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Lactam inhibiting Streptococcus mutans growth on titanium
Materials Science and Engineering C 68 (2016) 837–841
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Lactam inhibiting Streptococcus mutans growth on titanium J.G. Xavier a, T.C. Geremias a, J.F.D. Montero a, B.R. Vahey d, C.A.M. Benfatti a, J.C.M. Souza a, R.S. Magini a, A.L. Pimenta b,c,⁎ a
Center for Research on Dental Implants (CEPID), School of Dentistry (ODT), Federal University of Santa Catarina (UFSC), Florianópolis/SC, 88040-900, Brazil Department of Biologia, ERRMECe, Université de Cergy Pontoise, 2, Av. Adolphe Chauvin 95302 Cergy, Pontoise, France c Integrated Laboratories Technologies (InteLab), Dept. Chemical and Food Engineering (EQA), Federal University of Santa Catarina (UFSC), Florianópolis/SC, 88040-970, Brazil d Herman Ostrow School of Dentistry of USC, 925 W 34 St, Los Angeles, CA 90089, United States b
a r t i c l e
i n f o
Article history: Received 30 October 2015 Received in revised form 28 June 2016 Accepted 5 July 2016 Available online 7 July 2016 Keywords: Antibiofilm activity Biofilms Streptococcus mutans Lactams Bacterial adhesion Modified Calgary device
a b s t r a c t The aim of this work was to analyze the activity of novel synthetic lactams on preventing biofilm formation on titanium surfaces. Titanium (Ti6Al4V) samples were exposed to Streptococcus mutans cultures in the presence or absence of a synthetic lactam. After 48 h incubation, planktonic growth was determined by spectrophotometry. Biofilm was evaluated by crystal violet staining and colony forming units (CFU·ml−1), followed by scanning electron microscopy (SEM). Results showed that the average of adhered viable cells was approximately 1.5 × 102 CFU/ml in the presence of lactam and 4 × 102 CFU/ml in its absence. This novel compound was considerable active in reducing biofilm formation over titanium surfaces, indicating its potential for the development of antimicrobial drugs targeting the inhibition of the initial stages of bacterial biofilms on dental implants abutments. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Oral biofilms are diverse, organized microbial communities adhering to a solid surface, such as tooth enamel and restorative materials, embedded in a matrix composed of polysaccharides, glycoproteins, nucleic acids and water [1–3]. The process of oral biofilm formation occurs in a sequential manner, with primary colonizers adhering to glycoprotein receptors present on the acquired pellicle minutes after cleaning [1]. Streptococci and Actinomyces are the major initial colonizers of oral surface [4] and during the first hours of biofilm formation, the early microbiota is dominated by Streptococci [5]. Biofilm develops over the conditioning film through the following steps: (1) transport of primary colonizers to the surface; (2) weak and reversible initial adhesion resulting from chemical interactions between microorganisms and surface; (3) strong adhesion of microorganisms to the surface, established by specific interactions (covalent, ionic or hydrogen bonds); (4) maturation, resulting in the growth and maturation of the biofilm [6]. Similar to natural teeth, the roughness on dental implants facilitate the acquisition and maturation of oral biofilms, leading to inflammation of the supporting tissues. Several studies reported a relationship between the oral microbiota accumulation on dental implant surfaces and the topography of the surface of dental implants [7]. Bacteria ⁎ Corresponding author at: Integrated Laboratories Technologies (InteLab), Dept. Chemical and Food Engineering (EQA), Federal University of Santa Catarina (UFSC), Florianópolis/SC, 88040-970, Brazil. E-mail address: [email protected] (A.L. Pimenta).
http://dx.doi.org/10.1016/j.msec.2016.07.013 0928-4931/© 2016 Elsevier B.V. All rights reserved.
