Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : October 29, 2021
Accepted : November 19, 2021
Published online : December 31, 2021
doi:10.24659/gsr.8.4_175

Original article

Glycated keratin promotes cellular aggregation and biofilm formation in
Staphylococcus aureus
Kyle Haasbroek, Masayuki Yagi, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Science,
Doshisha University, Kyoto, Japan

Abstract

Glycation is a non-enzymatic reaction that occurs under physiological conditions which contributes to the pathogenesis
aging and related diseases. Staphylococcus aureus is a frequent cause of infection, whose overabundance on the skin islinked to conditions involving elevated glycative stress such as atopic dermatitis and diabetes mellitus. The influence ofglycative stress on host-pathogen interactions is still poorly understood. Therefore, we conducted a small-scale observational
study of the human skin, alongside in vitro experiments to determine the effects on S. aureus of exposure to advanced
glycation endproducts (AGEs). In contrast to that of young adults, the elderly skin was characterized by significantly
increased fluorescent AGE content, a 10-fold increase in S. aureus abundance, and a 20% reduction in coagulase-negative
Staphylococcus carriage. Glycated keratin triggered the formation of large multicellular S. aureus aggregates in vitro. Under
static conditions, glycated keratin promoted biofilm formation in a dose-dependent manner (ED 50 = 0.64 mg/mL). AGE
accumulation in the skin may play a crucial role in dysbiosis and recurring S. aureus infection. Preventative treatments to
reduce glycative stress could reduce the risk of disease and avoid overuse of antibiotics.

KEY WORDS: Staphylococcus aureus, glycative stress, biofilm, aggregation, advanced glycation endproducts (AGEs),		skin microbiome.

Introduction

Glycation, also known as the Maillard reaction, isa non-enzymatic process that occurs between reducing
sugars and protein or lipids. In addition to the well-known
browning reaction that occurs during the cooking process,glycation also occurs under physiological conditions inliving organisms. The resulting products of these reactions
are called Advanced Glycation Endproducts (AGEs). AGEs
contribute to the aging process and the pathogenesis ofvarious age-associated illnesses such as diabetes mellitus 1, 2)
and neurodegenerative disease 3). When AGEs form inassociation with proteins, they disrupt protein function andmay cause aggregation 4). AGEs also activate the Receptor
for AGEs (RAGE), triggering an inflammatory response 5).
AGEs accumulate in the body over time and may interferewith a variety of processes from bone homeostasis 6) to thefunctioning of the immune system.

Aging of the skin also involves glycative stress 7, 8).
AGEs form crosslinks between collagen fibers, reducing theskin’s elasticity and forming wrinkles. The accumulation of
AGEs can also lead to discolouration 9), further contributing
to the physical signs of aging. Recently, beauty products anddietary supplements are sold which aim to inhibit glycation
and breakdown AGE cross-links and aggregates in the skinand throughout the body in order to maintain a healthy
and youthful appearance. However, skin aging is not allabout looks. The thinning and stiffening of the epidermidis
that comes with age weakens the skin’s barrier function,
increasing the risk of injury and infection.
S. aureus is a gram-positive Firmicute that frequently
inhabits the skin and nasal epithelia of humans, and is amajor pathogen that is a frequent cause of skin infections
in hospital and community settings, including antibiotic

Correspondence: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center / Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1- 3 Tatara Miyakodani, Kyotanabe, Kyoto, 610 - 0394 Japan
TEL & FAX: +81-774-65- 6394 e-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Haasbroek K, cygd2001@mail4.doshisha.ac.jp;
Yagi M, myagi@mail.doshisha.ac.jp

Glycative Stress Research 2021; 8 ( 4 ) : 175-182(c) Society for Glycative Stress Research_ 175 _

