-
treated
forearm
R
2
= 0.0417 R
2
= 0.0733 R
2
= 0.0699
P = 0.7313 P = 0.0140 P = 0.0939
β= 0.508 β= 1.000 β= 0.957
ACV-
treated
forearm
R
2
= 0.0845 R
2
= 0.0823
P = 0.0070 P = 0.0170
β= 1.000 β= 0.999
AD
subject
Water-
treated
forearm
R
2
= 0.0519
P = 0.4375
β= 0.294
ACV-
treated
forearm
(Continued)
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group. S.aureus was significantly more abundant on forearms of AD subjects compared to
healthy controls (intention-to-treat analysis: 10.74% vs 0.01% respectively, p <0.001; as-treated
analysis: 5.73% vs 0.01% respectively, p <0.001) (Table 3). There were also no significant dif-
ferences in microbiota composition between the water-treated and ACV-treated forearms of
healthy controls or AD subjects at baseline (intention-to-treat analysis: p = 0.468 for healthy
controls, p = 0.385 for AD subjects; as-treated analysis: p = 0.731for healthy controls, p = 0.438
for AD subjects) (Table 2, Figs 1B and 2A).
We then examined the effects of 2 weeks of daily ACV treatments. Fig 1B illustrates skin
microbiota significant comparisons on both an intention-to-treat and as-treated basis. Analy-
ses on an intention-to-treat basis showed no difference in skin bacterial microbiota in healthy
control subjects after 2 weeks of daily water or ACV treatments (PERMANOVA, p = 0.662and
p = 0.469, respectively) (Table 2), or in mean abundance of S.aureus on ACV-treated forearms
(Wilcoxon signed rank test, p = 0.349). There was also no difference in skin bacterial micro-
biome in AD subjects after 2 weeks of daily water or ACV treatments (PERMANOVA,
p = 0.295 and p = 0.601, respectively). Although the mean abundance of S.aureus on ACV-
treated forearms seems to decrease after 2 weeks of daily ACV treatment, such decrease was
mostly contributed by a few individuals with drastic drop of S.aureus abundance and was not
consistent among individuals. The median abundance of S.aureus on ACV-treated arms
showed less evident decrease, and the drop in the mean abundance was not significant (Wil-
coxon signed rank test, p = 0.105). Interestingly, after 2 weeks of daily treatments, the skin bac-
terial microbiome of forearms treated with water were not significantly different from those
treated with ACV in either healthy control subjects or AD subjects (PERMANOVA, p = 0.506
and p = 0.323, respectively). At 2 weeks, the skin bacterial microbiomes of healthy control sub-
jects were not significantly different from the skin bacterial microbiome of AD subjects (PER-
MANOVA, p = 0.182, 0.235, 0.154, and 0.106) (Table 2), although mean abundance of S.
aureus remained significantly higher on forearms of AD subjects (Wilcoxon rank sum test,
p<0.001) (Table 3). When analyzed on an as-treated basis where non-compliant subjects
were excluded, results were similar, except there was a significant change in skin bacterial
microbiome of forearms of AD subjects that were treated with water daily for 2 weeks
(p = 0.031) (Table 2). Power analysis using R package ‘micropower’ on as-treated data showed
Table 2. (Continued )
Day 14 Healthy
control
Water-
treated
forearm
R
2
= 0.0458 R
2
= 0.0486 R
2
= 0.0697 R
2
= 0.0661 R
2
= 0.0390 R
2
= 0.0698 R
2
= 0.0676
P = 0.4056 P = 0.0829 P = 0.0639 P = 0.2078 P = 0.4755 P = 0.0649 P = 0.1069
β= 0.591 β= 0.995 β= 0.983β= 0.595 β= 0.820 β= 0.990 β= 0.953
ACV-
treated
forearm
R
2
= 0.0446 R
2
= 0.0455 R
2
= 0.0740 R
2
= 0.0699 R
2
= 0.0681 R
2
= 0.0688
P = 0.6723 P = 0.3317 P = 0.0310 P = 0.0889 P = 0.1269 P = 0.1039
β= 0.452 β= 0.657 β= 1.000 β= 0.974 β= 0.990 β= 0.984
AD
subject
Water-
treated
forearm
R
2
= 0.0668 R
2
= 0.0736 R
2
= 0.0652 R
2
= 0.0625 R
2
= 0.0477
P = 0.1299 P = 0.0230 P = 0.0312 P = 0.3281 P = 0.5938
β= 0.988 β= 0.997 β= 0.851 β= 0.335 β= 0.172
ACV-
treated
forearm
R
2
= 0.0680 R
2
= 0.0749 R
2
= 0.0602 R
2
= 0.0585
P = 0.0979 P = 0.0410 P = 0.2500 P = 0.2969
β= 0.984 β= 0.990 β= 0.339 β= 0.437
Key:
Yellow: Statistically significantly different (p <0.05).
Orange: Statistically significantly different (p <0.05), but more than one factor has changed between the two groups being compared.
