Biofilm formation on oral piercings

Abstract

PURPOSE: Biofilms on oral piercings might serve as a bacterial reservoir in the host and lead to bacteraemia and even septic
complications. The use of piercing materials less susceptible to biofilm accumulation could contribute to alleviation of problems.
The present study aimed to assess biofilm formation on four commercially available, surface characterized piercing materials
in vitro (polytetrafluoroethylene, titanium, stainless steel, and polypropylene). MATERIAL AND METHODS: Autoclave-sterilized piercings
were surface characterized by X-ray photoelectron spectroscopy, contact angle measurements, and atomic force microscopy. Biofilms
were grown for 1, 4, or 20 h on the piercing surfaces by immersion in pooled human whole saliva. Colony forming units (CFUs)
were determined. For fluorescence microscopy, samples were stained with DAPI. RESULTS: All four piercing materials included
showed significant amounts of biofilm after 20 h of incubation. Biofilm formation was significantly lowest on polytetrafluoroethylene
piercings (p < 0.001), and was mainly determined by wettability – which was significantly lowest for polytetrafluoroethylene (p < 0.001) – and the prevalence of carbon- and oxygen-rich components. Surface roughness measurements showed no statistically
significant differences, but standard deviations were rather high. High standard deviations are caused by substantial pits
and elevations and are due to poor machining quality in the manufacture of the piercings. CONCLUSION: Biofilm formation on
oral piercings was mainly determined by surface free energy and the prevalence of carbon- and oxygen-rich components, and
was significantly lowest on polytetrafluoroethylene piercings. The findings indicate that oral piercings might serve as a
reservoir of potentially pathogenic bacterial species.

Full text

J. Stomat. Occ. Med. (2010) 3: 171–176
DOI 10.1007/s12548-010-0059-z
Printed in Austria
© Springer-Verlag 2010
Biofilm formation on oral piercings
Ines Kapferer
1
, Christoph Steiner
1,
*, Ulrike Beier
1
, Natalia Schiefermeier
2
,
Markus Nagl
3
, Frederik Klauser
4
1
Department of Operative Dentistry, Dental School, Innsbruck Medical University, Innsbruck, Austria
2
Division of Cell Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria
3
Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria
4
Institute of Physical Chemistry, University of Innsbruck, Innsbruck, Austria
Received July 21, 2010; accepted after revision September 29, 2010
Purpose: Biofilms on oral piercings might serve as a bacterial
reservoir in the host and lead to bacteraemia and even septic
complications. The use of piercing materials less susceptible
to biofilm accumulation could contribute to alleviation of
problems. The present study aimed to assess biofilm forma-
tion on four commercially available, surface characterized
piercing materials in vitro (polytetrafluoroethylene, titanium,
stainless steel, and polypropylene).
Material and methods: Autoclave-sterilized piercings
were surface characterized by X-ray photoelectron spectros-
copy, contact angle measurements, and atomic force micros-
copy. Biofilms were grown for 1, 4, or 20 h on the piercing
surfaces by immersion in pooled human whole saliva. Colony
forming units (CFUs) were determined. For fluorescence
microscopy, samples were stained with DAPI.
Results: All four piercing materials included showed
significant amounts of biofilm after 20 h of incubation. Bio-
film formation was significantly lowest on polytetrafluoro-
ethylene piercings (p< 0.001), and was mainly determined by
wettability –which was significantly lowest for polytetrafluor-
oethylene (p< 0.001) –and the prevalence of carbon- and
oxygen-rich components. Surface roughness measurements
showed no statistically significant differences, but standard
deviations were rather high. High standard deviations are
caused by substantial pits and elevations and are due to poor
machining quality in the manufacture of the piercings.
Conclusion: Biofilm formation on oral piercings was
mainly determined by surface free energy and the prevalence
of carbon- and oxygen-rich components, and was significant-
ly lowest on polytetrafluoroethylene piercings. The findings
indicate that oral piercings might serve as a reservoir of
potentially pathogenic bacterial species.
