Light-activated conjugated polymer nanoparticles to defeat pathogens associated with bovine mastitis.

Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (∼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (∼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.

Complete photodynamic inactivation of Pseudomonas aeruginosa biofilm with use of potassium iodide and its comparison with enzymatic pretreatment.

Pseudomonas aeruginosa, a gram-negative bacterium, accounts for 7% of all hospital-acquired infections. Despite advances in medicine and antibiotic therapy, P. aeruginosa infection still results in high mortality rates of up to 62% in certain patient groups. This bacteria is also known to form biofilms, that are 10 to 1000 times more resistant to antibiotics compared to their free-floating counterparts. Photodynamic Inactivation (PDI) has been proved to be an effective antimicrobial technique for microbial control. This method involves the incubation of the pathogen with a photosensitizer (PS), then, a light at appropriated wavelength is applied, leading to the production of reactive oxygen species that are toxic to the microbial cells. Studies have focused on strategies to enhance the PDI efficacy, such as a pre-treatment with enzymes to degrade the biofilm matrix and/or an addition of inorganic salts to the PS. The aim of the present study is to evaluate the effectiveness of PDI against P. aeruginosa biofilm in association with the application of the enzymes prior to PDI (enzymatic pre-treatment) or the addition of potassium iodide (KI) to the photosensitizer solution, to increase the inactivation effectiveness of the treatment. First, a range of enzymes and PSs were tested, and the best protocols for combined treatments were selected. The results showed that the use of enzymes as a pre-treatment was effective to reduce the total biomass, however, when associated with PDI, mild bacterial reductions were obtained. Then, the use of KI in association with the PS was evaluated and the results showed that, PDI mediated by methylene blue (MB) in the presence of KI was able to completely eradicate the biofilm. However, when the PDI was performed with curcumin and KI, no additive reduction was observed. In conclusion, out of all strategies evaluated in the present study, the most promising strategy to improve PDI against P. aeruginosa biofilm was the use of KI in association with MB, resulting in eradication with 108 log bacterial inactivation.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for the septic mortality of burn patients. Objective: High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections and will be investigated in this study. Methods: Antibacterial effects of a light-emitting diode array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540 J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: aBL reduced the bacterial number [2.51-3.56 log10 colony-forming unit (CFU)/mL], whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. aBL reduced biofilm formation by 60-66%. PRT or CA treatment showed reductions by 25% or 13%. Here, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extend than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

Assessment of multispecies biofilm growth on root canal dentin under different radiation therapy regimens.

OBJECTIVES: To assess the growth of a multispecies biofilm on root canal dentin under different radiotherapy regimens. MATERIALS AND METHODS: Sixty-three human root dentin cylinders were distributed into six groups. In three groups, no biofilm was formed (n = 3): NoRT) non-irradiated dentin; RT55) 55 Gy; and RT70) 70 Gy. In the other three groups (n = 18), a 21-day multispecies biofilm (Enterococcus faecalis, Streptococcus mutans, and Candida albicans) was formed in the canal: NoRT + Bio) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. The biofilm was quantified (CFUs/mL). Biofilm microstructure was assessed under SEM. Microbial penetration into dentinal tubules was assessed under CLSM. For the biofilm biomass and dentin microhardness pre- and after biofilm growth assessments, 45 bovine dentin specimens were distributed into three groups (n = 15): NoRT) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. RESULTS: Irradiated specimens (70 Gy) had higher quantity of microorganisms than non-irradiated (p = .010). There was gradual increase in biofilm biomass from non-irradiated to 55 Gy and 70 Gy (p < .001). Irradiated specimens had greater reduction in microhardness after biofilm growth. Irradiated dentin led to the growth of a more complex and irregular biofilm. There was microbial penetration into the dentinal tubules, regardless of the radiation regimen. CONCLUSION: Radiotherapy increased the number of microorganisms and biofilm biomass and reduced dentin microhardness. Microbial penetration into dentinal tubules was noticeable. CLINICAL RELEVANCE: Cumulative and potentially irreversible side effects of radiotherapy affect biofilm growth on root dentin. These changes could compromise the success of endodontic treatment in oncological patients undergoing head and neck radiotherapy.

Biofilm formation by Listeria monocytogenes from the meat processing industry environment and the use of different combinations of detergents, sanitizers, and UV-A radiation to control this microorganism in planktonic and sessile forms.

