Exploring different photodynamic therapy parameters to optimize elimination of Enterococcus faecalis in planktonic form

Accepted Manuscript Title: Exploring different photodynamic therapy parameters to optimize elimination of Enterococcus faecalis planktonic forms Authors: Janir Alves Soares, Suelleng Maria Cunha Santos Soares, Rudys Rodolfo de Jesus Tavarez, Claudia de Castro Rizzi, Silvana Cristina Gama Vaz Rodrigues, Etevaldo Matos Maia Filho, Manoel Brito-J´unior, Rodrigo Dantas Pereira, Paula Prazeres Magalh˜aes, Luiz de Macˆedo Farias PII: DOI: Reference:

S1572-1000(17)30483-0 https://doi.org/10.1016/j.pdpdt.2018.03.009 PDPDT 1139

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

22-10-2017 13-3-2018 28-3-2018

Please cite this article as: Soares JA, Soares SMCS, de Jesus Tavarez RR, Rizzi CdC, Rodrigues SCGV, Filho EMM, Brito-J´unior M, Pereira RD, Magalh˜aes PP, Farias LdM, Exploring different photodynamic therapy parameters to optimize elimination of Enterococcus faecalis planktonic forms, Photodiagnosis and Photodynamic Therapy (2010), https://doi.org/10.1016/j.pdpdt.2018.03.009 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.

Exploring different photodynamic therapy parameters to optimize elimination of Enterococcus faecalis planktonic forms

Janir Alves Soaresa, [email protected] Suelleng Maria Cunha Santos Soaresa, [email protected]

Claudia de Castro Rizzib, [email protected]

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Silvana Cristina Gama Vaz Rodriguesb, [email protected] *Etevaldo Matos Maia Filhob, [email protected] Manoel Brito-Júniorc, [email protected]

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Rodrigo Dantas Pereirad, [email protected]

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Paula Prazeres Magalhãesd, [email protected] Luiz de Macêdo Fariasd, [email protected]

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Rudys Rodolfo de Jesus Tavarezb, [email protected]

Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri,

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Diamanteina, Minas Gerais, Brazil.

Postgraduate program in Dentistry, University CEUMA, São Luís, Maranhão, Brazil

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Rua Josué Montelo, Nº 1, Renascença II, São Luís, Maranhão, Brazil. CEP: 65075-120. Departamento f dentistry, State University of Mantes Claros. Montes Claros, Minas Gerais,

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Brazil. d

Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas

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Gerais, Belo Horizonte, Brazil.

* Corresponding author at: Postgraduate program in Dentistry, University CEUMA, São Luís, Maranhão, Brazil

Rua Josué Montelo, Nº 1, Renascença II, São Luís, Maranhão, Brazil. CEP: 65075-120. Phone/fax: XX 98 3214-4127 Cell phone: XX 98 981803085; E-mail: [email protected]

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Highlights: . In vitro the susceptibility of planktonic forms of E. faecalis was

significantly optimized by energy dose, volume of suspension and

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cycles of PDT.

. Volume of bacterial suspension similar to root canal can be

. PDT has a promising perspective as an adjunct in the intracanal

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reduced in logarithmically scale with the application of PDT cycles

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antisepsis

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Abstract

Background: The failure of endodontic treatment is linked to the presence of

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Enterococcus faecalis in the root canals. The scope of study was to evaluate the influence of the energy dose and frequency of photodynamic therapy (PDT

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cycles), as well as the volume of bacterial suspensions (BS) in the elimination of Enterococcus faecalis planktonic forms. Methods: In four successive assays

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BS of Enterococcus faecalis ATCC 19433 were irradiated with a diode laser (40 mW) using the photosensitizer (PS) methylene blue (MB) (0.005 µg/mL). Group 1 - Effect of energy dose: 100µL of BS and 100µL of PS were irradiated by 1, 2.5, 5, 7.5 and 10 minutes. Group 2 - Effect of PDT cycles: The BS received 1, 2, 3 or 4 PDT cycles (in each cycle 100mL of PS was added and irradiated by 2.5 minutes). Group 3 - Effect of energy dose and bacterial suspension volume:

10µL of BS and 10µL of PS were irradiated similar to group 1. Group 4 - Effect of energy dose, bacterial suspension volume and PDT cycles: 10µL of BS and 10 µL of PS were irradiated according to group 2. The laser source and MB isolated represented the controls. Results: The mean log reduction after separate applying laser light and MB were 0.01 and 0.07, respectively. It was

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found that wells with 100µL of BS irradiated with 2.4 to 24 J of energy did not

cause significant bacterial elimination (p>0.05), on the other hand PDT cycles

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above 12 J increased significantly bacterial elimination (p<0.05). In 10µL wells irradiation from 12 J of energy provided higher bacterial elimination (p<0.05) which combined with PDT cycle resulted in the logarithmic elimination of E.

