Enterococcus faecium and Enterococcus faecalis in endodontic infections: antibiotic resistance profile and susceptibility to photodynamic therapy

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1 Laser Dent Sci (2017) 1: ORIGINAL ARTICLE Enterococcus faecium and Enterococcus faecalis in endodontic infections: antibiotic resistance profile and susceptibility to photodynamic therapy Ana Carolina Chipoletti Prado 1 & Patrícia Pimentel De Barros 1 & Jéssica Diane Dos Santos 1 & Luciane Dias De Oliveira 1 & Claudio Antônio Talge Carvalho 2 & Marcia Carneiro Valera 2 & Antonio Olavo Cardoso Jorge 1 & Juliana Campos Junqueira 1 Received: 16 January 2017 /Accepted: 17 October 2017 /Published online: 30 October 2017 # Springer International Publishing AG 2017 Abstract Introduction Enterococcus faecium has become an important microorganism in nosocomial infections with great ability to acquire antibiotic resistance. However, little is known about their presence on the oral cavity. Therefore, our objective was to verify the presence of E. faecium and E. faecalis in endodontic infections and compare their susceptibility to conventional antibiotics and photodynamic therapy. Methods We performed 38 collections from the root canals of different patients. Positive Enterococcus agar samples were submitted to phenotypic and genotypic testing for speciesspecific confirmation. The isolates identified as E. faecium and E. faecalis were tested for susceptibility to antibiotics by the E-test method. After that, the isolates were evaluated for susceptibility to photodynamic therapy (PDT) using methylene blue and gallium arsenide aluminum laser with a wavelength of 660 nm and fluence of 39.5 J/cm 2 (energy of 15 J and time of 428 s). Results Cultures positive for E. faecalis were found in 22 patients (58%). Among these patients, only two had E. faecium in mixed infections with E. faecalis. In the isolates of E. faecalis, 27% were resistant to antibiotics, including tetracycline, ciprofloxacin, and azithromycin. The isolates of E. faecium showed no resistance to the antibiotics tested. Both the isolates of E. faecium and E. faecalis exhibit significant susceptibility to PDT, including the isolates resistant to antibiotics. The reductions achieved by PDT ranged of 2.76 to 4.31 log 10 for E. faecalis strains and of 3.93 to 4.33 log 10 for E. faecium strains. Conclusion E. faecium showed lower prevalence in endodontic infections and higher susceptibility to antibiotics when compared to E. faecalis. In in vitro assays, PDT had a significant antimicrobial activity for both strains. Keywords Enterococcus faecalis. Enterococcus faecium. Antimicrobials. Photodynamic therapy * Juliana Campos Junqueira juliana@fosjc.unesp.br AnaCarolinaChipolettiPrado anacarolinachipoletti@hotmail.com Patrícia Pimentel De Barros barrosdnapp@yahoo.com.br Jéssica Diane Dos Santos jessicadiane.santos@yahoo.com.br Luciane Dias De Oliveira luciane@fosjc.unesp.br Claudio Antônio Talge Carvalho claudiotalge@fosjc.unesp.br 1 2 Marcia Carneiro Valera marcia@fosjc.unesp.br Antonio Olavo Cardoso Jorge olavojorge@fosjc.unesp.br Department of Biosciences and Oral Diagnosis, Institute of Science and Technology, UNESP Univ Estadual Paulista, Francisco José Longo 777, São Dimas, São José dos Campos, SP , Brazil Department of Restorative Dentistry, Institute of Science and Technology, UNESP Univ Estadual Paulista, São José dos Campos, SP, Brazil

2 92 Laser Dent Sci (2017) 1:91 99 Introduction Enterococcus spp. are often related to important infectious diseases of great medical interest, including bacteremia, sepsis, urinary tract infections, wound infections, meningitis, and endocarditis, mainly in immunosuppressed and hospitalized patients [1, 2]. During the decade of 1980 and in the beginning of 1990, over than 90% of nosocomial enterococcal infections were caused by Enterococcus faecalis and5to10%by E. faecium. However, due to the high capacity to acquire resistance to antibiotics, E. faecium became responsible for 38 to 75% of the enterococcal infections. Currently, E. faecium is one of the most important microorganisms in nosocomial infections [3 5]. In dentistry, Enterococcus spp. can be associated with chronic periodontal diseases and endodontic infections [6, 7].The presence of enterococci within the root canal has been associated with persistent endodontic and periapical infections, being E. faecalis the most prevalent specie [8 11]. E. faecalis play an important role in post-treatment diseases because this specie can show high resistance to conventional antimicrobial agents, such as sodium hypochlorite, chlorhexidine, and calcium hydroxide [12 14]. In addition, certain clinical isolates of E. faecalis recovered from root canal infections demonstrated resistant to several