This study aimed to evaluate the ability of biofilm formation by L. monocytogenes from the meat processing industry environment, as well as the use of different combinations of detergents, sanitizers, and UV-A radiation in the control of this microorganism in the planktonic and sessile forms. Four L. monocytogenes isolates were evaluated and showed moderate ability to form biofilm, as well as carried genes related to biofilm production (agrB, agrD, prfA, actA, cheA, cheY, flaA, sigB), and genes related to tolerance to sanitizers (lde and qacH). The biofilm-forming isolates of L. monocytogenes were susceptible to quaternary ammonium compound (QAC) and peracetic acid (PA) in planktonic form, with minimum inhibitory concentrations of 125 and 75 ppm, respectively, for contact times of 10 and 5 min. These concentrations are lower than those recommended by the manufacturers, which are at least 200 and 300 ppm for QAC and PA, respectively. Biofilms of L. monocytogenes formed from a pool of isolates on stainless steel and polyurethane coupons were subjected to 14 treatments involving acid and enzymatic detergents, QAC and PA sanitizers, and UV-A radiation at varying concentrations and contact times. All treatments reduced L. monocytogenes counts in the biofilm, indicating that the tested detergents, sanitizers, and UV-A radiation exhibited antimicrobial activity against biofilms on both surface types. Notably, the biofilm formed on polyurethane showed greater tolerance to the evaluated compounds than the biofilm on stainless steel, likely due to the material's surface facilitating faster microbial colonization and the development of a more complex structure, as observed by scanning electron microscopy. Listeria monocytogenes isolates from the meat processing industry carry genes associated with biofilm production and can form biofilms on both stainless steel and polyurethane surfaces, which may contribute to their persistence within meat processing lines. Despite carrying sanitizer tolerance genes, QAC and PA effectively controlled these microorganisms in their planktonic form. However, combinations of detergent (AC and ENZ) with sanitizers (QAC and PA) at minimum concentrations of 125 ppm and 300 ppm, respectively, were the most effective.

Efficacy of a novel remotely-generated ultrasonic root canal irrigation system for removing biofilm-mimicking hydrogel from a simulated isthmus model.

AIM: To evaluate the efficacy of a novel ultrasonic irrigation device, remotely-generated irrigation with a non-invasive sound field enhancement (RINSE) system, in removing biofilm-mimicking hydrogel from a simulated isthmus model and compare it with sonically- and ultrasonically-activated irrigation systems. METHODOLOGY: A polycarbonate root canal model containing two standardized root canals (apical diameter of 0.20 mm, 4% taper, 18 mm long with a coronal reservoir) connected by three isthmuses (0.40 mm deep, 2 mm high, 4 mm long) was used as the test model. The isthmuses were filled with a hydroxyapatite powder-containing hydrogel. The canals were filled with irrigant, and the models were randomly assigned to the following activation groups (n = 15): EndoActivator (EA), ultrasonically activated irrigation (UAI), and RINSE system (RS). Syringe irrigation (SI) with a 30G needle served as the control. Standardized images of the isthmuses were taken before and after irrigation, and the amount of hydrogel removed was determined using image analysis software and compared across groups using anova (p < .05). RESULTS: Hydrogel removal was significantly higher with the RS (83.7%) than with UAI, EA, or SI (p ≤ .01). UAI (69.2%) removed significantly more hydrogel than SI and EA (p < .05), while there was no significant difference between SI (24.3%) and EA (25.7%) (p = .978). CONCLUSIONS: RINSE system resulted in the most hydrogel removal, performing better than UAI or EA. The effect of RS was also not reliant on the insert or tip entering the pulp chamber or root canal, making it particularly useful in conservative endodontics.

Photothermally responsive chitosan-coated iron oxide nanoparticles for enhanced eradication of bacterial biofilms.

