Regimen and different surfaces interfere with photodynamic therapy on Candida albicans biofilms

https://doi.org/10.1016/j.mimet.2020.106080Get rights and content

Highlights

  • Photodynamic therapy efficacy depends on substrates where biofilm is formed

  • Repeated applications of aPDT interfered on the Candida ability to form hyphae

  • Candida biofilms formed on acrylic resin were susceptible to photodynamic therapy

Abstract

The aim of this study is to compare antimicrobial photodynamic therapy (aPDT) against Candida albicans biofilms formed on two different substrates - acrylic resin or bottom of polystyrene plate; and two aPDT application regimens – twice-daily over the course of 48 h or single treatment after 48 h biofilm formation. C. albicans SN425 biofilms cultivated on Roswell Park Memorial Institute medium were incubated for 5 min with toluidine blue O (44 μM) used as a photosensitizer before red light (635 nm; 175.2 J/cm2) exposure for 2 min. As negative control, ultrapure water, and as positive control 0.12% chlorhexidine (CHX) were used. Biofilms were analyzed for colony forming units (CFU) and cells morphology by confocal scanning laser microscopy. Single treatment and twice-daily aPDT on polystyrene plate and single treatment on acrylic resin did not significantly reduce the CFU (p > 0.05); in contrast, twice-daily aPDT on acrylic resin has reduced C. albicans below the detection limit, similarly to CHX treatment. Single aPDT treatment on polystyrene plate and on the resin presented a bulky and homogeneous biofilm predominantly formed by pseudohyphae. In contrast, in the resin group, the biofilm treated twice-daily with aPDT was predominantly formed by yeast cells, whilst pseudohyphae were occasionally visible. In conclusion, biofilms formed on polystyrene plates are more resistant to aPDT than biofilms formed on acrylic resin. Moreover, applying aPDT twice-daily reduces C. albicans biofilm development on acrylic resin and is a better approach against C. albicans biofilms than one single application on the mature biofilm.

Introduction

Candida albicans is usually a harmless commensal fungus present in the normal microbiota of oral cavity, gastroitestinal tract and vagina (Gulati and Nobile, 2016; Zarnowski et al., 2014), however, it may cause infections in immunocompromised situations due to its great adaptability to different host niches (Sardi et al., 2013). The major virulence of C. albicans is its ability to form biofilms that are organized communities of cells embedded in a matrix of extracellular polymers (Nobile et al., 2012). The development process of biofilm involves four basic stages: attachment and colonization of yeast-form on the surface; growth and proliferation of yeast-form cells; biofilm maturation with growth of pseudohyphae and extensive hyphae concomitant with the production of extracellular matrix material; and finally, biofilm dispersal and controlled detachment [4]. C. albicans frequently colonize dentures due to its capacity to adhere on inner surfaces and it may cause biofilm infections, such as denture stomatitis (DS) (Puryer, 2017).

Denture stomatitis is an inflammatory process of the mucosa commonly observed in denture wearers which is characterized by erythema of the oral mucosal tissues and it is rarely associated with severe pain or discomfort (Puryer, 2017; Yarborough et al., 2016). The treatments for DS involve good oral hygiene, denture cleaning procedures, topical or systemic antifungal agents, discontinuation of nocturnal denture wearing habit, and eventually denture replacement (Emami et al., 2014; Puryer, 2017). However, the treatment is challenging once high recurrence rates of infection have frequently been reported (Emami et al., 2014). Therefore, alternatives approaches to treat DS have been studied, such as antimicrobial Photodynamic Therapy (aPDT), either alone or in conjunction with other treatments. The aPDT is a phototherapy with an association between a nontoxic photosensitizer (PS) and a source of light capable to form singlet oxygen and reactive oxygen species, which may irreversibly affect the cell metabolic activities as well as damage essential cell components, such as the cytoplasmic membrane, resulting in death (de Freitas et al., 2017; Fumes et al., 2018; Huang et al., 2018).

