Accepted Manuscript

Title: Comparison of antibacterial effect of Photodynamic

therapy using Indocyanine Green (Emundo) with 2%
Metronidazole and 2% Chlorhexidine Gel on Porphyromonas
Gingivalis (an in-Vitro Study)

Author: Reza Fekrazad Kasra Karamifar Abbas Bahador

PII: S1572-1000(16)30031-X
DOI: http://dx.doi.org/doi:10.1016/j.pdpdt.2016.04.003
Reference: PDPDT 760

To appear in: Photodiagnosis and Photodynamic Therapy

Received date: 31-12-2015

Revised date: 5-4-2016
Accepted date: 10-4-2016

Please cite this article as: Fekrazad Reza, Karamifar Kasra, Bahador Abbas.Comparison
of antibacterial effect of Photodynamic therapy using Indocyanine Green
(Emundo) with 2% Metronidazole and 2% Chlorhexidine Gel on Porphyromonas
Gingivalis (an in-Vitro Study).Photodiagnosis and Photodynamic Therapy
http://dx.doi.org/10.1016/j.pdpdt.2016.04.003

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Comparison of antibacterial effect of Photodynamic therapy using Indocyanine Green
(Emundo) with 2% Metronidazole and 2% Chlorhexidine Gel on Porphyromonas
Gingivalis (an in-Vitro Study)

Kasra Karamifar, DDS, MS1 , Reza Fekrazad2, Abbas Bahador3

1
Department of Enodontics, Faculty of Dentistry, Shiraz Branch, Islamic Azad
University, Shiraz, Iran
2
Periodontology Department, Dental Faculty - Laser Research Center in Medical
Sciences, AJA University of Medical Sciences, Tehran, Iran
3
Department of Microbiology, Laser Research Center, Dental Research Center,
Dentistry Research Institute, School of Medicine, Tehran University of Medical
Sciences,
Highlights

 Antimicrobial photodynamic therapy with Emundo reduced the amount of P. gingivalis

significantly.
 The use of metronidazole 2% in Orabase and 2% CHX gel performed better in terms of reducing
the amount of the bacteria than Laser + Emundo
 Antimicrobial photodynamic therapy with Emundo may provides an effective way to eliminate
P. gingivalis without adverse systemic effects
Abstract

Background: The treatment of periodontal disease focuses on eradication or

suppression of the pathogenic microbiota within the periodontal pocket. There are some
mechanical and chemical ways, and recently antimicrobial photodynamic therapy to
eliminate the bacteria.

The aim of this in vitro study was to compare the effects of 2% chlorhexidine gel, 2%
metronidazole in Orabase, antimicrobial photodynamic therapy with Emundo solution
and Emundo solution on P.gingivalis.

Methods: The antibacterial activities of 2% CHX gel, 2% Metronidazole in Orabase,

Emundo + Laser (an infra-red laser diode of 810 nm, 300 mW, continuous mode
radiation, 30 s and energy density of 11.5 J/cm2), and Emundo against P.gingivalis
were tested in vitro using two different methods; (1) Counting CFU/mL and (2) agar
diffusion test (ADT). Data of CFU/mL were analyzed using one way ANOVA and Post
hoc Tukey test (p<0.05). Data obtained from ADT test were analyzed using Kruskal-
Wallis.

Results: The percentage of colony-forming units reduction was (99.75, 99.98) in

Metronidazole, (99.66, 99.85) in CHX, and (96.68, 96.68) in Laser + Emundo (96.68,
96.68) groups after 24 and 48 hours, respectively. The amount of bacterial reduction in
terms of log10 CFU/mL was decreased in the Metronidazole, CHX, Laser + Emundo
and, Emundo groups, respectively after 48 hours (p<0.05). The inhibition zone radius
was decreased in CHX, Metronidazole and, Laser + Emundo, respectively (p<0.05).

Conclusions: It can be concluded that within the limitations of this in-vitro study,
although photodynamic therapy with Emundo reduced the amount of P. gingivalis
significantly, the use of metronidazole 2% in Orabase performed better in terms of
reducing the amount of the bacteria. Also, the antibacterial effect of 2% CHX gel on P.
gingivalis was significantly more than photodynamic therapy with Emundo.

Key words:

Antimicrobial photodynamic therapy; Chlorhexidine; Emundo; Metronidazole; Orabase;

Porphyromonas gingivalis
Corresponding Author:

Dr Kasra Karamifar, Department of Endodontics, Dental School, Shiraz Azad University,

Shiraz, Iran.