associated with failing implants are similar to those responsible for periodontal disease [8,9]. This inflammation is due to pathogenic bacteria and opportunistic microorganisms that colonize the peri-implant soft tissue-implant interface and adjacent dentition [10,11], and affect the integrity of approximately 24% of dental implants, becoming the main cause of implant failure after 5 years [12]. Considering the number of failures that lead to the removal of the implants and patient suffering, infections associated with dental implanted-supported systems represent a serious global public health concern. Currently, the control of oral biofilms is achieved by conventional systemic antibiotic therapies, which decrease the symptoms of infection by eliminating the circulating planktonic microbial population, although is ineffective against those that remain within the biofilm [2, 13]. When organized in biofilms, bacteria are able to survive against traditional antibiotics treatments at concentrations up to a thousand times higher than those used to kill their planktonic counterparts [14], by that emphasizes the need to develop therapies aiming the inhibition of biofilm formation. The discovery that bacterial-communication systems, such as quorum-sensing (QS) which orchestrate important temporal events during the infection process, has provided novel insights for the treatment of bacterial infections [15]. Therefore, identifying substances that are able to inhibit bacterial adherence and biofilm formation constitutes a promising approach for the development of a new generation of antimicrobial drugs targeted to bacterial virulence rather than bacterial viability, with the advantage of imposing a low selection pressure on the bacterial populations, thus
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avoiding development of resistance [16]. Amongst biofilm inhibitors reported in the current literature, natural halogenated furanones, isolated from the marine red algae Delisea pulchra, appear to be the most effectives. These compounds have been shown to prevent biofilm formation in Escherichia coli and Bacillus subtilis [17]. A similar activity was reported for a synthetic furanone, which is able to inhibit E. coli biofilm formation by 80% [18]. These discoveries have prompted other groups to investigate the biological activities of structure related lactones. Our group has been investigating the biological activity of analogues of natural lactones, such as rubrolides and lactams, against biofilm formation. A recent report stated that, in general, lactams are more active against biofilms than their precursors, thus designating this class of molecules as good candidates for the development of a new generation of anti-microbial drugs targeting biofilm inhibition [19]. The potential of these compounds as inhibitors of bacterial biofilm was tested against Enterococcus faecalis, Staphylococcus epidermidis and Pseudomonas aeruginosa and results confirmed that these classes of compounds to be very active [19,20]. The purpose of this present study was to assess the potential of a synthetic lactam, a rubrolide analogue, to inhibit biofilm formation of Streptococcus mutans on titanium surface. A significant decrease in bacterial adherence over titanium surfaces in the presence of the tested lactam is indicative of the antibiofilm activity of this compound. 2. Materials and methods 2.1. Inhibitor compound The synthesized new rubrolide analogue was prepared and converted into their corresponding lactam by Pereiras's group, at the Department of Chemistry of Federal University of Viçosa (Brazil). The compound was fully characterized by IR, NMR (1H and 13C), COSY and HETCOR bidimensional experiments, and NOE difference spectroscopy experiments [19] and gently donated for further studies. 2.2. Titanium samples preparation Cylindrical samples of a titanium alloy (Ti6Al4V) with dimensions of 15 × 8 × 5 mm were obtained from a bar using a precision cutting machine (Isomet 1000 Buehler®, USA). Samples were ground in an automatic polishing under water lubrication with SiC paper to 1200 mesh. After the polishing procedure, the samples were cleaned with isopropyl alcohol for 10 min and rinsed in distilled water in an ultrasonic bath for 5 min. Cylinders were kept in a dehumidifying chamber for 24 h. 2.3. Bacterial strains and growth conditions S. mutans ATCC 25175 was grown under microaerophilic conditions for 48 h at 37 °C on Brain Heart Infusion (BHI) agar plates (Bacto®, Difco), supplemented with 3 g/l of yeast extract (Bacto®, Difco) and 200 g/ of sucrose (Bacto®, Difco, USA). For biofilm assays, S. mutans cells were inoculated in Tryptic Soy Broth (TSB) (Bacto®, Difco) supplemented with 3 g/ of yeast extract and 200 g/l of sucrose, and incubated for 18 h at 37 °C. After incubation, cells were harvested by centrifugation at 5000 rpm for 10 min at 4 °C, and washed twice in a phosphate buffer solution (PBS). 2.4. Biofilm formation and analysis After growth and harvest following the conditions described above, S. mutans cells were re-suspended in TSB medium supplemented with mucin (2,5 g/l), peptone (5 g/l), urea (1 g/l), yeast extract (2 g/l) and sucrose (200 g/) to obtain a suspension with an optical density (OD630) of 0.6, corresponding to approximately 1 × 108 CFU/ml. OD630 was measured using a spectrophotometer (BioTek®, USA) [21,22].
