European Journal of Oral Sciences

Volume 115 Issue 1 Page 77-80, February 2007

To cite this article: Philipp Müller, Bernhard Guggenheim, Patrick R. Schmidlin (2007)
Efficacy of gasiform ozone and photodynamic therapy on a multispecies oral biofilm in
vitro
European Journal of Oral Sciences 115 (1) , 77–80 doi:10.1111/j.1600-
0722.2007.00418.x
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Efficacy of gasiform ozone and photodynamic therapy on a

multispecies oral biofilm in vitro
 Philipp Müller1,
 Bernhard Guggenheim2,
 Patrick R. Schmidlin1
 1
Clinic of Preventive Dentistry, Periodontology and Cariology; 2Institute for Oral
Biology, Section for Oral Microbiology and General Immunology, Center for
Dental and Oral Medicine and Maxillofacial Surgery, University of Zurich, Zurich,
Switzerland

Patrick R. Schmidlin, Center for Dental and Oral Medicine, Plattenstrasse 11, 8032
Zurich, Switzerland.
Telefax: +41–44–6344308
E-mail: patrick.schmidlin@zzmk.unizh.ch
Müller P, Guggenheim B, Schmidlin PR. Efficacy of gasiform ozone and photodynamic therapy on a
multispecies oral biofilm in vitro . Eur J Oral Sci 2007; 115: 77–80. © 2007 The Authors. Journal
compilation © 2007 Eur J Oral Sci

Abstract

Ozone gas and photodynamic therapy (PDT) have been claimed to be antimicrobially
effective. This study assessed their antimicrobial potential in vitro. Mature six-species
oral biofilms were treated as follows (n = 9 per group): (i) a 60-s application of
gasiform vacuum-ozone or vacuum alone (on wet or air-dried biofilm samples); (ii) PDT
(i.e. methylene blue in combination with or without a diode soft laser, and a soft laser
alone); or (iii) antimicrobial solutions: immersion of biofilms for 60 s in 0.2 and 2%
chlorhexidine or in 0.5 and 5% hypochlorite solution. Treatment with chlorhexidine or
hypochlorite served as a positive control, whereas untreated samples served as
negative controls. Colony-forming units on blood agar were counted. Only the 5%
hypochlorite solution was able to totally eliminate the microorganisms in the biofilm.
The observed reduction of viable counts by vacuum-ozone application and PDT was less
than one log10 step. Under the conditions of the current study, gasiform ozone and PDT
had a minimal effect on the viability of microorganisms organized in a cariogenic
biofilm.

Dental caries is a major oral health problem that affects 60–90% of schoolchildren and
the vast majority of adults (1). Etiologically, it represents complex interactions among
the oral microbiota, diet, dentition, and the oral environment (2). Bacteria are crucial
for the initiation and progression of carious lesions. They function as a microbial
community called dental plaque, which reflects a typical example of a microbial biofilm
(3).

The prevention of caries (primary prevention), and the control of disease progression
(secondary prevention), focus mainly on mechanical and/or chemical biofilm reduction
(4–6). Beyond chemical substances available in simple and inexpensive solutions with
known antimicrobial potential (e.g. chlorhexidine or hypochlorite) (7–10), modern
sophisticated and expensive tools, such as the use of ozone and photodynamic therapy
(PDT), have entered the field of dentistry.

High-dose ozone gas and PDT have been claimed to offer rapid and effective disinfection
of colonized dental tissues (11–14). These in vitro studies, however, used monospecies
biofilms grown on different substrates. In contrast, there is evidence that high-dose
ozone gas and PDT have less effect on microorganisms that are gathered in a more
complex, multispecies, biofilm, and it is not clear whether these new antimicrobial
treatment strategies are also effective on multispecies biofilms. Therefore, it was the
aim of the present investigation to compare, on a mature six-species biofilm (which
represents a well-established and validated cariogenic in vitro model) (15), the
antimicrobial efficacy of one single application of gasiform ozone and PDT with
commonly used antiseptics.

Material and methods

Disc preparation

Freshly extracted bovine permanent central lower incisors, free of defects and/or cracks
when trans-illuminated, were selected for this study. Labial tooth surfaces were
brushed, applying a pressure of 250 g, with a toothbrush (ParoM39; ESRO, Thalwil,
Switzerland) and a toothpaste-slurry for 30 min [6 g of Depurdent (Wild, Basel,
Switzerland); 10 g of artificial saliva; and eight drops of a silicone antifoam (Art. Nr.
85390; Fluka, Buchs, Switzerland) to remove debris and cementum. Discs with a
diameter of 7 mm were cut from the mid-labial aspect of each tooth using a trephine.
These discs were thinned, with preservation of the natural enamel surface, to 2 mm in
height and then autoclaved for 20 min.