Fax: +98- 713- 8308200

Email: kasra.karamifar@gmail.com
Introduction

The treatment of periodontal disease focuses on elimination of the pathogenic

microbiota within the periodontal pocket. Mainly Porphyromonas gingivalis
(P.gingivalis), Tannerella forsythensis, and the spirochete Treponema denticola are the
most common cause of periodontal diseases. [1] P.gingivalis is regarded as periodontal
pathogen and associated with periodontal disease. [2] This bacteria are found in patients
with aggressive as well as chronic periodontitis [3], and acute phase of periodontitis. [4]

There are mechanical and chemical as well as systemic and local ways such as tooth
brushing, scaling, drug therapy, and photodynamic therapy to eliminate the bacteria. [5, 6]
Mechanical debridement usually leads to clinical improvement in periodontal health;
however, some sites do not respond as well as anticipated. The persistence of
pathogenic species such as P.gingivalis, Actinobacillus actinomycetemcomitans,
Prevotella intermedia, Bacteroids forsythus, and Peptostreptococcus micros is thought
to prolong chronicity of inflammation and progression of attachment loss. [7, 8] In these
cases, systemic or topical antibiotic treatment can be used to help improving clinical
results over mechanical debridement alone. [5, 9] Some systemic antibiotics have been
used to treat periodontal disease. [10] Locally delivered antibiotics may be effective for
treating acute periodontitis. [4] Although antibiotics were used alone in treating
periodontitis in some studies, [11, 12] the use of systemic antibiotics for the treatment of
these cases has a risk of adverse drug reaction and increased selection of multiple
antibiotic-resistant organisms. Chlorhexidine mouthwash is effective in reducing the
bleeding on probing index and can be used along with periodontal treatments for
treating gingivitis and periodontitis. [13] Systemic use of Metronidazole is one of the
choices for treating periodontal diseases. This bactericidal drug damages bacterial and
protozoal DNA, leading to cell death and is effective against P.gingivalis.[14, 15]

Photodynamic therapy (PDT) as the light-induced inactivation of cells, microorganisms,

or molecules, [16] involves the combination of light, usually through the use of a diode
laser, a photosensitizer and singlet oxygen. The photosensitizer is capable of absorbing
light of a specific wavelength and transforming it into useful energy. [17] They can
produce lethal cytotoxic agents when absorbing light and selectively destroy cells. [17]
Antibacterial PDT has been used for reducing the bacterial load or eradicating certain
periodontal pathogens. [18, 19] The cytotoxic product, usually singlet oxygen, can act
locally which makes it ideal for the local application of PDT without endangering distant
molecules, cells, or organs. [20] It has been shown in an animal study that PDT was
advantageous in reducing the periodontal signs of redness and bleeding on probing,
and P.gingivalis was significantly suppressed. [21] Antimicrobial photodynamic therapy
can be used as a potential alternative to antibiotics for reduction of microbiota in the oral
cavity and avoid caries progression. [22] PDT has several advantages as it is a non-
invasive, repeatable method with high target specificity (localization). Also, development
of bacterial resistance after multiple treatments is unlikely. [23, 24]

Indocyanine green affects the target tissue/cell through its photodynamics (PDT) and
mainly photothermal effect (PTT).[25] PTT can damage cells by increasing intracellular
temperature when ICG is used as photosensitizer. [26] Most of the light energy absorbed
by this photosensitizer is converted to heat. [27] ICG green has low toxicity to non-target
host tissue and the mode of action seems to be nonspecific to bacterial species and
performs its bactericidal effect through oxidative stress.[28] Considering this method,
using indocyanine green-base photosensitizers (such as Emundo periogreen) have
been shown beneficial for fungal and bacterial elimination. [29, 30] Although the efficacy of
PDT for microbial disinfection on oral pathogens has been investigated previously, [31-35]
further studies seem necessary for achieving the best antimicrobial protocol.

The null hypothesis was that the antibacterial effect of antimicrobial photodynamic
therapy with Emundo solution was better than 2% chlorhexidine gel and 2%
metronidazole in Orabase.

The aim of this in vitro study was to compare the effects of 2% chlorhexidine gel, 2%
metronidazole in Orabase, antimicrobial photodynamic therapy with Emundo solution
and Emundo solution on P.gingivalis.