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faecalis (p<0.05). Conclusions: The energy dose, the volume of the bacterial

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of Enterococcus faecalis planktonic forms.

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suspension and, especially, the PDT cycles optimized the bacterial elimination

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Keywords: Enterococcus faecalis, Photodynamic therapy, Root canal therapy.

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Introduction

Effective elimination of microorganisms in infected root canals represents a

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constant challenge in clinical practice [1-3]. In chronic endodontic infections the microorganisms are organized in an extracellular polysaccharide matrix forming bacterial biofilms [1,2]. Unlike planktonic forms, in this structuring the elimination of the infectious process requires high doses of drugs or the microorganisms become refractory to conventional treatment protocols [2,3]. Despite

technological advances in chemomechanical preparation including new instruments and irrigation devices persistent microorganisms in both biofilm and planktonic form are often recovered in root canals after this initial disinfection [4, 5]. Because these remaining microorganisms can reestablish the original

intracanal disinfection approaches have been recommended [7, 8].

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infection [1], and prevent periradicular tissue healing [6], complementary

Microbiologically, endodontic retreatments are often associated with the

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presence of E. faecalis in biofilms, however the its susceptibility in planktonic form helps to understand possible strategies involved in its elimination.

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The photodymanic therapy (PDT) has the potential to be used as an

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adjunct disinfection strategy during endodontic therapy [9-14]. The antimicrobial

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PDT employs a dye (photosensitizer) in resonance with the wavelength of low

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intensity visible light [15]. The photosensitizers are activated to a short-lived excited state that then converts to a long-lived triplet state, which can generate

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free radicals or superoxide ions resulting from hydrogen or electron transfer

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(Type I reaction) and can produce singlet oxygen (1O2) (Type II reaction), which rapidly kill microorganisms [16]. It has been shown that PDT eliminates

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endodontic pathogens [17] but optimal parameters for their complete photoinactivation remain undefined [11, 12, 18-20]. The elimination of microorganisms by PDT can be influenced by light

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parameters as energy dose [19, 21-23] and power density [17-19, 24]. In addition, the amount of microbial cells in planktonic suspension [12, 23], biofilm cell aggregates [11, 24] and microorganism’s species [12, 18, 24, 25] can affect the PDT effectiveness. In current state of the art the irradiation protocols used in PDT trials are very diverse and their results are sometimes conflicting [22].

Thus, an effort should be made to sequentially assess same quantitative parameters for improved understanding of antibacterial PDT performance. Therefore, the aim of this study was to evaluate different PDT parameters to optimize elimination of Enterococcus faecalis in vitro. We hypothesized that increasing energy dose combined with periodical light and

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enhance the elimination of Enterococcus faecalis planktonic forms.

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photosensitiver application in reduced bacterial suspension volume could

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Materials and methods

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In the initial experiment the growth curve of E. faecalis ATCC 19433 was

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performed over a 24-hour period through the inoculation of brain heart infusion

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broth (BHI, Dfico, MI, Detroit) and the amount of microorganisms in colonyforming units (CFU)/mL was measured (Figure 1). Based on a series of

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previous pilot studies the main parameters of energy dose, bacterial suspension

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volume and PDT cycles were stablished. Therefore in all experiments, bacterial suspensions obtained from 4.5 hour

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inoculums from Enterococcus faecalis (ATCC 19433) cultured in brain heart infusion broth were used in a concentration of 108 (CFU/mL). A multi-functional laser diode unit of aluminum gallium-arsenic (Ga-Al-As) (Twin Flex II -

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MMOptics, São Paulo, Brazil), with central wavelength (λ) 660 nm and power (P) of 40 mW was used as a light source. The amount of energy deposited was obtained by the formula: energy (J) = power (mW) x time (s). The red light was distributed by using a flexible tapered fiber tip with diameter of 400 µm (Chimiolux, Belo Horizonte, Minas Gerais, Brazil). Helical movements were

performed with the fiber inserted in the bacterial suspension during irradiation. The photosensitizer methylene blue (MB) was used in an aqueous solution at a concentration of 500 μg/mL (Chimiolux ). Samples containing bacterial suspensions (100-µL, or 10-µL) received a similar volume of the photosensitizer. Ten samples (n=10) were used for each assay. Only 4 wells

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per plate were used in an equidistant way to avoid interference with the laser

source (Figure 2). All procedures were performed in a laminar flow hood, while

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maintaining the aseptic chain. E. faecalis monoinfection was confirmed by Gram staining and catalase test.