Polystyrene plates with 24 wells were used in this study to develop a modified device by molding a silicone membrane (Rhodorsil, RTV 4407A/B, 5 mm thick) inside the plate lids. After silicone polymerization, titanium samples were inserted into the membrane, inside slots spaced so as to fit the wells of the plate when the lid was placed over it. This set (silicone membrane containing titanium samples) was then sterilized in an autoclave at 121 °C for 20 min, and used for the biofilm inhibition assays. Sterile titanium samples, fixed in the plate lids as described above, were placed into 24 well plates containing 2 ml of S. mutans suspension in TSB medium, in the presence or absence of 87.5 μg/ml of the inhibitor compound, lactam U27, and incubated for 48 h at 37 °C, under microaerophilic conditions. 200 μl of the culture media were used to quantify planktonic growth by OD630 determination on a microplate reader (BioTek®, USA). For biofilm quantification, titanium samples recovered from the 24 well plates after incubation with S. mutans in the presence or absence of U27, were washed twice with PBS. Adherent bacteria were detached from the titanium surfaces by proteinase K treatment. Samples were then placed on 24-well plates containing 1 ml of PBS and 1% proteinase K (Sigma-Aldrich®), and were incubated at 37 °C for 60 min [23]. After proteinase K treatment, a physical method was used to increase biofilm detachment. Adherent bacteria were removed by vortex treatment for 1 min [24]. The suspension was then diluted (up to 10−6) in PBS and plated on BHI agar to quantify CFUs/ml. These experiments were performed in triplicate and carried out in three independent assays. Biofilm quantification by crystal violet (CV) staining assay was performed as previously described [21,25,26]. In brief, titanium samples were transferred for new 24-well plates and washed twice with PBS. Samples were subsequently immersed in 1 ml of methanol for 15 min for biofilm fixation. Methanol was removed, samples were dried at room temperature and 1 ml of CV (1%) was added to stain the bacterial biofilm for 5 min. Titanium samples were then dip-washed in distilled water, dried at room temperature, and transferred to new 24-well plates containing 1 ml of 1% sodium dodecyl sulfate (SDS) [26]. 200 μl of the suspension was used to determine the OD540 in a microplate reader. All experiments were carried out in triplicate. 2.5. Surface analysis The evaluation of surface roughness by mechanical profilometry was performed using a Profilometer (DektakXT®). The length of analysis was 2 mm, cut-off 0.25 mm and a speed of 30 s (Ra ~ 0.4 μm). Roughness values were obtained considering the parameter Ra, which is the arithmetic average between the heights of the peaks and valleys of the actual surface profile. For microscopic analyses, surfaces covered with biofilms were washed twice in PBS and fixed in glutaraldehyde 2% for 5 min. Then, surfaces were washed three times in PBS, and dehydrated through a series of graded ethanol solutions (50, 70, 80, 90, and 100%). Samples covered with S. mutans biofilms were sputter-coated with gold, and analyzed by Scanning Electron Microscopy (JEOL JSM-6390LV, Japan) at 15 kV. 2.6. Statistical analysis Results were statistically analyzed by Student's t-test for biofilm inhibition activity and Mann-Whitney Test for spectrophotometric quantification of crystal violet retained in biofilms with a significance level of p b 0.05. 3. Results 3.1. Planktonic growth As shown in Fig. 2, OD630 values for planktonic growth showed no significant statistical difference in the presence or absence of lactam,
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indicating that, addition of the tested lactam to the culture media did not affect S. mutans growth. 3.2. Biofilm analysis Biofilm inhibition activity was assessed by CFU/ml countings after bacterial disaggregation from the titanium surfaces. The average of adhered viable cells in the biofilm was approximately 1.5 × 102 CFU/ml in the presence of lactam and 4 × 102 CFU/ml in its absence (Fig. 1B). These results indicate a significant reduction (p b 0.05) of bacterial attachment to the titanium surface in the presence of lactam. Spectrophotometric quantification of crystal violet retained in the biofilms was also used to assess biofilm biomass on the Ti surfaces. Statistical analysis of results (Fig. 3) confirm the inhibitory effect of the lactam on biofilm development on the titanium surface. Scanning Electron Microscopy (SEM) images of the titanium surfaces revealed the morphology of S. mutans biofilms formed, as shown in Fig. 5A–D. S. mutans colonies observed were more widely separated as agglomerates on the titanium surface. A significant amount of biofilm accumulation was noticed in samples immersed in media where no U27 was added (Fig. 5C) contrasting with titanium surfaces incubated in the presence of the biofilm inhibitor, which appeared free of S. mutans colonies (Fig. 5D). Surface irregularities, such as deep depressions, probably originated from the manufacturing process, were susceptible to accumulation of S. mutans (Fig. 5B). The presence of mucin adhered to the titanium surface, associated with the S. mutans colonies, possibly provided the initial pellicle for further biofilm adhesion (Fig. 5A). 4. Discussion In the present study, results from the biofilm analysis support the hypothesis that the modified synthetic lactam tested is effective in suppressing S. mutans biofilm development directly on the titanium surface. These results were confirmed by both CFU counting and crystal violet quantification, indicating a significant reduced quantity of adhered, viable cells in the presence of U27 lactam (Figs. 3 and 4). Statistical analysis of the OD readings of biofilms formed over titanium surfaces in the presence of modified lactams indicate a significant inhibition of biofilm formation when compared to biofilms formed in the absence of the inhibitor and corroborates a previous report, where these compounds were shown to actively inhibit biofilm formed over polystyrene surfaces [19]. Planktonic growth rates of S. mutans in the presence of the tested lactam showed no statistical significant difference when compared
Fig. 1. Schematic illustration on the samples attached to the novel culture plate.