Method and Materials

Microorganism and growth conditions

Lyophilized P.gingivalis (ATCC33277, obtained from Rayen Biotechnology Co. Ltd.,

Tehran, Iran) was rehydrated in brain heart infusion (BHI) broth (Merck, Darmstadt,
Germany) supplemented with hemin (5 μg/ml) and vitamin K (1 μg/ml); (Both items were
purchased from Sigma-Aldrich, Steinheim, Germany) and incubated in anaerobic
atmosphere at 37◦C for 48 h in anaerobic glove box that contains an atmosphere of H2,
CO2, and N2. For experiments requiring cultures on plates, cultures grown in
supplemented BHI (SBHI) broth were transferred onto brain heart infusion (BHI) agar
plates.
Fresh colonies of P.gingivalis from SBHI Agar plates were suspended in SBHI broth,
and bacterial density was visually adjusted to a turbidity of 0.5 McFarland standard
reagents. The exact density (CFU/mL) of each suspension was verified on SBHI agar
plates.

Study design
The experiments were conducted in five groups: 1) Chlorhexidine Digluconate 2%
(Chlorhexidina S; FGM, Joinville, SC, Brazil) (CHX) (n=7), 2) Laser + EmunDo®
(LEm)(n=7), 3) EmunDo® (Em) (n=7), 4) Metronidazole 2% (Unichem Laboratories Ltd.,
Maharashtra, India) in Orabase® (ConvaTec Limited, Middlesex, UK) (Met)(n=7) and 5)
Control (n=3)

Photosensitizer and Light source

For evaluating the photo elimination by PDT, EmunDo® (indocyanine green) solution
(A.R.C laser GmbH, Nurnberg, Germany) in combination with an infra-red laser diode of
810 nm (A.R.C laser GmbH, Nurnberg, Germany) with EmunDo® transgingival
handpiece with recommended parameters of manufactures: 300 mW, continuous mode
radiation, 30 s and energy density of 11.5 J/cm2 was used. Light device was placed in a
fixed vertical position, tangent and 1 cm away from the surface of the suspension which
was the same in all the study groups.

Antibacterial procedure for counting CFU/mL

In each group, 200 µL of Pg suspension was added to eppendorf test tubes. In the
CHX group, 200 µL of chlorhexidine digluconate 2% was added to suspension and
vortexed for 30 seconds. In the LEm and Em groups, 200 µL of Emundo was added to
each eppendorf test tubes, vortexed for 30 seconds, and kept in dark environment for 5
minutes before irradiation letting time to photosensitizers absorption by the bacterial
cells. In the LEm group, laser irradiation was performed for 30 seconds and then kept in
the dark environment. Irradiation was performed in a laminar flow hood (Besat, Tehran,
Iran) in the dark, under aseptic condition. After the treatment, the samples were
overnight incubated and the samples were then serially diluted in PBS. In order to
evaluate the bacterial viability, 50 µL of each dilution was cultured on Mueller Hinton
agar and incubated for 24 h at 37◦C in a partial atmosphere of 5% CO2. After
incubation, the number of colony forming units per milliliter (CFU/mL) was determined
by a blind examiner. In the Met group, metronidazole was mixed with Orabase under
aseptic condition in order to obtain 2% weight ratio. The membrane-enclosed immersion
test (MEIT) method [36] was used. Briefly, each gram of the mixture was placed in a
permeable membrane and sewn with suture. The membranes were floated in 1 mL of
bacterial suspension in a tube and vortexed for 30 seconds. The amount of CFU/mL
was 4575000.00 in initial inoculum. The CFU/mL was calculated after 24 and 48 hours.
The amounts of CFU/mL are shown in Table 1 and 2 after 24 and 48 hours.

Agar diffusion test

Another method was also carried out to assess antimicrobial effects of 2% CHX gel, 2%
Metronidazole in Orabase, Emundo + Laser, and Emundo against P.gingivalis, the Agar
diffusion test (ADT) method [36]. Briefly, fresh bacterial colonies were suspended in SBHI
broth and their turbidity was adjusted to 0.5Mc‑Farland standard, corresponding to
approximately 1.5 × 108 CFU/ml. To achieve a lawn of bacterial growth, 100 µl of the
bacterial suspension was evenly distributed onto the surface of a BHK plate using a
sterile glass spreader. After inoculation, wells were made by removal of agar with a
puncher at equidistant points. Agar plates were prepared and 5 wells were punched out
for placing the Metronidazole mixture and CHX. The punched out holes were sealed for
inhibiting the leakage of the tested materials especially CHX. The holes were filled with
tested samples.

