Microscopic entities, microorganisms that drastically affect human health need to be thoroughly investigated. A biofilm is an architectural colony of microorganisms, within a matrix of extracellular polymeric substance that they produce. Biofilm contains microbial cells adherent to one-another and to a static surface (living or non-living). Bacterial biofilms are usually pathogenic in nature and can cause nosocomial infections. The National Institutes of Health (NIH) revealed that among all microbial and chronic infections, 65% and 80%, respectively, are associated with biofilm formation. The process of biofilm formation consists of many steps, starting with attachment to a living or non-living surface that will lead to formation of micro-colony, giving rise to three-dimensional structures and ending up, after maturation, with detachment. During formation of biofilm several species of bacteria communicate with one another, employing quorum sensing. In general, bacterial biofilms show resistance against human immune system, as well as against antibiotics. Health related concerns speak loud due to the biofilm potential to cause diseases, utilizing both device-related and non-device-related infections. In summary, the understanding of bacterial biofilm is important to manage and/or to eradicate biofilm-related diseases. The current review is, therefore, an effort to encompass the current concepts in biofilm formation and its implications in human health and disease.

Bacterial biofilm and associated infections

Photodynamic therapy (PDT), also known as photoradiation therapy, phototherapy, or photochemotherapy, involves the use of a photoactive dye (photosensitizer) that is activated by exposure to light of a specific wavelength in the presence of oxygen. The transfer of energy from the activated photosensitizer to available oxygen results in the formation of toxic oxygen species, such as singlet oxygen and free radicals. These very reactive chemical species can damage proteins, lipids, nucleic acids, and other cellular components. Applications of PDT in dentistry are growing rapidly: the treatment of oral cancer, bacterial and fungal infection therapies, and the photodynamic diagnosis (PDD) of the malignant transformation of oral lesions. PDT has shown potential in the treatment of oral leukoplakia, oral lichen planus, and head and neck cancer. Photodynamic antimicrobial chemotherapy (PACT) has been efficacious in the treatment of bacterial, fungal, parasitic, and viral infections. The absence of genotoxic and mutagenic effects of PDT is an important factor for long-term safety during treatment. PDT also represents a novel therapeutic approach in the management of oral biofilms. Disruption of plaque structure has important consequences for homeostasis within the biofilm. Studies are now leading toward selective photosensitizers, since killing the entire flora leaves patients open to opportunistic infections. Dentists deal with oral infections on a regular basis. The oral cavity is especially suitable for PACT, because it is relatively accessible to illumination.

Photodynamic Therapy in Dentistry

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

https://tandfonline.com/doi/pdf/10.1080/1040841X.2016.1208146

Critical review on biofilm methods

Nanoparticles are normally considered to be of a size no greater than 100 nm, and the exploitation of their unique attributes to combat infection has increased markedly over the past decade. The potential of nanoparticles to control the formation of biofilms within the oral cavity, as a function of their biocidal, anti-adhesive, and delivery capabilities, is now coming under close scrutiny. Possible uses as constituents of prosthetic device coatings, as topically applied agents, and within dental materials are being explored. The latest insights into the application of nanoparticles in the control of oral infections, including their use in photodynamic therapy, will be discussed in this review. In particular, the use of nanoparticulate silver, copper, zinc, silicon, and their oxides will be considered in relation to their effects on bacterial populations. The recent interest in the applications of nanoparticulate polymers and calcium phosphates will also be assessed. Particular attention will be paid to the toxicity issues surrounding the potential impact of nanoparticles on oral and other tissues.

The Use of Nanoparticles to Control Oral Biofilm Formation

New antibacterial strategies are required in view of the increasing resistance of bacteria to antibiotics. One promising technique involves the photodynamic inactivation of bacteria. Upon exposure to light, a photosensitizer in bacteria can generate singlet oxygen, which oxidizes proteins or lipids, leading to bacteria death. To elucidate the oxidative processes that occur during killing of bacteria, Staphylococcus aureus was incubated with a standard photosensitizer, and the generation and decay of singlet oxygen was detected directly by its luminescence at 1,270 nm. At low bacterial concentrations, the time-resolved luminescence of singlet oxygen showed a decay time of 6 ± 2 μs, which is an intermediate time for singlet oxygen decay in phospholipids of membranes (14 ± 2 μs) and in the surrounding water (3.5 ± 0.5 μs). Obviously, at low bacterial concentrations, singlet oxygen had sufficient access to water outside of S. aureus by diffusion. Thus, singlet oxygen seems to be generated in the outer cell wall areas or in adjacent cytoplasmic membranes of S. aureus. In addition, the detection of singlet oxygen luminescence can be used as a sensor of intracellular oxygen concentration. When singlet oxygen luminescence was measured at higher bacterial concentrations, the decay time increased significantly, up to ≈40 μs, because of oxygen depletion at these concentrations. This observation is an important indicator that oxygen supply is a crucial factor in the efficacy of photodynamic inactivation of bacteria, and will be of particular significance should this approach be used against multiresistant bacteria.