Fig. 2. Quantification of planktonic growth of S. mutans (TSB, 48 h at 37 °C) in the presence or absence of lactam.
with the control with no lactam. This result indicates that the synthetic U27 lactam not only inhibit structured biofilm formation by S. mutans on the treated titanium surface, but importantly, it does not have a bactericide/bacteriostatic effect in the surrounding media, which is a good indication that no systemic effect of the drug should be expected. These results are in agreement with recent reports on the effects of the quorum sensing inhibitor furanone C-30 on biofilm formation by S. mutans and the luxS mutant strain, showing that synthetic furanone C-30 does not affect S. mutans growth whilst being effective against biofilm formation by both wild type and mutant strain [27]. Results obtained here, showing reduced CFU countings in the biofilm detached from titanium surfaces exposed to the tested lactam (approximately 1.5 × 102 CFU/ml in the presence of lactam and 4 × 102 CFU/ml in its absence), are in agreement with those previously reported, where three synthetic lactams, derived from furanones, tested against biofilm of E. faecalis grown over an implantable polymeric material (PLGAHA), showed significant biofilm inhibition rates [28]. In the same study, the cytotoxicity evaluation of the lactam U27 against human cells has been performed using MTT assay, and revealed a high viability rate (N 80%) of cells in the presence of this lactam [28]. The biofilm culture device used in this study, in which a silicone membrane was used to fix the titanium samples to the culture plate, allowed the microorganisms to grow on fixed structures positioned in
Fig. 3. S. mutans biofilm formation (TSB, 48 h at 37 °C) over titanium surfaces, in the presence or absence of lactam, measured by colony forming units (CFU/ml) (Student's ttest with a significance level of p b 0.05).
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5. Conclusion
Fig. 4. S. mutans biofilm formation (TSB, 48 h at 37 °C) over titanium surfaces, in the presence or absence of lactam, measured by CV colorimetric method (Mann-Whitney Test with a significance level of p b 0.05).
Treatment of biofilms-associated oral diseases with traditional pharmacologic and systemic antibiotic therapies lacks direct, localized activity, mostly due to low diffusion rate of the antibiotics through the entire biofilm biomass. This observation, along with the emerging global concern about bacterial resistance to antibiotics, is leading to a prompt shift of drug targets from bacterial survival to pathogenicity control, involving bacterial adherence to surfaces as well as signaling systems controlling bacterial group behavior of populations organized in biofilms. In the present study the potential of synthetic lactam (U27) as inhibitor of bacterial biofilm formation against S. mutans was tested. Results showed a significant decreased accumulation of the biofilm on the titanium surface in the presence of lactam U27, indicating that coating dental implant surfaces with such biofilm inhibitor compounds could be a promising solution for the prevention of initial biofilm adhesion on dental implant surfaces. Additional studies are currently being performed to analyze the effect of modified synthetic lactams on the development of multispecies biofilms over titanium surfaces.
Conflict of interest the polystyrene lid coincident with the culture wells. The use of this device enhances the homogeneity of sample treatment, facilitates their manipulation with greater accuracy, enhances reproducibility, thus favorizing the standardization of microbiological tests on titanium. It is important to note that results presented here were obtained using a monospecies biofilm as a model. Similar studies involving multispecies biofilms are currently underway in order to complement and further validate the potential use of lactams as biofilm inhibitors in implantable materials, under conditions that better mimic the oral environment.
The authors have no conflicts of interest to disclose. There were no sources of funding that could have influenced the outcome of this work.
Acknowledgments The authors acknowledge the financial support provided by the government research funding agencie CNPq (SET-B 350091/2016-1) (Brazil).
Fig. 5. Scanning Electron Micrograph (SEM) of titanium surfaces after exposition to S. mutans cultures on biofilm formation assay (48 h in TSB, 37 °C). S. mutans colonies are only visible on titanium surfaces in the absence of the biofilm inhibitor (A, B, C). (D) Lactam-treated titanium surface free of S. mutans colonies.