This work developed a pH/NIR responsive antibacterial agent (CS-FeNPs) composed of chitosan (CS) and Fe3O4 nanoparticles (FeNPs). CS triggers bacterial attraction through surface charge, while Fe acts as a photothermal agent (PTA). The CS-Fe NPs exhibited antibacterial and antibiofilm activity against both bacteria (G+/G-). However, higher activity was observed against bacteria (G-) due to electrostatic interactions. The CS-FeNPs bind with the bacterial membrane through electrostatic interactions and disturb bacterial cells. Later, in an acidic environment, CS-FeNPs bind with bacterial membrane, and NIR irradiation leads the antibacterial activity. CS-FeNPs exhibited a potential photothermal conversion efficiency (η) of 21.53 %. Thus, it converts NIR irradiation into heat to kill the bacterial pathogen. The CS-FeNPs were found to be less cytotoxic with great antibacterial efficiency on planktonic bacteria and their biofilm, which indicates that they deserve to develop potential and safe treatment strategies for the treatment of bacterial infections.

Organic Photo-antimicrobials: Principles, Molecule Design, and Applications.

The emergence of multi-drug-resistant pathogens threatens the healthcare systems world-wide. Recent advances in phototherapy (PT) approaches mediated by photo-antimicrobials (PAMs) provide new opportunities for the current serious antibiotic resistance. During the PT treatment, reactive oxygen species or heat produced by PAMs would react with the cell membrane, consequently leaking cytoplasm components and effectively eradicating different pathogens like bacteria, fungi, viruses, and even parasites. This Perspective will concentrate on the development of different organic photo-antimicrobials (OPAMs) and their application as practical therapeutic agents into therapy for local infections, wound dressings, and removal of biofilms from medical devices. We also discuss how to design highly efficient OPAMs by modifying the chemical structure or conjugating with a targeting component. Moreover, this Perspective provides a discussion of the general challenges and direction for OPAMs and what further needs to be done. It is hoped that through this overview, OPAMs can prosper and will be more widely used for microbial infections in the future, especially at a time when the global COVID-19 epidemic is getting more serious.

UV and bacteriophages as a chemical-free approach for cleaning membranes from anaerobic bioreactors.

Anaerobic membrane bioreactor (AnMBR) for wastewater treatment has attracted much interest due to its efficacy in providing high-quality effluent with minimal energy costs. However, membrane biofouling represents the main bottleneck for AnMBR because it diminishes flux and necessitates frequent replacement of membranes. In this study, we assessed the feasibility of combining bacteriophages and UV-C irradiation to provide a chemical-free approach to remove biofoulants on the membrane. The combination of bacteriophage and UV-C resulted in better log cells removal and ca. 2× higher extracellular polymeric substance (EPS) concentration reduction in mature biofoulants compared to either UV-C or bacteriophage alone. The cleaning mechanism behind this combined approach is by 1) reducing the relative abundance of Acinetobacter spp. and selected bacteria (e.g., Paludibacter, Pseudomonas, Cloacibacterium, and gram-positive Firmicutes) associated with the membrane biofilm and 2) forming cavities in the biofilm to maintain water flux through the membrane. When the combined treatment was further compared with the common chemical cleaning procedure, a similar reduction on the cell numbers was observed (1.4 log). However, the combined treatment was less effective in removing EPS compared with chemical cleaning. These results suggest that the combination of UV-C and bacteriophage have an additive effect in biofouling reduction, representing a potential chemical-free method to remove reversible biofoulants on membrane fitted to an AnMBR.

Photoinactivation of Pseudomonas aeruginosa Biofilm by Dicationic Diaryl-Porphyrin.

In recent years, antimicrobial photodynamic therapy (aPDT) has received increasing attention as a promising tool aimed at both treating microbial infections and sanitizing environments. Since biofilm formation on biological and inert surfaces makes difficult the eradication of bacterial communities, further studies are needed to investigate such tricky issue. In this work, a panel of 13 diaryl-porphyrins (neutral, mono- and di-cationic) was taken in consideration to photoinactivate Pseudomonas aeruginosa. Among cationic photosensitizers (PSs) able to efficiently bind cells, in this study two dicationic showed to be intrinsically toxic and were ruled out by further investigations. In particular, the dicationic porphyrin (P11) that was not toxic, showed a better photoinactivation rate than monocationic in suspended cells. Furthermore, it was very efficient in inhibiting the biofilms produced by the model microorganism Pseudomonas aeruginosa PAO1 and by clinical strains derived from urinary tract infection and cystic fibrosis patients. Since P. aeruginosa represents a target very difficult to inactivate, this study confirms the potential of dicationic diaryl-porphyrins as photo-activated antimicrobials in different applicative fields, from clinical to environmental ones.

Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro.

Root canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

Development of Antimicrobial Phototreatment Tolerance: Why the Methodology Matters.

Due to rapidly growing antimicrobial resistance, there is an urgent need to develop alternative, non-antibiotic strategies. Recently, numerous light-based approaches, demonstrating killing efficacy regardless of microbial drug resistance, have gained wide attention and are considered some of the most promising antimicrobial modalities. These light-based therapies include five treatments for which high bactericidal activity was demonstrated using numerous in vitro and in vivo studies: antimicrobial blue light (aBL), antimicrobial photodynamic inactivation (aPDI), pulsed light (PL), cold atmospheric plasma (CAP), and ultraviolet (UV) light. Based on their multitarget activity leading to deleterious effects to numerous cell structures-i.e., cell envelopes, proteins, lipids, and genetic material-light-based treatments are considered to have a low risk for the development of tolerance and/or resistance. Nevertheless, the most recent studies indicate that repetitive sublethal phototreatment may provoke tolerance development, but there is no standard methodology for the proper evaluation of this phenomenon. The statement concerning the lack of development of resistance to these modalities seem to be justified; however, the most significant motivation for this review paper was to critically discuss existing dogma concerning the lack of tolerance development, indicating that its assessment is more complex and requires better terminology and methodology.

Surface barrier discharges for Escherichia coli biofilm inactivation: Modes of action and the importance of UV radiation.

Cold plasma generated in air at atmospheric pressure is an extremely effective antimicrobial agent, with proven efficacy against clinically relevant bacterial biofilms. The specific mode of bacterial inactivation is highly dependent upon the configuration of the plasma source used. In this study, the mode of microbial inactivation of a surface barrier discharge was investigated against Escherichia coli biofilms grown on polypropylene coupons. Different modes of exposure were considered and it was demonstrated that the long-lived reactive species created by the plasma are not solely responsible for the observed microbial inactivation. It was observed that a synergistic interaction occurs between the plasma generated long-lived reactive species and ultraviolet (UV) photons, acting to increase the antimicrobial efficacy of the approach by an order of magnitude. It is suggested that plasma generated UV is an important component for microbial inactivation when using a surface barrier discharge; however, it is not through the conventional pathway of direct DNA damage, rather through the synergistic interaction between liquid in the biofilm matrix and long-lived chemical species created by the discharge.

Potentiated antimicrobial blue light killing of methicillin resistant Staphylococcus aureus by pyocyanin.

As antimicrobial resistance continues to threaten the efficacy of conventional antibiotic therapy, it is paramount that we investigate innovative approaches to treat infectious diseases. In this study, we investigated the antimicrobial capabilities of the innovative combination of antimicrobial blue light (aBL; 405 nm wavelength) with the Pseudomonas aeruginosa pigment pyocyanin against methicillin resistant Staphylococcus aureus (MSRA. We explored the effects of different radiant exposures of aBL and increasing concentrations of pyocyanin against planktonic cells and those within biofilms. In addition, we investigated the effect of the aBL/pyocyanin on the endogenous staphyloxanthin pigment, as well as the role of hydrogen peroxide and singlet oxygen scavenging in the efficacy of this combination. Lastly, we investigated the potential for the aBL/pyocyanin to reduce the MRSA burden within a proof-of-principle mouse abrasion infection model. We found pyocyanin to be a powerful potentiator of aBL activity under all in vitro conditions tested. In addition, we serendipitously discovered the capability of the aBL/pyocyanin combination to bleach staphyloxanthin within colonies of MRSA. Furthermore, we established that singlet oxygen is an important mediator during combined aBL/pyocyanin exposure. Moreover, we found that the combination of aBL and pyocyanin could significantly reduce the viability of MRSA within a proof-of-principle early onset MRSA skin abrasion infection. Exposure to the treatment did not have deleterious effects on skin tissue. In conclusion, the combination of aBL and pyocyanin represents a potentially powerful therapeutic modality for the treatment of infections caused by MRSA.

Targeted Photodynamic Treatment of Bacterial Biofilms Using Curcumin Encapsulated in Cells and Cell Wall Particles.

