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.

Segmentation of hyphae and yeast in fungi-infected tissue slice images and its application in analyzing antifungal blue light therapy.

Candida albicans is a pathogenic fungus that undergoes morphological transitions between hyphal and yeast forms, adapting to diverse environmental stimuli and exhibiting distinct virulence. Existing research works on antifungal blue light (ABL) therapy have either focused solely on hyphae or neglected to differentiate between morphologies, obscuring potential differential effects. To address this gap, we established a novel dataset of 150 C. albicans-infected mouse skin tissue slice images with meticulously annotated hyphae and yeast. Eleven representative convolutional neural networks were trained and evaluated on this dataset using seven metrics to identify the optimal model for segmenting hyphae and yeast in original high pixel size images. Leveraging the segmentation results, we analyzed the differential impact of blue light on the invasion depth and density of both morphologies within the skin tissue. U-Net-BN outperformed other models in segmentation accuracy, achieving the best overall performance. While both hyphae and yeast exhibited significant reductions in invasion depth and density at the highest ABL dose (180 J/cm2), only yeast was significantly inhibited at the lower dose (135 J/cm2). This novel finding emphasizes the importance of developing more effective treatment strategies for both morphologies.

We studied the effects of blue light therapy on hyphal and yeast forms of Candida albicans. Through image segmentation techniques, we discovered that the changes in invasion depth and density differed between these two forms after exposure to blue light.

Photoinactivation of microorganisms using bacteriochlorins as photosensitizers.

In recent years, some microorganisms have shown resistance to conventional treatments. Considering this increase in resistant pathogens, treatment alternatives are needed to promote greater treatment efficiency. In this sense, antimicrobial photodynamic therapy (aPDT) has been an alternative treatment. This technique uses a photosensitizer that is activated by light with a specific wavelength producing reactive species, leading to the death of pathogenic microorganisms. In this study, bacteriochlorophyll derivatives such as bacteriochlorin metoxi (Bchl-M) and bacteriochlorin trizma (Bchl-T) obtained from purple bacterium (Rhodopseudomonas faecalis), were evaluated as photosensitizers in the aPDT. Photodynamic inactivation (PDI) of the microorganisms Staphylococcus aureus, Micrococcus luteus, Candida albicans and Pseudomonas aeruginosa was investigated with both bacteriochlorins (Bchl-M and Bchl-T) at different concentrations (1, 15 and 30 µM for S. aureus; 1, 15, 30, 45, 60 and 75 µM for M. luteus; 30, 60, 90, 105, 120 and 150 µM for C. albicans; and 200 µM for P. aeruginosa) and different doses of light (20 and 30 J/cm2 for S. aureus and M. luteus; 30 and 45 J/cm2 for C. albicans; and 45 J/cm2 for P. aeruginosa) to inactivate them. Both photosensitizers showed good activation against S. aureus and for M. luteus, we observed the inactivation of these microorganisms at approximately 3 log, showing to be a good photosensitizers for these microorganisms.

In vitro evaluation of the cis-[Ru(phen)2(pPDIp)]2+⁎⁎ complex for antimicrobial photodynamic therapy against Sporothrix brasiliensis and Candida albicans.

BACKGROUND: Photodynamic therapy (PDT) activates a photosensitizer by visible light to generate cytotoxic oxygen species that lead to cell death. With proper illumination, PDT is often used in applications on superficial and sub-surface lesions. Sporotrichosis infection occurs by Sporothrix fungi which causes a skin wound, worsened by Candida albicans infections. This study investigated the photosensitizing efficiency of the Ru(phen)2(pPDIp)(PF6)2 complex, RupPDIp, against S. brasiliensis and C. albicans. MATERIAL AND METHODS: RupPDIp efficiency against these fungi was tested using 450 nm (blue light and 36 J/cm2) and 525 nm (green light, 25.2 J/cm2) at 0.05-20 µM concentrations. To ensure PDT effectiveness, control groups were tested in the absence and in the presence of RupPDIp under light irradiation and in the dark. RESULTS: RupPDIp eliminated both fungi at ≤5.0 µM. Green light showed the best results, eliminating S. brasiliensis and C. albicans colonies at RupPDIp 0.5 µM and 0.05 µM, respectively. CONCLUSION: RupPDIp is a promising photosensitizer in aPDT, eliminating 106 CFU/mL of both fungi at 450 nm and 525 nm, with lower light doses and concentrations when treated with the green light compared to the blue light.