J.G. Xavier et al. / Materials Science and Engineering C 68 (2016) 837–841
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Lactam inhibiting Streptococcus mutans growth on titanium J.G. Xavier a, T.C. Geremias a, J.F.D. Montero a, B.R. Vahey d, C.A.M. Benfatti a, J.C.M. Souza a, R.S. Magini a, A.L. Pimenta b,c,⁎ a
Center for Research on Dental Implants (CEPID), School of Dentistry (ODT), Federal University of Santa Catarina (UFSC), Florianópolis/SC, 88040-900, Brazil Department of Biologia, ERRMECe, Université de Cergy Pontoise, 2, Av. Adolphe Chauvin 95302 Cergy, Pontoise, France c Integrated Laboratories Technologies (InteLab), Dept. Chemical and Food Engineering (EQA), Federal University of Santa Catarina (UFSC), Florianópolis/SC, 88040-970, Brazil d Herman Ostrow School of Dentistry of USC, 925 W 34 St, Los Angeles, CA 90089, United States b
a r t i c l e
i n f o
Article history: Received 30 October 2015 Received in revised form 28 June 2016 Accepted 5 July 2016 Available online 7 July 2016 Keywords: Antibiofilm activity Biofilms Streptococcus mutans Lactams Bacterial adhesion Modified Calgary device
a b s t r a c t The aim of this work was to analyze the activity of novel synthetic lactams on preventing biofilm formation on titanium surfaces. Titanium (Ti6Al4V) samples were exposed to Streptococcus mutans cultures in the presence or absence of a synthetic lactam. After 48 h incubation, planktonic growth was determined by spectrophotometry. Biofilm was evaluated by crystal violet staining and colony forming units (CFU·ml−1), followed by scanning electron microscopy (SEM). Results showed that the average of adhered viable cells was approximately 1.5 × 102 CFU/ml in the presence of lactam and 4 × 102 CFU/ml in its absence. This novel compound was considerable active in reducing biofilm formation over titanium surfaces, indicating its potential for the development of antimicrobial drugs targeting the inhibition of the initial stages of bacterial biofilms on dental implants abutments. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Oral biofilms are diverse, organized microbial communities adhering to a solid surface, such as tooth enamel and restorative materials, embedded in a matrix composed of polysaccharides, glycoproteins, nucleic acids and water [1–3]. The process of oral biofilm formation occurs in a sequential manner, with primary colonizers adhering to glycoprotein receptors present on the acquired pellicle minutes after cleaning [1]. Streptococci and Actinomyces are the major initial colonizers of oral surface [4] and during the first hours of biofilm formation, the early microbiota is dominated by Streptococci [5]. Biofilm develops over the conditioning film through the following steps: (1) transport of primary colonizers to the surface; (2) weak and reversible initial adhesion resulting from chemical interactions between microorganisms and surface; (3) strong adhesion of microorganisms to the surface, established by specific interactions (covalent, ionic or hydrogen bonds); (4) maturation, resulting in the growth and maturation of the biofilm [6]. Similar to natural teeth, the roughness on dental implants facilitate the acquisition and maturation of oral biofilms, leading to inflammation of the supporting tissues. Several studies reported a relationship between the oral microbiota accumulation on dental implant surfaces and the topography of the surface of dental implants [7]. Bacteria ⁎ Corresponding author at: Integrated Laboratories Technologies (InteLab), Dept. Chemical and Food Engineering (EQA), Federal University of Santa Catarina (UFSC), Florianópolis/SC, 88040-970, Brazil. E-mail address: [email protected] (A.L. Pimenta).
http://dx.doi.org/10.1016/j.msec.2016.07.013 0928-4931/© 2016 Elsevier B.V. All rights reserved.