Efficient inactivation and removal of pathogenic biofilms in food and biomedical environments remain a significant challenge for food safety applications and medical facilities. This research aims to develop food-grade microcarriers for the targeted delivery of a photosensitizer, curcumin, and photodynamic inactivation of a model pathogenic bacterial biofilm. The microcarriers evaluated in this study include alive yeast cell carriers, deactivated yeast cell carriers, and yeast cell wall particles. The microcarriers were evaluated based on the encapsulation yield of a model photosensitizer (curcumin), binding of the microcarriers to biofilms, and inactivation of the bacteria in the biofilms. The results illustrate that the combination of binding affinity, encapsulation yield, and the intracellular composition of the microcarriers influenced the overall inactivation of bacteria in the biofilms. All of the selected compositions achieved more than 93% inactivation of the bacteria in the biofilm using the photodynamic treatment, and the yeast cell wall particles with curcumin achieved over 99% inactivation of the bacteria in the biofilm matrix. In addition, all of the selected compositions demonstrated significant potential to remove the biofilm from the plastic surface, suggesting the role of binding affinity of the microcarriers in removal of the biofilm from surfaces. Overall, this study developed biomaterial formulations for targeted photodynamic inactivation and potential removal of biofilms.

(-)-Tetrahydroberberrubine∙acetate accelerates antioxidant potential and inhibits food associated Bacillus cereus in rice.

A protoberberine alkaloid, (-)-tetrahydroberberrubine∙acetate (THBA) was assessed for its antioxidant potential and ability to inhibit the growth of a food hazard bacterium Bacillus cereus in vitro and in situ. THBA displayed significant and dose-dependent cellular antioxidant potential against hydrogen peroxide-induced oxidative stress in NIH 3T3 fibroblast cells and decreased the ROS levels as well as increased the expression levels of SOD1 and SOD2 enzymes. The inhibitory spectrum of THBA confirmed its mechanistic role in the disruption of the membrane integrity of B. cereus as evidenced by the results of time-inactivation, cell membrane integrity, NPN membrane uptake, membrane potential, and electron microscopy analyses. Moreover, THBA inhibited biofilm formation by B. cereus and disrupted pre-established biofilms on a glass surface. Furthermore, THBA was also able to inhibit B. cereus in raw rice with a significant amount of reduction in CFU counts, suggesting its potential role as a natural antioxidant and antimicrobial agent.

Evidence-Based Review of Antibiofilm Agents for Wound Care.

Significance: Biofilms in vivo are small densely packed aggregations of microbes that are highly resistant to host immune responses and treatment. They attach to each other and to nearby surfaces. Biofilms are difficult to study and identify in a clinical setting as their quantification necessitates the use of advanced microscopy techniques such as confocal laser scanning microscopy. Nonetheless, it is likely that biofilms contribute to the pathophysiology of chronic skin wounds. Reducing, removing, or preventing biofilms is thus a logical approach to help clinicians heal chronic wounds. Recent Advances: Wound care products have demonstrated varying degrees of efficacy in destroying biofilms in in vitro and preclinical models, as well as in some clinical studies. Critical Issues: Controlled studies exploring the beneficial role of biofilm eradication and its relationship to healing in patients with chronic wounds are limited. This review aims to discuss the mode of action and clinical significance of currently available antibiofilm products, including surfactants, dressings, and others, with a focus on levels of evidence for efficacy in disrupting biofilms and ability to improve wound healing outcomes. Future Directions: Few available products have good evidence to support antibiofilm activity and wound healing benefits. Novel therapeutic strategies are on the horizon. More high-quality clinical studies are needed. The development of noninvasive techniques to quantify biofilms will facilitate increased ease of research about biofilms in wounds and how to combat them.

Antimicrobial photodynamic therapy against Propionibacterium acnes biofilms using hypericin (Hypericum perforatum) photosensitizer: in vitro study.

