Controlling bacterial biofilms growth through photodynamic action

doi:10.21203/rs.3.rs-2247986/v1

Download PDF

Article

Controlling bacterial biofilms growth through photodynamic action

https://doi.org/10.21203/rs.3.rs-2247986/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Changes in microbial growth kinetics and biofilm formation capacity were evaluated in bacterial cells remaining from PDT. Curcumin (CUR) used as photosensitizer, shows penetration and photodynamic effects on biofilm layers, evaluated considering parameters such as the biofilm complexity (BC), and formation time (FT) of the biofilm. Microbial growth and biofilm reformulation capacity were modified in cells remaining after partial photodynamic action. The photosensitizer (PS) permeation per bacterial biofilm layer showed to be dependent on the FT and BC in addition to the incubation time (IT) of the PS. Post-PDT effects showed (1) a delay in the onset of the exponential phase; (2) limitation of the maximum microbial amount; (3) slowdown of the growth rate. The use of PDT shows to be promising for biofilm control remnants presents slowdown and modification on the growth patterns and penetration of CUR is facilitated through the complex structure of the biofilm.

Biological sciences/Microbiology/Antimicrobials

Biological sciences/Microbiology/Clinical microbiology

Anatomical evidence of mixed microbial biofilms on the surface and within the removed tonsillar crypts in recurrent pharyngotonsillitis PT is one of the explanations for treatment failures¹. The microbial biofilm contains metabolic products from microorganisms at different stages of development². The biofilm-associated cells clump together during maturation and communicate using chemical signals to protect and expand the microbial community³. The extracellular matrix formed (extracellular polysaccharides, DNA and proteins) provides an environment of low penetration of substances to the biofilm with up to 1000 times more described resistance to antibiotics when compared to planktonic cells ⁴.

The ability of Staphylococcus aureus to form biofilms in hosts emanates from chronic and recalcitrant diseases, being one of the most frequent pathogens in PT ^5,6. For the effectiveness of the treatment of infections containing biofilms, the association and complex use of antibiofilm techniques is necessary, which normally aim at: removal of infected tissues; selection of active and penetrating antibiotics as well as their combinations; administration of anti-quorum sensing ⁷, or application of biofilm dispersing agents. In the conventional treatment of bacterial infections, with the oral administration of antibiotics, the penetration of the drug into the multicellular biofilm is slow and incomplete, as it presents a complex and altered biochemical microenvironment ^8,9. Therefore, in view of this, the presence of persistent cells of microbial biofilms characterizes the chronicity of infections and constitute a greater difficulty in the treatment.

Photodynamic therapy (PDT) has been evaluated as a potential antimicrobial treatment for infections against this form of antimicrobial resistance (biofilms) as well as for multidrug resistant strains^10–12. Avoiding the reformulation of the biofilm based on the understanding of post-PDT effects on cells remaining from the treatment, as well as specifically delivering PS in different situations of biofilm development, that is, homogeneously, may circumvent situations of antimicrobial resistance.

Antimicrobial PDT in vitro, in vivo and clinical research studies, mainly in oral cavities of inflammation and infections, has been carried out^13–15. The evaluation of the effectiveness of this treatment for respiratory infections is in the ascendancy¹⁶. Initial studies on the treatment of respiratory tract infections are catching up to clinical research in the upper respiratory tract as in PT, and initial evidence of the effectiveness of the treatment is being verified¹⁷. For the photodynamic reaction to occur reactive oxygen species (ROS) are formed from two types of reactions: with the FSs transferring electrons to organic substrates, biomolecules or directly to molecular oxygen (type 1 reaction); or in the type II reaction transferring energy directly to molecular oxygen, exciting it to its highly reactive singlet state (1O₂). PDT studies for microbial disruption and death in biofilms using different PSs and light doses and conjugated to mechanical methods (sonification) have been widely described in the literature¹⁸. The PDT protocols used show that the complete removal of biofilms is difficult to be found, especially in tissues in vivo. Illumination, even with blue light at 450 nm (in vitro) of microbial biofilms during PDT, reaches the full thickness of the biofilm for the effectiveness of the in vitro process¹⁹. Therefore, this study was directed to create solutions in the face of technology gaps regarding biofilm formation by bacterial cells remaining from PDT, and to understand the penetration of curcumin (PS) in the optimization of the treatment. The understanding of curcumin permeation in biofilms, formed from bacterial cells remnants of PDT, to achieve an effective photodynamic action for translation in clinical research has not yet been described. As well as analyzing its antimicrobial potential in biofilm layers characterized by permeation in specific regions of this complex resistance system. The dynamics of the SF homogeneity of bacterial cells at different stages of development in the biofilm must be considered for the adequate treatment of infections using PDT.