Analyzing Efficacy and Safety of Anti-Fungal Blue Light Therapy via Kernel-Based Modeling the Reactive Oxygen Species Induced by Light.

OBJECTIVE: The goal of this study is to investigate the efficacy, safety, and mechanism of ABL for inactivating Candida albicans (C. albicans), and to determine the best wavelength for treating candida infected disease, by experimental measurements and dynamic modeling. METHODS: The changes in reactive oxygen species (ROS) in C. albicans and human host cells under the irradiation of 385, 405, and 415 nm wavelengths light with irradiance of 50 mW/cm2 were measured. Moreover, a kernel-based nonlinear dynamic model, i.e., nonlinear autoregressive with exogenous inputs (NARX), was developed and applied to predict the concentration of light-induced ROS, whose kernels were selected by a newly developed algorithm based on particle swarm optimization (PSO). RESULTS: The ROS concentration was increased respectively about 10-12 times in C. albicans and about 3-6 times in human epithelial cells by the ABL treatment with the same fluence of 90 J/cm2. The NARX models were respectively fitted to the data from the experiments on both types of cells. Besides, four different kernel functions, including Gaussian, Laplace, linear and polynomial kernels, were compared in their fitting accuracies. The errors with the Laplace kernel turned out to be only 0.2704 and 0.0593, as respectively fitted to the experimental data of the C. albicans and human host cells. CONCLUSION: The results demonstrated the effectiveness of the NARX modeling approach, and revealed that the 415 nm light was more effective as an anti-fungal treatment with less damage to the host cells than the 405 or 385 nm light. SIGNIFICANCE: The kernel-based NARX model identification algorithm offers opportunities for determining the effective and safe light dosages in treating various fungal infection diseases.

Latex membranes with methylene blue dye for antimicrobial photodynamic therapy.

The search for new materials that can be applied in the treatment of injured human tissues has led to the development of new dressings. Membranes have potential as dressing materials because they can be fitted to and interact with the tissue surface. In this study, we analyze the morphological properties and wettability of latex membranes, along with the incorporation of the photosensitizer methylene blue, in the context of the utility of the membranes in curative applications involving photodynamic therapy (PDT). It was observed that deposition of the photosensitizer into latex membranes increased both the surface roughness and wettability. Antifungal testing indicated that antimicrobial PDT assisted by the latex membranes incorporating methylene blue effectively inactivated Candida albicans.

Synthesis of Co3S4-SnO2/polyvinylpyrrolidone-cellulose heterojunction as highly performance catalyst for photocatalytic and antimicrobial properties under ultra-violet irradiation.

In this work, we present Co3S4-SnO2 supported polyvinylpyrrolidone-cellulose (PVPCS) nano-structure for Lidocaine degradation. The nanostructure was characterized by various techniques i.e. morphological and optical ones. The results have demonstrated that Co3S4-SnO2 nanocomposites were evenly supported on the PVPCS. Moreover, the photocatalysis performances of the catalysts were investigated under ultra-violet (UV) light irradiation. The nano-structure Co3S4-SnO2/PVPCS composite (98.72%) revealed the highest photocatalysis performance as compared to SnO2 nanoparticles, and Co3S4-SnO2 nanocomposites. The photo-stability of nano-structure Co3S4-SnO2/PVPCS composite was characterized using cyclic catalytic experimental. Results demonstrated a substantially stable performance of the nano-structure Co3S4-SnO2/PVPCS composite. The biological properties of Co3S4-SnO2/PVPCS composite were investigated through the antibacterial (versus Staphylococcus aureus, and Escherichia coli) and antifungal studies (Candida albicans). As the results declared, Co3S4-SnO2 nanocomposites have substantial biological properties as compared to SnO2 nanoparticles, and Co3S4-SnO2 nanocomposites.

Photodynamic inactivation of Candida albicans and Candida tropicalis with aluminum phthalocyanine chloride nanoemulsion.