AGEs and Staphylococcus aureus Biofilms

resistant varieties (i.e., MRSA). Carriage of the bacterium is
a risk factor for post-surgery infection 10, 11). S. aureus can also
cause bacterial pneumonia, particularly in co-infection withviruses such as Influenza A 12, 13) and SARS-CoV-2 14).
During infection S. aureus often forms biofilms:
communities of bacteria that live in a viscous extracellularmatrix consisting of polysaccharides, proteins, and eDNA.
The viscosity of the biofilm, the density of cells, presenceof protective enzymes, and changes in metabolic state ofresident bacteria all contribute to increased resistance toantibiotics and host immune response 15). S. aureus readily
forms biofilms on catheters and medical implants which canlead to serious infection 16).
In addition to medical plastics, S. zaureus is also known
to form biofilms on the skin itself. Skin lesions resulting
from poorly treated diabetes mellitus often form on
extremities where they are readily colonized, leading to staph
infection. S. aureus abundance increases while the diversityof commensal and/or mutualistic Staphylococcus species islost on the feet of diabetic patients 17). Staphopain and otherproteases are secreted which further breakdown the skinbarrier, worsening the risk of systemic infection. Other skinconditions associated with elevated glycative stress, such as
atopic dermatitis 18, 19) and psoriasis 20, 21) are also characterizedby colonization with S. aureus 22, 23), which forms biofilms inskin lesions and exacerbates the severity of accompanying
symptoms.
While the effects of glycative stress on aging andthe pathogenesis of various diseases has come to be better
understood in the recent years, its interactions with bacteria
are still poorly understood. While it has been known for sometime that AGEs or Maillard Reaction Products in culture
media may influence the growth of some microorganisms
under laboratory conditions 24), the implications for humanhealth in more complex systems are largely unknown.
Among the research that has been published, there areindications that glycative stress can be a factor in infectious
disease. Glycation of the bladder epithelium increases thebinding affinity of E. coli 25), facilitating bacterial adhesion
and infection, partially explaining the increased frequency
of UTIs in diabetes. Dietary AGEs may impact the gut
microbiome 26), and coffee melanoidins have been reported tomodulate adhesion by H. pylori in the stomach 27).
In our previous research 28) we found that AGEs formed
from glucose, fructose, and proteins found in skin (collagen I
and keratin) exhibited growth inhibitory effects on planktonic
cultures of Staphylococcus epidermidis, a ubiquitous skininhabitant with a variety of beneficial effects on skin healthand immunity. We noted the formation of cellular aggregatesfollowing exposure to AGEs, and hypothesized that this may
be related to biofilm formation. Considering the associationof S. aureus skin colonization and disorders involving
elevated glycative stress, we hypothesized that AGEs may
modulate the pathogenicity of S. aureus by promoting biofilm
formation. The aim of the present study was to examine theeffects of glycative stress on S. aureus. We conducted a smallscale sampling of the elderly skin in order to observe agerelated changes in abundance and carriage of S. aureus, andexamined the effects of AGEs on biofilm formation in vitro.

Materials and Methods
Human Study Design

A human study was carried out to examine the coagulasenegative Staphylococcus vs. Staphylococcus aureus makeupof the skin microbiome of healthy young adults and theelderly. Volunteers were recruited into two age groups: ayoung cohort of 20-30 years of age, and an elderly cohort of
65 or greater years of age. Participants with diagnosed skinconditions or use of topical steroids or antibiotics within
30 days of testing were excluded to eliminate potential
confounding effects. Participants were asked to refrainfrom the use of skin creams or sunscreen lotion on thedays of testing, and to avoid the use of antimicrobial soaps.
Testing consisted of bacterial sampling via swabbing andskin autofluorescence measurement of the volar forearm.
Doshisha University Ethics Review Committee on
Research involving Human Subjects approved of the study(application number #17085). Participants provided written
informed consent prior to taking part in the study.

Bacterial Sampling
Bacterial skin samples were collected by swabbing
using sterile cotton swabs. Sterile swabs were dipped in
extraction buffer (0.15 M NaCl and 0.01% Tween20) thenrubbed against a 4 cm 2 section of the volar forearm for 15
seconds. Samples were immediately plated onto mannitol
salt agar and incubated at 37 °C for up to 72 hours. Control
swabs were exposed to the air in the sampling room andthen applied to sample plates to test for contamination.

Skin Autofluorescence
Fluorescent AGE content in the skin was measured usinga TruAGE Scanner (Morinda, UT, USA). Participants rested
their arms on the device, exposing the volar forearm to thesensor, and the mean value of three readings was recorded.
Fluorescent AGE readings were taken after swabbing toprevent potential cross-contamination of skin bacteria bycontact with the device.

Bacterial Strains
S. epidermidis NBRC100911 (ATCC 14990) and S. aureus
NBRC 100910 (ATCC 12600) were purchased from the
National Institute of Technology and Evaluation Biological
Resource Center ( Tokyo, Japan).