antibiotics recommended for dental procedures, such as benzylpenicillin, ampicillin, clindamycin, and tetracycline [15], cephalosporins [8], azithromycin, and erythromycin [12]. Based on this context, various studies have been developed to investigate the efficacy of alternative approaches against E. faecalis, including antimicrobial photodynamic therapy (PDT) [16 18, 20 25]. PDT is a therapeutic strategy that involves the combination of a nontoxic photosensitizer (PS) and a harmless visible light source [19]. The excited PS reacts with molecular oxygen to produce highly reactive oxygen species, which induce injury and death of microorganisms [20]. It has been established that PS, which possess a cationic charge, can rapidly bind to or penetrate into bacterial cells. Therefore, these compounds demonstrate a high degree of selectivity for killing microorganisms and little toxicity toward host mammalian cells [21]. For this reason, the action of different PSs has been the subject of widespread research, mainly phenothiazine dyes, porphyrins, and phthalocyanines [22]. Currently, the association of methylene blue (MB), with lasers at red wavelengths ( nm), has been widely used for bacterial management in endodontic treatment by PDT [16 18, 20 25]. In the presence of a light source, permeability to MB can vary according to the structural components present on the surface of the microorganisms, reducing their antimicrobial effects. In general, the literature shows that Grampositive bacteria are more susceptible to the action of PDT compared to Gram-negative bacteria [25 28]. This is due to differences in the physiology of these microorganisms, since Gram-positive bacteria have a relatively porous outer membrane formed by a thicker layer of peptidoglycan and lipoteichoic acid. This feature allows a greater diffusion of the MB within the microbial cells, since they can be eliminated by lower concentration of this dye and lower doses of radiation, which explains the greater susceptibility of E. faecalis to PDT [20 25]. On the other hand, the outer membrane of Gram-negative bacteria is thinner and complex, being formed by a heterogeneous composition of proteins with porin function, lipopolysaccharides, and lipoproteins. Due to the characteristics of their cell walls, Gram-negative bacteria are less permeable to MB reducing their action and therefore are more resistant to PDT than Gram-positive species [25 30]. In vitro [16 18, 20 25] and clinical [31] studies have supported the use of PDT to eliminate microorganisms in a root canal, particularly E. faecalis, a multiple drug resistance microorganism often associated with endodontic treatment failure. Although the role of E. faecalis in endodontic infections had been extensively studied, little is known about the presence of E. faecium and its role in endodontic infections. Thus, the objective of this study was to verify the presence of E. faecium and E. faecalis in endodontic infections and compare their susceptibility to conventional antibiotics and PDT. Materials and methods Patient selection This study was approved by the Ethics Committee (Process: ) of the Institute of Science and Technology of Univ. Estadual Paulista (ICT/Unesp), and informed consent was obtained from all subjects. We selected 38 patients with clinical and radiographic signs compatible of diagnosis and need for endodontic treatment at the Endodontic clinics of ICT/Unesp and the Faculty of Pindamonhangaba (FAPI). Patients aged 21 to 73 years, comprising 18 males and 20 females, participated in the study. The patients were submitted to anamnesis and clinical examination with complete panoramic and periapical dental radiographic evaluation. Patients with general diseases and those who had used antibiotics in the past 3 months were excluded from the study. The tooth type and pulp stages (pulpal vitality or necrosis) that could be correlated with microbial findings were recorded for each patient. Microbiological collection The collection was performed on teeth that showed the following characteristics: teeth with temporary or definitive restorations, absence of periodontal pockets deeper than 4 mm, and the possibility of adequate absolute isolation. The