Emundo was added to the broth and kept in a dark place. The bacteria were inoculated
on the plates. After 5 minutes, laser irradiation was performed for 30 seconds on 5
different sites. The radius of the laser beam was adjusted to the radius of the punched
holes in other groups.

Control samples were remained without any further changes during the period of
examination.

Data analysis

Kolmogorov-Smirnov test was performed to evaluate the normality distribution. The

results of the CFU/mL were log10-transformed and analyzed by one way analysis of
variance (ANOVA) and Post hoc Tukey test in SPSS statistical software version 20.
Statistical significance was defined as p<0.05. Data obtained from ADT test were
analyzed using Kruskal-Wallis.

Results

Colony forming units (CFU/mL)

The percentage of colony-forming units reduction was (99.75, 99.98) in Metronidazole,

(99.66, 99.85) in CHX, and (96.68, 96.68) in Laser + Emundo (96.68, 96.68) groups
after 24 and 48 hours, respectively. The means and standard deviations of the number
of log10CFU/mL obtained for the studied groups after treatments for 24 and 48 hours.
Metronidazole reduced the CFU/mL significantly more than the other groups. Also, CHX
reduced CFU/mL significantly more than the LEm group. Emundo did not significantly
reduce the CFU/mL compared to the control group and no significant reduction of
colonies was seen. Furthermore, the reduction in the CFU/mL was significantly more in
48 hours than 24 hours in the CHX and metronidazole group (p<0.05). Obviously, there
was no change in the LEm group as there was no further irradiation to the samples (Fig.
1, 2 & Table 1, 2).

Agar diffusion test

The radius of the inhibition zone was measured in millimeter (mm) which was
significantly greater in CHX group than metronidazole group (p<0.05). In the LEm
group, there was no bacterial growth in the sites of irradiation. Emundo alone could not
produce any inhibition zone. (Fig. 3 & Table 3)

Discussion

A low patient compliance rate, [37] a requirement for frequent dosing to maintain
therapeutic drug concentrations in the serum and crevicular fluid, side effects , and drug
interactions [38] are some drawbacks of systemically administered antibiotics.
Supplemental use of local antibiotics, local antiseptic drugs, and systemic antibiotics
may have additional benefits to mechanical debridement. [39, 40] This method can provide
the greatest benefit to patients who do not respond to debridement alone. [41]

Metronidazole is a bactericidal nitroimidazole. Reduction of their nitro group results in

the production of short-lived cytotoxic intermediates, which damages bacterial and
protozoal DNA, leading to cell death. [14, 15] Metronidazole was used in this study as it is
the drug of choice in the treatment of periodontal diseases. [42] Biofilm-associated P.
gingivalis may be resistant to metronidazole at concentrations which are usually
attained by systemic administration. [43] Orabase is composed of gelatin, pectin and
sodium carboxymethylcellulose in plastibase (plasticized hydrocarbon gel). It has
adhesive properties and remains in intimate contact with mucous membranes of the
mouth and gums. [44] Some locally applied antibiotics such as Atridox, are effective
against anaerobic pathogens and can be placed gently into periodontal pockets where
bacteria thrive and cause infection. [45] Orabase was used as a delivery vehicle for
metronidazole. Same method might be used in the treatment of periodontal diseases as
higher concentration of metronidazole can be in close contact with the bacteria
(P.gingivalis) with minimum possibility of systemic adverse effects; however, some
limitations may be encountered when using in the oral cavity of patients with acute
periodontitis.

As metronidazole with Orabase has a creamy consistency the membrane-enclosed

immersion test (MEIT) method [36] was used. The volume of the bacterial suspension
was increased to 1 mL in this group with the same CFU/mL so that immersion of the
samples can be done.
EmunDo® is a photosensitizer which includes mainly indocyanine green dye.
Indocyanine green (ICG) is a water-soluble tricarbocyanine dye that strongly absorbs in
near infra-red region. ICG has FDA approval for medical diagnostic studies, [46, 47] a
high absorption cross-section in the infra-red region at 810 nm leading to more
penetration depth in compare to many other sensitizers. [48, 49] The effects of Emundo on
some bacteria were studied. [29, 50] The bactericidal effect of PDT on P.gingivalis using
ICG-loaded nanospheres photosensitizer with an 810 nm diode laser was examined
and significant reduction was seen. [51] In our study, significant reduction of the
P.gingivalis after irradiation was observed after 24 and 48 hrs.