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Group 1 – Effect of Energy Dose

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following experimental groups were defined:

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As a function of energy dose, bacterial suspension volume and PDT cycles, the

96-well microtiterplates (NuncTM, Roskilde, Denmark) containing 100-µL of

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bacterial suspension received similar volume of the photosensitizer. After preirradiation for 1 minute, 50 wells containing microbiological samples were

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equally distributed among the experiments: control, E1, E2, E3 and E4. Upon

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irradiation for 1, 2.5, 5, 7.5 and 10 minutes was deposited 2.4, 6, 12, 18 and 24 J energy doses, respectively. In microbiological processing 100-µL aliquots were 10-fold diluted in saline and spread onto brain heart infusion agar (BHI-A;

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Difco, Detroit, MI) media in duplicate. The plates were incubated for 48 hours at 37°C, the culture were examined and the colony forming units (CFU)/mL) were estimated.

Group 2 - Effect of PDT Cycles

The initial samples and procedures were similar to group 1. However, PDT cycles were applied after the pre-irradiation time. Each PDT cycle consisted of: i) addition of 100-µL of the photosensitizer and ii) irradiation for 2.5 minutes. Thus, 1, 2, 3 and 4 PDT cycles were applied. In the control group, the samples

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were irradiated for 1 minute and processed microbiologically. Group 3 - Effect of Energy Dose and Bacterial Suspension Volume

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Eppendorf tubes (200-μl) containing 10-μl of bacterial suspension received 10μL of the photosensitizer using graduated and calibrated volumetric

micropipette. After pre-irradiation time (1 minute), the samples were irradiated.

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The distribution of the groups, irradiation exposure and energy dose were

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similar to group 1. Microbial 10-μL aliquots were diluted into Eppendorf tubes

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containing 90-µL of saline and processed microbiologically.

Group 4 - Effect of Energy Dose, Bacterial Suspension Volume and PDT

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Cycles

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The initial samples and procedures were similar to group 3. However, after preirradiation time, PDT cycles were applied as described in group 2 and

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processed microbiologically.

Statistical analysis Preliminary data from bacterial reduction were subjected to the test of normality (Shapiro-Wilk test). To verify the influence of the energy dose, PDT frequency,

bacterial suspension volume in eliminating E. faecalis were used the one way ANOVA with post hoc Tukey test. The statistical program used was the SPSS 23.0 (IBM, Armonk, NY, USA) with a level of significance of 5%. Results The number of CFUs (CFU/ml-1) before and after control and experimental

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procedures were transformed into logarithm (Log10). Tables 1 shows the Log

reduction of all control and experimental groups. The mean log reduction after

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separate applying light and methylene blue (controls) were 0.01 log and 0.07

log, respectively. Wells with 100-µL of bacterial suspension irradiated with 2.4 to 24 J of energy did resulted in progressive bacterial log reduction ranging from

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0.21 at 1.51 (p>0.05). On the other hand PDT cycles with energy above from 12

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J provided significant log reduction (ranging from 0.87 at 1.76, p<0.05). Wells

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with 10-µL irradiation resulted in gradual bacterial reduction to energy above

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from 6 J (ranging 0.39 to 2.13, p <0.05) while this wells irradiated with PDT

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cycle resulted in the logarithmic elimination of E. faecalis (ranging from 0.39 to 3.04, p<0.05). Overall, the efficacy of PDT varied in scale statistically significant

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according to the energy dose increasing, reducing of the volume of the bacterial suspension and mainly when the outpout power was deposited in the form of

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cycles (Figure 3).

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Table 1. Bacterial Log reduction of wells containing 100-μL and 10-μL bacterial suspension containing 108 CFU/mL, irradiated by Twin Flex II device, with power of 40 mW.

In this figure it can be observed that the progressive increase of energy provided proportional increase of the bacterial reduction as a function of the PDT cycles and the reduction of the volume of the bacterial suspension.