Considering that in the PDT of infections located in human tissues, the total removal of microbial cells never occurs, the possibilities of restructuring the biofilm from remaining PDT cells were considering in this article. And even if these cells are able to restructure the biofilm, we described how we can deal with this situation. Therefore, in this study, we investigated the penetration and consumption of PS in biofilm layers under photodynamic effects and interrogated its relationship with the biofilm reforming power in structured microbial communities (Fig. 1).

2.1. Microrganisms

The microbial species used was S. aureus. The strains ATCC 25925, methicillin-resistant S. aureus (isolated from a clinical sample of a patient with pharyngotonsillitis-Brazil, USA300 ATCC TCH1516 [USA300-HOU-MR] and RN4220 obtained through BEI Resources, NIAID, NIH were used. These cultures evaluate the antimicrobial effects of photodynamic inactivation in planktonic cultures and biofilms. The cultures were stored in a -80°C freezer in broth (Brain Heart Infusion - BHI) at a concentration of 50% by final volume containing glycerol (40%). Bacteria (1 mL) grown and maintained in a nutrient medium (BHI) was cultivated for 16 h on 9 mL culture medium in a shaker (150–200 rpm), at 37 ± 0.1°C (pre-inoculum).

2.2. Growth curve

S. aureus cells from planktonic and biofilm culture post-PDT were pre-incubated under identical conditions to their initial culture for biofilm formation (item 2.3.1). Three colonies remaining from the treatment under the PDT conditions described above (item 2.2) for each condition (planktonic and biofilm) were isolated from Petri dishes containing BHI Agar after incubation at 37°C for 24 h and seeded in BHI broth at 37°C. Samples of 100 µL were taken and placed in 1 cm wide cuvettes and read in the UV-VIS Spectrophotometer. Measurements were performed every 60 min for 16 hours. Optical density calibration was performed using the growth curve by counting colonies in CFU/mL (y = 0.5033, R² = 0.995).

2.3. Biofilms

The microorganism was first separated from the pre-inoculum culture medium by centrifugation, performing successive (two) washes with PBS phosphate buffer followed by centrifugation at 5000 rpm and 15 min. Ninety percent of the liquid broth (BHI) and 10% of the inoculum (initial concentration of 10⁸ CFU/mL) were added to multiwell plates (1000 µL) and confocal dishes (100 µL) ²¹.Ninety percent of the liquid broth (BHI) and 10% of the inoculum (initial concentration of 10⁸ CFU/mL) were added to multiwell plates (1000 µL) and confocal dishes (100 µL). The solution was homogenized six times with a pipette of 1000 and 100 µL in each well for the different volumes. The biofilm formation time (FT) was 24 hours (time 1) and 48 hours (time 2). The culture medium was changed every 24 hours. The biofilms formed were characterized by bacterial colony count (CFU/mL).

2.4. Photodynamic inactivation

The stock solution of CUR [HOC6H3(OCH3) CH = CHCO]2CH2 (Sigma-Adrich) was prepared in 5 M ethyl alcohol and diluted in distilled water at the concentrations of interest. The experiments were composed of three control groups: control (bacteria), dark (bacteria + FS), light (bacteria + light); and PDT (bacteria + FS + light). For the PDT and dark groups, the bacteria were incubated with a concentration of 5mM CUR with a 15 min IT. The light and PDT groups were submitted to light doses at 450 nm with a total exposure of 70 mW/cm² and two rates of 40 J/cm² (PDT40) and 80 J/cm² (PDT80) using the 450 nm LED device (Biotable® - produced by MM Optics - Brazil)²⁰.For the PDT and dark groups, the bacteria were incubated with a concentration of 5mM CUR in 0.1% alcohol (from a standard solution of 5M) with a 15 min IT. The samples were seeded in a Petri dish containing BHI agar to count the colony-forming units per milliliter (CFU/ml). The optical density (OD) was measured at 660 nm in a spectrophotometer.