Introduction
The trend in oral piercing has grown in popularity over the
past decade. Oral piercing mostly involves lips, tongue, and
cheeks, with the tongue being the most commonly pierced
intraoral site [28]. A variety of complications have been
reported and can be categorized as early (acute) and late
(chronic) [11]. Early complications include bacterial infec-
tion, pain, swelling, prolonged bleeding, and difficulties in
swallowing, speech, and mastication [11]. Late complica-
tions include recurrent infections, gingival trauma, local-
ized periodontitis, chipped and fractured teeth, persistent
difficulties in oral functions, and swallowing of the device
[11]. A number of studies have been conducted to assess the
prevalence of these complications [7, 16–18, 23, 25, 27].
However, many biologic questions on these foreign bodies
remain unaddressed, one of which being biofilm formation.
Biofilms on oral piercing may serve as a bacterial reservoir
and lead to systemic bacteraemia and even septic compli-
cations. Several case reports have been published on life-
threatening systemic infections after oral piercing: Infective
endocarditis [3], breast abscess [6], epidural abscess [10],
chorioamnionitis [21], herpes simplex virus hepatitis [25],
hepatitis C virus infection [15], toxic shock syndrome due to
Staphylococcus aureus infection [4], and cerebellar brain
abscess [26] are rare, but dangerous complications. Addi-
tionally, biofilms on oral piercings might serve as reservoirs
for bacteria associated with periodontitis, due to the anaer-
obic condition in the piercing channel. The first microbio-
logical analysis of piercing biofilms was conducted by
Ziebolz et al. [34]. The microbiological analysis of tongue
piercing sites showed that jewellery can provide a reservoir
for periodontopathogenic bacteria. The incidence and the
severityoflocalinfectionsaswellassystemicproblems
could depend on the extent and the bacterial composition of
the biofilm formed on the piercing’s surface. The use of
piercing materials less susceptible to biofilm accumulation
could hence contribute to alleviation or even prevention of
problems. However, at the time being, there is no reference
available regarding the surface plaque retention of different
materials used as oral jewellery.
*Author contributed equally to this work.
Correspondence: Ines Kapferer, Reichenauerstraße 46, 6020 Innsbruck,
Austria. E-mail: ines.kapferer@gmx.net
J. Stomat. Occ. Med. ©Springer-Verlag Biofilm formation on oral piercings 3/2010 171
original article

Oral piercings are made of different materials, usually
metals such as stainless steel or titanium. Steel as well as
titanium is well known for good mechanical properties, high
corrosion resistance and excellent biocompatibility [12]. To-
day these desirable properties are also achievable by Teflon
(PTFE) and polypropylene (Bormed
TM
), which are heat and
radiation sterilizable polymers designed for medical applica-
tions [1, 2]. Materials such as gold and silver –popular in
other body regions –are only of scarce utilization in the oral
cavity, and thus have not been taken into consideration in
this study.
It has been demonstrated that initial adhesion of bacte-
ria to a biomaterial surface is influenced by the surface
physico-chemical properties, such as surface roughness,
surface free energy (SFE) and elemental surface composition
[8, 13, 22, 30], of both the bacterial cell and the substratum.
Rough surfaces promote biofilm formation and maturation,
and high-energy surfaces are known to collect more oral
biofilm and to bind it more strongly [30]. The charge of the
surface influences electrostatic interactions and bacterial
adhesion, too [22]. In the oral cavity, natural and artificial
surfaces are immediately covered with a so-called acquired
pellicle consisting mainly of saliva-derived proteins. The
pellicle serves as adhesion site for the pioneering bacteria.
The adsorption of pellicle proteins is a selective process and
is largely affected by the material characteristics. Therefore,
the bacterial adhesion is –via the selectivity of molecule
adsorption –indirectly influenced by the material
characteristics.