3 Laser Dent Sci (2017) 1: procedures for sample collection were based on the study of Gomes et al. [32]. Before the coronal opening for endodontic treatment rinsing with chlorhexidine gluconate solution (0.12%) was performed for 1 min. Restorations and carious lesions were removed and the teeth were individually isolated with a rubber dam. Antisepsis was performed using firstly 30% hydrogen peroxide and then 2.5% sodium hypochlorite. The solution was inactivated with 5% sodium thiosulfate for 30 s. After the exposure of the pulp tissue, the samples were collected by the use of four sterile absorbent paper points with numbering compatible to the root canal (Endopoints Ind. Com. Ltda., Brazil) for 60 s. Next, all the absorbent paper points were transferred into tubes containing 3 ml of buffered saline (PBS, ph 7.2). The tubes were maintained on ice for a maximum period of 3 h and transported to the Microbiology Laboratory. Isolation and identification of samples The samples were homogenized by vortexing for 60 s and diluted in phosphate buffered saline. Aliquots of 0.1 ml of the dilutions (10 1,10 2 and 10 3 ) were transferred to plates containing M-enterococcus agar (Difco, Detroit, USA) (in duplicate). The plates were incubated at 37 C for 24 h. After growth, colonies with different characteristics in each plate were isolated and submitted to phenotypic and genotypic identification testing. Phenotypic identification was carried out using the Rapid ID 32 Strep system (Bio Merieux, France) following the manufacturer s recommendations. The strains identified as E. faecalis and E. faecium were submitted to species-specific confirmation by the multiplex PCR method [33]. Reference strains of E. faecalis (ATCC4083) and E. faecium (ATCC6569) were included in all experiments. Antibiotic resistance profile The clinical strains identified as E. faecalis and E. faecium were subjected to the E-test method (BIODISK AB, Solna, Sweden) according to manufacturer s recommendations. The following antibiotics were tested: amoxicillin, amoxicillin + clavulanic acid, erythromycin, azithromycin, tetracycline, and ciprofloxacin. The antibiotic vancomycin was also included in order to identify vancomycin-resistant enterococci (VRE) in the oral cavity. The isolates were considered susceptible or resistant according to breakpoint values by the recommended CLSI document M100-S21 (Wayne, PA, USA). Susceptibility to photodynamic therapy (PDT) For the PDT experiment, we selected five isolates of E. faecalis sensitive to antibiotics (isolates 1, 2, 5, 8 and 14), five isolates resistant to at least one antibiotic (isolates 3, 6, 9, 15 and 18) and two isolates of E. faecium (isolates 4.2 and 7.3). The methodology for PDT was performed according to Souza et al. [16]. Methylene blue (Synth, São Paulo, Brazil), at a concentration of 0.1 mg/ml, was used for the sensitization of Enterococcus strains. The photosensitizer was prepared by dissolution of the dye in physiological solution (0.85% NaCl) and filtration through a sterile 0.22 μm Millipore membrane (São Paulo, Brazil). After filtration, the photosensitizer solution was stored in the dark. The light source used was a gallium aluminum arsenide (GaAlAs) laser (Easy Laser, Clean Line, Taubaté, Brazil) with a wavelength of 660 nm, output power of W, spot size area of cm 2 and fluence of 39.5 J/cm 2 (energy of 15 J and time of 428 s). The illuminated area was 0.38 cm 2. The optical output of the laser unit was measured before, halfway through, and after the experiment. The temperature at the bottom of the 96-well microtiter plates (Costar Corning, New York, NY, USA) was monitored using an infrared thermometer (MX4, Raytek, Sorocaba, São Paulo, Brazil), and no increase in temperature was observed after irradiation. Standardized suspensions of 10 6 cells/ml were dosed in the spectrophotometer (B582, Micronal, São Paulo, Brazil) at a wavelength of 760 nm and added to a 96- well flat bottom microtiter plate (Costar Corning, New York, NY, USA). Two experimental groups were evaluated: control group with absence of photosensitizer and laser (P L ) and PDT group with presence of photosensitizer and laser (P+L+). In the PDT group, 0.1 mg/ml of the methylene blue was added in each well, and the plates were incubated for 5 min on an orbital shaker (Solab Piracicaba, Brazil). Subsequently, the contents of the wells were subjected to irradiation according to the protocol described above. Each assay was performed in aseptic conditions within a laminar flow chamber and with ambient lights turned off. A black mask with a hole matching the diameter of the wall opening minimized artefacts related to light scattering during the irradiation procedure. After irradiation, 0.1 ml of serial dilutions were seeded in duplicate on Brain Heart Infusion (BHI) agar plates (Difco, Detroit, USA) for counting colony forming units (CFU/mL). The experiments were performed three times at different moments (n =8). Statistical analysis The data for CFU/ml were converted to logarithmic form and analyzed using Student s t test. The statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc., CA, USA). P values < 0.05 were considered significant. The percentage of CFU/ml reduction for both Enterococcus species was calculated considering the group PDT (P+L+) in relation to the control group (P L ).

