Removal of Dental Biofilms with an Ultrasonically Activated Water Stream

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DOI: 10.1177/0022034515589284 · Source: PubMed
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Abstract
Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis. Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and biofilms exposed to the water stream alone without ultrasonic activation (P < 0.05). The water stream alone showed no statistically significant difference in removal compared with the untreated control (P = 0.24). High-speed videography demonstrated a rapid rate (151 mm(2) in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a potentially effective method for biofilm removal and improved oral hygiene. © International & American Associations for Dental Research 2015.
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Journal of Dental Research
2015, Vol. 94(9) 1303 –1309
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DOI: 10.1177/0022034515589284
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Research Reports: Biological
Introduction
The oral cavity provides an optimal environment for the colo-
nization and proliferation of a diverse array of microorganisms
(Aas et al. 2005; Zaura et al. 2009). The most prevalent are
bacteria, which exist primarily as a biofilm, commonly known
as dental plaque, on the tooth surface. The accumulation of
dental biofilm plays a key role in the pathogenesis of a range of
oral diseases, including gingivitis, periodontitis, and caries
(Aspiras et al. 2010).
Streptococcus mutans is a major cariogenic constituent of
the supragingival biofilm due, in part, to its ability to grow and
metabolize optimally at low pH (von Ohle et al. 2010). This
gives it the ability to outcompete noncariogenic commensal
species, thus altering microbial homeostasis in favor of the pro-
liferation of acidogenic and aciduric microbial species and the
establishment of a disease state (Marsh 2003; Falsetta et al.
2012; Lemos et al. 2013). Most control strategies, therefore,
focus on preventing the proliferation of dental biofilm through
frequent removal by mechanical oral hygiene procedures, usu-
ally in combination with chemical detrifrices (Brading and
Marsh 2003; Forssten et al. 2010; Marsh 2010). However, even
with good oral hygiene practices, such as regular brushing,
flossing, water jets, and high-velocity water drops, biofilms can
still accumulate on hard-to-reach places on the tooth surface.
Studies have previously demonstrated that the passage of a
water-air interface over a solid surface can entrain bacteria and
589284JDRXXX10.1177/0022034515589284Journal of Dental ResearchRemoval of Dental Biolms with an Ultrasonically Activated Water Stream
research-article2015
1National Institute for Health Research Southampton Respiratory
Biomedical Research Unit, Southampton Centre for Biomedical
Research, University Hospital Southampton NHS Foundation Trust,
Southampton, UK
2Centre for Biological Sciences, Faculty of Natural and Environmental
Sciences and Institute for Life Sciences, University of Southampton,
Southampton, UK
3National Centre for Advanced Tribology, Faculty of Engineering and
Institute for Life Sciences, University of Southampton, Southampton, UK
4Chemistry, University of Southampton, Southampton, UK
5Southampton Nanofabrication Centre Electronics & Computer Science,
University of Southampton, Southampton, UK
6Faculty of Engineering and the Environment, University of Southampton,
Southampton, UK
7Institute of Sound and Vibration Research, University of Southampton,
Southampton, UK
8Departments of Microbial Infection and Immunity and Orthopaedics,
Center for Microbial Interface Biology, The Ohio State University,
Columbus, OH, USA
A supplemental appendix to this article is published electronically only at
http://jdr.sagepub.com/supplemental.
Corresponding Author:
P. Stoodley, Center for Microbial Interface Biology, The Ohio State
University, 716 Biomedical Research Tower, 460 West 12th Ave,
Columbus, OH 43210, USA.
Email: pstoodley@gmail.com
Removal of Dental Biofilms with an
Ultrasonically Activated Water Stream
R.P. Howlin1,2, S. Fabbri3, D.G. Offin4, N. Symonds3, K.S. Kiang5,
R.J. Knee2, D.C. Yoganantham2, J.S. Webb1,2, P.R. Birkin4,
T.G. Leighton6,7, and P. Stoodley3,8
Abstract
Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral
environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis.
Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of
bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was
evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii
ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface
and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed
videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a
statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and
biofilms exposed to the water stream alone without ultrasonic activation (P < 0.05). The water stream alone showed no statistically
significant difference in removal compared with the untreated control (P = 0.24). High-speed videography demonstrated a rapid rate
(151 mm2 in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from
machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a
potentially effective method for biofilm removal and improved oral hygiene.