The objectives of the present investigation were (i) to
characterize the surface physico-chemical properties of four
commercially available piercing systems in their sterilized
state and (ii) to quantitatively assess bacterial adhesion on
these piercing surfaces.
Materials and methods
Materials
Commercially available lip piercings of four commonly used
materials (titanium, PTFE, Bormed
TM
, and stainless steel)
were selected for the present study, three samples for each
material. The intraoral disc (diameter: 5 mm, gauge: 1.2 mm)
of each piercing was cut off and used for further experiments.
All discs were packaged individually and sterilized (121C,
20 min, steam autoclaving machine) prior to experiments.
Surface roughness, surface chemistry, and surface
free energy
For atomic force microscopy (AFM) (Nanoscope IVa Dimen-
sion 3100 AFM, Digital Instruments, Santa Barbara, CA, USA),
three samples of each piercing material were analyzed.
Surface plots were made of each measurement to provide
a three-dimensional perspective of the surface, from which
the mean surface roughness (R
a
) was computed. R
a
repre-
sents the average distance of the roughness profile to the
center plane of the profile. The surface area ratio expresses
the ratio between the surface area (taking the z height into
account) and the area of the flat x, y plane.
The surface elemental and chemical compositions of
sterilized piercing discs were determined by X-ray photoelec-
tron spectroscopy (XPS) (Thermo MultiLab 2000 spectrome-
ter equipped with a monochromatic Al X-ray source
1486.6 eV). To remove the first few atomic layers, the samples
were analyzed as received and after a 5 min Argon sputter
cleaning treatment.
For surface free energy, 1 ml of double distilled water was
dispensed manually on the surfaces of the discs and photo-
graphed immediately using a Moticam 2000 CCD camera
(Motic
+
) mounted on a Wild Heerbrugg stereomicroscope.
Contact angles were measured between a tangent to the
water drop and the substratum surface.
Biofilm formation and analysis
Human whole saliva from five healthy volunteers of both
sexes was collected into ice-chilled beakers after stimula-
tion by chewing paraffin. After the saliva was pooled,
phenylmethylsulfonylfluoride as a protease inhibitor was
added to a concentration of 1 mM to reduce protein
breakdown. Each piercing disc was individually immersed
in 2 ml of freshly pooled saliva, and biofilms were allowed
to grow on the piercing surfaces at 37C for 1, 4, or 20 h in
an aerobic incubator. Three independent experiments
were done.
The piercing discs with adhering biofilms were sonicat-
ed for 15 min in individual Eppendorf tubes containing 1 ml
PBS to disperse adhering bacteria. Aliquots of 50 ml were
spread onto tryptic soy agar plates in duplicate. Plates were
incubated at 37C under aerobic conditions, and CFUs were
counted after 48 h.
For fluorescence microscopy, samples were fixed in 4%
paraformaldehyde (Sigma-Aldrich) for 10 min, brieflywashed
with distilled water and stained with DAPI (40,6-Diamidino-2-
phenylindole dihydrochloride, Sigma-Aldrich; concentration
1:50000) for 20 min, followed by washing in distilled water.
Images were taken with an AxioImager M1.2 microscope (Carl
Zeiss Micro-Imaging, Inc.) equipped with a CoolSnapHQ2
CCD camera (Photometrics) and the AxioVision Release4.5
SP1 software (Carl Zeiss Micro-Imaging, Inc.).
Statistical analysis
Statistical analysis was carried out using one-way analysis
of variance (ANOVA) with Turkey post-test analysis.
Significance was set at p< 0.05 (VassarStats
+
). Linear regres-
sion analysis was performed to identify the surface properties
most predictive for biofilm formation on sterilized piercings.
Probability levels of 0.05 or less were considered to indicate
statistical significance (VassarStats
+
).