Growth Conditions
Planktonic bacterial cultures were grown in Tryptic Soy
Broth (TSB) at 37 °C, with 250 rpm shaking. Solid media
cultures were grown on either Tryptic Soy Agar or Mannitol
Salt Agar at 37 °C. All growth media used was purchasedfrom Becton, Dickinson, and Company (MD, USA).
Bacterial viability of planktonic cultures wasdetermined by plate-counting after serial dilution; Tryptic
Soy Agar plates were inoculated in triplicate and incubatedat 37 °C for up to 72 hours with daily colony counting.
Turbidity assay (OD600) was found to poorly correlate with_ 176 _Glycative Stress Research

Staphylococcus overnight stock and thoroughly vortexed
before pipetting 100 µL aliquots of each sample into the wellsof a sterile un-treated 96-well plate (8 replications per sample).
The outer wells of each plate were filled with purified waterto prevent evaporation of test wells. Plates were then sealed
and incubated at 37 °C for 48 hours before staining with 1.0
mg/mL crystal violet solution for 10 minutes. After staining,plates were gently washed with water twice and left to dry
overnight. Each dried and stained biofilm well was solubilizedusing 200 µL of 30 % glacial acetic acid solution. Finally,
the absorbance of 125 µL aliquots were measured at 587
nm using a Varioskan Flash Multimode Microplate Reader(Thermo Fisher Scientific, MA, USA).

viable cell concentration in post-logarithmic growth phasecultures containing glycated keratin, and was omitted fromthe results.

Glycation Model
Glycated keratin solutions used in testing were
produced by incubating proteins with glycating agentsin accordance with the recipes outlined by 29). The final
concentration of the solution consisted of 50 mM phosphatebuffer (pH 7.4), 0.60 mg/mL keratin (Nacalai Tesque, Kyoto,
Japan), and 40 mM glucose. Solutions were incubated at
60 °C for 10 days to accelerate the glycation process. Afterincubation, ultrafiltration was performed using Amicon
Ultra Centrifugal Filters according to the manufacturer’s
instructions (Merck Millipore Ltd, Cork, Ireland) to remove
unreacted glucose. Protein concentration was measured by
BCA protein Assay (Thermo Fisher Scientific, MA, USA).

Logistic curve-fitting was performed in R 31) using theDose Response Curve package 32).

Quantification of Cellular Aggregation

Statistical Analysis

Planktonic cultures were supplemented with either
milliQ (control) or 0.5 mg/mL glycated keratin (AGE +)and incubated for 24 hours. After incubation, samples werevortexed and 2 µL aliquots were collected, transferred tomicroscope slides, and flame fixed. Slides were observed
via brightfield microscopy, and images were captured at
a maximum magnification of 400X. Image analysis was
performed using ImageJ. Briefly, images were converted toblack and white and size of cells and cellular aggregates wasmeasured (size represented by area in µm 2 ).

Simple descriptive statistical analysis was performed
using Excel (Microsoft). Between group statistical difference
was calculated using Student’s T-test. For multiple
comparisons, One-way ANOVA was performed with an α of
0.05, followed by Tukey’s test using SPSS Statistics (IBM).

Biofilm Assay

For the purposes of comparative analysis, participants
were divided into two cohorts by age: one group for those
20-30 years of age (hereafter Young Cohort) and one forthose aged 65 years or older (hereafter Elderly Cohort). As
outlined in Table 1, the groups were characterized by thefollowing observations:

Dose-Response Curve Fitting

Results

S. aureus Abundance on Elderly Skin

Biofilm assay was performed as previously reported 30).
1 mL solutions of TSB were prepared, with the desired
amount of glycated keratin added per sample type. Eachsolution was inoculated with 1:100 volume (10 µL) of
Table 1. Skin sampling data.
AGE compound
Age (years)
Skin Autofluorescence (units)

Young Cohort (20-30 years)

Elderly Cohort (65 + years)

p value

23. 5 ± 2.1
1.6 ± 0. 2

79.0 ± 5.7
2.8 ± 0. 5

2.02E -26 ***3.24E - 09 ***Abundance (CFU/Sample)
Coagulase-Negative Staphylococcus
S. aureus
Total Colony Forming Units
S. aureus Proportion (%)

50.8 ± 73.0
5. 5 ± 6.6

56. 3 ± 77.3
12.0 ± 14.9

61.4 ± 94.6
55.9 ± 71.6

117. 3 ± 132.2
50.0 ± 36.8

7.35E-01

1.70E- 02 *1.40E-01

1.30E-03 **Detection Rate (%)
Coagulase-Negative Staphylococcus
S. aureus

100

81

69

81

4. 9 7E - 0 2 *4. 4 9E - 01

Coagulase-negative Staphylococcus was differentiated from S. aureus by colour after growth on Mannitol Salt Agar selective media. The young cohort
consisted of 13 volunteers, and the elderly 21. Mean ± standard deviation. Statistical difference calculated by Student’s t-test. *p < 0.05, **p < 0.01,*** p < 0.001.