associated with failing implants are similar to those responsible for periodontal disease [8,9]. This inflammation is due to pathogenic bacteria and opportunistic microorganisms that colonize the peri-implant soft tissue-implant interface and adjacent dentition [10,11], and affect the integrity of approximately 24% of dental implants, becoming the main cause of implant failure after 5 years [12]. Considering the number of failures that lead to the removal of the implants and patient suffering, infections associated with dental implanted-supported systems represent a serious global public health concern. Currently, the control of oral biofilms is achieved by conventional systemic antibiotic therapies, which decrease the symptoms of infection by eliminating the circulating planktonic microbial population, although is ineffective against those that remain within the biofilm [2, 13]. When organized in biofilms, bacteria are able to survive against traditional antibiotics treatments at concentrations up to a thousand times higher than those used to kill their planktonic counterparts [14], by that emphasizes the need to develop therapies aiming the inhibition of biofilm formation. The discovery that bacterial-communication systems, such as quorum-sensing (QS) which orchestrate important temporal events during the infection process, has provided novel insights for the treatment of bacterial infections [15]. Therefore, identifying substances that are able to inhibit bacterial adherence and biofilm formation constitutes a promising approach for the development of a new generation of antimicrobial drugs targeted to bacterial virulence rather than bacterial viability, with the advantage of imposing a low selection pressure on the bacterial populations, thus
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avoiding development of resistance [16]. Amongst biofilm inhibitors reported in the current literature, natural halogenated furanones, isolated from the marine red algae Delisea pulchra, appear to be the most effectives. These compounds have been shown to prevent biofilm formation in Escherichia coli and Bacillus subtilis [17]. A similar activity was reported for a synthetic furanone, which is able to inhibit E. coli biofilm formation by 80% [18]. These discoveries have prompted other groups to investigate the biological activities of structure related lactones. Our group has been investigating the biological activity of analogues of natural lactones, such as rubrolides and lactams, against biofilm formation. A recent report stated that, in general, lactams are more active against biofilms than their precursors, thus designating this class of molecules as good candidates for the development of a new generation of anti-microbial drugs targeting biofilm inhibition [19]. The potential of these compounds as inhibitors of bacterial biofilm was tested against Enterococcus faecalis, Staphylococcus epidermidis and Pseudomonas aeruginosa and results confirmed that these classes of compounds to be very active [19,20]. The purpose of this present study was to assess the potential of a synthetic lactam, a rubrolide analogue, to inhibit biofilm formation of Streptococcus mutans on titanium surface. A significant decrease in bacterial adherence over titanium surfaces in the presence of the tested lactam is indicative of the antibiofilm activity of this compound. 2. Materials and methods 2.1. Inhibitor compound The synthesized new rubrolide analogue was prepared and converted into their corresponding lactam by Pereiras's group, at the Department of Chemistry of Federal University of Viçosa (Brazil). The compound was fully characterized by IR, NMR (1H and 13C), COSY and HETCOR bidimensional experiments, and NOE difference spectroscopy experiments [19] and gently donated for further studies. 2.2. Titanium samples preparation Cylindrical samples of a titanium alloy (Ti6Al4V) with dimensions of 15 × 8 × 5 mm were obtained from a bar using a precision cutting machine (Isomet 1000 Buehler®, USA). Samples were ground in an automatic polishing under water lubrication with SiC paper to 1200 mesh. After the polishing procedure, the samples were cleaned with isopropyl alcohol for 10 min and rinsed in distilled water in an ultrasonic bath for 5 min. Cylinders were kept in a dehumidifying chamber for 24 h. 2.3. Bacterial strains and growth conditions S. mutans ATCC 25175 was grown under microaerophilic conditions for 48 h at 37 °C on Brain Heart Infusion (BHI) agar plates (Bacto®, Difco), supplemented with 3 g/l of yeast extract (Bacto®, Difco) and 200 g/ of sucrose (Bacto®, Difco, USA). For biofilm assays, S. mutans cells were inoculated in Tryptic Soy Broth (TSB) (Bacto®, Difco) supplemented with 3 g/ of yeast extract and 200 g/l of sucrose, and incubated for 18 h at 37 °C. After incubation, cells were harvested by centrifugation at 5000 rpm for 10 min at 4 °C, and washed twice in a phosphate buffer solution (PBS). 2.4. Biofilm formation and analysis After growth and harvest following the conditions described above, S. mutans cells were re-suspended in TSB medium supplemented with mucin (2,5 g/l), peptone (5 g/l), urea (1 g/l), yeast extract (2 g/l) and sucrose (200 g/) to obtain a suspension with an optical density (OD630) of 0.6, corresponding to approximately 1 × 108 CFU/ml. OD630 was measured using a spectrophotometer (BioTek®, USA) [21,22].