Results
Surface characterization
Contact angle measurements were significantly different
between all tested materials (p< 0.01). Surface free energy
(wettability) was the lowest for PTFE (88.78±0.40), followed
172 3/2010 Biofilm formation on oral piercings ©Springer-Verlag J. Stomat. Occ. Med.
original article

by Bormed
TM
(81.26±0.16), titanium (78.56±0.15), and
stainless steel (61.90±1.09).
The mean surface roughness was the highest for titani-
um (range 84–148 nm), followed by Bormed
TM
(range 63–
194 nm), PTFE (range 62–448 nm), and stainless steel (range
56–163 nm). The profiles obtained from AFM revealed dif-
ferences in surface morphology (Fig. 1).
The results of XPS analysis are displayed in Fig. 2.
Small amounts of surface contaminants, referred to as
‘other’in the image, were detected on all as received
surfaces except on PTFE. All these contaminants vanished
after removing the first few atomic layers with argon
sputter treatment.
Biofilm analysis
After 1h of incubation, Bormed
TM
and titanium piercings had
significantly higher numbers of CFU attached to the surface
(5.20 ±0.25 a nd 5 .19 ±0.29, respectively) than steel and PTFE
(4.90 ±0.11 and 4.89 ±0.11, respectively; p¼0.01). After 4 and
20 h of incubation, Bormed
TM
continuously revealed the high-
est numbers of CFU. However, the differences between Bor-
med
TM
(5.20 ±0.26 and 5.34 ±0.26, respectively), titanium
(5.19 ±0.10 and 4.77 ±0.26, respectively), and steel piercings
(5.11 ±0.31 and 5.11 ±0.20, respectively) were only small. At
both time points, biofilm formation was highly significantly
lower on PTFE piercings (4.88 ±0.30 and 4.65 ±0.26; p<0.001).
0.0
1.0
2.0
3.0
4.0
5.0
Z (µm)
0.0
1.0
2.0
3.0
4.0
5.0
Z (µm)
0.0
1.0
2.0
3.0
4.0
5.0
Z (µm)
0.0
1.0
2.0
3.0
4.0
5.0
Z (µm)
Steel Titanium
PTFE
Bormed TM
Y (µm)
X (µm) X (µm)
X (µm)
X (µm)
Y (µm)
Y (µm)
Y (µm)
Fig. 1: Atomic force microscopy (AFM), large area scans (50 mm50 mm) of Ti, steel, PTFE, and Bormed
TM
. All materials showed clear surface
roughening and prominent pittings of variable pore sizes. The surfaces of Ti and steel piercings were characterized by clearly visible striations, likely
to be the result of machining. Bormed
TM
and PTFE piercings are produced by a casting process. For Bormed
TM
, this results in a fibrillar surface
100
Elemental composition (%)
90
80
70
60
50
40
30
20
10
0
BormedTM BormedTM
sputtered
PTFE PTFE
sputtered
Steel
sputtered
Titanium Titanium
sputtered
Steel
Other
Al
Ti
Cr
Co
Fe
F
C
O
Fig. 2: X-ray photoelectron spectroscopy (XPS). The surface elemental and chemical compositions of samples were analyzed as received and after a
5 min Argon sputter cleaning treatment, sufficient to remove the first few atomic layers. The oxygen content was lower for Bormed
TM
and PTFE
samples than for steel and Ti. Small amounts of surface contaminants, referred to as ‘other’in the image, were detected on all untreated surfaces
except on PTFE. All these contaminants were removed after the argon sputter treatment
J. Stomat. Occ. Med. ©Springer-Verlag Biofilm formation on oral piercings 3/2010 173
original article

After 20 h incubation, differences in biofilm formation were
clearly visible in fluorescence microscopy (Fig. 3).