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that the effect size of compositional change in microbiota before and after water or ACV treat-
ment is very close to zero, resulting in a median power of 0.48 (ranging from 0.17 to 0.85) in
PERMANOVA. To get a statistical power >0.9 to detect compositional change before and
after water or ACV treatment similar to what was observed in this study, at least 15 patients
are required for each group. On the other hand, the effect size in microbiota between AD
patients and healthy controls ranges from 0.003 to 0.022, yielding a power >0.9 in
PERMANOVA.
Kruskal-Wallis test by ranks of Shannon diversity indices revealed no significant differences
in microbiota diversity of any forearms at any point in time (p = 0.474, Fig 2B).
Discussion
We found AD subjects have different skin microbiota composition compared to healthy con-
trols at baseline. In particular, Staphylococcus was more abundant in AD subjects, while Halo-
monas,Delftia,Massilia,Cutibacterium,Shewanella,Leuconostoc,and Sphingomonas were less
abundant, findings consistent with the literature [4,10]. However, although prior studies had
found that healthy controls have higher skin microbiome diversity compared to AD subjects
[5], we did not find any significant differences in skin bacterial microbiota diversity between
AD subjects and healthy controls at baseline. As expected, at baseline there was no difference
between skin bacterial microbiota of forearms to be treated with water and forearms to be
treated with ACV in either healthy controls or AD subjects.
In both intent-to-treat and as-treated analysis, healthy control subjects showed no differ-
ence from baseline skin bacterial microbiome after 2 weeks of either daily water treatments or
daily ACV treatments. This is consistent with prior studies showing that the skin microbiota
exhibits temporal stability despite environmental disruptions in healthy adults [48,49]. There-
fore, 2 weeks of daily 10-minute treatments with dilute ACV was not sufficient to change the
skin bacterial microbiome of healthy controls. However, we also found that the skin micro-
biota and mean abundance of S.aureus on forearms of AD subjects showed no significant
changes after 14 days of daily ACV treatment, and the forearms treated with ACV were not
significantly different from those treated with water. These results were surprising, as we had
hypothesized that the AD skin bacterial microbiome would be more susceptible to changes
due to environmental perturbations such as daily ACV treatments, and that ACV would have
antimicrobial properties against S.aureus. The fact that we did not see a change shows that AD
skin microbiomes may be more resilient to environmental perturbations than anticipated. Of
note, previous studies have found that antiperspirant use significantly changes the axillary skin
microbiome [50,51] and cosmetic products affect the diversity of the facial skin microbiome
Fig 2. Skin microbiota composition and diversity. A, Stacked bar plots of bacterial taxonomic compositions in the skin microbiome, as-treated. B,
Shannon diversity plot, as-treated.
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Table 3. Mean and median abundance of Staphylococcus aureus on ACV-treated forearms. The median abundances are shown in the parentheses.
Intention-to-treat As-treated
Healthy control AD participant Healthy control AD participant
Baseline 0.01% (0.00%) 10.74% (1.16%) 0.01% (0.00%) 5.73% (1.06%)
2 weeks 0.05% (0.01%) 6.37% (0.81%)
†
0.05% (0.02%) 3.61% (0.59%)
†
pairwise comparison vs healthy control at baseline, p <0.05 by Wilcoxon rank-sum test.
† pairwise comparison vs healthy control at 2 weeks, p <0.05 by Wilcoxon rank-sum test.
https://doi.org/10.1371/journal.pone.0252272.t003
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[52]. Water treatments also did not produce significant differences in skin bacterial micro-
biome compared to dilute ACV in either controls or AD subjects, suggesting that water treat-
ment was equivalent to dilute ACV treatment.
In spite of lack of significant changes in the skin bacterial microbiome after 2 weeks of ACV
soaks in either healthy controls or AD subjects compared to baseline, we do note that after 2
weeks there were no longer any significant differences in skin bacterial microbiome between
healthy controls and AD subjects, and no significant differences in skin bacterial microbiome
diversity either. This finding suggests that although water and ACV treatments did not pro-
duce a statistically significant change in skin bacterial microbiome over the 2-week timespan,
the treatments did cause a small enough change to cause the skin bacterial microbiomes of AD
subjects to become more similar to the skin bacterial microbiomes of healthy controls. When
analyzed on an as-treated basis, 2 weeks of daily water treatments caused a significant change
in skin bacterial microbiome in AD subjects, and the skin bacterial microbiome in AD subjects
became similar to that of healthy controls. This suggests that hydration of the skin is important
in maintaining the microbiome of AD patients. This significance is lost when analyzing the data
on an intention-to-treat basis, so the burden of treatment should be considered carefully. Not
all patients will be able to adhere to a daily 10-minute soaking regimen; this may not be a valu-
able treatment option except in very motivated patients. Daily compresses that allow the subject
to retain mobility during treatment time or relaxing full-body baths may be more acceptable
options. It might be possible to observe significant effects if patients were treated more inten-
sively or for longer times, but our study non-adherence rate of 3 of 11 AD subjects and 1 of 11
healthy controls missing 3 or more days of treatment over 14 days suggests that more intense
interventions would only be possible for very highly motivated patients. It is also possible that
our study was underpowered. However, given that the magnitude of the effects we observed
were small, it would be reasonable to question the clinical significance of quantitatively small
albeit statistically significant effects. A conservative interpretation of our observations would
suggest that the effects of the ACV soak regimen used in our study are small in any case.
