Rodrigues, C.F.

PharmD and Researcher

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.

Lactic acid increases the susceptibility of Candida albicans to fluconazole

Alves R; Mota S; Silva S; Rodrigues, CF; Brown AJp; Henriques M; Casal M; Paiva S; Berchet V.
XIX National Congress of Biochemistry, Guimarães, Portugal, December, 2016 - ORAL PRESENTATION
Candida spp. often inhabit niches that are glucose-limited but rich in alternative carbon sources, such as lactate or acetate, an ability that contributes to cells’ virulence. In glucose-poor niches, Candida albicans cells express JEN1 and JEN2 genes encoding the carboxylic acids transporters Jen1 and Jen2, respectively, which have been reported to be important in the early stages of infection. In this work, we aimed at analysing biofilm formation and antifungal drug resistance of C. albicans cells grown either in the presence of glucose or lactic acid. Additionally, we tested the involvement of Jen1 and Jen2 on these processes. Our results show that biofilm formation and susceptibility to fluconazole depend on the carbon source used. Wild-type and jen1jen2 lactic acid-grown cells formed more biofilm biomass, with predominance of yeast cells, than the ones grown in glucose. In the presence of this sugar a hyphae network is observed only for wild-type cells. In the presence of lactic acid, a jen1jen2 mutant strain exhibited a more compact biofilm with higher resistance to fluconazole when compared to the wild type. In the case of planktonic cells, the phenotype was exactly the opposite; the double mutant strain was more susceptible to fluconazole in lactic acid containing media. These findings show that carboxylic acids transporters have an important role in biofilm formation and in the acquisition of resistance to antifungal drugs, supporting the view that adaptation of Candida cells to the carbon source present in host niches affects their pathogenicity. 

 

In vitro study of antifungal drug effect in a tissue conditioner

Assunção B; Henriques, M; Rodrigues, CF; Vaz P; Figueiral H; Braga AC; Fernandes S.
IV International Conference on Biodental Engineering, Porto, Portugal, June, 2016- ORAL PRESENTATION
Tissue conditioners are generally used in acrylic removable dentures to improve the fit of the prosthesis and to allow support tissues recovery. It is mostly by the elastic property of the conditioners, which translates the performance of the "cushion" effect by the forces exerted during mastication, that is obtained the tissue recovery. However, these tissue conditioners are also well known for its poor physical properties - easy sensitivity degradation and colonization by microorganisms. Thus, their characteristics favor the accumulation of plaque and colonization by species of Candida, which can irritate the oral tissues and promote the development of denture stomatitis, threatening the oral health of patients with removable dental prosthesis. It was shown that these same acrylic resin when placed at the base of the prosthesis, could act as a reservoir for microorganisms and have the potential for biofilm formation.
 

Candida glabrata’s biofilms response to amphotericin B

Rodrigues, CF; Silva, SC; Salgado JM; Abrunhosa L; Venâncio A; Azeredo, J; Henriques, M
FEMS 2015 - 6th Congress of European Microbiologists, Maastricht, The Netherlands, The Netherlands, 2015 - ORAL PRESENTATION
Candida species are responsible for recurrent human infections, mostly in immunocompromised patients, due to their high vulnerability. Candida glabrata has been showing to have a major role in these infections being the second most prevalent species involved in human fungemia. Amphotericin B (AmB), a common antifungal drug, is a hospital-environment exclusive polyene, normally being efficient when used to fight candidiasis. 
Objectives: To infer about the influence of AmB in Candida glabrata biofilms formation and its effect on matrix composition and ERG genes expression.

AmB reduces biofilms of C. glabrata.
ERG genes seem to be less involved than the matrix composition in C. glabrata biofilms response to AmB. 
C. glabrata biofilms matrices respond with an increase of carbohydrates, particularly β-1,3 glucans, and with a decrease of total proteins. 
New matrix artifact component was found: ergosterol.
The present work supports the theory of multifaceted mechanisms developed by C. glabrata biofilms as response to the presence of AmB.
 