Keywords: bacteria, caries, dental hygiene, infection control, microbiology, Streptococcus mutans
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© International & American Associations for Dental Research 2015
1304 Journal of Dental Research 94(9)
provide effective biofilm cleaning (Gomez-Suarez et al. 2001;
Parini and Pitt 2006). This can be achieved with the passage of
a microbubble stream, occasionally combined with ultrasonic
agitation, to generate significant surface tension and shear
forces for mechanical-based cleaning (Parini and Pitt 2005;
Halford et al. 2012). Recently, a novel cleaning system has
been developed that uses the acoustic activation of bubbles
within a free flow of water to generate an ultrasonically acti-
vated stream (UAS) (Leighton et al. 2011). The forces acting
on individual gas bubbles cause them to coalesce and move
over the surface or be trapped within pits and fissures within
the substratum (Leighton 1994; Doinikov 2001; Stricker et al.
2013), where the motion and cavitation dynamics of the bub-
bles create local shear and pressure, contributing to cleaning
efficacy (Rooney 1970). This has been demonstrated in oral
models (Leighton 1994; O’Leary et al. 1997; Lea et al. 2005)
using standard dental ultrasonic equipment but never with
contact-free technologies such as UAS. Particularly with
respect to the pits and recesses of a surface, the entrapment of
dynamic gas bubbles produces highly effective cleaning that
may not be achieved with a normal water stream (Offin et al.
2014). This study aims to evaluate the efficacy of UAS as a
novel approach to dental biofilm removal.
Materials and Methods
Bacteria and Biofilm Growth Conditions
Overnight cultures of S. mutans UA159 (ATCC 700610),
Actinomyces naeslundii ATCC 12104, and Streptococcus ora-
lis ATCC 9811 were grown in brain heart infusion (BHI;
Sigma-Aldrich, St. Louis, MO, USA) broth at 37 °C (for
S. mutans, BHI was supplemented with 2% sucrose [Sigma-
Aldrich] and cultures were grown at 5% CO2). Each culture
was diluted in fresh media to an optical density value corre-
sponding to 106 colony-forming units (CFU)/mL. The adjusted
culture was used as an inoculum to assess UAS cleaning on a
variety of increasingly complex surfaces with different rough-
ness and material properties. Biofilms were grown on all sur-
faces for 72 h at 37 °C (with 5% CO2 for S. mutans biofilm
growth) in a humidified incubator with media changes per-
formed every 24 h.
The UAS Device
We used a benchtop prototype of the StarStream UAS device
(Leighton 2011) (Ultrawave Precision Ultrasonic Cleaning
Equipment, Cardiff, UK). The device generates a stream of
water at 2.1 L/min (±0.2 L/min) from a 10-mm diameter circu-
lar orifice, down which an ultrasonic field is projected. The
device also creates bubble clouds, which impinge on the sam-
ple and spread laterally, and clean from the shear they generate
(Leighton 1994). Biofilms were positioned 1 cm downstream
from the orifice and exposed to a continuous stream of UAS
for 10 s at room temperature.
Removal of Biofilms from Flat Surfaces
Using an UAS
Glass slides were sterilized by autoclaving at 121 °C for
20 min. The slides were immersed vertically in a tube contain-
ing 40 mL of a 106 CFU/mL culture of either S. mutans,
A. naeslundii, or S. oralis, and biofilms were grown as described
above.
Following UAS exposure with the water stream positioned
perpendicular to the surface, the slides were fluorescently
stained with Live/Dead Baclight (Invitrogen, Carlsbad, CA,
USA) in the dark for 20 min. Following a rinse in Hank’s buff-
ered salt solution (HBSS; Sigma-Aldrich) for 5 s, the slides
were imaged using an inverted Leica DMI600 SP5 confocal
scanning laser microscope (CSLM; Leica Microsystems,
Milton Keynes, UK). Image analysis was carried out using the
image analysis package COMSTAT (www.comstat.dk)
(Heydorn et al. 2000). Assays were performed in duplicate (n =
4 image stacks per repeat) and statistical analysis performed
using the Mann-Whitney rank sum tests for nonnormally
distributed data and difference considered significant where
P < 0.05.