There are a number of antiseptic agents that has been used for optimizing the treatment
outcome of the periodontal diseases such as CHX. CHX has some drawbacks including
bitter taste, teeth staining, non-selective effect, and allergic reactions which are more
significant when using 2% than lower concentrations. [52, 53] CHX has been shown to be
effective at reducing the viability of P.gingivalis in biofilms. [54] Either 0.12% or 0.2%
chlorhexidine digluconate is usually used as mouthwash. Although chlorhexidine
digluconate 2% was used in this study, 2% metronidazole was significantly more
effective. This was in contrast to Noiri et al. [54] results which may be due to the fact that
P.gingivalis was in planktonic state in our study. In the CHX group, inhibition zone was
significantly greater which may be due to better diffusion in the agar than metronidazole
in Orabase. As Emundo could not produce any inhibition zone, it seems that this
photosensitizer when used alone may have minimal cytotoxic effects when comes in
contact with such bacterial cells.

Antimicrobial PDT with the aid of Emundo was less effective than CHX and
metronidazole which may be due to less exposure time than the other groups. However,
this method has no effect on the change of taste, and tooth staining comparing to CHX.
Furthermore, there may be no risk of systemic adverse effects comparing to
metronidazole as Emundo was applied locally and also effective locally which was
confirmed by agar diffusion test. The effects of aPDT are immediate and local but may
be more expensive comparing to the other methods. Furthermore, aPDT may act
selectively and locally in the sites of infection while in the other groups there is a risk of
systemic absorption of the drugs. Metronidazole and CHX effects where continuous
during the study time while laser effects happened during only 30 seconds. As ICG
effects are mainly through PTT and increasing intracellular temperature, [25, 26]
increasing the time intervals for laser may provide better outcomes. The adsorption of
metronidazole via Orabase through periodontal pocket may be hampered in this method
in clinical cases with pain and acute periodontitis as the application of the drug may be
painful. These three methods were not evaluated simultaneously in previous studies.
Further studies should focus on changing the PDT factors such as time and mode of
irradiation, biofilm environment, and side effects in order to achieve better outcomes.
The null hypothesis was rejected as the antibacterial effect of 2% chlorhexidine gel and
2% metronidazole in Orabase were better than that of antimicrobial photodynamic
therapy with Emundo solution.

Conclusion

It can be concluded that although photodynamic therapy with Emundo reduced the
amount of P. gingivalis significantly, the use of 2% metronidazole in Orabase performed
better in terms of reducing the amount of the bacteria. Also, the antibacterial effect of
2% CHX gel on P. gingivalis was significantly more than photodynamic therapy with
Emundo considering the conditions of this study.

Acknowledgement

This article was based on the thesis submitted by the second author to the Faculty of
Dentistry at AJA University. We would like to thank AJA University for providing the
funds and facilities for carrying out this research.

Conflict of interest statement

We declare that we have no conflict of interest.