Synergistic Antimicrobial Interaction Of Honey And Bacteriophage In Escherichia coli Biofilms 2

Ribeiro H; Oliveira A; Silva AC; Silva AD; Rodrigues, CF; Melo LDR; Henriques A; Sillankorva S.
I Simpósio Bioquímica Aplicada, Braga, Portugal, May, 2017
Wound colonization by biofilms-forming bacteria is one of the main obstacles to the treatment of chronic wounds, causing a number of biological and financial problems. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix, adhered to inert or living surfaces, blocking antibiotics and patient’s immune cells from reaching bacteria. Bacterio(phages), viruses infecting exclusively bacteria, and honey are being considered as valuable alternatives to treat a variety of infections (Table 1). Phages are harmless to mammalian cells, specific for target bacteria therefore not affecting commensal microflora, have the ability to selfreplicate as long as the host is present, and are effective against antibiotic resistant bacteria. Honey is a complex substance with broad spectrum antimicrobial activity, essentially attributed to the high sugar content, low pH, the presence of hydrogen peroxide and methylglyoxal that reacts with important biological molecules (RNA, DNA and proteins). Honey had yet the potential to promote tissue regeneration, cicatrization, decrease inflammation, improving wound healing. In this work the combination of those two antimicrobial agents was considered in the control E. coli biofilms.

The Portuguese honey PF2 50% (w/v) showed great antibiofilm effect with 6-log cell reduction in 24 h-old biofilms. 
A synergistic effect was observed with PF2 25% (w/v) and EC3a in 24 and 48 h-old biofilms. 
48 h-old biofilms were more difficult to combat than 24 h-old biofilms. 
This is a promising strategy for biofilm control that will be further tested with other pathogens and using ex vivo models.

Synergistic Antimicrobial Interaction Of Honey And Bacteriophage In Escherichia coli Biofilms

Ribeiro H; Oliveira A; Silva AC; Silva AD; Rodrigues, CF; Melo LDR; Henriques A; Sillankorva S.
X Encontro Nacional de Estudantes de Bioquímica (X ENEBIOQ), Braga, Portugal, April, 2017
Wound colonization by biofilms-forming bacteria is one of the main obstacles to the treatment of chronic wounds, causing a number of biological and financial problems. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix, adhered to inert or living surfaces, blocking antibiotics and patient’s immune cells from reaching bacteria. Bacterio(phages), viruses infecting exclusively bacteria, and honey are being considered as valuable alternatives to treat a variety of infections (Table 1). Phages are harmless to mammalian cells, specific for target bacteria therefore not affecting commensal microflora, have the ability to selfreplicate as long as the host is present, and are effective against antibiotic resistant bacteria. Honey is a complex substance with broad spectrum antimicrobial activity, essentially attributed to the high sugar content, low pH, the presence of hydrogen peroxide and methylglyoxal that reacts with important biological molecules (RNA, DNA and proteins). Honey had yet the potential to promote tissue regeneration, cicatrization, decrease inflammation, improving wound healing. In this work the combination of those two antimicrobial agents was considered in the control E. coli biofilms.

The Portuguese honey PF2 50% (w/v) showed great antibiofilm effect with 6-log cell reduction in 24 h-old biofilms. 
A synergistic effect was observed with PF2 25% (w/v) and EC3a in 24 and 48 h-old biofilms. 
48 h-old biofilms were more difficult to combat than 24 h-old biofilms. 
This is a promising strategy for biofilm control that will be further tested with other pathogens and using ex vivo models.
 
 

Impact of alternative carbon sources and antifungal treatment on Candida glabrata biofilms' transcription profile

 

Alves, R; Kastora, S; Pinho E; Rodrigues, CF; Silva, S; Casal M; Brown, AJp; Henriques, M; Paiva S.
XIX National Congress of Biochemistry, Guimarães, Portugal, December, 2016
Candida glabrata is considered a major opportunistic fungal pathogen of humans and has emerged as a leading cause of nosocomial fungal infections. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that Candida can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of these fungi. Our recent study (Mota et al., 2015) supported this view by demonstrating that acetic acid influences C. glabrata behavior in biofilm formation, antifungal drug resistance and phagocytosis; and suggesting a potential role of putative carboxylate transporters on these processes. In order to extend our studies and provide a comprehensive view of the C. glabrata biofilms’ response to alternative carbon sources and antifungal treatment, we performed comparative transcriptomics analyses using RNA-sequencing. Our data support the view that adaptative responses of Candida cells to the types of carbon source present in host niches affects the virulence of these fungal cells through multifarious mechanisms (Brown et al., 2014). Finally, elucidating the effect of local nutrients and pH environment on drug resistance can potentially provide new and effective treatment strategies for C. glabrata infections such as vaginal candidiasis.
 