Acne vulgaris is the most recurring skin condition in the world, causing great harm to the physical and psychological well-being of many patients. Antimicrobial photodynamic therapy (aPDT) has broad therapeutic applicability. The purpose was to evaluate in vitro the photodynamic inactivation against Propionibacterium acnes (P. acnes) biofilms by using different concentrations of hypericin (Hypericum perforatum) photosensitizer associated with different energies of low-level laser. The biofilms were placed in 96-well microplates with a 6.4-mm diameter surface, by using standard suspensions (2 × 107 CFU/mL) and grown in brain heart infusion broth (BHI) for 48 h in anaerobic chamber. Subsequently, the control group received application of 0.9% sterile saline solution for 3 min; the photosensitising groups received hypericin at concentrations of 5 and 15 µg/mL for 3 min; the laser groups received irradiation of energies of 3 and 5 J (660 nm, continuous output, 100 mW, 30 and 50 s and 100 J/cm2 and 166 J/cm2, respectively); the aPDT groups received 5 and 15 µg/mL concentrations of hypericin associated with energies of 3 and 5 J of low-level laser irradiation. After the biofilms were broken up and seeded for CFU counting. The results showed a reduction in P. acnes biofilms after aPDT emphasising that 15 µg/mL hypericin associated with 3 and 5 J laser irradiation reduced biofilms by 14.1 and 27.9%, respectively. In addition, all groups of aPDT demostrated statistically significant reductions. In vitro photodynamic inactivation against P. acnes biofilms using different concentration of hypericin photosensitizer associated with different energies of low-level laser promoted effective antimicrobial action.

Efficacy of antimicrobial photodynamic therapy with Rose Bengal and blue light against cariogenic bacteria.

OBJECTIVE: We evaluated the effect of antimicrobial photodynamic therapy (a-PDT) with Rose Bengal and blue light LED on bacteria that initiate and promote dental caries. DESIGN: Colony forming units of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis, and Lactobacillus salivarius under planktonic and biofilm conditions were counted after a-PDT treatment using Rose Bengal and blue light LED. In addition, cariogenic bacteria from saliva and dental plaques from ten volunteers were used for evaluation of a-PDT treatment. RESULTS: We found that a-PDT using Rose Bengal at > 10 µg/mL had antimicrobial effects on oral Gram-positive S. mutans, S. sobrinus, S. sanguinis, and L. salivarius under both planktonic and biofilm conditions. The effect was also observed for cariogenic bacteria that formed biofilms containing water-insoluble glucans, through which the bacteria are firmly attached to the tooth surface. Moreover, a-PDT led to a marked reduction in cariogenic bacteria in saliva and dental plaques. CONCLUSION: a-PDT could be a useful approach for controlling dental caries in dental surgery.

Light Energy Dose and Photosensitizer Concentration Are Determinants of Effective Photo-Killing against Caries-Related Biofilms.

Caries-related biofilms and associated complications are significant threats in dentistry, especially when biofilms grow over dental restorations. The inhibition of cariogenic biofilm associated with the onset of carious lesions is crucial for preventing disease recurrence after treatment. This in vitro study defined optimized parameters for using a photosensitizer, toluidine blue O (TBO), activated via a red light-emitting diode (LED)-based wireless device to control the growth of cariogenic biofilms. The effect of TBO concentrations (50, 100, 150, and 200 µg/mL) exposed to light or incubated in the dark was investigated in successive cytotoxicity assays. Then, a mature Streptococcus mutans biofilm model under sucrose challenge was treated with different TBO concentrations (50, 100, and 150 µg/mL), different light energy doses (36, 108, and 180 J/cm2), and different incubation times before irradiation (1, 3, and 5 min). The untreated biofilm, irradiation with no TBO, and TBO incubation with no activation represented the controls. After treatments, biofilms were analyzed via S. mutans colony-forming units (CFUs) and live/dead assay. The percentage of cell viability was within the normal range compared to the control when 50 and 100 µg/mL of TBO were used. Increasing the TBO concentration and energy dose was associated with biofilm inhibition (p < 0.001), while increasing incubation time did not contribute to bacterial elimination (p > 0.05). Irradiating the S. mutans biofilm via 100 µg/mL of TBO and ≈180 J/cm2 energy dose resulted in ≈3-log reduction and a higher amount of dead/compromised S. mutans colonies in live/dead assay compared to the control (p < 0.001). The light energy dose and TBO concentration optimized the bacterial elimination of S. mutans biofilms. These results provide a perspective on the determining parameters for highly effective photo-killing of caries-related biofilms and display the limitations imposed by the toxicity of the antibacterial photodynamic therapy's chemical components. Future studies should support investigations on new approaches to improve or overcome the constraints of opportunities offered by photodynamic inactivation of caries-related biofilms.