 References

[1] Socransky SS, Haffajee AD. Evidence of bacterial etiology: a historical perspective. Periodontology
2000. 1994;5:7-25.
[2] Taubman M, Haffajee A, Socransky S, Smith D, Ebersole J. Longitudinal monitoring of humoral
antibody in subjects with destructive periodontal diseases. Journal of periodontal research.
1992;27:511-21.
[3] López NJ. Occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and
Prevotella intermedia in progressive adult periodontitis. Journal of periodontology. 2000;71:948-54.
[4] Umeda M, Tominaga Y, He T, Yano K, Watanabe H, Ishikawa I. Microbial Flora in the Acute Phase of
Periodontitis and the Effect of Local Administration of Minocycline*. Journal of periodontology.
1996;67:422-7.
[5] Ellen RP, McCulloch CA. Evidence versus empiricism: rational use of systemic antimicrobial agents for
treatment of periodontitis. Periodontology 2000. 1996;10:29-44.
[6] de Oliveira RR, Schwartz-Filho HO, Novaes Jr AB, Taba Jr M. Antimicrobial photodynamic therapy in
the non-surgical treatment of aggressive periodontitis: a preliminary randomized controlled clinical
study. Journal of periodontology. 2007;78:965-73.
[7] Bragd L, Dahlén G, Wikström M, Slots J. The capability of Actinobacillus actinomycetemcomitans,
Bacteroides gingivalis and Bacteroides intermedius to indicate progressive periodontitis; a retrospective
study. Journal of clinical periodontology. 1987;14:95-9.
[8] Slots J, Emrich LJ, Genco RJ, Rosling BG. Relationship between some subgingival bacteria and
periodontal pocket depth and gain or loss of periodontal attachment after treatment of adult
periodontitis. Journal of clinical periodontology. 1985;12:540-52.
[9] Loesche W, Syed S, Morrison E, Kerry G, Higgins T, Stoll J. Metronidazole in Periodontitis: I. Clinical
and Bacteriological Results after 15 to 30 Weeks*. Journal of periodontology. 1984;55:325-35.
[10] Walker CB, Karpinia K, Baehni P. Chemotherapeutics: antibiotics and other antimicrobials.
Periodontology 2000. 2004;36:146-65.
[11] Lindhe J, Liljenberg B, Adielson B, Börjesson I. Use of metronidazole as a probe in the study of
human periodontal disease. Journal of clinical periodontology. 1983;10:100-12.
[12] López NJ, Gamonal JA, Martinez B. Repeated metronidazole and amoxicillin treatment of
periodontitis. A follow-up study. Journal of periodontology. 2000;71:79-89.
[13] Herrera D. Chlorhexidine mouthwash reduces plaque and gingivitis. Evidence-based dentistry.
2013;14:17-8.
[14] Müller M. Mode of action of metronidazole on anaerobic bacteria and protozoa. Surgery.
1983;93:165.
[15] SLOTS J, Ting M. Systemic antibiotics in the treatment of periodontal disease. Periodontology 2000.
2002;28:106-76.
[16] Von Tappeiner H, Joldlbauer A. On the effect of photodynamic (fluorescent) substances on protozoa
and enzymes. Arch Klin Med. 1904;80:427-87.
[17] Sharman WM, Allen CM, van Lier JE. Photodynamic therapeutics: basic principles and clinical
applications. Drug discovery today. 1999;4:507-17.
[18] Wilson M, Dobson J, Harvey W. Sensitization of oral bacteria to killing by low-power laser radiation.
Current microbiology. 1992;25:77-81.
[19] Pfitzner A, Sigusch BW, Albrecht V, Glockmann E. Killing of periodontopathogenic bacteria by
photodynamic therapy. Journal of periodontology. 2004;75:1343-9.
[20] Moan J, BERG K. The photodegradation of porphyrins in cells can be used to estimate the lifetime of
singlet oxygen. Photochemistry and photobiology. 1991;53:549-53.
[21] Sigusch BW, Pfitzner A, Albrecht V, Glockmann E. Efficacy of photodynamic therapy on
inflammatory signs and two selected periodontopathogenic species in a beagle dog model. Journal of
periodontology. 2005;76:1100-5.
[22] Baptista A, Kato IT, Prates RA, Suzuki LC, Raele MP, Freitas AZ, et al. Antimicrobial Photodynamic
Therapy as a Strategy to Arrest Enamel Demineralization: A Short‐Term Study on Incipient Caries in a Rat
Model†. Photochemistry and photobiology. 2012;88:584-9.
[23] Maisch T. Anti-microbial photodynamic therapy: useful in the future? Lasers in medical science.
2007;22:83-91.
[24] Kömerik N, Wilson M. Factors influencing the susceptibility of Gram‐negative bacteria to toluidine
blue O‐mediated lethal photosensitization. Journal of applied Microbiology. 2002;92:618-23.
[25] Engel E, Schraml d, Maisch T, Kobuch K, König , Szeimies -M, et al. Light-induced decomposition
of indocyanine green. Investigative ophthalmology & visual science. 2008;49:1777-83.
[26] Reichel E, Puliafito CA, Duker JS, Guyer DR. Indocyanine green dye-enhanced diode laser
photocoagulation of poorly defined subfoveal choroidal neovascularization. Ophthalmic Surgery, Lasers
and Imaging Retina. 1994;25:195-201.
[27] Holzer W, Mauerer M, Penzkofer A, Szeimies R-M, Abels C, Landthaler M, et al. Photostability and
thermal stability of indocyanine green. Journal of Photochemistry and Photobiology B: Biology.
1998;47:155-64.
[28] Parker S. The use of diffuse laser photonic energy and indocyanine green photosensitiser as an
adjunct to periodontal therapy. British dental journal. 2013;215:167-71.
[29] Fekrazad R, Khoei F, Hakimiha N, Bahador A. Photoelimination of< i> Streptococcus mutans</i> with
two methods of photodynamic and photothermal therapy. Photodiagnosis and photodynamic therapy.
2013;10:626-31.
[30] Fekrazad R, Barghi VG, Mir APB, Shams-Ghahfarokhi M. In vitro photodynamic inactivation of
Candida albicans by phenothiazine dye (new methylene blue) and Indocyanine green (EmunDo®).
Photodiagnosis and photodynamic therapy. 2015;12:52-7.