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Discussion

In our study the elimination of E. faecalis planktonic forms was

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significantly affected by the energy dose and the frequency of PDT, as well as by the bacterial suspension volume. Moreover, the strategic combination of

these variables provided logarithmic elimination of microorganisms, thereby the

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hypothesis tested was accepted. In the control groups, there was a virtual

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reduction in the viability of E. faecalis after exposure to energy dose of the 2.4

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J, of order 0.01 Log. Exposure to MB resulted in reduction of 0.07 Log. Soukos

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et al. (2006) [24] exposing to the methylene blue only verified reduced

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bactericidal effect (5.5%), while employing PDT with laser source of 1 W outpout power they reached 53% of lethal effect. In our study, there was a

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progressive reduction of E. faecalis as a function of continued energy dose deposition. Such irradiation reduced planktonic E. faecalis compared to laser

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light alone and MB alone. The log reduction of E. faecalis increased linearly with the energy dose up to 4.5 J [23]. Williams et al. (2009) [19] reported similar

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findings against Streptococcus mutans with dose up to 2.4 J. In a recent study, the gradual increasing of the energy at to 18.8 J/cm2

led to greater reduction of metabolic activity of planktonic cells [22]. However we see that continuous exposure to laser light resulted in the progressive loss of methylene blue staining. These occurrences suggest decrease of effectiveness

of PS and it awakened to the need for renewal. Thus the idea of repeated cycles of PDT optimized the reduction of bacterial load using similar dose of energy. These encouraging results suggest that bacterial death maintain stronger relation with the active principles of PS, which can be attributed to the: i) increased generation of singlet oxygen and free radical oxygen;

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ii) the oxygen depletion could be partially overcome by reducing or fractionating the light delivery [26] and;

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iii) continued exposure of microorganisms to high concentration of bactericidal PDT principles.

Regarding the use of devices with higher power, Soukos et al. (2006) [24]

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highlighted that it can cause a rapid consumption of molecular oxygen and to

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reduce the bactericidal efficacy of PDT. They hypothesized that using

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methylene blue at 6.25 mg/mL associated with light exposure times up to 10

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minutes will led to complete eradication of microorganisms. We agree with this

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hypothesis, especially bearing in mind the elimination of microorganisms in root canals and especially those located in the dentinal tubules, which is low oxygen

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tension [21].

The 10-μL volume was used in reference to the mean volume of a root

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canal of a permanent tooth [4]. In this study, the PDT was more effective when a smaller bacterial suspension volume (10-μL) was irradiated for 10 minutes.

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Thus, the reduction in volume increased the elimination of planktonic E. faecalis. The uninterrupted irradiation with PDT caused decrease of 2.13 Log, while the repeated PDT cycles reduced 3.04 Log under an energy dose of 24 J. Hypothetically, a lower volume of bacterial suspension favors the availability of a greater amount of energy per bacterial cell. In addition, the bacterial cells took

more PS by successive applications of PDT cycles. Consequently this association would tend to eliminate more bacterial cells. Until then attempts failed in the complete eradication intracanal of the heavy load of E. faecalis containing 108-109 CFU/mL [3, 12, 24]. Whereas the PDT has no prospect of replacing the chemomechanical preparation as

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aforamentioned [19] and that in clinical practice, the chemomechanical

preparation eliminates the main bulk of microorganisms [1, 4]. Thus, it was

eradication of these microorganisms, respectively.

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found that at 3 and 7 minutes at doses of 18 J and 42 J, gave complete

Enterococcus faecalis is Gram-positive, non-sporing, facultative

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anaerobe frequently selected for experimental studies of endodontic disinfection

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[14, 27-29] because it is often associated with endodontic treatment failure [9,

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30] and expressions commonly resistance to the irrigants and calcium

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hydroxide intracanal [7, 31]. Such microorganisms forming biofilms in root canal

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and colonize dentinal tubules [12, 18]. Moreover, Enterococcus faecalis are the gold standard in assay photodynamic therapy [12, 18, 21, 23]. Have a lower

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susceptibility to PDT compared to other microorganisms commonly isolated from root canals, such as the Actinomyces israelli, Fusobacterium nucleatum,

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Porphyromonas gingivalis, Prevotella intermedia [12], Streptococcus anginosus [10],Proteus mirabilis and Pseudomonas aeruginosa [13].

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Compared to other proposals for antisepsis complementary to

biomechanical preparation, the PDT several advantages present: i) photodynamic inactivation of microorganisms by topical application of photosensitizer and light restricts the action of highly reactive oxygen species and neutralizes systemic effects on non-pathogenic bacterial flora [32];

ii) the low power laser does not generate unwanted temperature rise effect in periodontal ligament [24]; iii) there are no possibilities of microbial resistance since, unlike antibiotics, PDT involves multiple biomolecules targets in microorganisms [16]; iv) the PDT can inactivate pathogens without endodontic affecting host

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cell viability [20];

v) being rapid and effective the endodontic treatment can be completed

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in the same session.