Polystyrene plates with 24 wells were used in this study to develop a modified device by molding a silicone membrane (Rhodorsil, RTV 4407A/B, 5 mm thick) inside the plate lids. After silicone polymerization, titanium samples were inserted into the membrane, inside slots spaced so as to fit the wells of the plate when the lid was placed over it. This set (silicone membrane containing titanium samples) was then sterilized in an autoclave at 121 °C for 20 min, and used for the biofilm inhibition assays. Sterile titanium samples, fixed in the plate lids as described above, were placed into 24 well plates containing 2 ml of S. mutans suspension in TSB medium, in the presence or absence of 87.5 μg/ml of the inhibitor compound, lactam U27, and incubated for 48 h at 37 °C, under microaerophilic conditions. 200 μl of the culture media were used to quantify planktonic growth by OD630 determination on a microplate reader (BioTek®, USA). For biofilm quantification, titanium samples recovered from the 24 well plates after incubation with S. mutans in the presence or absence of U27, were washed twice with PBS. Adherent bacteria were detached from the titanium surfaces by proteinase K treatment. Samples were then placed on 24-well plates containing 1 ml of PBS and 1% proteinase K (Sigma-Aldrich®), and were incubated at 37 °C for 60 min [23]. After proteinase K treatment, a physical method was used to increase biofilm detachment. Adherent bacteria were removed by vortex treatment for 1 min [24]. The suspension was then diluted (up to 10−6) in PBS and plated on BHI agar to quantify CFUs/ml. These experiments were performed in triplicate and carried out in three independent assays. Biofilm quantification by crystal violet (CV) staining assay was performed as previously described [21,25,26]. In brief, titanium samples were transferred for new 24-well plates and washed twice with PBS. Samples were subsequently immersed in 1 ml of methanol for 15 min for biofilm fixation. Methanol was removed, samples were dried at room temperature and 1 ml of CV (1%) was added to stain the bacterial biofilm for 5 min. Titanium samples were then dip-washed in distilled water, dried at room temperature, and transferred to new 24-well plates containing 1 ml of 1% sodium dodecyl sulfate (SDS) [26]. 200 μl of the suspension was used to determine the OD540 in a microplate reader. All experiments were carried out in triplicate. 2.5. Surface analysis The evaluation of surface roughness by mechanical profilometry was performed using a Profilometer (DektakXT®). The length of analysis was 2 mm, cut-off 0.25 mm and a speed of 30 s (Ra ~ 0.4 μm). Roughness values were obtained considering the parameter Ra, which is the arithmetic average between the heights of the peaks and valleys of the actual surface profile. For microscopic analyses, surfaces covered with biofilms were washed twice in PBS and fixed in glutaraldehyde 2% for 5 min. Then, surfaces were washed three times in PBS, and dehydrated through a series of graded ethanol solutions (50, 70, 80, 90, and 100%). Samples covered with S. mutans biofilms were sputter-coated with gold, and analyzed by Scanning Electron Microscopy (JEOL JSM-6390LV, Japan) at 15 kV. 2.6. Statistical analysis Results were statistically analyzed by Student's t-test for biofilm inhibition activity and Mann-Whitney Test for spectrophotometric quantification of crystal violet retained in biofilms with a significance level of p b 0.05. 3. Results 3.1. Planktonic growth As shown in Fig. 2, OD630 values for planktonic growth showed no significant statistical difference in the presence or absence of lactam,
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indicating that, addition of the tested lactam to the culture media did not affect S. mutans growth. 3.2. Biofilm analysis Biofilm inhibition activity was assessed by CFU/ml countings after bacterial disaggregation from the titanium surfaces. The average of adhered viable cells in the biofilm was approximately 1.5 × 102 CFU/ml in the presence of lactam and 4 × 102 CFU/ml in its absence (Fig. 1B). These results indicate a significant reduction (p b 0.05) of bacterial attachment to the titanium surface in the presence of lactam. Spectrophotometric quantification of crystal violet retained in the biofilms was also used to assess biofilm biomass on the Ti surfaces. Statistical analysis of results (Fig. 3) confirm the inhibitory effect of the lactam on biofilm development on the titanium surface. Scanning Electron Microscopy (SEM) images of the titanium surfaces revealed the morphology of S. mutans biofilms formed, as shown in Fig. 5A–D. S. mutans colonies observed were more widely separated as agglomerates on the titanium surface. A significant amount of biofilm accumulation was noticed in samples immersed in media where no U27 was added (Fig. 5C) contrasting with titanium surfaces incubated in the presence of the biofilm inhibitor, which appeared free of S. mutans colonies (Fig. 5D). Surface irregularities, such as deep depressions, probably originated from the manufacturing process, were susceptible to accumulation of S. mutans (Fig. 5B). The presence of mucin adhered to the titanium surface, associated with the S. mutans colonies, possibly provided the initial pellicle for further biofilm adhesion (Fig. 5A). 4. Discussion In the present study, results from the biofilm analysis support the hypothesis that the modified synthetic lactam tested is effective in suppressing S. mutans biofilm development directly on the titanium surface. These results were confirmed by both CFU counting and crystal violet quantification, indicating a significant reduced quantity of adhered, viable cells in the presence of U27 lactam (Figs. 3 and 4). Statistical analysis of the OD readings of biofilms formed over titanium surfaces in the presence of modified lactams indicate a significant inhibition of biofilm formation when compared to biofilms formed in the absence of the inhibitor and corroborates a previous report, where these compounds were shown to actively inhibit biofilm formed over polystyrene surfaces [19]. Planktonic growth rates of S. mutans in the presence of the tested lactam showed no statistical significant difference when compared
Fig. 1. Schematic illustration on the samples attached to the novel culture plate.