The in vitro susceptibilities of Candida albicans and Candida tropicalis to Antimicrobial Photodynamic Treatment with aluminum phthalocyanine chloride in nanoemulsion (ClAlPc/NE) were investigated. PS concentration- and fluence-dependent cell survival after APDT were compared before and after unbound extracellular PS had been washed out. The PS uptake and its subcellular localization were also determined. Exposure to light in the absence of the PS and treatment with the PS in the absence of light did not kill the fungi. APDT with ClAlPc/NE resulted in a reduction of five orders of magnitude in viability for C. albicans and between four and five orders of magnitude for C. tropicalis. Washing the cells to remove unbound PS before light exposure did not impair fungal inactivation, suggesting that cell photosensitization was mainly carried out by cell bound ClAlPc. The degree of ClAlPc uptake was dependent on its concentration. Internalization of ClAlPc by C. albicans and C. tropicalis was confirmed by confocal fluorescence microscopy that showed the PS does not penetrate the nucleus and instead accumulates in specific regions of the cytoplasm. Our results show that incorporating the water-insoluble ClAlPc into a nanoemulsion leads to an efficient formulation capable of photoinactivating both Candida species.

Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans.

In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicansIMPORTANCECandida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

Tribenzoporphyrazines with dendrimeric peripheral substituents and their promising photocytotoxic activity against Staphylococcus aureus.

Infectious diseases constitute a serious problem for human health and life. Although many bacterial and fungal infections can be successfully cured by commonly used antibiotics, a new threat emerges in the form of microbial resistance. For this reason, researchers try to find not only new active pharmaceutical ingredients for conventional antibiotherapy but also try to develop new strategies of microbial inactivation. Photodynamic antimicrobial chemotherapy, which relies on reactive oxygen species generated in situ in the presence of a photosensitizer and with the light of an appropriate wavelength, is one of them. Porphyrazines have been considered as potential photosensitizers for anticancer and antimicrobial photodynamic therapy. In this study, three tribenzoporphyrazines with dendrimeric peripheral substituents were subjected to in vitro antimicrobial photocytotoxicity study. One magnesium(II) tribenzoporphyrazine with peripheral 3,5-bis(3,5-dimethoxybenzyloxy)benzylsulfanyl substituents was synthesized and subjected to physicochemical characterization using NMR, UV-Vis, and mass spectrometry techniques. In photochemical studies this molecule revealed moderate singlet oxygen generation ability (ΦΔDMF = 0.12, ΦΔDMSO = 0.13). The other two magnesium(II) tribenzoporphyrazines applied in the biological study were 4-[3,5-di(hydroxymethyl)phenoxy]butylsulfanyl-substituted tribenzoporphyrazine and 4-[3,5-bis(benzyloxy)benzyloxy]phenyl-substituted tribenzopyrazinoporphyrazine. For the assessment, three microbial strains were chosen: Gram-positive bacteria Staphylococcus aureus ATCC 25923, Gram-negative bacteria Escherichia coli ATCC 25922, and fungal strain Candida albicans ATCC 10231. Very high activity against Staphylococcus aureus at low 10-6 M concentration was recorded for magnesium(II) tribenzoporphyrazines with peripheral 3,5-bis(3,5-dimethoxybenzyloxy)benzylsulfanyl and 4-[3,5-di(hydroxymethyl)phenoxy]butylsulfanyl substituents with calculated log reductions of 4.4 and 4.8, respectively. It is worth noting that magnesium(II) tribenzoporphyrazine with 4-[3,5-di(hydroxymethyl)phenoxy]butylsulfanyl substituents revealed also 3.2 log reduction in bacterial growth at the concentration 10-7 M.

Antimicrobial Blue Light Inactivation of Microbial Isolates in Biofilms.

BACKGROUND AND OBJECTIVES: Biofilms cause more than 80% of infections in humans, including more than 90% of all chronic wound infections and are extremely resistant to antimicrobials and the immune system. The situation is exacerbated by the fast spreading of antimicrobial resistance, which has become one of the biggest threats to current public health. There is consequently a critical need for the development of alternative therapeutics. Antimicrobial blue light (aBL) is a light-based approach that exhibits intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the antimicrobial effect of this non-antibiotic approach against biofilms formed by microbial isolates of multidrug-resistant bacteria. STUDY DESIGN/MATERIALS AND METHODS: Microbial isolates of Acinetobacter baumannii, Candida albicans, Escherichia coli, Enterococcus faecalis, MRSA, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Proteus mirabilis were studied. Biofilms were grown in microtiter plates for 24 or 48 hours or in the CDC biofilm reactor for 48 hours and exposed to aBL at 405 nm (60 mW/cm2 , 60 or 30 minutes). The anti-biofilm activity of aBL was measured by viable counts. RESULTS: The biofilms of A. baumannii, N. gonorrhoeae, and P. aeruginosa were the most susceptible to aBL with between 4 and 8 log10 inactivation after 108 J/cm2 (60 mW/cm2 , 30 minutes) or 216 J/cm2 (60 mW/cm2 , 60 minutes) aBL were delivered in the microplates. On the contrary, the biofilms of C. albicans, E. coli, E. faecalis, and P. mirabilis were the least susceptible to aBL inactivation (-0.30, -0.24, -0.84, and -0.68 log10 inactivation, respectively). The same aBL treatment in biofilms developed in the CDC biofilm reactor, caused -1.68 log10 inactivation in A. baumannii and -1.74 and -1.65 log10 inactivation in two different strains of P. aeruginosa. CONCLUSIONS: aBL exhibits potential against pathogenic microorganisms and could help with the significant need for new antimicrobials in clinical practice to manage multidrug-resistant infections. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Quinine Improves the Fungicidal Effects of Antimicrobial Blue Light: Implications for the Treatment of Cutaneous Candidiasis.