Biofilm preparation

Actinomyces naeslundii OMZ 745, Veillonella dispar ATCC 17748T (OMZ 493),

Fusobacterium nucleatum KP-F2 (OMZ 596), Streptococcus sobrinus OMZ 176, S. oralis
SK248 (OMZ 607), and Candida albicans OMZ 110 were used as inocula for biofilm
formation (15,16). Biofilms were grown in 24-well polystyrene cell culture plates on 108
bovine enamel discs (17). In brief, discs were preconditioned (pellicle-coated) in
processed whole unstimulated saliva and then covered with 1.6 ml of substrate
composed of 70% saliva + 30% of filter-sterilized fluid universal medium supplemented
with 67 mmol/L Sørensen's buffer, pH 7,2 (‘modified fluid universal medium’, mFUM)
(18,19). The cell culture plate wells were inoculated with mixed cell suspensions
(200 µl), prepared from equal volumes and densities of each species, and then the
culture plates were incubated anaerobically at 37°C. At 16.5, 20.5, 24.5, 40.5, 44.5,
48.5, and 64.5 h, the biofilms were washed by three consecutive dips in 2 ml of sterile
physiological saline (1 min per dip, room temperature). The medium was changed, after
dipping, at 16.5 and 40.5 h.

Antimicrobial treatment of biofilm-coated discs

The different treatments (as shown in Table 1) were performed on a sterile clean-bench.
The treatments were repeated three times, using triplicate samples, within one
experiment. To assess the inhibitory action of the vacuum ozone delivery system
(Healozone; KaVo Dental, Biberach/Riss, Germany), the discs were removed from the
wells, placed in sterile Petri dishes, and immediately exposed to the gas for 60 s by
placing a sterile silicone cup (diameter 8 mm), attached to the hand piece, over the wet
biofilm-coated specimens. The same treatment was performed on biofilms that had
been air-dried for 10 s. Vacuum application alone, without ozone generation, was
facilitated by a switch mechanism on the ozone device. This treatment was performed
on wet and dried biofilms.

The disinfecting potential of the PDT was assessed by fully covering the discs with
methylene blue for 60 s. Following the manufacturer's protocol, the discs were rinsed by
dipping for 60 s in 6 × 2 ml of sterile physiological saline solution (immersion time per
dip: 10 s). The discs were then radiated for 60 s by a soft laser (Helbo TheraLite Laser;
Helbo Photodynamic Systems, Grieskirchen, Austria) with a wavelength of 665 nm and
a power output of 75 mW. Afterwards, the discs were rinsed by being double-dipped
sequentially in 3 × 2-ml samples of fresh physiological saline (immersion time per dip:
10 s). The following controls were used as references: immersion of biofilms for 60 s in
0.2 and 2% chlorhexidine digluconate and in 0.5 and 5% hypochlorite solution. Biofilm-
coated discs treated with sodium hypochlorite solution were placed in sodium thiosulfate
for 30 s to stop the action of the sodium hypochlorite. A mixture of L-α-lecithin (Fluka)
in 3% Tween 80 was used to inactivate chlorhexidine. Untreated discs served as
positive controls. All discs were then rinsed by being double-dipped sequentially in
3 × 2-ml samples of fresh physiological saline (immersion time per dip: 10 s).

Harvesting the biofilms

To harvest adherent cells, each disc was transferred to a sterile 50-ml polypropylene
tube containing physiological saline (1 ml, room temperature) and vortexed vigorously
for 2 min. The suspensions were then transferred to sterile 6-ml polystyrene tubes and
sonicated for 5 s at 30 W.

Examination of harvested cells

Serial dilutions (10−2 to 10−5) of sonicated cells were prepared in physiological saline,
and aliquots (50 µl) were spirally plated (Spiral System, Model D; Spiral Systems,
Cincinnati, OH, USA) onto plates of Columbia blood agar base (Oxoid, Ltd., Basingstoke,
UK) supplemented with 5% (v/v) hemolyced human blood (CBA). After 72 h, the
number of colony-forming units (CFUs) was counted with the aid of a stereomicroscope.

Data presentation and statistical analysis

Mean values and corresponding 95% confidence intervals (95% CI) were calculated. To
determine the differences between test and control values, the one sample t-test was
used, given the hypothesis that the true difference is equal to zero (i.e. no treatment
influence occurred). One-way analysis of variance (ANOVA), together with the posthoc
Scheffé test, was applied to establish the differences between the experimental
treatments. Significance was set at 95%.