In addition, S. mutans biofilms were grown in 9-cm, prester-
ilized Petri dishes as described above. The UAS device was
positioned centrally over the Petri dish and the biofilm exposed
to UAS action or the water stream alone without ultrasonic acti-
vation with the water flow perpendicular to the surface.
Representative photographs were taken for observation of gross
biofilm removal. Each assay was performed in duplicate.
High-Speed Camera Assessment of S. mutans
Removal Using an UAS from an Interproximal
Space Model
To simulate the interproximal (IP) space of the teeth, 2
S. mutans biofilm-colonized slides were placed inside a rect-
angular plastic holder in parallel with a gap of 1 mm. The IP
space holder was then placed under the device, and a high-
speed camera (1,000 f/s; Motion Pro X3, IDT, Tallahassee,
FL, USA) equipped with a Nikon (Tokyo, Japan) 105-mm
f/2.8 VR G lens was used to capture the removal of the biofilm
due to the UAS and the water stream alone without ultrasonic
activation. In this assay, the water flow was run parallel to the
biofilm. Representative videos can be found in the online sup-
plementary material. Each experiment was performed in
duplicate. High-speed videos were postprocessed with ImageJ
software (National Institutes of Health, Bethesda, MD, USA).
S. mutans biofilm clearance zone (CZ) was quantified by mea-
suring the CZ area (ACZ) in each frame every 300 ms. Then,
the averaged ACZ values (n = 2) with the relative SD were
plotted as a function of the time. Statistical analysis was
performed using an unpaired t test to compare normally dis-
tributed data means and difference considered significant
where P < 0.05.
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© International & American Associations for Dental Research 2015
Removal of Dental Biofilms with an Ultrasonically Activated Water Stream 1305
Surface Roughness Following UAS Exposure
Glass slides and hydroxyapatite (HA) coupons were exposed
to the UAS for 10 s and 10 min continuously under the same
conditions described above. Following exposure, the surface
profiles were measured 2-dimensionally using the contact trac-
ing system provided by the Taylor Hobson Talysurf 120L
(Leicester, UK). The evaluation lengths were set at 5 and 40 mm
for the HA coupons and glass slide, respectively, with a mea-
surement speed of 0.5 mm/s. The primary raw data were fil-
tered following the rules and procedures given in BS EN ISO
4288:1998. The characteristic wavelength of the profile filter
λc was set at 0.8 and 0.08 mm for the HA coupons and glass
slides, respectively. Surface roughness (Ra/µm) was deter-
mined in experimental triplicate, and statistical analysis was
performed using an unpaired t test to compare normally dis-
tributed data means and difference considered significant
where P < 0.05.
Removal of Biofilm from Artificial Rough Surface
Using an UAS
Using a Loadpoint Microace 3 dicing saw (Swindon, UK),
micro-grooves were cut into standard microscope glass slides
to a uniform depth of 150 µm to a lattice configuration (period
spacing: 500 µm × 760 µm, 760 µm × 1 mm, and 500 µm ×
1 mm). The glass slides were then reduced in size to 15 mm ×
15 mm using the dicing saw and rinsed in acetone and isopro-
panol to remove any organic residues, followed by dehydration
at 200 °C for 30 min using a conventional oven. Following
autoclaving at 121 °C for 20 min to sterilize, the slides were
immersed in 4 mL of 106 CFU/mL and S. mutans, A. naeslun-
dii, and S. oralis biofilms grown as described previously.
Following exposure to the UAS or water stream alone with
the water flow positioned perpendicular to the surface, the
slides were immersed in a primary fixative of 0.15 M sodium
cacodylate buffer (pH 7.2) containing 3% glutaraldehyde and
0.15% Alcian blue 8GX for 24 h at 4 °C. A 1-h rinse in 0.15 M
cacodylate buffer was performed at room temperature, and the
biofilms were then postfixed in a secondary fixative containing
1% osmium tetraoxide in 0.15 M cacodylate buffer (pH 7.2) for
1 h. Following a further 1-h rinse in cacodylate buffer, the bio-
films were dehydrated through an ascending ethanol series
(50%, 70%, 80%, 95%, and 100% [twice]) prior to critical point
drying and gold-palladium sputter coating and imaged using an
FEI Quanta 200 Scanning Electron Microscope (Hillsboro, OR,
USA).