2.4.1. Multiple cycles

Biofilms from different conditions (mono and multispecies) were washed with 1 mL of PBS for 1 min to remove planktonic and PDT-killed bacteria. Then, the adhered surviving biofilm cells were resubmitted to optimal conditions for cell growth following sub-section 2.2. The biofilm received three doses of PDT every 24h, indicated as a second PDT after 48h and a third PDT session after 72h from the first PDT, thus simulating a treatment with three sessions of therapy with 24h intervals, observing the continuous growth of the cells that were not killed in the treatments.

2.5. Confocal microscopy

Confocal images were collected on an Olympus Fluoview 3000 microscope with an inverted microscope with Second Generation IX83 Zero Drift Compensator (IX3-ZDC2) laser-based autofocus with a 60× PlanApo (1.4 NA), 120 WD (µm) oil immersion objective. Excitation was from 405 and 488 nm solid-state Diode Lasers 50 and 20 mW Ar ion laser/fiber (Melles Griot, Tokyo, Japan).

2.6. Statistical analyses

Statistical analyses were performed by one-way analysis of variance (ANOVA) for rate and growth comparisons (control vs treatment) to assess the reduction in growth rate and biofilm formation capacity after PDT and differences between treatment control groups, respectively. Analyzes were performed using Qtiplot SPSS version 5.9.8. P values ≤ 0.05 were considered statistically significant.

The analysis of microbial growth provides a way of assessing metabolic viability by measuring multiplication rates. The rate of microbial multiplication over time determines the potential of an original cell to produce new offspring under ideal conditions of nutrition, temperature, pH, and oxygen. Post-treatment cells from planktonic S. aureus cultures (Fig. 2 (a)) and biofilm treatments (Fig. 2 (b)) were exposed to ideal conditions for their growth. Bacterial growth delay occurred in planktonic cells and post-PDT biofilms as they did not grow at the regular rate observed in the curves of untreated bacteria controls. We observed a 2h difference in the Lag phase between S. aureus (control) and the post-PDT group (80J/cm2) (henceforth, PDT80), which corresponds to the interval of low cell reproduction after the start of cultivation. However, this phase was a dynamic, organized, adaptable, and evolutionary period that prepares bacteria for cell division²². We observed that in both cases (planktonic and biofilm), the exponential phase (where the number of live cells was greater than dead cells) was maintained up to 8h; however, the microbial OD (nm) presented values of 0.10 and 0.078 for post-treatment PDT80 in planktonic cells and biofilms, respectively, i.e. 0.046 and 0.068 less than the control at its maximum point of inclination, 5h growth. The bacterial number per unit time calculated by the graph area was reduced from 1 to 0.25 and 0.18 (nm*t) at the maximum point of its exponential phase (8h).

The absorption of CUR using blue light (450 nm) was studied both so that its intended application is on the surface of tonsils and to spare the underlying tissues with photodamage using a light source of 70 mW/sq cm with an optimized light dose of 80 J/cm² in 20 min of lighting. The CUR irradiance and incubation time protocol can be adapted in clinical research [19] with a lighting device (tip) and can be designed for greater power, thus reducing the lighting time in patients [20].

The post-PDT effects showed different effects for planktonic bacteria or in biofilms: (1) a delay in the onset of the exponential phase with the increase of the lag phase in microbial growth; (2) limits the maximum microbial amount; (3) it slows down the growth rate. Post-PDT effects on planktonic bacteria cannot be extrapolated to cells in communities (biofilms). Therefore, an analysis of FS permeation and effects of subsequent PDT sessions on biofilms were evaluated.

The biofilm contains microbial cells in various stages of growth. Exponential phase cells are found in the surface layer, and dormant cells are found in in deeper layers (latent or stationary phase – Fig. 3 (V)). Biofilm regions chemically altered by the production of metabolites in the stress zone (I) make it difficult for antimicrobial substances to penetrate (Fig. 3 – a and c). Understanding how the penetration of CUR occurs by layer (Fig. 3 - b) and its consumption in these regions could help understand mechanisms of antimicrobial treatments. We performed a layered CUR localization study of the bacterial biofilms of two S. aureus strains (mono and multi-species) at two incubation times (5 and 45 min). In Fig. 3, we observe the penetration of CUR through layers 1 (d), 2 (e), 3 (f), and 4 (g) following 5 min of incubation, and layers 1 (h), 2 (i), 3 (j) and 4 (k) following 45 min of incubation for the simplest form of biofilm formed, denoted by X1 (single species (X) and X min for formation (1)).