_ 17 7 _AGEs and Staphylococcus aureus Biofilms

Growth Inhibitory Effect of Glycated Keratin
As S. aureus is increased on the elderly skin incomparison to S. epidermidis, we initially hypothesized thatdifferential responses to AGE exposure between the species
could be responsible for the observed difference in abundanceon the aged skin; if S. aureus has greater resistance toglycative stress, it could outcompete S. epidermidis in a
glycated skin environment. As we have previously reported 28),
S. epidermidis growth was inhibited by exposure to exogenous
glycated protein in vitro. Here we replicated the previous
S. epidermidis test using S. aureus strain NBRC100910,
observing the effects of glucose-keratin exposure. As shownin Fig. 1, addition of glycated keratin to growth media results
in a 0.9 log reduction of S. epidermidis compared to a 1.1 log
reduction of S. aureus after 24 hours. Both species are
similarly vulnerable to glycative stress from exogenous
glycated proteins. Thus, the differences observed on thehuman skin are likely not due a direct difference invulnerability to glycative stress by S. aureus.

Cellular Aggregation
During growth of S. aureus in media containing glycatedproteins, the formation of large macroscopic cellularaggregates was observed, settling in a large cloudy mass atthe bottom of culture tubes. Microscope images of cellularaggregates of S. epidermidis and S. aureus can be seenin Fig. 2- a. When observed microscopically, the initial

1.0E+10
S. aureus***

1.0E+ 0 9

CFUs/mLThe Young Cohort consisted of 13 volunteers with amean age of 23.5 ± 2.07 years, and a mean fluorescent AGE
score of 1.60 ± 0.16. The Elderly Cohort consisted of 21
volunteers with a mean age of 79.0 ± 5.75 years and a mean
fluorescent AGE score of 2.77 ± 0.47. General bacterial
abundance was estimated by a total count of colony forming
units in each sample. The mean bacterial abundance in theyoung cohort was 56.31 ± 77. 30 CFU, while in the elderly
cohort it was 117.30 ± 132.20 CFU. Although nearly a twofold increase, the difference was not statistically significant
due to the large amount of variation between individuals.
Coagulase-negative Staphylococcus mean abundance was50.85 ± 73.04 and 55.94 ± 71.59 in the young and elderly
cohorts respectively, not significantly different. However,
the mean S. aureus abundance represents the main betweengroup difference, with mean abundances of 5.46 ± 6.6 4
CFU in the young cohort which were significantly lesscompared to 55.94 ± 71.59 CFU in the elderly cohort ( p =0.017).
The relative abundance of S. aureus as a proportion of
total CFUs was calculated to normalize the data, with theresults given as a percentage of S. aureus colonies per sample.
Using these criteria, the relative abundance of S. aureus
was 12.01 ± 14.87 % and 49.97 ± 36.83 % in the young
and elderly samples respectively. This difference was also
statistically significant ( p = 0.001).
The S. epidermidis carriage rate was 100 % in the
Young Cohort and 81.0 % in the Elderly Cohort, a differencewith borderline significance (p = 0.049). S. aureus carriage
rates were 69.2 % and 81. 0 % respectively, but the difference
observed was not significant (p = 0.449).

S. epidermidis***

1.0E+ 0 8

1.0E+ 07

1.0E+ 0 6
Control

Glycated
KeratinControl

Glycated
KeratinFig. 1. Growth inhibitory effect of glycated keratin.

24 -hour cell viability of planktonic cultures of S. epidermidis
and S. aureus in media with 1.0 mg/mL glycated keratin. n =3. Mean ± standard deviation. Statistical difference calculatedby Student’s t-test. *** p < 0.001.

formation of small clumps of up to a dozen cells differentiatedglycated keratin cultures from controls in the logarithmic
growth phase, roughly 5 hours after inoculation. Image
analysis conducted on samples collected after 24 hours ofgrowth ( Fig. 2-b) showed aggregates in glycated keratin
cultures were significantly larger than those in control
cultures: roughly 3-fold larger for S. epidermidis, and 8-foldfor S. aureus ( p < 0.001). Aggregate size was substantiallysmaller in S. epidermidis samples, and was not apparent
macroscopically.