[31] Lima JP, Sampaio de Melo MA, orges F, Teixeira AH, Steiner‐Oliveira C, Nobre dos Santos M, et al.
Evaluation of the antimicrobial effect of photodynamic antimicrobial therapy in an in situ model of
dentine caries. European journal of oral sciences. 2009;117:568-74.
[32] Meisel P, Kocher T. Photodynamic therapy for periodontal diseases: state of the art. Journal of
Photochemistry and Photobiology B: Biology. 2005;79:159-70.
[33] Pereira CA, Romeiro RL, Costa ACBP, Machado AKS, Junqueira JC, Jorge AOC. Susceptibility of
Candida albicans, Staphylococcus aureus, and Streptococcus mutans biofilms to photodynamic
inactivation: an in vitro study. Lasers in medical science. 2011;26:341-8.
[34] Rolim JP, De-Melo MA, Guedes SF, Albuquerque-Filho FB, De Souza JR, Nogueira NA, et al. The
antimicrobial activity of photodynamic therapy against< i> Streptococcus mutans</i> using different
photosensitizers. Journal of Photochemistry and Photobiology B: Biology. 2012;106:40-6.
[35] Mahdi Z, Habiboallh G, Mahbobeh NN, Mina ZJ, Majid Z, Nooshin A. Lethal effect of blue light-
activated hydrogen peroxide, curcumin and erythrosine as potential oral photosensitizers on the
viability of Porphyromonas gingivalis and Fusobacterium nucleatum. Laser therapy. 2015;24:103.
[36] Bahador A, Pourakbari B, Bolhari B, Hashemi FB. In vitro evaluation of the antimicrobial activity of
nanosilver-mineral trioxide aggregate against frequent anaerobic oral pathogens by a membrane-
enclosed immersion test. Biomedical journal. 2015;38:77.
[37] Loesche W, Grossman N, Giordano J. Metronidazole in periodontitis (IV). The effect of patient
compliance on treatment parameters. Journal of clinical periodontology. 1993;20:96-104.
[38] Slots J, Rams TE. Antibiotics in periodontal therapy: advantages and disadvantages*. Journal of
clinical periodontology. 1990;17:479-93.
[39] Hanes PJ, Purvis JP. Local anti-infective therapy: pharmacological agents. A systematic review.
Annals of periodontology. 2003;8:79-98.
[40] Haffajee AD, Socransky SS, Gunsolley JC. Systemic anti-infective periodontal therapy. A systematic
review. Annals of periodontology. 2003;8:115-81.
[41] Slots J. Systemic antibiotics in periodontics. Journal of periodontology. 2004;75:1553-65.
[42] Pangsomboon K, Kaewnopparat S, Pitakpornpreecha T, Srichana T. Antibacterial activity of a
bacteriocin from< i> Lactobacillus paracasei</i> HL32 against< i> Porphyromonas gingivalis</i>. Archives
of Oral Biology. 2006;51:784-93.
[43] Wright T, Ellen R, Lacroix JM, Sinnadurai S, Mittelman M. Effects of metronidazole on
Porphyromonas gingivalis biofilms. Journal of periodontal research. 1997;32:473-7.
[44] ORABASE PROTECTIVE PASTE
1991.
[45] Stoller NH, Johnson LR, Trapnell S, Harrold CQ, Garrett S. The pharmacokinetic profile of a
biodegradable controlled-release delivery system containing doxycycline compared to systemically
delivered doxycycline in gingival crevicular fluid, saliva, and serum. Journal of periodontology.
1998;69:1085-91.
[46] Kuo W-S, Chang Y-T, Cho K-C, Chiu K-C, Lien C-H, Yeh C-S, et al. Gold nanomaterials conjugated with
indocyanine green for dual-modality photodynamic and photothermal therapy. Biomaterials.
2012;33:3270-8.
[47] Nagahara A, Mitani A, Fukuda M, Yamamoto H, Tahara K, Morita I, et al. Antimicrobial
photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine
green‐loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis. Journal of
periodontal research. 2013;48:591-9.
[48] Sawa M, Awazu K, Takahashi T, Sakaguchi H, Horiike H, OhJi M, et al. Application of femtosecond
ultrashort pulse laser to photodynamic therapy mediated by indocyanine green. British journal of
ophthalmology. 2004;88:826-31.
[49] Bäumler W, Abels C, Karrer S, Weiss T, Messmann H, Landthaler M, et al. Photo-oxidative killing of
human colonic cancer cells using indocyanine green and infrared light. British journal of cancer.
1999;80:360.
[50] Meister J, Hopp M, Schäfers J, Verbeek J, Kraus D, Frentzen M. Indocyanine green (ICG) as a new
adjuvant for the antimicrobial photo-dynamic therapy (aPDT) in dentistry. SPIE BiOS: International
Society for Optics and Photonics; 2014. p. 89290T-T-10.
[51] Nagahara A, Mitani A, Fukuda M, Yamamoto H, Tahara K, Morita I, et al. Antimicrobial
photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine
green‐loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis. Journal of
periodontal research. 2013.
[52] Nayak S, Ankola A, Metgud S, Bolmal U. Effectiveness of mouthrinse formulated from ethanol
extract of Terminalia chebula fruit on salivary Streptococcus mutans among 12 to 15 year old school
children of Belgaum city: A randomized field trial. Journal of Indian Society of Pedodontics and
Preventive Dentistry. 2012;30:231.
[53] Malhotra R, Grover V, Kapoor A, Saxena D. Comparison of the effectiveness of a commercially
available herbal mouthrinse with chlorhexidine gluconate at the clinical and patient level. Journal of
Indian Society of Periodontology. 2011;15:349.
[54] Noiri Y, Okami Y, Narimatsu M, Takahashi Y, Kawahara T, Ebisu S. Effects of chlorhexidine,
minocycline, and metronidazole on Porphyromonas gingivalis strain 381 in biofilms. Journal of
periodontology. 2003;74:1647-51.
Figure 1. Log CFU/mL of tested groups after 24 hours. It shows significant difference
between the groups and significant reduction of bacteria in Met, CHX, and LEM
comparing to control group.
Figure 2. Log CFU/mL of tested groups after 48 hours. It shows the significant reduction
of bacteria in Met, CHX, LEm groups.