PDT occurs with the absorption of blue light by CUR. We observed the formation of porosity in the bacterial biofilm resulting from PDT. Layer 2 of the biofilm represents the passage of CUR through the otherwise antimicrobial impermeant stress zone (Fig. 3 – I) (layer 1).

The quantification of CUR penetration by layers (1, 2, 3, and 4 referenced in Fig. 3 - b) was performed for four types of biofilms formed: only one strain of S. aureus (X) and two strains of S. aureus (XY) at two FT (1 and 2) referring to 24 hours (time 1) and 48 hours (time 2).

The absence of bacterial cells appeared white in Fig. 4 for X1 (a) and XY2 (b) for layer 2 on Fig. 4 (c) (layer 2 at 5 min) shows the reduction of post-PDT porosity for more complex biofilms with longer formation time (2). We observed that with less time for biofilm formation, there is a greater permeation in layers 1 and 2 and greater cellular penetrability with 5 min of CUR incubation (Fig. 4 (d)), quantified by the green pixels from its fluorescence region. The PS IT was dependent on the penetration response and showed an increase in layer 3 permeation by 45 min (Fig. 4. (e)). We observed that increasing biofilm formation time interferes with the CUR's permeation, with the reduction of cell incorporation from layer 1 to layer 4 in the first 5 min and increasing the homogeneity between layers 1, 2, and 3 in 45 min. The schemes (f) and (g) of Fig. 4 represent the trends of CUR permeation by layers (1 and 2 – superficial) and (3 and 4 – deep) of the biofilm represented by interference of the study variables with increases (↑), reductions (↓), and not influential (ࣧ). The reduction of the PS IT with the increase of the biofilm formation time (FT) maintained the CUR in the more superficial layers of the biofilm (Fig. 4 (f)). However, increasing the IT to 45 min increased the incorporation of CUR in layers 3 and 4, mainly for low biofilm BC with less TF.

In addition to studying the dynamics of curcumin in biofilms, we propose increasing the treatment dose in these systems. In Fig. 5 we observed the growth of biofilms with doses of PDT every 24h. The cells killed by the treatment were removed from the biofilm and resubmitted to ideal conditions for microbial growth. Reformation of the microbial biofilm was observed every 24h and then new treatments (new PDT sessions) were performed (24, 48, and 72h after the first session). For each microbial biofilm (different microorganisms) controls are presented in the absence of photosensitizer and light with the application of only light and only curcumin referred to as light and dark, respectively in the SI (Fig. 5). One PDT session was performed every 24 hours (3 sessions), which showed a way to avoid biofilm reform. One application of PDT (PS + lighting) corresponding to one treatment session (PDT). Three PDT (sequential sessions after 24h) were indicated in Fig. 5 and their microbial growth before each session was observed.

Breakdown of resistance in biofilms by PDT was evaluated in three subsequent sessions evaluating the recovery of the bacterial population 24h after each treatment. Bacterial quantification was performed by evaluating viable cells of S. aureus [Log (CFU/mL)] (black), MRSA (red), and S. aureus + MRSA (green). Control groups (bacteria only), light (bacteria and light), dark (bacteria and CUR), PDT (bacteria, CUR and light) and biofilm formation after 24h after each PDT session are shown in Fig. 5. More significant death effects were observed for S. aureus in mono-strain compared to multi-strains. We observed that repeated applications of PDT on biofilms prevented the biofilm from returning to its original form. Therefore, after each PDT the film grows less in 24h and in the case of biofilms containing MRSA more deaths were caused in subsequent sessions. The panel with the sequential PDT is slowing down biofilm growth.