While there are several in vitro investigations (Costa et al., 2012; de Figueiredo Freitas et al., 2017; Dovigo et al., 2013; Matthes De Freitas-Pontes et al., 2014; Panariello et al., 2019a; Panariello et al., 2019b; Pellissari et al., 2016) assessing the efficacy of aPDT against C. albicans, there is not a standardized biofilm model to study the effects of aPDT on the denture materials. An effective comparison between results from different studies is difficult due to the lack of a standardized model and can easily lead to dubious conclusions.

Among diverse biofilm growth conditions, such as static or flowing environment, single species or multi species models, concentration of yeast used in the initial inoculum, growth media and substrate, the last one plays an important role in the attachment process that may influence the aPDT efficacy (Donlan, 2002). It is common to see studies using the bottom of polystyrene plates as a substrate to grow C. albicans biofilms and assess aPDT efficacy (Costa et al., 2012; Dovigo et al., 2013; Panariello et al., 2019a; Panariello et al., 2019b; Pellissari et al., 2016), and there are protocols that use biofilm models on acrylic resin to simulate DS conditions[11,12], considering acrylic resins are the material used to make dentures. However, little is known about how different in vitro C. albicans biofilm models can interfere with aPDT results. Treatment regimen is also a factor to consider when applying aPDT. Therefore, the aims of this study are (1) to compare the aPDT outcomes in cell viability of C. albicans biofilms formed on the bottom of polystyrene plates or on acrylic resin; and (2) to compare the effects of two different regimens of aPDT on C. albicans biofilms, when applied only once after 48 h biofilm formation or twice-daily over the course of 48 h.

Section snippets

Experimental design

In this experimental in vitro study different substrates and treatment regimens were tested to learn how they interfere in the aPDT effects against C. albicans biofilms. The bottom of polystyrene plates or acrylic resin surfaces were used for the biofilm growth and the aPDT was applied only once at the end of the experimental period or twice – daily during the experiment (Fig. 1). The biofilms formed in both substrates were evaluated for colony forming units count (CFU/mL) and cell morphology

CFU

A significant interactions between treatment regimens – twice daily and single treatment – and different treatments – negative control, CHX and aPDT – were observed by the two-way ANOVA in acrylic resin substrate (p < 0.0001) and in polystyrene bottom plates (p < 0.0001). Both treatment regimens, only once at the end of the experimental period or twice-daily aPDT application on biofilms formed on the bottom of polystyrene plates did not significantly reduce the CFU/mL (p > 0.05). Chlorhexidine

Discussion

Based on the inconsistency of results seen in different C. albicans biofilm in vitro models in the literature mostly related to the efficacy of antimicrobial treatments, the present study focused on comparing aPDT on biofilms formed on two different substrates, acrylic resin or bottom of polystyrene plate, as well as two aPDT application regimens once at the end of biofilm formation or twice-daily during the course of biofilm formation.

When the acrylic resin was used as substrate, one-time aPDT

Conclusion

In conclusion, biofilms formed on the bottom of polystyrene plates are more resistant to aPDT than biofilms formed on acrylic resin. Moreover, applying aPDT twice-daily is a better approach to reduce C. albicans biofilm formation on acrylic resin than applying aPDT when the biofilm is already formed.

Acknowledgment

We thank Dr. Alexander D. Johnson, Department of Microbiology and Immunology, UCSF, for his kind donation of the strains used in this study.

Funding

This study was supported in part by the Coordination of Improvement of Higher Education Personnel (Capes) – Brazil. Finance code - 88881.189555/2018–01.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Bruna Albuquerque Garcia and Beatriz Helena Dias Panariello. The first draft of the manuscript was written by Bruna Albuquerque Garcia and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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      However, there was more variability as far as the type and concentrations photosensitizers were concerned. Nevertheless, majority of the photosensitizers were effective in reducing the Candida count following exposure to light [19,29–33,35–50]. Given that, to date, there is insufficient evidence to ascertain the optimal type and the most effective concentration of photosensitizer.

    1

    Departament of Oral Biology, College of Dentistry University of Florida. 1395 Center Dr., Gainesville, FL 32610, USA.

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