4 94 Laser Dent Sci (2017) 1:91 99 Results Patient data and microbiological findings Cultures positive for E. faecalis were found in 58% of the patients analyzed (22/38), and only two patients showed E. faecium in mixed infections with E. faecalis. Among the 38 patients analyzed, 31 showed primary infections and 7 had secondary infections. In primary infections, 54.83% patients were positive for E. faecalis (17/31). In relation to secondary infections, E. faecalis was found in 71.42% patients (5/7). In the patients with primary infections (31), 20 showed necrotic pulp and 11 presented pulpal vitality. Positive cultures for Enterococcus were observed in 60% of patients with necrotic pulp (12/20) and 45% of patients with vital pulp (5/11). Both isolates of E. faecium were isolated from primary infections with necrotic pulp (Table 1). Among the 38 teeth analyzed, the teeth involved were 14 incisors, 4 canines, 10 premolars and 10 molars. Positive cultures were found in 64% of incisors (9/14), of 50% canines (2/4), 40% of premolars (4/10), and 70% of molars (7/10). The results obtained in the identification tests by phenotypic (API 20STREP) and genotyping (multiplex PCR) methods were similar for the most of isolates. Among the 22 isolates of E. faecalis, 16 showed similar results in both phenotypic and genotypic methods. However, in six isolates, the identification by genotypic methods not confirmed the results obtained in phenotypic method. In addition, it was verified that API 20STREP method was unable to identify E. faecium isolates (Table 1). Analysis of antibiotic resistance In the antibiotic susceptibility tests, 27.3% (6/22) of the E. faecalis isolates showed resistance to antibiotics. The resistance observed was 14% (3/22) for tetracycline, 9% (2/22) for ciprofloxacin, and 4.5% (1/22) for azithromycin. Among the results, we noted that 32% (7/22) of the E. faecalis isolates had an intermediate sensitivity to vancomycin, exhibiting a MIC greater than the breakpoint concentration for resistance to this antimicrobial, and values of 6 24 μg/ml, very close to resistance ( 32 μg/ml). The E. faecium isolates showed no resistance to the antibiotics tested, but showed intermediate sensitivities for erythromycin and azithromycin (Table 2). Analysis of the susceptibility to photodynamic therapy (PDT) The isolates of E. faecalis and E. faecium showed a significant reduction in the number of viable cells (CFU/mL) after PDT application (Figs. 1 and 2). The clinical isolates of E. faecalis and E. faecium showed reductions of 2.76 to 4.31 log 10 and 3.93 to 4.33 log 10, respectively. In relation to reference strains, PDT reached reduction of 4.07 log 10 for E. faecalis and 4.33 for E. faecium. The percentage of CFU/mL reduction ranged from 85 to 99% for clinical isolates of E. faecalis and 95 to 99% for E. faecium. Discussion Firstly, this study investigated the presence of the genus Enterococcus in 38 teeth with endodontic infections. E. faecalis was found in 58% of the patients. In primary and secondary infections, E. faecalis was found, respectively, in and 71.42%. According to the literature, E. faecalis is often found in root canals with chronic apical periodontitis after endodontic treatment [11, 32, 34, 35]. In systematic review, Zhang et al. [11] reported that the rate of E. faecalis by both culture and PCR methods was higher in persistent infections compared with untreated chronic periapical periodontitis. The high prevalence of E. faecalis in persistent periapicopatias may be due to its resistance to disinfection procedures used during the initial endodontic therapy. In addition, E. faecalis are able to invade root canals during or after endodontic treatments of coronary micro-leakages and to survive in conditions of low nutrient concentrations [36, 37]. Recently, E. faecium has