The results presented demonstrate alteration of cellular mechanisms of post-PDT responses. This approach showed less adaptive changes under ideal microbial growth conditions ^23,24. The delay in microbial growth was observed in post-PDT conditions, which may result from metabolic deficiency effects ²⁵ due to the initial phase (lag phase) the bacteria are metabolically active but not dividing. In Fig. 2 (a and b), PDT surviving bacteria (biofilm (a) and planktonic (b)) show changes in the growth curve in terms of quantity and slope in the exponential phase, indicating failure in the recovery of viable cells.²⁶; however, it can decrease not only because of limited availability of nutrients but also because of decreases in cellular catabolic capacity. The cellular responses to reactive oxygen species, generated by PDT, are extracellular signals of oxidative and osmotic stress²⁷. These signals can eventually modify transitional regulatory systems, which affect physiological and morphological changes that do not allow organisms to adapt to multiplication rates effectively. Photodynamic activity is dependent on antimicrobial response factors such as the location of PS in the cell and the signaling network when in biofilms, which may interfere with the recovery of fragile bacteria ^28,29. PS typically interacts with lipids and proteins at the plasma membrane and cytoplasmic level, generating changes in the chemistry of DNA and RNA after light absorption³⁰. These perturbations present information that alters metabolism and culminate in adjustment mechanisms that can alter enzymatic regulation, which is fundamental in controlling the transcription of genes that regulate metabolism. The remaining effects of PDT that affect planktonic cells were not corresponding in biofilms, and the possibilities for this to occur are due to (1) distribution of PS; (2) oxygenation within the biofilm; (3) light penetration.

The cellular communication system between bacterial cells in different growth phases within the biofilm configures stress regions with altered environments that help to protect against antimicrobials. Therefore, incorporating PS by the microbial cell is the first step for antimicrobial photodynamic action. The cellular penetration of PS in biofilms is related to the permeation in different layers by depth. The first layer of a biofilm is a matrix of hydrated extracellular polymeric substances formed by polysaccharides, proteins, nucleic acids, and lipids, which collectively provide a low penetration environment for antimicrobials ^9,31. We observed with the porosity in the second layer of the biofilm (Fig. 4 – a and b) that with the rapid permeation of the exopolymeric matrix (layer 1) by CUR, oxidative stress induced by PDT was observed in the biofilm within 5 min of IT of the PS. The permeation dynamics were altered in microbial systems with the diversity of bacterial strains and their maturity. However, the time of biofilm formation was a variable that impedes the penetration of curcumin more than when compared to the number of strains. This stress may destabilize the cohesive and three-dimensional polymeric network that interconnects and temporarily immobilizes biofilm cells. We did not observe the desired permeability in the layer for more BC with 45 min of PS IT. The ability of light at 450 nm to penetrate multiple bacterial cell layers was described by Zangirolami et al. ¹⁹ and collaborated with the data in Fig. 4, where we observed the formation of porosity in layers 2 of 4 of the biofilms.

Antimicrobial PDT was used on biofilms in three sessions in Fig. 5, and we observed cellular recolonization at lower levels for post-PDT biofilm formation. Therefore, with the addition of PDT treatments (second and third session), it was possible to maintain a reduced number of microorganisms in the biofilm that could help immunological activity in cases of infections. Considering that biofilms can prevent some immune response by presenting physical barrier that reduce phagocytic uptake by immune cells, and thereby suppress the activation of cellular immune response regulators³², decreasing this barrier through PDT treatment may provide an elegant mechanism to facilitate immune recognition and defense. From this study, we propose to use the possibility of increasing the number of sessions in the treatment of pharyngotonsillitis for application in cases of bacterial plaques in the tonsils ³³. It is possible with sequential PDT to decrease biofilm growth.

In this study, we intend to raise theoretical bases to be adapted to the treatment of pharyngotonsillitis that has been carried out under the protocol with the regulation of the incubation time and insertion of new PDT sessions in cases of bacterial plaque in the tonsils.

The investigation of PS penetration and consumption in biofilm layers under photodynamic effects and questioning the relationship with the biofilm reforming power in microbial communities were found in this study. This could lead to antimicrobial applications in persistent cells of microbial biofilms, such as those that characterize the chronicity of PTs, which constitutes a greater difficulty in the treatment. The use of PDT may therefore alter future clinical study protocols for enhancing PS penetration into biofilms as well as using strategies to prevent biofilm reformation from PDT remnant cells.