Biofilm Production
Suspecting biofilm formation from the observanceof cellular aggregates, we performed photometric assays
in order to quantitatively measure the amount of S. aureus
biofilm formation in response to glycated keratin AGEs.
Biofilm absorbance was measured at 587 nm after 48 hoursof stationary incubation.
Fig. 3-a demonstrates the biofilm response of S. aureus
to glycated and un-glycated keratin at a concentration of
0.5 mg/mL. Sterile TSB was also tested to ensure there was
no interference during the crystal violet staining, producing
negligible absorbance values (not shown). For glucose
conditions, an additional 2.5mg/mL of glucose was added to
TSB, resulting in a final glucose concentration of 5.0 mg/mL.
Heated keratin, produced following the same procedure
as glycated keratin, but without glucose, did not differ
significantly from control. All sample varieties resulted insignificantly less biofilm formation than glycated keratin,
which produced the strongest response at 1. 9. Exposure to_ 178 _Glycative Stress Research

Biofilm Dose-Response

glycated keratin resulted in a roughly 10-fold increase inabsorbance compared to heated, un-glycated keratin (p <0.001).
The biofilm response of S. epidermidis and S. aureus
are compared in Fig. 3-b. While glycated keratin produced
a significant increase in biofilm formation in both species,
S. aureus produced significantly more biofilm thanS. epidermidis at the same dosage (p < 0.001).

a)b)

Glycated Keratin

Control

The dose-response curve of S. aureus biofilm production
in response to glucose-keratin exposure was also examined.
The resulting curve, shown in Fig. 4, reveals a classic
sigmoidal relationship with a midpoint of 0.6 4 mg/mL of
glucose-keratin ( R 2 = 0.9998). Biofilm abundance rises
rapidly at increasing dosage below 1 mg/mL, before beginningto plateau at an absorbance value of roughly 3.0 above
baseline between a dosage of 2 – 5 mg/mL.

S. epidermidis

20 µm

20 µm

20 µm

20 µm

Aggregate Size (µm2 )Absorbance (587 nm)

S.aureus

15 0

***

125
10 0

S. aureus

75

2
50
25
0

1

S. epidermidis***

Control Glycated Control
Keratin

Glycated
Keratinn.s.

Fig. 2. Glycated keratin induced cellular aggregation.
a: Representative bright-field microscopy images of S. aureus and S. epidermidis cellular aggregation after 24 hours under planktonic
conditions. Images captured at 400X magnification. b: Mean bacterial aggregate size. Samples were vortexed and 2 µL inoculates were0transferred to microscope slides. n = 10. Mean ± standard deviation. Statisticaldifference determined by Student’s t-test. *** p < 0.001.
Glycated
Keratin

HeatedKeratinControlb)2

Absorbance (587 nm)

Absorbance (587 nm)a)Glucose

1

n.s.

0

2

n.s.
S. aureus
S. epidermidis

10
Glycated
Keratin

GlucoseHeatedKeratinControl Glycated Control
KeratinControl

Glycated
KeratinFig. 3. Glycated keratin induced biofilm formation.
a: S. aureus biofilm formation in response to 0. 5 mg/mL of keratin. Glucose conditions contain 5.0 mg/mL of glucose, double theconcentration of standard TSB. b: Difference in biofilm formation between S. epidermidis and S. aureus. n = 8. Mean ± standard deviation.
rbance (587 nm)

Bars indicate standard deviation. Statistical difference was calculated via One-way ANOVA followed by Tukey’s test. p < 0.001.

2n.s.
S. aureus

1_ 179 _

S. epidermidis

AGEs and Staphylococcus aureus Biofilms

Absorbance (587 nm)

3.0
2.5
2.0

.

1.5
1.0
0.5
0.0
0

1

2

3

4

Glycated Keratin Concentration (mg/mL)

Fig. 4. Glycated keratin dose-response relationshipDose-response relationship of S. aureus biofilm formation and > 10 kDA glucose-keratin concentration. Biofilms were
stained with crystal violet and absorbance was measured at 587 nm after 48 hours of growth under static conditions at
37 °C. Each data point represents the mean of eight samples. Midpoint = 0.64 ± 0.03. Upper limit = 3.36 ± 0.12. R2 = 0.99.