Removal of S. mutans Biofilms from a Typodont
Model Using an UAS
To re-create a realistic anatomical geometry of patient dental
architecture in vitro, S. mutans biofilms were grown on the
molars of a training typodont (A-PZ periodontal dental model
4030025; Frasaco GmbH, Tettnang, Germany) (Rmaile et al.
2014). The typodont teeth were autoclave sterilized and
immersed in 5 mL of a 106 CFU/mL culture of S. mutans and
biofilms grown as described previously. After this time, the
teeth were removed using sterile tweezers and repositioned
into the typodont and exposed to the UAS and water stream
alone without ultrasonic activation with the water flow posi-
tioned perpendicular to the tooth crown. Following this, the
teeth were removed from the typodont and immersed in 0.5%
crystal violet (Sigma-Aldrich) for 10 min. Poststaining, the
surface was dipped and gently rinsed in deionized water to
remove excess stain prior to photographing to observed gross
biofilm removal. To visualize removal from the teeth at the
micro-scale, subsequent repeats were performed where the
teeth were fixed as described above for scanning electron
microscopy (SEM).
Figure 1. Removal of oral biofilms using an ultrasonically activated
water stream (UAS). (A) Images show the zone of clearing of
Streptococcus mutans UA159 biofilms grown in Petri dishes following 10-s
exposure using the water stream alone without ultrasonic activation
and the UAS, relative to an untreated control. In both cases, the water
stream was positioned in the center of the plate. (B) Representative
confocal scanning laser microscopy (CSLM) images of residual S. mutans
UA159 biofilms following exposure to the UAS and water stream alone
for 10 s, relative to an untreated control following Live/Dead Baclight
fluorescent staining. Scale bars: 25 µm. (C) Graph shows COMSTAT
analysis of residual mean biofilm mass with standard error bars of
S. mutans UA159, Actinomyces naeslundii ATCC 12104, and Streptococcus
oralis ATCC 9811 biofilms following a 10-s exposure to the UAS and
the water stream alone as identified by Live/Dead Baclight fluorescent
staining and CSLM (n = 8 with assay performed in duplicate). * and **
indicate corresponding data showing a statistically significant difference
(P < 0.01).
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© International & American Associations for Dental Research 2015
1306 Journal of Dental Research 94(9)
Results
Gross S. mutans biofilm removal from Petri dishes was demon-
strated as a larger (50.8-cm2) zone of clearing from the center
of the plate covering almost the entire plate diameter following
10-s exposure to the UAS, relative to the water stream alone
without ultrasonic activation (3.5 cm2; Fig. 1A). The water
stream alone showed no removal of biofilm from the center of
the plate at the initial water stream impact site and was
indistinguishable from untreated controls. Biofilm removal
with the water stream alone was noted only at the edge of the
plate, possibly due to water streaming around the plate edge.
A more detailed inspection by confocal microscopy showed
that the UAS was significantly more effective at removing bio-
films grown on simple flat surfaces (Fig. 1B) than the water
stream alone. COMSTAT analysis of S. mutans biofilm removal
showed that water stream treatment alone caused a 0.10 log
reduction (20.7%) in biomass from 21.8 µm3/µm2 to 17.3 µm3/
µm2 and a 0.17 log reduction (33.8%) in average thickness
from 25.3 µm to 16.7 µm, although these reductions were not
statistically different (P = 0.24). The UAS caused a further
2.3 log reduction in biomass to 0.08 µm3/µm2 (99.5% reduction
compared with the untreated control) and a 2.9 log reduction in
thickness to 0.02 µm (99.9% reduction), which was statisti-
cally significant (P = 0.002). Similarly, the water stream alone
was unable to elicit a statistically significant reduction of
A. naeslundii biofilms (P = 0.645) compared with the control,
while biofilm removal with the UAS was significantly greater
than the water stream alone (P < 0.001). However, the water
stream alone, without UAS activation, resulted in a significant
reduction in mean S. oralis biofilm mass relative to controls
(P = 0.001), equivalent to a 99.95% reduction, suggesting
weak surface attachment of S. oralis in this assay.