Effects of surface physico-chemical properties
on biofilm formation
Effects of the investigated physico-chemical properties on the
number of CFU were investigated by linear regression ana-
lysis. CFU showed a negative correlation to surface free
energy after exclusion of Bormed
TM
from analyses: the lower
the contact angle, the higher the bacterial adhesion (R
2
¼
0.999, p¼0.014). CFU correlated to surface roughness
(R
2
¼0.998, p¼0.020), again after exclusion of Bormed
TM
(R
2
¼0.8461, p¼0.8). Bormed
TM
, titanium, and steel pier-
cings showed a linear increase in the number of adhering
bacteria with increasing roughness, whereas for PTFE there
was no such trend observed. Surface fluorine content had a
negative correlation with CFU (R
2
¼0.977, p¼0.011). Sur-
face oxygen content correlated significantly with SFE (R
2
¼
0.837, p¼0.042).
Discussion
With infections being one of the most frequent complications
[19], biofilm formation on oral piercing is a fundamental
issue. Additionally, biofilms on oral piercings might serve as a
bacterial reservoir for systemic infections. The piercing pro-
cedure exposes the piercee to a high risk of infection because
the oral cavity harbours a huge amount of bacteria [5]. The
high vascularity of the area is a further aspect to be consid-
ered. The present study was designed to quantify bacterial
colonization and visualize biofilm formation on surface
characterized piercings. Freshly pooled human whole saliva
was used as a source of bacteria for biofilm formation to
closely simulate in vivo conditions.
One pivotal outcome of this study is that all four piercing
materials included showed in general significant amounts of
biofilm after 20 h of incubation, confirmed by fluorescence
microscopy (Fig. 3) as well as bacterial cultivation. Biofilm
formation was significantly lower on commercially available
PTFE piercings compared to all other materials. These find-
ings indicate that piercings might serve as an important
reservoir of potentially pathogenic bacterial species when
placed in the oral cavity. Piercees should be informed about
the microbiological contamination of oral piercings, and
about possible side effects such as recurrent infections or
systemic bacteriaemia.
Surface roughness
There is a general agreement that rough surfaces accumulate
considerably more biofilm in relation to the number of
bacteria, the thickness, the area covered, and the maturity
–that is more rods, spirochetes, and motile organisms –as
smooth surfaces [30, 32]. On a rough surface, bacteria are
better protected against shear forces, facilitating the estab-
lishment of an irreversible attachment [32]. Moreover, the
surface area for attachment is increased [32].
Surface roughness measurements of the investigated
piercings showed no statistically significant differences, but
standard deviations were rather high. Roughness differ-
ences between the piercings of one material group and
consequently the high standard deviations are partially
caused by several substantial pits and elevations on the
surface (Fig. 1) and are due to poor machining quality in the
manufacture of the piercings, where optimization of rough-
ness does not seem to be a crucial procedure. With dimen-
sions of 0.5 to 1 mm, streptococci –common oral bacteria –
can easily hide in these pits. Oral piercing is a personal
decision, therefore, if patients cannot be convinced of
removing their piercings, they should at least choose studs
of high quality.
Surface free energy
The surface free energy (wettability) directly influences pro-
tein adsorption on biomaterial surfaces [32]. The impact of
the substratum surface free energy remains on the whole
after the pellicle formation [32]. Thus, the surface free energy
properties are transferred through the adsorbed protein layer
[32]. Bacteria that have a high surface free energy themselves
selectively adhere better to high-energy (hydrophilic) sur-
faces, whereas bacteria with a low surface free energy adhere
better to low-energy (hydrophobic) surfaces [29, 33]. The
majority of the oral bacteria possess a high surface free
energy (Streptococcus mitis and some Streptococcus milleri
strains for example are an exception with a low surface free
energy) [31]. Hydrophobic materials thus are less prone to
PTFE Titanium
BormedTM
Steel
Fig. 3: Fluorescence microscopy after 20h incubation. The piercing
discs with adhering biofilms were stained with DAPI. PTFE showed the
lowest number of adhering bacteria, which was highly significant
(p¼0.0004)
174 3/2010 Biofilm formation on oral piercings ©Springer-Verlag J. Stomat. Occ. Med.
original article

biofilm accumulation in the oral cavity, which explains the
reduced bacterial growth on the hydrophobic, low-
energy PTFE piercings in our study (88.78±0.40). Steel
(61.90±1.09), titanium (78.56±0.15), and Bormed
TM
(81.28±0.16) in contrast showed smaller contact angles,
that is a higher surface free energy (hydrophilic), and conse-
quently more biofilm formation.