become one of the most important microorganisms in nosocomial and systemic infections [2 5], but few studies have reported the presence of E. faecium associated with endodontic infections [35, 37, 38]. In this study, E. faecium was found in only two patients (5.2%). Ferrari et al. [37] also reported a low prevalence of E. faecium in primary infections using culture and biochemical testing identification methods. However, some studies verified a high prevalence of E. faecium in persistent endodontic infections through identification using molecular methods (DNA-DNA hybridization checkerboard) [35, 38]. Our study pioneered the detection of E. faecium in endodontic infections using the polymerase chain reaction (PCR). Zhan et al. [11] described the importance of conventional PCR as well as real time qpcr in studies related to the presence of Enterococcus in primary and secondary endodontic infections, emphasizing its specificity and sensitivity. In this study, the presence of E. faecium was found in mixed infections with E. faecalis in primary endodontic infections. In a heterogeneous bacterial community, different bacterial species often establish synergism interactions to induce mutual growth through quorum sensing communication. The bacterial species also exchange genetic information favoring drug resistance and increased virulence [39]. However, until now the interactions between E. faecium and E. faecalis were not investigated. In recent years, the Enterococcus species have received increased attention due to their capacity to acquire resistant

5 Laser Dent Sci (2017) 1: Table 1 Number of cultures positive for Enterococcus spp. collected from root canals Patients Pulp vitality Infection Cultures* API PCR 1 Necrosis Primary + 97% E. faecalis E. faecalis 2 Necrosis Primary + 87% E. faecalis E. faecalis 3 Necrosis Primary + 93% E. faecalis/3.5% E. faecium E. faecalis 4 Necrosis Primary + Isolate % E. faecalis E. faecalis Isolate 4.2 Aerococcusviridans/Leuconostocspp E. faecium 5 Vital Primary + 87% E. faecalis E. faecalis 6 Vital Primary + Aerococcusviridans/Leuconostocspp E. faecalis 7 Necrosis Primary + Isolate 7.1 E. faecalis/aerococcusurinae/streptococcus E. faecalis uberis/streptococcusagalactiae Isolate 7.3 E. faecalis/a. urinae/s. uberis/s. agalactiae E. faecium 8 Necrosis Primary + 99% E. faecalis E. faecalis 9 Necrosis Secondary + 72% E. faecalis E. faecalis 10 Vital Primary 11 Necrosis Primary 12 Necrosis Secondary 13 Vital Primary 14 Necrosis Primary + 87% E. faecalis/13% S. uberis E. faecalis 15 Necrosis Primary + 89% E. faecalis/5,4% S. uberis E. faecalis 16 Necrosis Primary 17 Necrosis Primary 18 Necrosis Secondary + 49% E. faecalis/37% E. faecium/14% S. uberis E. faecalis 19 Necrosis Primary 20 Necrosis Primary 21 Vital Primary 22 Necrosis Primary 23 Necrosis Secondary + 87% E. faecalis/11% S. uberis E. faecalis 24 Necrosis Primary + 87% E. faecalis/11% S. uberis E. faecalis 25 Necrosis Secondary + A. viridians/e. faecium/s. uberis E. faecalis 26 Necrosis Primary + 99% E. faecalis E. faecalis 27 Necrosis Primary + 87% E. faecalis/11% S. uberis E. faecalis 28 Necrosis Primary 29 Vital Primary + 48% E. faecalis E. faecalis 30 Necrosis Secondary + 99% E. faecalis E. faecalis 31 Vital Primary 32 Vital Primary 33 Vital Primary 34 Necrosis Primary 35 Necrosis Secondary 36 Vital Primary + 87% E. faecalis E. faecalis 37 Necrosis Primary + 48% E. faecalis/37% E. faecium E. faecalis 38 Vital Primary + 72% E. faecalis E. faecalis *Positive (+) and negative ( ) cultures for Enterococcus spp. to several antibiotics, including erythromycin, tetracycline, chloramphenicol, azithromycin, and vancomycin [10, 38, 40]. In our study, susceptibility to antibiotics was assayed by the E-test method that provides easy handling, interpretation,