Results

To determine the antimicrobial activity of the ozone and PDT applications, we compared
the number of CFU obtained from the negative (sterile physiological saline) and the
positive (5% hypochlorite solution) controls. The results are summarized in Table 2.

Hypochlorite, at a concentration of 5%, effectively eliminated all bacteria. Hypochlorite

at a concentration of 0.5%, and chlorhexidine at a concentration of 2%, caused a
reduction in the viable counts of > 1 log10 (P = 0.05). These solutions exhibited a
significantly (P = 0.05) increased antimicrobial potential compared with 0.2%
chlorhexidine, gasiforme ozone, and PDT, which reduced the microbiota of the biofilm
by less than one order of magnitude. These test treatments did not statistically differ
among each other (P = 0.05).

Discussion

The present in vitro study failed to demonstrate any effective reduction of microbiota in
a multispecies biofilm by gasiform ozone or PDT after one single application of 60 s.

Bacteria in biofilms display increased resistance to antimicrobial agents (20, 21). For
instance, to eliminate S. sanguinis in biofilms, it was necessary to administer a
concentration of chlorhexidine 10–50 times higher than that required to eliminate
S. sanguinis in planktonic form (22). The age of the biofilm is also a significant factor:
older biofilms (72 h) of S. sanguinis have been reported to be more resistant to
chlorhexidine than younger (24 h) biofilms (23). The established 64.5-h biofilm used in
the current study can be viewed as a relatively stable system that is resistant to
antiseptic challenges. Such simplified models, reflecting the most salient features of
more complex ecosystems, can help to clarify the effectiveness of disinfectant treatment
strategies under standardized laboratory conditions.

Under the current conditions, only the 5% sodium hypochlorite solution was able to
eliminate the biofilm completely, whereas 0.5% hypochlorite and 2% chlorhexidine
caused significant, but limited, reductions in the viable counts. The single application of
chlorhexidine at a concentration of 0.2% for 60 s failed to demonstrate a significant
effect. Thus, the antimicrobial efficacy of the solutions under investigation was dose
dependent. This confirms the results of previous studies that also demonstrated a
concentration dependency of the antimicrobial effect, especially of sodium hypochlorite
(24). Confocal microscopy of in situ established plaque showed that chlorhexidine only
affected the outer layers of cells in 24- and 48-h natural biofilms (25), suggesting either
quenching of the agent at the biofilm surface or a lack of active moiety penetration
(26).

Ozone is a strong oxidizer of cell walls and cytoplasmatic membranes of bacteria and is
considered to be one of the best bactericidal, antiviral, and antifungal agents (27). After
overnight incubation of single-species biofilms of S. mutans and S. sobrinus, the
application of 13.3 ml s−1 of ozone for 10 s killed > 99% of bacteria (13). In contrast, in
the present study, exposing established 64.5-h multispecies biofilms to gasiform ozone
for 60 s did not result in any major reductions in biofilm viability. Our results suggest
that well-established biofilms are resistant to ozone application, which is in accordance
with other findings showing that ozone had an antibacterial effect on planktonic
Enterococcus faecalis cells and those suspended in fluid, but little effect when embedded
in biofilms (28). The same was the case with PDT. Previous work has shown that PDT is
capable of killing oral bacteria in planktonic cultures (29), in plaque scrapings (30), and
on mono-species biofilms (31) in vitro.

The results of this study show that the matrix-embedded microbial populations in a
biofilm are well protected towards antimicrobial agents. Even sophisticated tools, such
as an ozone gas delivery device or PDT, were not able to reduce significantly, or
completely eliminate, bacteria in the biofilm. Therefore, mechanical or additional
chemical disruption of biofilms still appears to be imperative before gasiform ozone or
PDT can effectively be applied (32). Furthermore, one must be aware that a single
application of these in-office techniques, which are used by dentists, cannot be
compared with the antimicrobial effect of mouthrinses, such as 0.2% chlorhexidine,
which can be repeatedly applied in home care treatments.

Based on the current results, it may be concluded that dentists should not uncritically
rely on strongly promoted high-tech bactericidal tools. Gasiform ozone and PDT are
currently not a valid option to be eligible as a supportive measure in cases where
mechanical removal of bacteria is not possible (e.g. around orthodontic brackets,
fissures or in residual dentin caries). It must be borne in mind that an established
biofilm population, even when reduced by 90%, will regrow within hours. The efficiency
of these methods must be considerably improved before these technologies can be
recommended to clinicians.

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