Rodrigues, C.F.

PharmD and Researcher

The Effectiveness of Voriconazole in Therapy of Candida glabrata’s Biofilms Oral Infections and Its Influence on the Matrix Composition and Gene Expression

Rodrigues CF; Gonçalves B; Rodrigues ME; Silva S, Azeredo J, Henriques M. Mycopathologia. 2017 Apr 24. doi: 10.1007/s11046-017-0135-7.
Candida glabrata is one of most prevalent yeast in fungal infections, especially in immunocompromised patients. Its azole resistance results in a low therapeutic response, particularly when associated with biofilms. The main goal of this work was to study the effectiveness of voriconazole (Vcz) against C. glabrata biofilms oral pathologies, as esophageal or oropharyngeal candidiasis. Antifungal susceptibilities were determined in pre-formed 24-h-biofilms and ERG genes expression was determined by qRT-PCR. Protein quantification was performed using BCA® Kit, carbohydrate was estimated according to the Dubois assay and β-1,3 glucans concentration were determined using Glucatell® kit. Finally, ergosterol, Vcz, and fluconazole (Flu) concentrations within the biofilm matrices were determined by RP-HPLC. Results showed that C. glabrata biofilms were more susceptible to Vcz than to Flu and that ERG genes expression evidenced an overexpression of the three ERG genes in the presence of both azoles. The matrix content presented a remarked decrease in proteins and an increase in carbohydrates, namely β-1,3 glucans. Ergosterol was successfully detected and quantified in the biofilm matrices, with no differences in all the considered conditions. Vcz demonstrated better diffusion through the biofilms and better cell penetration capacities, than Flu, indicating that the structure of the drug molecule fully influences its dissemination through the biofilm matrices. This work showed that Vcz is notably more effective than Flu for the treatment of resistant C. glabrata oral biofilms, which demonstrates a clinical relevance in its future use for the treatment of oropharyngeal/esophageal candidiasis caused by this species.


Candida glabrata Biofilms: How Far Have We Come?

Rodrigues CF; Rodrigues ME; Silva S; Henriques M. Journal of Fungi, 2017, 3(11) 
Infections caused by Candida species have been increasing in the last decades and can result in local or systemic infections, with high morbidity and mortality. After Candida albicans, Candida glabrata is one of the most prevalent pathogenic fungi in humans. In addition to the high antifungal drugs resistance and inability to form hyphae or secret hydrolases, C. glabrata retain many virulence factors that contribute to its extreme aggressiveness and result in a low therapeutic response and serious recurrent candidiasis, particularly biofilm formation ability. For their extraordinary organization, especially regarding the complex structure of the matrix, biofilms are very resistant to antifungal treatments. Thus, new approaches to the treatment of C. glabrata’s biofilms are emerging. In this article, the knowledge available on C. glabrata’s resistance will be highlighted, with a special focus on biofilms, as well as new therapeutic alternatives to control them.


Candida Species Biofilms' Antifungal Resistance

Silva S; Rodrigues CF; Araújo D; Rodrigues ME; Henriques M. Journal of Fungi, 2017, 3(8).

Candida infections (candidiasis) are the most prevalent opportunistic fungal infection on humans and, as such, a major public health problem. In recent decades, candidiasis has been associated to Candida species other than Candida albicans. Moreover, biofilms have been considered the most prevalent growth form of Candida cells and a strong causative agent of the intensification of antifungal resistance. As yet, no specific resistance factor has been identified as the sole responsible for the increased recalcitrance to antifungal agents exhibited by biofilms. Instead, biofilm antifungal resistance is a complex multifactorial phenomenon, which still remains to be fully elucidated and understood. The different mechanisms, which may be responsible for the intrinsic resistance of Candida species biofilms, include the high density of cells within the biofilm, the growth and nutrient limitation, the effects of the biofilm matrix, the presence of persister cells, the antifungal resistance gene expression and the increase of sterols on the membrane of biofilm cells. Thus, this review intends to provide information on the recent advances about Candida species biofilm antifungal resistance and its implication on intensification of the candidiasis.

Oral mucositis caused by Candida glabrata biofilms: failure of the concomitant use of fluconazole and ascorbic acid

Rodrigues CF; Henriques M. Ther Adv Infect Dis. 2017 Jan; 4(1): 10–17. doi:  10.1177/2049936116684477
Objectives: Candida glabrata is becoming one of the most prevalent pathogenic yeasts in cases of oral diseases. Mucositis is an recurrent oral infection in immunocompromised patients, and the actual guidelines recommend the use of fluconazole (Flu) for many cases. However, the azole resistance by C. glabrata is renowned, causing a reduced therapeutic response, especially when it occurs in biofilms. In this study, we performed an in vitro evaluation of an alternative pharmacotherapy for C. glabrata biofilm infections, combining ascorbic acid (AA) with Flu. AA is recognized for degrading β-glucans, an important compound of the biofilm matrices, which prevent drug diffusion.
Materials and Methods: Thus, routine clinical 30 or 40mg/l doses of Flu were applied to C. glabrata biofilms simultaneously with 200 or 300mg/l of AA.
Results: The results showed that this combination effectively promoted the degradation of the biofilm network, but unfortunately, also stimulated the growth of the yeasts population due to release of several glucose monomers during β-glucans hydrolysis.
Conclusions: As a result, it was proven that, contrary to what happens in treatment of bacterial infection, AA should not be used together with Flu in the treatment of oral mucositis caused by Candida.