 

The chemical structure of the biofilm matrices of Candida glabrata induces resistance to antifungal drugs

Rodrigues, CF; Rodrigues ME; Silva, SC; Azeredo, J; Henriques, M
BIOFILMS'7- Internation Congress of Biofilms, Porto, Portugal, June, 2016.
Candida infections are often associated to biofilms and consequently to high resistance to the most common drugs. These resistance mechanisms are not only associated with the biofilm yeast physiology, but also with the presence of a barrier imposed by the biofilm matrix. However, the biochemical role of the biofilm components remains very unclear. Therefore, this work intends to further enlighten the effect of antifungal agents on C. glabrata biofilm resistance. As a good biofilm former, Candida glabrata ATCC2001 was selected to this study. Several antifungal drugs, belonging to different groups, were used in this work, namely fluconazole, amphotericin B, caspofungin and micafungin and their effect on biofilm matrix was assessed. Biofilm matrix chemical composition and structure was evaluated by analytical methods. As expected, C. glabrata biofilms were resistant to the antifungals used in an agent-dependent manner. The results showed significant differences in polysaccharides and proteins contents, in the matrix of biofilms formed in the absence and in the presence of the drugs. Moreover, the diffusion through the matrix was evaluated demonstrating that different agents, even belonging to the same group, present very different profiles, explaining the different tolerance registered. So, with this study we confirmed that C. glabrata biofilm’s resistance to antifungal drugs is a very complex mechanism, where the matrix plays a major role.

The biofilm matrices of C. glabrata ATCC2001 strongly changes when in contact to antifungal drugs: protein profile changes and β-1,3 glucans  production increases.
It is known that β-1,3 glucans are linked to the antifungal resistance, by making it difficult to the drugs to diffuse through the biofilm matrices and reach the yeast cells. This change in the matrix composition may be an attempt of the cells to obtain a denser matrix, unflexible and more protective for their environment.
 

Fluconazole vs Voriconazole: Candida glabrata’s biofilms response to different azoles

Rodrigues, CF; Gonçalves B; Rodrigues ME; Silva, SC; Azeredo, J; Henriques, M
TIMM 2015 - 7th Congress on Trends in Medical Mycology, Portugal, October, 2015.
Candida glabrata is the second most prevalent yeast in fungal infections, especially in immunocompromised and/or hospitalized patients. The azole resistance within this species is very well-known and results in a low therapeutic response of C. glabrata infections, particularly when associated with biofilms.
Objective - To understand the different efficacies of two azoles against C. glabrata biofilms: - fluconazole (Flu), a long time used drug - voriconazole (Vcz), a latest drug used only in hospitals.

The pattern of C. glabrata biofilm resistance to Flu and Vcz is effectively different;
The cell genetic alterations in genes encoding for the ergosterol biosynthesis exists but is still unable to   explain the different resistance profiles obtained for these two agents;
There were alterations in the matrix composition in the presence of the azoles, but there was not found a direct correlation with Flu and Vcz efficacy; 
The diffusion of the drugs within the biofilms is azole’s dependent, with Vcz having a better diffusion and a better activity against C. glabrata biofilms than Flu, explaining its enhanced performance. 
 

How do Candida glabrata's biofilms respond to antifungal drugs?

Rodrigues, CF; Rodrigues ME; Silva S; Henriques M.
TIMM 2015 - 7th Congress on Trends in Medical Mycology,, Lisboa, Portugal, 2015.
Candida species are responsible for recurrent human infections, mostly in immunocompromised patients, due to their high vulnerability.  Candida glabrata has been shown to have a major role in these infections being the second most prevalent species involved in human fungemia.
Objective: To understand the effect of three different antifungal agents – Fluconazole (Flu), Amphotericin B (AmB) and Caspofungin (Csf) - in C. glabrata’s biofilm formation, specially their role on matrix composition.
 
The pattern of C. glabrata biofilm response to Flu, AmB and Csf is clearly different;
The three agents had diverse effects on C. glabrata’s biofilm formation;
Matrices´ composition display variations when exposed to different antifungal agents, and these differences depend on the drug is used;
AmB, and especially Csf, were confirmed in this study, to be the most effective pharmacoterapies for eradication of C. glabrata infections associated to biofilms.