BACKGROUND AND OBJECTIVE: Candida albicans is an opportunistic fungal pathogen of clinical importance and is the primary cause of fungal-associated wound infections, sepsis, or pneumonia in immunocompromised individuals. With the rise in antimicrobial resistance, it is becoming increasingly difficult to successfully treat fungal infections using traditional antifungals, signifying that alternative non-traditional approaches must be explored for their efficacy. STUDY DESIGN/MATERIALS AND METHODS: We investigated the combination of antimicrobial blue light (aBL) and quinine hydrochloride (Q-HCL) for improved inactivation of C. albicans, in vitro and in vivo, relative to either monotherapy. In addition, we evaluated the safety of this combination therapy in vivo using the TUNEL assay. RESULTS: The combination of aBL (108 J/cm2 ) with Q-HCL (1 mg/mL) resulted in a significant improvement in the inactivation of C. albicans planktonic cells in vitro, where a 7.04 log10 colony forming units (CFU) reduction was achieved, compared with aBL alone that only inactivated 3.06 log10 CFU (P < 0.001) or Q-HCL alone which did not result in a loss of viability. aBL + Q-HCL was also effective at inactivating 48-hour biofilms, with an inactivation 1.73 log10 CFU at the dose of 108 J/cm2 aBL and 1 mg/mL Q-HCL, compared with only a 0.73 or 0.66 log10 CFU by aBL and Q-HCL alone, respectively (P < 0.001). Transmission electron microscopy revealed that aBL + Q-HCL induced morphological and ultrastructural changes consistent with cell wall and cytoplasmic damage. In addition, aBL + Q-HCL was effective at eliminating C. albicans within mouse abrasion wounds, with a 2.47 log10 relative luminescence unit (RLU) reduction at the dose of 324 J/cm2 aBL and 0.4 mg/cm2 Q-HCL, compared with a 1.44 log10 RLU reduction by aBL alone. Q-HCL or nystatin alone did not significantly reduce the RLU. The TUNEL assay revealed some apoptotic cells before and 24 hours following treatment with aBL + Q-HCL. CONCLUSION: The combination of aBL + Q-HCL was effective at eliminating C. albicans both in vitro and in vivo. A comprehensive assessment of toxicity (cytotoxicity and genotoxicity) is required to fully determine the safety of aBL + Q-HCL therapy at different doses. In conclusion, the combination of aBL and Q-HCL may be a viable option for the treatment of cutaneous candidiasis. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Secretory Vesicle Clustering in Fungal Filamentous Cells Does Not Require Directional Growth.

During symmetry breaking, the highly conserved Rho GTPase Cdc42 becomes stabilized at a defined site via an amplification process. However, little is known about how a new polarity site is established in an already asymmetric cell-a critical process in a changing environment. The human fungal pathogen Candida albicans switches from budding to filamentous growth in response to external cues, a transition controlled by Cdc42. Here, we have used optogenetic manipulation of cell polarity to reset growth in asymmetric filamentous C. albicans cells. We show that increasing the level of active Cdc42 on the plasma membrane results in disruption of the exocyst subunit Sec3 localization and a striking de novo clustering of secretory vesicles. This new cluster of secretory vesicles is highly dynamic, moving by hops and jumps, until a new growth site is established. Our results reveal that secretory vesicle clustering can occur in the absence of directional growth.

Characterizing the Antimicrobial Properties of 405 nm Light and the Corning® Light-Diffusing Fiber Delivery System.