Figure 3. Inhibition zone of tested groups measured in millimeters (from left to right;
CHX, Met, LEm and Em).

Groups N Minimum Maximum Mean Std. Deviation Mean

Reduction
Percentage
Control 3 4575000.00 6452000.00 5453333.3333 944268.14694 0
Met 7 8600.00 21200.00 13550.0000 4329.78059 99.75
CHX 7 13700.00 23700.00 18000.0000 3255.76412 99.66
LEm 7 28100.00 43200.00 34942.8571 5340.67946 99.36
Em 7 2178000.00 4524000.00 3007285.7143 812759.02831 44.85

Table 1. CFU/mL after 24 h (values less than p<0.05 was considered significant). All P-
values are less than 0.001 except for comparison of Met and CHX (P = 0.314).

Groups N Minimum Maximum Mean Std. Deviation Mean

Reduction
Percentage
Control 3 4575000.00 6452000.00 5453333.3333 944268.14694 0
Met 7 200.00 1700.00 678.3333 559.62190 99.98
CHX 7 5600.00 10200.00 8028.5714 1496.34475 99.85
LEm 7 28000.00 42900.00 34997.4210 5340.67945 96.36
Em 7 2177800.00 4525000.00 3007394.2357 812759.02831 44.85

Table 2. CFU/mL after 48 h, Control, Em, and LEm had little changes between two time
intervals (values less than p<0.05 was considered significant). All P-values are less
than 0.001 except for comparison of control and EM (P = 0.046).

Groups N Minimum Maximum Mean Std. Deviation

Met 7 18.00 19.00 18.3333 0.51640
CHX 7 20.00 24.00 21.8571 1.77281
LEm 7 3.00 4.00 3.5000 0.57735
Em 7 0 0 0 0

Table 3. Inhibition zone of tested groups in millimeter.