The concentration we chose of 0.5% AA is nearly 10-fold above the 1:80 (0.06% AA) ACV
bath preparations recently recommended by Lee and Jacobs [53]. 0.5% AA is comparable to
the MIC of 0.312% AA against methicillin-sensitive S.aureus (MSSA) and 0.625% AA against
methicillin-resistant S.aureus (MRSA) [32]. While concentrations of 2.5% AA have been used
in burn centers to prevent P.aeruginosa wound infections, concentrations above 3% AA have
been associated with pain and itching [39]. In our study, S.aureus colonization persisted after
14 days of 10-minute soaks with 0.5% AA by PCR analysis, indicating that 0.5% AA from ACV
has no lasting biocidal activity on AD skin already colonized with S.aureus. Although we did
not test for MRSA in this study, the lack of efficacy of 0.5% AA was possibly due to subject col-
onization with resistant S.aureus strains.
This study has several limitations. First, this study examined a fairly small, homogenous
group of patients, as the average ages of AD and control subjects were 20.6 and 28.8 years,
respectively. Of note, the AD group of subjects included some participants of adolescent age,
while the control group of subjects were all adults (over age 18). The skin microbiota shifts as
people move from childhood to adulthood due to changing levels of sebum production, lipid
content, pH, and hair growth, and so there are different microbial signatures associated with
AD at different ages [54]. Therefore, this study may be less applicable to children. Further
studies among a larger and more diverse group of patients are needed to further characterize
the full effect of topical ACV treatments on skin microbiomes. Second, this study was
unblinded due to the intrinsic appearance and odor of dilute ACV. Third, our study analyzed
a single brand and dilution of ACV. Future studies should examine different concentrations of
AA from different sources. Lastly, this study focused on the bacterial microbiome. It is well-
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Apple cider vinegar soaks in atopic dermatitis
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known that other organisms, such as Malessezia, are important components of the skin micro-
biome that may also provoke inflammatory reactions and contribute to skin diseases such as
atopic dermatitis [55]. Therefore, future studies should include a fungal ITS analysis as well.
In conclusion, apple cider vinegar is prominent among emerging natural remedies used in
AD in spite of sparse evidence [53]. Our results show that even though daily soaks in 0.5%
ACV do not change skin bacterial microbiomes significantly compared to water and are not
likely a useful agent to affect skin-colonizing S.aureus, they may cause AD skin microbiome to
become more similar to controls. Further studies are needed to explore whether ACV at differ-
ent concentrations can promote a healthier skin microflora and modify disease severity.
Acknowledgments
We would like to thank the clinical faculty and staff in the Department of Dermatology at the
University of Virginia for their assistance in recruiting patients for our study. We would also
like to thank Ms. Katherine Boguszewski, our clinical research coordinator, for her assistance
with administrative tasks. Most importantly, we would like to thank our 22 atopic dermatitis
patients and healthy control subjects who volunteered their time to participate in our study.
Author Contributions
Conceptualization: Richard H. Flowers, Ann L. Kellams, DeVon C. Preston, Barrett J. Zlotoff,
Julia A. Wisniewski, Steven L. Zeichner.
Data curation: Lydia A. Luu, Yingnan Gao, Martin Wu, Sofia Gasperino, Steven L. Zeichner.
Formal analysis: Lydia A. Luu, Yingnan Gao, Martin Wu, Sofia Gasperino, Steven L.
Zeichner.
Funding acquisition: Richard H. Flowers, Martin Wu, Ann L. Kellams, Barrett J. Zlotoff, Julia
A. Wisniewski, Steven L. Zeichner.
Investigation: Lydia A. Luu, Martin Wu, Barrett J. Zlotoff, Julia A. Wisniewski, Steven L.
Zeichner.
Methodology: Lydia A. Luu, Richard H. Flowers, Julia A. Wisniewski, Steven L. Zeichner.
Project administration: Lydia A. Luu.
Resources: Lydia A. Luu, Richard H. Flowers, Julia A. Wisniewski, Steven L. Zeichner.
Software: Yingnan Gao, Martin Wu, Julia A. Wisniewski, Steven L. Zeichner.
Supervision: Richard H. Flowers, Martin Wu, Barrett J. Zlotoff, Julia A. Wisniewski, Steven L.
Zeichner.
Validation: Lydia A. Luu, Yingnan Gao, Martin Wu.
Visualization: Lydia A. Luu, Richard H. Flowers, Julia A. Wisniewski, Steven L. Zeichner.
Writing – original draft: Lydia A. Luu, Richard H. Flowers, Julia A. Wisniewski.
Writing – review & editing: Lydia A. Luu, Richard H. Flowers, Yingnan Gao, Martin Wu,
Ann L. Kellams, Barrett J. Zlotoff, Julia A. Wisniewski, Steven L. Zeichner.
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