https://www.pnas.org/content/pnas/104/17/7223.full.pdf

The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria

OBJECTIVES The purpose of this study was to evaluate the clinical plaque disclosing agent erythrosine as a photosensitizer in the photodynamic killing of the oral bacterium Streptococcus mutans grown as a biofilm. METHODS S. mutans biofilms of 200 microm thickness were grown in a constant-depth film fermenter. In addition to determining localization of the photosensitizer within biofilms using confocal laser scanning microscopy (CLSM), we compared the bacterial killing efficacy of erythrosine with that of two well-characterized photosensitizers, methylene blue (MB) and photofrin. Incubations were carried out with each photosensitizer (22 microM), and irradiation was for 15 min using a 400 W white light source. RESULTS The CLSM results showed that erythrosine is taken up into S. mutans biofilms, where it is associated with the biomass of the biofilm rather than the fluid-filled channels and voids. Comparison of the cell killing efficacy of erythrosine in S. mutans biofilms of different ages showed that erythrosine was 1-2 log(10) more effective at killing biofilm bacteria than photofrin and 0.5-1 log(10) more effective than MB. The results were statistically significant (P < 0.01). Photodynamic therapy (PDT) with all three photosensitizers was increasingly effective as biofilm age increased, suggesting that temporal changes in biofilm architecture and composition affect susceptibility to PDT. CONCLUSIONS PDT using erythrosine as photosensitizer shows excellent potential as a treatment for oral plaque biofilms.

Erythrosine is a potential photosensitizer for the photodynamic therapy of oral plaque biofilms

Biofilm is the predominant mode of life for bacteria and today it is implicated in numerous human diseases. A growing body of scientific and clinical evidence now exists regarding the presence of biofilm in wounds. This review summarizes the clinical experiences and in vivo evidence that implicate biofilm in delayed wound healing. The various mechanisms by which biofilm may impede healing are highlighted, including impaired epithelialization and granulation tissue formation, and reduced susceptibilities to antimicrobial agents and host defenses. Strategies to manage biofilm and encourage progression to wound healing are discussed; these include debridement and appropriate antimicrobial therapies which may be improved upon in the future with the emergence of anti-biofilm technologies.

/pdf/biofilm-delays-wound-healing-a-review-of-the-evidence-4t8uoprj1o.pdf

Biofilm delays wound healing: A review of the evidence

Objectives We aimed to increase the bacterial cell killing efficacy of erythrosine-mediated photodynamic therapy (PDT) of Streptococcus mutans biofilms by fractionating the delivered light dose into a series of shorter pulses. Methods S. mutans biofilms of 200 microm thickness were grown in a constant-depth film fermenter (CDFF). Biofilms were incubated with 22 microM erythrosine before being irradiated with white light for increasing periods of time. We also used light dose fractionation to deliver the same overall dose of light in a series of shorter pulses separated by dark periods. Bacterial cell killing as a result of each killing protocol was quantified by colony counting. Results A 2 log10 of bacterial cell killing was achieved with 5 min of continuous white light irradiation. For time periods longer than 5 min the amount of killing increased more slowly, which was probably due to photobleaching of the erythrosine. Fractionation of the light dose into 5 x 1 min doses separated by dark recovery periods of 5 min increased the amount of bacterial killing by 1 log10 compared with 5 min continuous irradiation. Further fractionation of the light dose into 10x 30 s doses separated by 2 min recovery periods resulted in 3.7 log10 of cell kill, an improvement of 1.7 log10 compared with the continuous irradiation protocol. Conclusions Erythrosine-mediated PDT of S. mutans biofilms can be further enhanced by fractionation of the applied light dose.

/pdf/enhancement-of-erythrosine-mediated-photodynamic-therapy-of-25ygrwgjke.pdf

Enhancement of erythrosine-mediated photodynamic therapy of Streptococcus mutans biofilms by light fractionation

Significance: Biofilms have been implicated in a variety of wound complications. Recent Advances: Research has confirmed that biofilms form in wounds of patients experiencing delayed healing and may be a precursor to infection. Critical Issues: Research into the strength of this association is still in its infancy. Is biofilm formation a cause of these complications, a step toward them, or a signal that unresolved factors injuring tissue or delaying healing are setting the stage for biofilm formation, infection, and healing delay? To qualify biofilms for use in informing clinical practice decisions, biofilm characteristics supporting those decisions need standardized definitions and valid evidence that they predict or diagnose healing or infection outcomes. Literature searches of relevant terms reviewed biofilm definitions and validation of their role in predicting and diagnosing delayed wound healing or infection. Future Directions: Further research is needed to provide a rapid accurate technique to identify and characterize biofilms in ways that optimize their validity in diagnosing or screening patient risk of infection or delayed healing and to inform clinical decisions. This research will help validate biofilm's capacity to support wound care clinical practice decisions and establish their importance in guiding clinical practice.

/pdf/clinical-biofilms-a-challenging-frontier-in-wound-care-1yihjrufa7.pdf

Clinical Biofilms: A Challenging Frontier in Wound Care.

Recognition of the existence of biofilm in chronic wounds is increasing among wound care practitioners, and a growing body of evidence indicates that biofilm contributes significantly to wound recalcitrance. While clinical guidelines regarding the involvement of biofilm in human bacterial infections have been proposed, there remains uncertainty and lack of guidance towards biofilm presence in wounds. The intention of this report is to collate knowledge and evidence of the visual and indirect clinical indicators of wound biofilm, and propose an algorithm designed to facilitate clinical recognition of biofilm and subsequent wound management practices.

Daniel G. Metcalf

Papers

Erythrosine is a potential photosensitizer for the photodynamic therapy of oral plaque biofilms

Biofilm delays wound healing: A review of the evidence

Enhancement of erythrosine-mediated photodynamic therapy of Streptococcus mutans biofilms by light fractionation

Clinical Biofilms: A Challenging Frontier in Wound Care.

A clinical algorithm for wound biofilm identification