BACKGROUND AND OBJECTIVES: Hospital-acquired infections (HAIs) and multidrug resistant bacteria pose a significant threat to the U.S. healthcare system. With a dearth of new antibiotic approvals, novel antimicrobial strategies are required to help solve this problem. Violet-blue visible light (400-470 nm) has been shown to elicit strong antimicrobial effects toward many pathogens, including representatives of the ESKAPE bacterial pathogens, which have a high propensity to cause HAIs. However, phototherapeutic solutions to prevention or treating infections are currently limited by efficient and nonobtrusive light-delivery mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Here, we investigate the in vitro antimicrobial properties of flexible Corning® light-diffusing fiber (LDF) toward members of the ESKAPE pathogens in a variety of growth states and in the context of biological materials. Bacteria were grown on agar surfaces, in liquid culture and on abiotic surfaces. We also explored the effects of 405 nm light within the presence of lung surfactant, human serum, and on eukaryotic cells. Pathogens tested include Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, and Escherichia coli. RESULTS: Overall, the LDF delivery of 405 nm violet-blue light exerted a significant degree of microbicidal activity against a wide range of pathogens under diverse experimental conditions. CONCLUSIONS: The results exemplify the fiber's promise as a non-traditional approach for the prevention and/or therapeutic intervention of HAIs. Lasers Surg. Med. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Gelatin nanoparticles loaded methylene blue as a candidate for photodynamic antimicrobial chemotherapy applications in Candida albicans growth.

Gelatin nanoparticles (GN) with an intrinsic antimicrobial activity maybe a good choice to improve the effectiveness of photodynamic antimicrobial chemotherapy (PACT). The aim of this study was to development gelatin nanoparticles loaded methylene blue (GN-MB) and investigate the effect of GN-MB in the Candida albicans growth by PACT protocols. The GN and GN-MB were prepared by two-step desolvation. The nanoparticulate systems were studied by scanning electron microscopy and steady-state techniques, the in vitro drug release was investigated, and we studied the effect of PACT on C. albicans growth. Satisfactory yields and encapsulation efficiency of GN-MB were obtained (yield = 76.0% ± 2.1 and EE = 84.0% ± 1.3). All the spectroscopic results presented here showed excellent photophysical parameters of the studied drug. Entrapment of MB in GN significantly prolongs it's in vitro release. The results of PACT experiments clearly demonstrated that the photosensitivity of C. albicans was higher when GN-MB was used. Gelatin nanoparticles loaded methylene blue-mediated photodynamic antimicrobial chemotherapy may be used against Candida albicans growth.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J·cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Fractal analysis and mathematical models for the investigation of photothermal inactivation of Candida albicans using carbon nanotubes.

Candida albicans is responsible for the majority of nosocomial infections affecting immunocompromised patients. Systemic antifungals may promote microbial resistance, which has led to the search for alternative treatments, such as photothermal therapy (PTT). PTT assumes that the interaction of electromagnetic radiation with a photothermal agent generates heat that can lead to the destruction of tumor cells and the death of microorganisms. Carbon nanotubes (CNTs) have the potential for applications in biomedical systems, including acting as controlled deliverers of drugs, biosensors and scaffolds for tissue engineering and regenerative medicine. Furthermore, the absorption of radiation by CNTs in the infrared region induces an increase in temperature, which makes CNTs candidates for photothermal agents. In this work, the photothermal inactivation of C. albicans was evaluated by multiple wall CNTs associated with laser radiation in the near-infrared region. The mechanisms that are involved in inactivation were evaluated through cell susceptibility studies and an analysis of microscopic images that are associated with mathematical models and fractal concepts. The results indicate that direct contact between the cells and CNTs without irradiation does not lead to cell death, whereas the laser-mediated process is effective in inactivation. The application of the laws of scale and fractal concepts indicate that in the control groups, there are two distinct regimes that are delimited by the mean diameter of the microorganisms, as described by the Eden model and by the quasi-Euclidean surface. For the irradiated groups, the surfaces present only one regime described by Kardar-Parisi-Zhang, KPZ. The analysis of the fractality of the system by mathematical models can help in the identification of new strategies for the inactivation of microorganisms.

In Vitro Effect of Er:YAG Laser on Different Single and Mixed Microorganisms Being Associated with Endodontic Infections.