Funding: The authors acknowledge the financial support of the Cepid/FAPESP Proc. Nº 13/07276-1 and CNPq/INCT Proc. Nº 573587/2008-6). Blanco K. C. thanks FAPESP for Post-doc grant 2019/13569-8 and 2021/09952-0. Components of this work were supported by the Texas A&M Clinical Science Translational Research Institute Pilot Grant CSTR2016-1, the Defense Advanced Research Projects Agency (DARPA) (HR001118A0025-FoF-FP-006), NIH (R21AI139738-01A1, 1R01AI141607-01A1, 1R21GM132705-01), and the National Science Foundation (DBI 1532188, NSF0854684). The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.

Data Availability Statement: We declare the availability of all data that supported all analyzes related to this study.

Competing Interests: The Authors declare no Competing Financial or Non-Financial Interests

Author Contributions: Conceptualization, Kate C. Blanco, Vladislav V Yakovlev and Vanderlei S. Bagnato; methodology, Kate C. Blanco, Jace A. Willis; formal analysis, Kate C. Blanco; investigation, Kate C. Blanco; resources, Jace A. Willis, Paul de Figueiredo, Vladislav V Yakovlev, Vanderlei S. Bagnato; data curation, Kate C. Blanco, Jace A. Willis, writing—original draft preparation, Kate C. Blanco, Jace A. Willis, Paul de Figueiredo, Vladislav V Yakovlev, Vanderlei S. Bagnato; writing—review and editing, Kate C. Blanco, Jace A. Willis, Paul de Figueiredo, Vladislav V Yakovlev, Vanderlei S. Bagnato; supervision, Vladislav V Yakovlev, Vanderlei S. Bagnato; project administration, Vladislav V Yakovlev, Vanderlei S. Bagnato; funding acquisition, Paul de Figueiredo, Vladislav V Yakovlev, Vanderlei S. Bagnato. All authors have read and agreed to the published version of the manuscript