6 96 Laser Dent Sci (2017) 1:91 99 Table 2 Antibiotic resistance profile of E. faecalis and E. faecium isolated from endodontic infections. MIC breakpoints (μg/ml) as recommended by CLSI (2011) were used to classify the isolates as sensitive (S), resistant (R), or susceptibility intermediarie (I) to the antibiotics: amoxicillin (AC) ( 8S, 16 R); amoxicillin + clavulanic acid (XL) ( 8S, 16 R) erythromycin (EM) ( 0.5 S, 8 R); azithromycin (AZ) ( 2S, 8R); tetracycline (TC) ( 4S, 16 R), Ciprofloxacin (CL) ( 1S, 4R), and vancomycin (VA) ( 4S, 32 R) Isolates MICμg/mLL AC XL EM AZ TC CI VA 1 0.4(S) 0.2(S) 0.4(S) 1.5(S) 3.0(S) 0.5(S) 2.0(S) 2 0.4(S) 0.25(S) 0.4(S) 1.0(S) 0.02(S) 3.0(I) 1.0(S) 3 0.4(S) 0.4(S) 3.0(I) 0.25(S) 16.0(R) 1.5(I) 6.0(I) (S) 0.25(S) 0.5(S) 0.25(S) 16.0(R) 0.07(S) 6.0(I) (S) 0.2(S) 6.0(I) 3.0(I) 0.13(S) 0.25(S) 1.5(S) 5 0.4(S) 0.1(S) 0.4(S) 0.2(S) 5.0(I) 1.5(I) 2.0(S) 6 0.5(S) 0.5(S) 3.0(I) 8.0(R) 0.4(S) 0.75(S) 1.5(S) (S) 0.1(S) 0.75(I) 1.0(S) 12.0(I) 0.25(S) 1.5(S) (S) 0.4(S) 0.05(S) 0.03(S) 0.25(S) 0.75(S) 1.0(S) 8 0.5(S) 1.0(S) 1.0(I) 3.0(I) 0.75(S) 1.0(S) 2.0(S) (S) 0.4(S) 0.4(S) 0.2(S) 6.0(I) 5.0(R) 2.0(S) (S) 0.5(S) 0.75(I) 1.0(S) 0.13(S) 1.5(I) 24.0(I) (S) 0.75(S) 1.5(I) 0.13(S) 0.4(S) 4.0(R) 24.0(I) (S) 0.5(S) 1.0(I) 0.25(S) 24.0(R) 0.75(S) 1.5(S) (S) 0.7(S) 0.5(S) 0.5(S) 0.13(S) 1.0(S) 3.0(S) (S) 2.0(S) 0.75(I) 1.5(S) 12.0(I) 0.38(S) 2.0(S) (S) 0.2(S) 0.5(S) 0.25(S) 0.25(S) 0.5(S) 0.5(S) (S) 0.5(S) 0.75(I) 1.5(S) 12.0(I) 0.5(S) 3.0(S) (S) 0.5(S) 0,5(S) 1.5(S) 0.25(S) 0.5(S) 1.0(S) (S) 0.25(S) 1.0(I) 1.0(S) 0.1(S) 0.5(S) 3.0(S) (S) 0.2(S) 1.5(I) 2.0(S) 0.05(S) 1.0(S) 6.0(I) (S) 0.12(S) 1.0(I) 1.0(S) 0.1(S) 0.75(S) 8.0(I) (S) 0.32(S) 0.5(S) 0.75(S) 0.5(S) 0.75(S) 6.0(I) (S) 0.25(S) 1.5(I) 2.0(S) 0.13(S) 0.5(S) 4.0(S) ATCC a 1.0(S) 0.4(S) 0.2(S) 0.03(S) 0.01(S) 6.0(R) 3.0(S) ATCC b 1.0(S) 1.5(S) 3.0(I) 12.0(R) 1.0(S) 0.4(S) 0.8(S) a E. faecalis ATCC4083 b E. faecium ATCC6569 and reliability [41]. E. faecalis isolates showed resistance of 14, 9 and 4.5% to tetracycline, ciprofloxacin and azithromycin, respectively, in agreement with previously reported results [12, 42 44]. Importantly, in this study we found 32% of the E. faecalis isolates with intermediate susceptibility to vancomycin that is an antibiotic recommended intravenously to treat a number of bacterial infections caused by resistant strains in hospitalized patients. The emergence of vancomycin-resistant enterococci infections has been associated with increased mortality and morbidity [45]. Therefore, the presence of E. faecalis in endodontic infections raises the question as to whether the oral cavity serves as a reservoir for virulent and resistant strains [46]. Anderson et al. [46] reported that E. faecalis strains isolated from oral biofilm or saliva revealed high percentages of virulence genes and resistance to antibiotics that were similar to clinical isolates from systemic infections. Due to the increasing of the resistance strains to antibiotics, in this study, we investigated the susceptibility of the E. faecalis and E. faecium isolates to photodynamic therapy. The strains of E. faecalis and E. faecium isolated from patients with endodontic infections showed a reduction around 3 to 4 log 10 when treated with PDT mediated by methylene blue and laser as a source of light. These findings agree with Misba et al. [25] that compared the efficacy of phenothiazinium dyes on Gram-positive and Gram-negative bacteria in planktonic cultures and biofilms. These authors found that antimicrobial photodynamic inhibition in planktonic cells were pronounced for E. faecalis. A reduction of 8 log 10 was found in bacterial count of E. faecalis after irradiation for 80s (630 nm). This difference can be explained by the morphology and physiology characteristics of these species. Gram-positive bacterium consists of a relatively simple envelope. Its cytoplasmic