Surface elemental composition
The surface elemental composition of the four materials was
determined by X-ray photoelectron spectroscopy. The high
sensitivity of this technique reveals the elemental composi-
tion of the outermost few atomic layers of the piercings,
which determine the interaction of the material with the
environment. This surface composition can differ from the
material’s bulk. Therefore the samples were analyzed firstly
as received and secondly after a short argon sputter treat-
ment, sufficient to remove layers eventually belonging to
physisorbed or chemisorbed species. Elements completely
removed after sputter etching can be regarded as surface
contaminants. Several of these were detected on as received
surfaces with the exception of PTFE piercings. The oxygen
content was lower for Bormed
TM
and PTFE samples (13.50%
and 9.98%, respectively) than for steel (23.07%) and titani-
um (28.29%). After sputter cleaning, the oxygen signal
vanishes on Bormed
TM
and PTFE, whereas it remains al-
most unchanged for steel and decreases to 13.34% for
titanium. This elucidates that oxygen extends to several
atomic layers on the metals, indicative of a distinctive oxide
coating, whereas it is limited to the first layer on Bormed
TM
and PTFE. There it belongs either to an oxide coating too or
to physisorbed oxygen containing molecular species. In any
case, the presence of oxygen on a surface is expected to
enhance the hydrophilic interactions with the environment
because of the high polarity of heterogeneous oxygen
bonds.
The lower number of attached oral bacteria with PTFE is
often explained by its low wettability [9]. Additionally, one of
its major components is fluorine (39.63%). The effect of
fluorine on the inhibition of initial bacterial attachment was
recently investigated [14]. The attachment of both hydrophil-
ic and hydrophobic bacterial strains was dramatically re-
duced when introducing fluorine into a polydimethylsiloxane
coating [14].
The outstanding bacterial colonization of Bormed
TM
piercings cannot be explained by surface roughness (the
linear regression analysis for R
a
and CFU was only signifi-
cant after exclusion of this material) or surface free energy.
The adhesion mechanism appears to lie rather in a chemi-
cal attraction between the microorganisms and the sub-
stratum. The surface of Bormed
TM
in water (or saliva) is
altered through oxidation, resulting in free carbonyl and
hydroxylgroupsonthesurface,offeringbacteriatheop-
portunity to attach [20]. The relative surface carbon content
of Bormed
TM
piercings (82.39%) was higher than of the
other investigated materials, and was significantly related to
CFU in our study. Additionally, the surface of Bormed
TM
contains sulfur, which is an essential element for bacterial
metabolism and could thus act as a chemical cue to attract
bacteria.
Conclusions
All piercing materials included showed in general significant
amounts of biofilm. However, biofilm formation was signifi-
cantly lowest on commercially available PTFE piercings. The
results of this study affirm the importance of good oral
hygiene to prevent postpiercing complications. Patients need
better information on the microbiological contamination of
oral piercings and potential complications such as recurrent
infections or systemic bacteriaemia. Additionally, they
should attend regular dental checks and receive professional
advice on cleaning their piercing jewellery regularly with a
toothbrush and/or a disinfectant.
Take-home message
Patients should be informed about the microbiological con-
tamination of oral piercings and possible side effects such as
recurrent infections or systemic bacteriaemia. If patients
cannot be convinced of removing their oral piercings, it is
important that they are fully aware of potential complications
and they should at least choose highly polished PTFE studs
instead of cheap custom jewellery.
Conflict of interest
The authors declare that there is no conflict of interest.
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