Further analysis using a high-speed camera of S. mutans
biofilm removal from glass slides in a model mimicking the
interproximal space showed a more rapid rate of biofilm
removal during 0 to 3 s of UAS exposure relative to the water
stream alone (Fig. 2; P < 0.5, n = 2). Within the first second of
exposure, the biofilm clearance zone area (ACZ) was 151 mm2,
relative to 80 mm2 with the water stream alone. The ACZ after a
period of 3 s was 139.5 mm2 (±32.03 mm2, n = 2) and 430.4
(±50.34 mm2, n = 2) for the water stream alone and the UAS,
respectively. Representative high-speed camera videos can be
found in the online supplementary material.
Figure 2. High-speed camera (1,000 f/s) imaging of Streptococcus
mutans UA159 biofilm removal, using a ultrasonically activated water
stream (UAS) and water stream alone, from glass slides placed in
an interproximal space model. Images show representative frames
from the high-speed camera at 0- and 3-s intervals. Scale bars: 5 mm.
Graph shows the mean area of biofilm clearance against time following
high-speed camera imaging of S. mutans biofilm removal using the UAS
and water stream alone. Data points represent the mean of duplicate
experimental repeats with standard error bars. * and ** represent
data ranges of 0 to 1 s and 0 to 3 s, showing a statistically significant
difference (P < 0.5).
Figure 3. Surface profile (Ra/µm) following exposure of clean glass
and hydroxyapatite surfaces to an ultrasonically activated water
stream (UAS) for 10 s and 10 min. Data represent the mean of assays
performed in experimental triplicate with standard deviation bars.
Data points represent the mean of duplicate experimental repeats with
standard error bars. * represents data showing a statistically significant
difference (P < 0.5).
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Removal of Dental Biofilms with an Ultrasonically Activated Water Stream 1307
Analysis of the effect of a UAS on
the underlying substratum was deter-
mined by 10-s and 10-min exposure
to glass slides (used in Figs. 1 and 2)
and HA coupons (Fig. 3). Exposure of
glass slides to the UAS had no signifi-
cant effect on Ra relative to the con-
trol (10 s: P = 0.246; 10 min: P =
0.468). There was also no statistically
significant difference in Ra relative to
controls of HA coupons exposed to
the UAS for 10 s (P = 0.544).
However, a 10-min exposure did elicit
a significant increase in Ra (P = 0.011)
from 0.72 to 1.15, equivalent to a
62.5% increase in surface Ra.
To further evaluate the effective-
ness of UAS biofilm removal from a
more complex surface, rough surfaces
were created with various micro-
groove configurations and S. mutans,
A. naeslundii, and S. oralis biofilms
grown to demonstrate broad-spectrum
bacterial species removal. SEM imag-
ing following exposure to the water
stream alone without ultrasonic acti-
vation showed no difference in residual biofilm relative to
untreated controls (Fig. 4). However, a dramatic reduction in
residual biofilm of all 3 bacterial strains was observed follow-
ing treatment with the UAS relative to the water stream and
untreated controls, with the rough surface showing no reduc-
tion in the efficacy of UAS mediated removal compared with
previous assays on flat surfaces. Importantly, for S. oralis, this
is in contrast to Figure 1, where the water stream alone was
highly effective at biofilm removal, confirming UAS efficacy
of hard-to-clean surfaces where the water stream alone was
inefficient.
Similarly, the UAS was also effective at removing biofilm
from teeth in a typodont training model representing a realistic
patient dental architecture (Fig. 5). Crystal violet (CV) staining
to assess gross biofilm removal again showed no noticeable
difference between the water stream alone and control treat-
ment groups, with a marked reduction in CV staining noted on
teeth exposed to the UAS. SEM analysis imaging of the teeth
to assess micro-scale removal of S. mutans biofilm revealed
only occasional single cells visible in the areas exposed to the
UAS. In contrast, the water stream alone showed comparable
residual biofilm to the untreated control.