Candida glabrata's recurrent infections: biofilm formation during Amphotericin B treatment

Rodrigues CF; Silva S; Azerede J; Henriques M. Letters in Applied Microbiology 2016 Aug;63(2):77-81. doi: 10.1111/lam.12600.
Candida species are responsible for recurrent human infections, mostly in immunocompromised patients, due to their high vulnerability. Candida glabrata has a major role in systemic candidiasis and Amphotericin B (AmB), a hospital environment exclusive polyene, is frequently used to treat this disease. Lately, however, clinical evidences of Candida recurrent infections during these treatments are being described, probably due to biofilms (re)formation during this therapy. Thus, this work aims at inferring if C. glabrata biofilms are still being formed during AmB treatment. For that, C. glabrata biofilms were formed in the presence of AmB and analysed by dry weight. Matrix composition was analysed quantifying carbohydrates and, specifically, β-1,3 glucans. Results demonstrated that, although in a lesser extent, C. glabrata is able to develop biofilms in the presence of AmB, with a thick extracellular matrix, with an increase on carbohydrates, especially β-1,3 glucans. Therefore, it is confirmed that complex biofilms of C. glabrata can be formed during an AmB treatment.
This study shows new insights regarding recurrent candidiasis. The authors demonstrated that Amphotericin B did not totally prevent the development of biofilms during Candida glabrata's infection treatment and that the change in the biofilm matrices may have a high responsibility for the fail in the treatment of systemic candidiasis.

Detection and Quantification of Fluconazole Within Candida glabrata Biofilms

Rodrigues CF; Silva S; Azeredo J; Henriques M. Mycopathologia, 2015. DOI 10.1007/s11046-015-9862-9 
Candida infections are often associated with biofilms and consequent high resistance to most common drugs (e.g. azoles). These resistance mechanisms are not only associated with the biofilm yeast physiology, but also with the presence of a diffusional barrier imposed by the biofilm matrix; however, the real biochemical role of the biofilm components remains very unclear. So, in order to further clarify this issue, we intend to determine, for the first time, fluconazole in biofilms within both supernatants and matrices. Candida biofilms were formed in the presence of fluconazole, and it was recovered from both supernatant and matrix cell-free fractions. Then, high-pressure liquid chromatography was used to identify and quantify the amount of drug that was present in the two fractions. Moreover, this study also showed that the presence of fluconazole in both fractions indicated that the drug administrated did not completely reach the cells, so this phenomena can easily be associated with lower biofilm susceptibility, since the drug administered did not completely reach the cells.

Effects of fluconazole in Candida glabrata biofilms and its relation with ABC transporters genes expression

Fonseca E, Silva S, Rodrigues CF, Alves CT, Azeredo J, Henriques M. Biofouling. 2014;30(4):447-57. doi: 10.1080/08927014.2014.886108.
Candida glabrata has emerged as the second most prevalent fungal pathogen and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms present a high tolerance to antifungal agents used in fungal infection treatment. The mechanisms of biofilm tolerance to antifungal agents remain poorly understood. Thus, the aim of this study was to evaluate the effects of fluconazole (FLU) in the formation and control of C. glabrata biofilms and its relation with the expression of genes encoding for ABC transporters, CDR1, SNQ2, and PDR1. For that, Minimal inhibitory concentrations values for seven C. glabrata strains were determined and the FLU effect against C. glabrata biofilms evaluated by total biomass quantification and CFUsviable cells enumeration. Matrices from biofilms were analyzed in terms of proteins, carbohydrates and DNA content. ABC transporters genes expression was analyzed for quantitative real-time PCR. Additionally to the high amounts of proteins and carbohydrates detected in the extracellular matrices in the presence of FLU, this work showed that the overexpression of efflux pumps is a possible mechanism of biofilm tolerance to FLU and this phenomenon alters the structure of C. glabrata biofilms by creating cell clusters.

Drug-delivery systems for green tea catechins for high stability and bioavailability.

Rodrigues, CF; Ascenção, K; Silva, FA; Sarmento, B; Oliveira, MB; Andrade, JC. Curr Med Chem. 2013;20(37):4744-57. DOI: 10.2174/09298673113209990158.
Numerous studies in humans, animal models and cell lines have suggested the potential benefits from the consumption of green tea polyphenols, including prevention of cancer and heart diseases. However these potential effects have been strongly limited by green tea catechins low bioavailability, which hinders the development of therapeutic applications. In this review formulations that are being proposed for delivery of green tea catechins are discussed. New delivery systems are presented as valid alternatives to overcome the limitations such as green tea catechins poor stability or intestinal absorption.

Candida glabrata: a review of its features and resistance.