membrane is surrounded by a porous cell wall that allows photosensitizer to diffuse inside. The Gram-negative bacterium cell envelope is more complex and relatively impermeable structure that consists of an inner cytoplasmic membrane and an outer membrane, which are separated by a peptidoglycan layer [25 28]. Komine and Tsujimoto [47] correlated the amount of singlet oxygen generated by different concentrations of activated methylene blue during PDT with the bactericidal effect against E. faecalis cells. They concluded that methylene blue at a concentration of 0.01%, when activated by diode laser with a wavelength of 660 nm and 200 mw of power, was able to generate the greatest amount of singlet oxygen that consequently resulted in a reduction of 3 to 4 log 10 in the number of CFU cells. However, similar reduction was obtained in our study using a higher concentration of methylene blue activated by diode laser with a lower power (35 mw). The end outcome of any photodynamic intervention is dependent on many factors like power density (energy fluence), exposure time, photosensitizer concentration and optical properties of tissues [22, 25 28]. In this study, methylene blue (MB) was chosen because of its great phototoxic efficiency against a wide range of microorganisms, requiring a smaller amount of energy to obtain the same result when compared to other photosensitizers. Moreover, methylene blue produces satisfactory results with remarkable ability to penetrate the polymer matrix of bacterial biofilms due to its hydrophilic nature, low molecular weight cationic nature and water solubility, which allows for greater adhesion to anionic structures, such as teichoic acid [48]. According to Rolim et al. [29], the use of methylene blue promotes the formation of oxygen singlet, which is the

7 Laser Dent Sci (2017) 1: Fig. 1 Effects of PDT on E. faecalis sensitive and resistant to antimicrobials used in dentistry for the following groups: P L (control group) and P+L+ (photosensitizer and light). a Mean and standard deviation of the data CFU/ml (log 10 )for standard and clinical strains of E. faecalis sensitive to antibiotics, ATCC4083, and clinical isolates 1, 2, 5, 8 and 14. b Mean and standard deviation of UFC/ml (log 10 ) for the clinical strains of E. faecalis resistant 3, 6, 9, 15 and 18. Student s t test was performed between P L and P+L+ (**p < 0.01, *** p <0.001) reactive species responsible for bacterial death, at a rate 1.3 times higher than toluidine blue. Regarding the light source, several laser systems can be utilized as additional tool in the disinfection of root canals. Low-power lasers, such as helium-neon (He-Ne) and diodes, are the most used light sources in PDT for microbial reduction of various oral bacteria and fungi [49]. Recently, LED has emerged as an alternative light source to replace the laser in PDT. Despite having a lower potency than lasers, LED are widely used in dental clinics as bleaching tools; moreover, they have shown potent activity in PDT, reduced weight and cost compared to laser, and greater flexibility in treatment irradiation time and easy operation [48, 49]. Recently, Romeo et al. [50] compared the antibacterial activity of Fig. 2 Effects of PDT on E. faecium sensitive to antimicrobials used in dentistry for the following groups: P L (control group) and P+L+ (photosensitizer and light). Mean and standard deviation of the data CFU/ml (log 10 ) for standard and clinical strains of E. faecium sensitive to antibiotics (ATCC6569, 4.2 and 7.3). Student s t test was performed between P L and P+L+ (**p < 0.01, *** p <0.001) potassium-titanyl phosphate (KTP) laser with a 980 nm diode laser on E. faecalis biofilms. The authors showed that KTP laser was more efficient than 980 nm diode laser reducing significantly the population of E. faecalis in