Discussion
As a key cariogenic species and a major risk factor for early
childhood caries and future caries development, as well as its
propensity to form biofilms, both in vitro and in vivo in the oral
cavity, S. mutans was chosen as the model organism for the
study (García-Godoy and Hicks 2008), in addition to
A. naeslundii and S. oralis, to demonstrate broad-spectrum
biofilm cleaning. Relative to a water stream flow of 2.1 L/min
(±0.2 L/min), ultrasonic activation of the same stream at the
same flow rate demonstrated a greater efficiency and rate of
biofilm removal from a variety of increasingly complex sur-
faces, including, importantly, machine-etched slides to provide
a consistent “rough” surface and molar teeth from a typodont
model. Importantly, typodont model teeth effectively repro-
duce the normal dental architecture, including the complexity
of the crown fissures where mechanical biofilm removal is
more challenging and, combined with the IP space, are the
most at-risk sites for caries development (Rugg-Gunn 2013).
UAS in a free water stream has several key and beneficial
features that make it effective at biofilm removal (Leighton
et al. 2011). First, effective cleaning can be achieved through
pure water alone under ambient conditions and does not require
chemical additives or the generation of high temperature. This
is of added benefit as the lack of antimicrobial additives
reduces the risk of antibiotic resistance developing and the risk
to patient health due to the high doses of antimicrobials some-
times required to clear oral biofilm infections (Larsen and
Fiehn 1996; Shani et al. 2000). Instead, the effectiveness of the
UAS is achieved due to the utilization of the ultrasonically
induced bubble activity and shear (Leighton et al. 2011). While
it is known that, for some substrates and some bacterial spe-
cies, the simple proximity of the passage of a nearby gas bub-
ble (e.g., rising under buoyancy) can cause detachment
(Gomez-Suarez et al. 2001), in this study, it is the ultrasoni-
cally induced volume and shape oscillations in the bubbles, as
well as the associated shear, that produce the significant
Figure 4. Scanning electron microscopy imaging of residual Streptococcus mutans UA159,
Actinomyces naeslundii ATCC 12104, and Streptococcus oralis ATCC 9811 biofilms, grown on
machine-etched glass slides to artificially and reproducibly mimic a rough surface, following
exposure to the ultrasonically activated water stream (UAS) and water stream alone for 10 s,
relative to untreated controls. Scale bars: 500 µm.
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© International & American Associations for Dental Research 2015
1308 Journal of Dental Research 94(9)
removal effect (Leighton et al. 2011). Importantly, since the
activated bubbles are in a free water stream, no direct contact
between the device and the oral surface is required, facilitating
access to hard-to-reach places. Second, the acoustic field
causes the bubbles to move to crevices and such surface struc-
tures, preferentially cleaning features that are normally more
difficult to clean (Leighton 1994; Offin et al. 2014).
Consequently, due to the complex oral cavity topography, this
approach has the potential to greatly contribute to improved
oral hygiene. Third, the area of biofilm removal in this study
was relevant in the context of dental hygiene and was achieved
over the relatively short time period of a few seconds. The
removal efficacy of laboratory-grown biofilms by UAS was
similar to that of microburst technology in which high-velocity
micro water drops generate high enough fluid shear to remove
significant amounts of biofilm from an interproximal space
model (Rmaile et al. 2014). However, the microdrops have the
advantage of using minimal volumes of water. In addition,
while not required for efficacy in this study, additives to the
water reservoir, such as fluoride with proven anticaries proper-
ties, may further enhance not just the immediate cleaning effi-
cacy but also long-term oral hygiene (Aspiras et al. 2010).
However, issues will need to be addressed regarding appli-
cation of UAS to oral health care. Future work should address
the influence of different surface materials (e.g., dental enamel
and dentin) on UAS efficacy. In addition, the influence of the
pellicle and salivary coating of a surface on UAS-mediated
biofilm clearance needs to be assessed. Existing studies sug-
gest that salivary mucins such as MUC5B decrease surface
attachment and biofilm formation of S. mutans, and so UAS
removal could be enhanced with a more representative oral
environment (Frenkel and Ribbeck 2015). Careful consider-
ation and future work will also be needed to assess the poten-
tial for tissue damage to the surrounding gingiva, but it is
expected that these can be overcome by optimizing exposure
time and power output to settings capable of maintaining the
efficacy of the device and alleviating the risk of damage to the
surrounding tissue. This is corroborated by data from this study
where effective biofilm removal without a detrimental effect to
the substratum was observed at short exposure times (10 s).