Rodrigues, CF; Silva, SC; Henriques, M. Eur J Clin Microbiol Infect Dis. DOI 10.1007/s10096-013-2009-3
Candida species belong to the normal microbiota of the oral cavity and gastrointestinal and vaginal tracts, and are responsible for several clinical manifestations, from mucocutaneous overgrowth to bloodstream infections. Once believed to be non-pathogenic, Candida glabrata was rapidly blamable for many human diseases. Year after year, these pathological circumstances are more recurrent and problematic to treat, especially when patients reveal any level of immunosuppression. These difficulties arise from the capacity of C. glabrata to form biofilms and also from its high resistance to traditional antifungal therapies. Thus, this review intends to present an excerpt of the biology, epidemiology, and pathology of C. glabrata, and detail an approach to its resistance mechanisms based on studies carried out up to the present.

Synergistic antimicrobial interaction between honey and phage against Escherichia coli biofilms.

 Oliveira A; Ribeiro H; Silva AC; Silva MD; Sousa JC; Rodrigues, CF; Melo LDR; Henriques A; Sillankorva S.
CEB Annual Meeting, Braga, Portugal, July, 2017
Chronic wounds that take months, years or may even never heal present a major biological and financial problem on both individual patients and the broader health system. Chronic wounds afford a hostile environment of damaged tissues that allow bacterial proliferation and further wound colonization. Wound colonization by bacterial biofilms is one of the main obstacles of chronic wounds healing. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix and adhered to an inert or living surface. Escherichia coli is among the most common colonizers of infected wounds and it is a prolific biofilm former. Living in biofilm communities, cells are protected, become more difficult to control and eradicate, and less susceptible to antibiotic therapy. Due to the vast increase of antibiotic resistant bacteria, there is a renewed interestin pre-antibiotic therapies. Years before the discovery of modern antibiotics, bacteriophages(phages) that are bacterial viruses, and beehive products such as honey were extensively used for their antimicrobial properties. Phages, are the natural bacterial enemies and have proven efficacy towards antibiotic-resistant bacteria, have self-replicating nature, do not interfere with the commensal flora and many studies acknowledge that phages can destroy, tovarying extent, mono and mixed biofilm populations. Honey, on the other hand, has a broad spectrum antibacterial activity against bacteria and its high viscosity provides a protective barrier against infections being suitable for skincare, promoting the wound healing, tissue regeneration and anti-inflammatory process. This work presents insights into the proceedings triggering E.coli biofilm control with phage, two Portuguese(PT) honeys and their combination, achieved through standard antimicrobial activity assays, zeta potential and flow cytometry studies and further visual insights sought by SEM and TEM microscopy
Synergistic and additive effects were perceived at 12h and 24h of phage-honey combined treatment.
Honey caused minor membrane perturbations to complete collapse and consequent discharge of cytoplasmic content, and phage completely destroyed cells leaving only vesicle-like structures and debris.
Portuguese honeys possess excellent anti biofilm activity and may be potential alternative therapeutic agents in biofilm-related wound infection.
The antiviral effect of honey limits the emergence of phage resistant phenotypes.
The use of diluted honey solutions is advantageous, not only due to a potential lower cost of treatment, but also might be therapeutically more desirable as a topical rinsing solution maximizing the tolerability and practicality of the delivery technique.
The pioneering combined delivery of phage and honey is thus a promising antimicrobial alternative towards E.coli.

Novel strategies to fight Candida infections: natural honey

Rodrigues ME; Fernandes L; Rodrigues, CF; Oliveira A; Henriques M. 
CEB Annual Meeting, Braga, Portugal, July, 2017
The incidence of Candida infections (Candidosis) has increased remarkably in the last years, being attributed to the rise in the elderly population, the increasing number of immunocompromised patients, and the widespread use of indwelling medical devices. Candida albicans remains as the most prevalent species of these infections, but a clear rise in the proportion of non-Candida albicans Candida (NCAC) species has been noted. These species have an inherent level of resistance to certain antifungal agents higher than C. albicans though their virulence factors are much less understood. A major virulence factor is the ability to adhere and to form biofilms in medical devices and host tissues, because of a higher tolerance to antifungal therapy. Consequently, there is an urgent need to develop new strategies to fight these infections. Natural compounds are attracting increased interest in this field, among which honey. AIM: To evaluate honey as a novel strategy to fight C. tropicalis infections
Novel strategies to fight Candida infections: natural honey.

A first screening of the treatment with four planktonic Candida species culture indicated higher efficiency of honey against C. tropicalis.
Honey is capable of 2-log reductions on C. tropicalis biofilms after 24h of treatment
The combination of honey with nystatin and chlorhexidine reduces the antifungal dosage typically required in clinical settings by 50%, with a total biofilm reduction of 3-log
with nystatin and 5-logs with chlorhexidine.
In mixed biofilms of C. tropicalis and P. aeruginosa, honey at 50% or above reduces yeast and bacterial biofilm by 2-log and 4-log, respectively.
Altogether, our results highlight the great potential of honey as an alternative or complimentary strategy for the control of Candida infections.