Discussion

Human StudyS. aureus carriage was higher in our participants thanis reported among the general population in other studies 33),
although the trend of increased carriage in the elderly was
replicated. These differences may be due to this study’s
focus on the skin. Skin sampling may be more likely tocapture transient carriage, overestimating S. aureus carriage
rates in comparison to the more common nasal sampling
methods. The sampling location of the volar forearm
may also have resulted in relatively low overall bacterial
abundance compared to more sebaceous regions. The innerforearm was used to measure skin autofluorescence as itis less exposed to UV damage; the same location was thus
used for the bacterial sampling. Healthy participants withno diagnosed skin disorders were selected to remove thepotential confounding effects and focus on the difference influorescent AGE skin content between the age groups.
The participants were not evenly distributed by sex,
and female participants outnumbered male. Regardless,there was no statistically significant difference observed inbacterial abundance or carriage between male and female
participants. While we were able to obtain statisticallysignificant data points, a larger sampling including the fullage range of the population would provide further evidence
for an age-related shift in skin S. aureus abundance.

Cellular Aggregation
Cellular aggregation can occur for a variety of reasons.
A similar response has been reportedly observed whencultivating Staphylococcus in synovial fluid 34), which occurs
due to the binding of fibrin/fibrinogen to cell surface
receptors which trigger cellular aggregation and biofilm
response. Aggregation may also occur in response to toxic
stimuli, such as exposure to sub-lethal concentrations ofantibiotics. The formation of cellular aggregates protects

constituent cells from antibiotics 35) and immune responseby physically shielding cells in the interior of aggregates, aswell as by decreasing the surface area-to-volume ratio of
the structure. We speculate that the glycative stress exerted
by exogenous AGEs triggers an aggregation response
rather than acting as a binding substrate for the nucleationof bacterial aggregates, as aggregation did not occur when
Staphylococcus was resuspended in carbon free non-growth
media and exposed to glycated keratin. However, we have
not ruled out the possibility that glycated keratin upregulates
the expression of cell surface binding receptors that are notexpressed under standard culture conditions.
When both Staphylococcus species were compared,
S. aureus demonstrated a stronger aggregation responsecompared to S. epidermidis at the same glycated keratin
dosage. A similar difference was also observed in the capacity
for biofilm formation. This may indicate that S. aureus isbetter able to respond to the effects of glycative stress andprotect itself, allowing it to better outcompete S. epidermidis
in environments under high levels of glycative stress, such as
the skin of those with diabetes or atopic dermatitis.

Potential Biofilm Formation Mechanisms
In S. aureus there are several physiological processes
that govern biofilm formation. As a natural process of theirlife- cycle, S. aureus may form biofilms from the presence ofsuitable binding substrates (e.g., fibronectin 36), collagen 37),
and keratin 38) depending on strain) for cell-surface adhesins
or high concentrations of neighboring cells.
Extracellular DNA can also act as a trigger of S. aureus
biofilms 39, 40). The eDNA released from damaged or autolysed cells becomes a major part of the biofilm itself, andupregulates biofilm formation in surviving cells. Membrane
damage by AGEs could lead to an increase in mortality andbiofilm formation.
In response to cellular stress, many bacteria form
biofilms to protect themselves from adverse environmental_ 18 0 _Glycative Stress Research

conditions or host immune response. S. aureus is known tomodulate its biofilm production in response to conditions
such as hyperoxia 41), anoxia and hypoxia 42, 43), pH 44), high salt
and suboptimal temperature 45), and sub-lethal concentrationsof alcohol 46). Based on our observations, we believe it islikely that glycative stress is also a factor in S. aureus biofilm
formation and regulation. Further investigation is necessary
to uncover the precise mechanism(s) by which glycatedproteins exert this stress. In future, we plan to examine theeffect of exposure to glycated proteins on the expressionof genes related to biofilm formation, such as the iac
locus which regulates polysaccharide intercellular adhesin
production 47).

Conclusion
The aging microbiome of the skin is characterized byincreasing abundance, in absolute and relative terms, ofS. aureus. In vitro, the addition of AGEs produced fromglucose and keratin triggered cellular aggregation andbiofilm production in S. aureus. The presence of elevatedconcentrations of AGEs in the skin (whether it be fromgeneral aging, metabolic syndrome/diabetes mellitus, or askin disorder) may exacerbate the formation of S. aureus

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Conflict of Interest
The authors declare that this research was conductedin the absence of any commercial or financial relationshipsthat could be construed as a potential conflict of interest.

Acknowledgments
This study was supported in part by IDF grant number
21003.

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