Longer exposure times of 10 min did cause an increase in sur-
face roughness on a hydroxyapatite surface; however, this
should be put into context of other studies where exposure of
2 min to toothbrushing using certain dentrifices produced a
much greater surface abrasion than observed with a 10-min
UAS exposure (Pascaretti-Grizon et al. 2013). In addition,
while the flow rate of 2.1 L/min used in this study provides
good surface area coverage, there is the issue of requiring rela-
tively large volumes of water, and thus miniaturization would
be desirable. The current flow rate is higher than commercially
available continuous or pulsed water irrigation shear-based
removal devices that generally operate on the order of a few
Figure 5. Representative images showing removal of Streptococcus mutans UA159 biofilms from molar teeth in a typodont training model, following
a 10-s exposure to the ultrasonically activated water stream (UAS) and water stream alone, relative to untreated controls. Left-hand column panels
show total residual biomass (blue/purple) as identified by crystal violet staining. Remaining panels show increasingly higher magnification scanning
electron microscopy images of the crown surface. White arrows indicate residual S. mutans biofilm on low-magnification images. This figure is available
in color online at http://jdr.sagepub.com.
at University of Southampton on September 8, 2015 For personal use only. No other uses without permission.jdr.sagepub.comDownloaded from
© International & American Associations for Dental Research 2015
Removal of Dental Biofilms with an Ultrasonically Activated Water Stream 1309
hundreds of mL/min (Rmaile et al. 2014). However, the use of
a UAS represents a potentially practical and effective method
for oral biofilm removal with the capacity to improve oral
hygiene.
Author Contributions
R.P. Howlin, contributed to conception, design, data acquisition,
analysis, and interpretation, drafted and critically revised the man-
uscript; S. Fabbri, contributed to data acquisition, analysis, and
interpretation, critically revised the manuscript; D.G. Offin,
N. Symonds, K.S. Kiang, R.J. Knee, D.C. Yoganantham, contrib-
uted to data acquisition, critically revised the manuscript;
J.S. Webb, contributed to conception, critically revised the manu-
script; P.R. Birkin, T.G. Leighton, contributed to conception,
design, and data interpretation, critically revised the manuscript;
P. Stoodley, conception, design, data analysis, and interpretation,
critically revised the manuscript. All authors gave final approval
and agree to be accountable for all aspects of the work.
Acknowledgments
This work was funded by the Royal Society Brian Mercer Award
scheme. The authors thank Prof. J. Barton from The University of
Southampton. Leighton and Birkin are named inventors of the
ultrasonically activated water stream technology on the patent and
on a standard licence deal between the University of Southampton
and Ultrawave Ltd. The other authors declare no potential con-
flicts of interest with respect to the authorship and/or publication
of this article.
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  • ... Cavitation has been investigated for ultrasonic cleaning in a range of industries, for example, to remove marine biofouling or food contamination (Chahine et al. 2016;Fink et al. 2017;Oulahal et al. 2007;Salta et al. 2016). Ultrasonic cavitation can also be used in the health care sector to remove biofilms, for example, oral biofilms on teeth and dental implants, biofilms on wounds and biofilms on medical instruments Chahine et al. 2016;Chen et al. 2007;Erriu et al. 2014;Felver et al. 2009;Howlin et al. 2015;Pishchalnikov et al. 2003;Rivas et al. 2012;Walmsley et al. 2010Walmsley et al. , 2013Wang and Cheng 2013). Koo et al. (2017) highlight the advantages of physical biofilm removal: It reduces the probability of antimicrobial resistance because the physical disruption means that less antimicrobial is required, and physical biofilm removal can be easily combined with various antimicrobial agents or nanoparticles. ...
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  • ... The use of antimicrobial solutions plays a key role in biofilm eradication among the antiplaque strategies by detaching and dissolving the biofilms and by facilitating the killing of biofilm microorganisms Howlin et al., 2015;Lecic et al., 2016). Because much of the biofilm consists of extracellular polymeric substance (EPS) (Xiao et al., 2012), an effective EPS dissolving process may facilitate biofilm eradication. ...
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