Objective: The purpose of this in vitro study was to evaluate the antimicrobial effect of activated irrigation with different modes of erbium-doped yttrium aluminum garnet (Er:YAG) laser application on microorganisms related to secondary endodontic infection. Background: Er:YAG laser has been recommended as an adjuvant tool for root canal disinfection during endodontic treatment. Materials and methods: Laser-activated irrigation (LAI) with 300 or 600 µm tips were tested with or without intermittent irrigation with 0.9% sodium chloride (NaCl) solution against different microorganisms (five single strains and dual species (Streptococcus gordonii combined with Actinomyces oris or Fusobacterium nucleatum) in root canals after 3 days of incubation. In a 21-day infection model, LAI was used together with intermittent rinsing with sodium hypochlorite (NaOCl) against the dual-species mixtures; here the incidence of microbial regrowth after up to 7 days was monitored. Results: In the 3-day root infection model, LAI protocols did not show any significant reduction of the microbial load when compared with manual irrigation with saline solution. In the 21-day infection, S. gordonii combined with A. oris were not detectable anymore after applying the LAI protocol with a 600 µm tip (30 mJ/10 pps) up to 7 days after treatment. Conclusions: Application of LAI with a 600 µm tip by using an Er:YAG laser might be advantageous in treatment of endodontic infections.

Effect of Photodynamic Therapy on Multispecies Biofilms, Including Streptococcus mutans, Lactobacillus casei, and Candida albicans.

Objective: The aim of this study was to evaluate the effect of photodynamic therapy (PDT) on multispecies oral caries biofilms composed of Streptococcus mutans, Lactobacillus casei, and Candida albicans. Background: The abovementioned microorganisms largely cause dental caries, especially early childhood caries (ECC), by synthesizing of acids in the presence of sugar. PDT is considered an effective process to remove oral biofilms, and erythrosine, an oral bacterial disclosing agent, is an ideal dye that can be used as a photosensitizer in PDT. However, until now, there are no studies that have reported the effect of erythrosine-mediated PDT on biofilms, including the three microorganisms. Methods: The biofilms were formed on hydroxyapatite discs, and erythrosine was used as the photosensitizer, diluted to a concentration of 40 µM for 3 min. Light was irradiated for 10 and 20 sec using a blue light-emitting diode dental curing light. After the experiment, the colony-forming units of each microbial group cultured on blood agar plates were counted, and a confocal laser-scanning microscope was used to evaluate the effect of PDT. Results: The counts of all three microorganisms significantly decreased in the PDT group compared with those in the control group. For S. mutans and L. casei, there was a larger decrease proportional to the amount of energy irradiated. Conclusions: Overall, PDT showed a significant antimicrobial effect against oral biofilms composed of the three microorganisms, suggesting its potential clinical application for infants with ECC.

The photo-activated and photo-thermal effect of the 445/970 nm diode laser on the mixed biofilm inside root canals of human teeth in vitro: A pilot study.

AIMS: 1) Evaluation of the photo-thermal (PT) and photo-activated (PAD) antibacterial effect of the 445/970 nm diode laser on E. faecalis, S. aureus and C. albicans mixed biofilms grown together inside root canals of human teeth. 2) Defining a potentially efficient clinical protocol for safe and predictable usage in endodontic procedures. METHODOLOGY: The root canals of 100 extracted human teeth with single straight canals were prepared with ProTaper NEXT files, sterilized, contaminated with a combination of three cultures (E. faecalis, S. aureus, C. albicans) and incubated for 15 days. The samples were randomly distributed into three groups (n = 20) and treated as follows: Group 1 (G1) - the 445 nm photo-thermal (PT) effect, Group 2 (G2) - a combination of the 445 nm and 970 nm PT effect, Group 3 (G3) - the 445 nm photo-activated (PAD) effect with 0.1% riboflavin, Group 4 (G4) - a combination of 3% sodium hypochlorite (NaOCl) and the 445 nm PAD effect. Four samples were used as positive control (non-treated) and four as a negative control. 12 aditional samples were used as a control for the G4 (3% NaOCl rinse without the laser). The number of viable microbes in each canal was determined by the colony forming unit (CFU) count. RESULTS: A statistically significant reduction in the microbial population after all treatments was observed (P < 0.001). Groups 2 and 3 showed similar results, both better than Group 1. Group 4 produced the best results. CONCLUSIONS: The 445 nm PAD protocol has a stronger antimicrobial effect than the 445 nm PT protocol. Prolonged exposure time to laser light and a combination of wavelengths (445/970 PT protocol) helps in the reduction of microbes. C. albicans appears to be more sensitive to laser irradiation than the other bacteria tested in this study. Following current results, tested laser protocols could be recommended for clinical usage but only as an adjunct to "classic" NaOCl rinse since alone they are not able to completely eradicate all microorganisms.