Chole, R. A. & Faddis, B. T. Anatomical Evidence of Microbial Biofilms in Tonsillar Tissues. Arch. Otolaryngol. Neck Surg. (2003) doi:10.1001/archotol.129.6.634.
Penesyan, A., Paulsen, I. T., Kjelleberg, S. & Gillings, M. R. Three faces of biofilms: A microbial lifestyle, a nascent multicellular organism, and an incubator for diversity. npj Biofilms Microbiomes 7, 1–9 (2021).
Andersen, J. B. et al. Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms. npj Biofilms Microbiomes 7, (2021).
Nadell, C. D., Drescher, K., Wingreen, N. S. & Bassler, B. L. Extracellular matrix structure governs invasion resistance in bacterial biofilms. ISME J. 9, 1700–1709 (2015).
Cavalcanti, V. P. et al. Staphylococcus aureus in tonsils of patients with recurrent tonsillitis: prevalence, susceptibility profile, and genotypic characterization. Brazilian J. Infect. Dis. 23, 8–14 (2019).
Archer, N. K. et al. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence 2, 445–459 (2011).
Mion, S. et al. Disrupting quorum sensing alters social interactions in Chromobacterium violaceum. npj Biofilms Microbiomes 7, 1–16 (2021).
Stewart, P. S. et al. Conceptual model of biofilm antibiotic tolerance that integrates phenomena of diffusion, metabolism, gene expression, and physiology. J. Bacteriol. 201, e00307-19 (2019).
Sharma, D., Misba, L. & Khan, A. U. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob. Resist. Infect. Control 8, 1–10 (2019).
Zangirolami, A. C. et al. Avoiding ventilator-associated pneumonia: Curcumin-functionalized endotracheal tube and photodynamic action. Proc. Natl. Acad. Sci. U. S. A. 117, 22967–22973 (2020).
Willis, J. A. et al. Photodynamic viral inactivation: Recent advances and potential applications. Appl. Phys. Rev. 8, 021315 (2021).
Willis, J. A. et al. Breaking down antibiotic resistance in methicillin-resistant Staphylococcus aureus: Combining antimicrobial photodynamic and antibiotic treatments. Proc. Natl. Acad. Sci. 119, e2208378119 (2022).
Vatansever, F. et al. Antimicrobial strategies centered around reactive oxygen species - bactericidal antibiotics, photodynamic therapy, and beyond. FEMS Microbiology Reviews at https://doi.org/10.1111/1574-6976.12026 (2013).
Idris, N. M. et al. In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers. Nat. Med. 18, 1580–1585 (2012).
Talebi, M., Taliee, R., Mojahedi, M., Meymandi, M. & Torshabi, M. Microbiological efficacy of photodynamic therapy as an adjunct to non-surgical periodontal treatment: A clinical trial. J. Lasers Med. Sci. 7, (2016).
Kassab, G. et al. Safety and delivery efficiency of a photodynamic treatment of the lungs using indocyanine green and extracorporeal near infra-red illumination. J. Biophotonics (2020) doi:10.1002/jbio.202000176.
Blanco, K. C., Inada, N. M., Carbinatto, F. M., Giusti, A. L. & Bagnato, V. S. Treatment of recurrent pharyngotonsillitis by photodynamic therapy. Photodiagnosis and Photodynamic Therapy at https://doi.org/10.1016/j.pdpdt.2017.01.187 (2017).
Fan, L., Muhammad, A. I., Ismail, B. B. & Liu, D. Sonodynamic antimicrobial chemotherapy: An emerging alternative strategy for microbial inactivation. Ultrason. Sonochem. 75, 105591 (2021).
Zangirolami, A. C., Carbinatto, F., Vollet Filho, J. D., Bagnato, V. S. & Blanco, K. C. Impact of light-activated curcumin and curcuminoids films for catheters decontamination. Colloids Surfaces B Biointerfaces 213, 112386 (2022).
Kate C. Blanco, Natalia M. Inada, Fernanda M. Carbinatto & Vanderlei S. Bagnato. Antimicrobial Efficacy of Curcumin Formulations by Photodynamic Therapy. J. Pharm. Pharmacol. 5, 506–511 (2017).
Soares, J. M., Silva, K. O. O., Inada, N. M., Bagnato, V. S. & Blanco, K. C. Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation. Photodiagnosis Photodyn. Ther. 29, (2020).
Bertrand, R. L. Lag phase is a dynamic, organized, adaptive, and evolvable period that prepares bacteria for cell division. J. Bacteriol. 201, e00697-18 (2019).
Peleg, M. & Corradini, M. G. Microbial growth curves: what the models tell us and what they cannot. Crit. Rev. Food Sci. Nutr. 51, 917–945 (2011).
Maier, R. M. & Pepper, I. L. Bacterial growth. in Environmental microbiology 37–56 (Elsevier, 2015).
Wang, J. D. & Levin, P. A. Metabolism, cell growth and the bacterial cell cycle. Nat. Rev. Microbiol. 7, 822–827 (2009).
Mason, C. A. & Egli, T. Dynamics of microbial growth in the decelerating and stationary phase of batch culture. in Starvation in bacteria 81–102 (Springer, 1993).
Finkel, T. Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15 (2011).
Madsen, J. S., Burmølle, M., Hansen, L. H. & Sørensen, S. J. The interconnection between biofilm formation and horizontal gene transfer. FEMS Immunol. Med. Microbiol. 65, 183–195 (2012).
Mahalingam, S. M., Ordaz, J. D. & Low, P. S. Targeting of a photosensitizer to the mitochondrion enhances the potency of photodynamic therapy. ACS omega 3, 6066–6074 (2018).
Castano, A. P., Demidova, T. N. & Hamblin, M. R. Mechanisms in photodynamic therapy: Part two - Cellular signaling, cell metabolism and modes of cell death. Photodiagnosis and Photodynamic Therapy at https://doi.org/10.1016/S1572-1000(05)00030-X (2005).
Flemming, H.-C. & Wingender, J. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633 (2010).
González, J. F., Hahn, M. M. & Gunn, J. S. Chronic biofilm-based infections: skewing of the immune response. Pathog. Dis. 76, fty023 (2018).
BLANCO, K. C.; SANTOS, S. S. ; BAGNATO, V. S. Turmeric and LED in the treatment of sore throat.https://www.isrctn.com/ISRCTN14862781?q=&filters=&sort=&offset=117&totalResults=19417&page=2&pageSize=100&searchType=basic-search (2020) doi:https://doi.org/10.1186/ISRCTN14862781.

(Not answered)

Download PDF

Version 1

posted

You are reading this latest preprint version

Controlling bacterial biofilms growth through photodynamic action

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Microrganisms

2.2. Growth curve

2.3. Biofilms

2.4. Photodynamic inactivation

2.4.1. Multiple cycles

2.5. Confocal microscopy

2.6. Statistical analyses

3. Results

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Status:

Version 1