Although we use planktonic forms instead of biofilms this work has its scientific relevance because it has proven that it is possible to eliminate

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logarithmic quantities of a resistant microorganism in vitro. Therefore, our

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results demonstrate that the antibacterial efficacy of PDT was associated at the

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quantum variables on the laser source, renewal of the photosensitizer and the

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cycle application. Soon, a suggestive PDT protocol combining PDT cycles and

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laser source with 100 mW outpout power could provide surprising antibacterial

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elimination from E. faecalis in root canals.

Conclusions

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The antibacterial efficacy of PDT increased progressively as a function of the

increment of energy dose as well as the decrease in the volume of the bacterial

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suspension. PDT cycles optimized in logarithmic scale the elimination of planktonic forms of Enterococcus faecalis.

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Figure captions

CUF/mL x 108 40 35 30

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25 20

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15 10 5 6

8 10 12 14 16 18 20 22 24 Time (Hours)

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Figure 1. Growth curve of Enterococcus faecalis ATCC 19433. Monoculture of Enterococcus faecalis in 12 hour BHI (Dfico) broth was transferred to a volumetric flask containing 200 mL of fresh BHI broth supplemented with yeast extract and incubated at 37°C. At regular intervals, over 24 hours in a laminar flow chamber, 0.1 mL of the inoculum was obtained, and after serial dilution in Eppendorf tubes containing 0.9 mL of 0.85% saline solution, were spread in Petri containing 20 ml of Tryptic Soy Agar followed by incubation at 37 °C. After 24 hours, the colony forming units (CFU)/mL) were counted. Figure 1 shows the lag phase (0 to 0.5h), log or exponential phase (1 to 4h), stationary phase (4.5 to 12h) and the cell death or decline phase (13 to 24h).

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B

C

Figure 2: Experiments performed with the laser source Twin Flex II: (A) 96-well microtiterplates containing bacterial suspension and photosensitizer methylene

blue (MB). (B) Irradiation of the inner shaft through the flexible fiber optic taper. (C) After 2.5 minutes of irradiation note the discoloration of MB.

Figure 3. Influence of frequency (PDT cycles of 2.5 min) and volume of the bacterial suspension in reducing (Log scale) of planktonic forms of

IP T

Enterococcus faecalis irradiated by the laser source Twin Flex II with power 40 mW at different energy doses. Different letters mean statistical differences

A

CC E

PT

ED

M

A

N

U

SC R

significant (p<0.05).

Table 1. Bacterial Log reduction of wells containing 100-μL and 10-μL bacterial suspension containing 108 CFU/mL, irradiated by Twin Flex II device, with power of 40 mW. PDT

Tim

y

cycles

e

dose

(2.5 min

(min

(J)

each)

)

Experiment s

Bacterial

Log

suspension

reduct

volume (μL)

ion

only light

0.01

100

methylene

0.07

1

E1

6

2.5

E2

12

E3

18

E4

24

Control

2.4

E1

6

E2

12

E3

Group 3 (Effect of energy

ED

E4

SC R

2.4

0.21

10

1.51

1

0.32

1

2.5

0.37

2

5

18

3

7.5

1.40

24

4

10

1.76 0.27

-

5

U N

Control

2.4

1

E1

6

2.5

12

E3

-

100

0.30 0.41 0.49

0.87

0.39 10

5

1.18

18

7.5

1.31

E4

24

10

2.13

Group 4

E1

6

1

2.5

0.39

(Effect of energy

E2

12

2

5

1.87

dose, bacterial

E3

18

3

7.5

PT

E2

100

7.5

A

Group 2 (Effect of PDT cycles)

Control

M

blue

Group 1 (Effect of energy dose)

IP T

Groups

Energ

dose and bacterial

A

CC E

suspension volume)

suspension volume and PDT cycles)

E4

24

4

10

10

2.90 3.04

Group 1: Well containing 100µL BS plus and 100µL PS after irradiated with 2.4 at 24 J provided bacterial reduction ranging from 0.21 to 1.51 log. Group 2: corresponded to the group 1 added of PDT cycles and were obtained bacterial reduction ranging 0.32 to 1.76 log. Group 3: Eppendorf tubes containing 10µL SB plus and10µL PS provided

bacterial reduction ranging from 0.27 to 2.13 log. Group 4: corresponded to the group 3

A

CC E

PT

ED

M

A

N

U

SC R

IP T

added of PDT cycles and were obtained bacterial reduction ranging 0.39 to 3.04 log.