Fig. 2. Quantification of planktonic growth of S. mutans (TSB, 48 h at 37 °C) in the presence or absence of lactam.
with the control with no lactam. This result indicates that the synthetic U27 lactam not only inhibit structured biofilm formation by S. mutans on the treated titanium surface, but importantly, it does not have a bactericide/bacteriostatic effect in the surrounding media, which is a good indication that no systemic effect of the drug should be expected. These results are in agreement with recent reports on the effects of the quorum sensing inhibitor furanone C-30 on biofilm formation by S. mutans and the luxS mutant strain, showing that synthetic furanone C-30 does not affect S. mutans growth whilst being effective against biofilm formation by both wild type and mutant strain [27]. Results obtained here, showing reduced CFU countings in the biofilm detached from titanium surfaces exposed to the tested lactam (approximately 1.5 × 102 CFU/ml in the presence of lactam and 4 × 102 CFU/ml in its absence), are in agreement with those previously reported, where three synthetic lactams, derived from furanones, tested against biofilm of E. faecalis grown over an implantable polymeric material (PLGAHA), showed significant biofilm inhibition rates [28]. In the same study, the cytotoxicity evaluation of the lactam U27 against human cells has been performed using MTT assay, and revealed a high viability rate (N 80%) of cells in the presence of this lactam [28]. The biofilm culture device used in this study, in which a silicone membrane was used to fix the titanium samples to the culture plate, allowed the microorganisms to grow on fixed structures positioned in
Fig. 3. S. mutans biofilm formation (TSB, 48 h at 37 °C) over titanium surfaces, in the presence or absence of lactam, measured by colony forming units (CFU/ml) (Student's ttest with a significance level of p b 0.05).
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5. Conclusion
Fig. 4. S. mutans biofilm formation (TSB, 48 h at 37 °C) over titanium surfaces, in the presence or absence of lactam, measured by CV colorimetric method (Mann-Whitney Test with a significance level of p b 0.05).
Treatment of biofilms-associated oral diseases with traditional pharmacologic and systemic antibiotic therapies lacks direct, localized activity, mostly due to low diffusion rate of the antibiotics through the entire biofilm biomass. This observation, along with the emerging global concern about bacterial resistance to antibiotics, is leading to a prompt shift of drug targets from bacterial survival to pathogenicity control, involving bacterial adherence to surfaces as well as signaling systems controlling bacterial group behavior of populations organized in biofilms. In the present study the potential of synthetic lactam (U27) as inhibitor of bacterial biofilm formation against S. mutans was tested. Results showed a significant decreased accumulation of the biofilm on the titanium surface in the presence of lactam U27, indicating that coating dental implant surfaces with such biofilm inhibitor compounds could be a promising solution for the prevention of initial biofilm adhesion on dental implant surfaces. Additional studies are currently being performed to analyze the effect of modified synthetic lactams on the development of multispecies biofilms over titanium surfaces.
Conflict of interest the polystyrene lid coincident with the culture wells. The use of this device enhances the homogeneity of sample treatment, facilitates their manipulation with greater accuracy, enhances reproducibility, thus favorizing the standardization of microbiological tests on titanium. It is important to note that results presented here were obtained using a monospecies biofilm as a model. Similar studies involving multispecies biofilms are currently underway in order to complement and further validate the potential use of lactams as biofilm inhibitors in implantable materials, under conditions that better mimic the oral environment.
The authors have no conflicts of interest to disclose. There were no sources of funding that could have influenced the outcome of this work.
Acknowledgments The authors acknowledge the financial support provided by the government research funding agencie CNPq (SET-B 350091/2016-1) (Brazil).
Fig. 5. Scanning Electron Micrograph (SEM) of titanium surfaces after exposition to S. mutans cultures on biofilm formation assay (48 h in TSB, 37 °C). S. mutans colonies are only visible on titanium surfaces in the absence of the biofilm inhibitor (A, B, C). (D) Lactam-treated titanium surface free of S. mutans colonies.
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