biofilms. Lasers when used for root canal disinfection are suggested to more effectively reduce bacteria located deep in the dentin than chemo-mechanical methods [51, 52]. E. faecalis is usually viable at high ph levels and is capable of invading dentinal tubules and surviving as a single species, where it adheres to collagen in the presence of human serum [53 55]. Furthermore, it penetrates deeply in dentinal tubules, up to 400 μm in vitro [53]. The eradication of this bacterium in the distant areas of the tubular root canal system is a major challenge in treatment regimens and is crucial for the long term preservation of endodontically treated teeth [55]. The use of lasers like low-power diode laser associated with optical fiber in endodontics is an innovative approach for disinfection, providing access to formerly unreachable parts of the tubular network, due to their ability to penetrate dental tissues better than irrigant solutions [17, 18, 20, 56, 57]. The superior bactericidal effect of diode laser irradiation could be attributed to its greater depth of penetration (up to 1000 μm into dentinal tubules) when compared to the penetration power of chemical disinfectants, which is limited to 100 μm [58]. It has been found that with progressive decrease in diameter of the deep dentinal tubules, the penetration of irrigants is restricted. However, laser irradiation with its inherent properties of light scattering, local intensity enhancement and attenuation allows light penetration deeper in the dentin tubules contributing to a superior antimicrobial efficacy [56 58].

8 98 Laser Dent Sci (2017) 1:91 99 The present research is the first study to compare the susceptibility of E. faecalis and E. faecium to PDT. We found that both species were equally susceptible to PDT with CFU/mL reductions of 3 to 4 log 10. The effects of PDT against E. faecalis strains have been extensively investigated in previous studies in which PDT caused significant reductions of the CFU/mL number [16 18, 20 25, 46 50, 52]. On the other hand, there is only one published study that investigated the effects of PDT on E. faecium [59]. The cited study was developed by our group, and the results showed that E. faecium strains sensitive to vancomycin were more susceptibility to PDT compared to vancomycin-resistant strains. The Gram-positive, facultative aerobe E. faecalis was used as a model organism in this study since it is strongly associated with different forms of periradicular disease, primary endodontic infections as well as persistent infections after failed endodontic treatment [24, 60]. A major characteristic of E. faecalis is its ability to withstand very high alkaline conditions ph 11.5 [52, 60], which elucidates the importance of alternative or supportive regimens like PDT for treatment of refractory endodontic infections [52]. These results suggest that PDT can be effective to treat mixed infections by E. faecalis and E. faecium in root canals and E. faecium was more susceptible to antibiotics than E. faecalis. However further studies should determine the extent of PDT disinfection in deep dentin tubules to increase endodontics outcomes. Role of funding source Supported by São Paulo Council of Research FAPESP, Brazil (Grant 2011/ ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil). Compliance with ethical standards Conflict of interest interests. The authors declare that they have no competing Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee (Process: ) of the Institute of Science and Technology of Univ. Estadual Paulista (ICT/Unesp). This article does not contain any studies with animals performed by any of the authors. Informed consent Informed consent was obtained from all individual participants included in the study. References 1. 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