Biomaterials surfaces capable of resisting fungal attachment and biofilm formation

Microbial attachment onto biomedical devices and implants leads to biofilm formation and infection; such biofilms can be bacterial, fungal, or mixed. In the past 15 years, there has been an increasing research effort into antimicrobial surfaces but the great majority of these publications present research on bacteria, with some reports also testing resistance to fungi. Very few studies have focused exclusively on antifungal surfaces. However, with increasing recognition of the importance of fungal infections to human health, particularly related to infections at biomaterials, it would seem that the interest in antifungal surfaces is disproportionately low. In studies of both bacteria and fungi, fungi tend to be the minor focus with hypothesized antibacterial mechanisms of action often generalized to also explain the antifungal effect. Yet bacteria and fungi represent two Distinct biological Domains and possess substantially different cellular physiology and structure. Thus it is questionable whether these generalizations are valid. Here we review the scientific literature focusing on surface coatings prepared with antifungal agents covalently attached to the biomaterial surface. We present a critical analysis of generalizations and their evidence. This review should be of interest to researchers of “antimicrobial” surfaces by addressing specific issues that are key to designing and understanding antifungal biomaterials surfaces and their putative mechanisms of action.     

Fungal Biofilms in the Clinical Lab Setting

Device-related infections are often associated with biofilms (microbial communities encased within polysaccharide-rich extracellular matrix) formed by pathogens on surfaces of these devices. Candida species are the most common fungi isolated from infections associated with catheters and dentures, and both Candida and Fusarium are commonly isolated from contact lens–related infections such as fungal keratitis. These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Drug resistance in fungal biofilms is multifactorial and phase-dependent; for example, efflux pumps mediate resistance in biofilms during early phase, whereas altered membrane sterol composition contributes to resistance in mature phase. Both substrate type and surface coatings play an important role in the pathogenesis of device-related fungal biofilms. Host immune cells influence the ability of Candida to form biofilms in vitro. This review summarizes recent advances in research on fungal biofilms and discusses their clinical relevance.     

Actividad de la micafungina contra las biopelículas de Candida

BackgroundMost recalcitrant infections are associated to colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial therapy.AimTo describe the antifungal of micafungin against fungal biofilms based in the scientific and medical literature of recent years.MethodsWe have done a bibliographic retrieval using the scientific terms “micafungin”, “activity”, “biofilm”, “Candida”, “Aspergillus”, “fungi”, “mycos”*, susceptibility, in PubMed/Medline from the National Library of Medicine from 2006 to 2009.ResultsMost current antifungal agents (amphotericin B and fluconazole) and the new azole antifungals have no activity against fungal biofilms. However, micafungin and the rest of echinocandins are very active against Candida albicans, Candida dubliniensis, Candida glabrata, and Candida krusei biofilms but their activities are variable and less strong against Candida tropicalis and Candida parapsilosis biofilms. Moreover, they have not activities against the biofilms of Cryptococcus y Trichosporon.ConclusionsThe activity of micafungin against Candida biofilms gives more strength to its therapeutic indication for candidaemia and invasive candidiasis associated to catheter, prosthesis and other biomedical devices.     

Purpurin Triggers Caspase-Independent Apoptosis in Candida dubliniensis Biofilms

Candida dubliniensis is an important human fungal pathogen that causes oral infections in patients with AIDS and diabetes mellitus. However, C. Dubliniensis has been frequently reported in bloodstream infections in clinical settings. Like its phylogenetically related virulent species C. albicans, C. Dubliniensis is able to grow and switch between yeast form and filamentous form (hyphae) and develops biofilms on both abiotic and biotic surfaces. Biofilms are recalcitrant to antifungal therapies and C. Dubliniensis readily turns drug resistant upon repeated exposure. More than 80% of infections are associated with biofilms. Suppression of fungal biofilms may therefore represent a viable antifungal strategy with clinical relevance. Here, we report that C. dubliniensis biofilms were inhibited by purpurin, a natural anthraquinone pigment isolated from madder root. Purpurin inhibited C. dubliniensis biofilm formation in a concentration-dependent manner; while mature biofilms were less susceptible to purpurin. Scanning electron microscopy (SEM) analysis revealed scanty structure consisting of yeast cells in purpurin-treated C. dubliniensis biofilms. We sought to delineate the mechanisms of the anti-biofilm activity of purpurin on C. Dubliniensis. Intracellular ROS levels were significantly elevated in fungal biofilms and depolarization of MMP was evident upon purpurin treatment in a concentration-dependent manner. DNA degradation was evident. However, no activated metacaspase could be detected. Together, purpurin triggered metacaspase-independent apoptosis in C. dubliniensis biofilms.     

Candida biofilms

In response to attachment to a surface, fungal cells produce biofilms, three-dimensional structures composed of cells surrounded by exopolymeric matrices. Surface attachment causes Candida albicans cells to enter a special physiological state in which they are highly resistant to antifungal drugs and express the drug efflux determinants CDR1, CDR2 and MDR1. C. albicans biofilms produced under different conditions differ in their cellular morphology and matrix content, which suggests that biofilms formed within a host, for example on indwelling medical devices, would also differ depending on the nature of the device and its location. The mechanisms by which surface attachment leads to biofilm formation are presently not understood.     

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.Download video file.^{(44M, mov)}     

A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.     

Fungal Biofilms: Relevance in the Setting of Human Disease

The use of indwelling medical devices is rapidly growing and is often complicated by infections with biofilm-forming microbes that are resistant to antimicrobial agents and host defense mechanisms. Fungal biofilms have emerged as a clinical problem associated with these medical device infections, causing significant morbidity and mortality. This review discusses the recent advances in the understanding of fungal biofilms, including the role of fungal surface components in adherence, gene expression, and quorum sensing in biofilm formation. We propose novel strategies for the prevention or eradication of microbial colonization of medical prosthetic devices.     

Biofilm formation by five species of Candida on three clinical materials

Most recalcitrant infections are associated with colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial. Fungi can form biofilms on biomaterials commonly used in clinical practice (intravascular catheters, dentures, heart valves, implanted devices, contact lenses and other devices) and are associated with infections.A variety of in vitro models using different substrates/devices have been described. These models have been used to investigate the effect of different variables, including flow, growth time, nutrients and physiological conditions on fungal biofilm formation, morphology and architecture.The purpose of our study is to analyze biofilm formation capacity by 84 strains of Candida spp. (23 C. albicans, 23 C. parapsilosis, 16 C. tropicalis, 17 C. glabrata and 5 C. krusei) on three materials used in medical devices and its quantification using a method based on viable cell count.Under the conditions of our study, all assayed Candida strains have been able to form biofilms. All species showed greater biofilm formation capacity on Teflon™, with the exception of C. glabrata which displayed higher biofilm formation capacity on PVC. Biofilm formation by Candida spp. varies depending on the type of material on which it grows and on the species and strain of Candida.The method we propose could be of great use to deepen scientific knowledge on this subject of remarkable clinical significance, considering the absence of standard biofilm formation and quantification techniques on the catheters and the level of difficulty associated to those available.     

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L

Background

Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies.

Methods

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide.

Results

Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species, and 7.81 to 62.5 μg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 × MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 × MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 × to 8 × MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol.

Conclusions

The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.     

Photodynamic inactivation for controlling Candida albicans infections

Antimicrobial photodynamic therapy (APDT) combines a non-toxic dye, termed photosensitizer, which is activated by visible light of appropriate wavelength which will produce reactive oxygen species (ROS). These ROS will react with cellular components inducing oxidative processes, leading to cell death. A wide range of microorganisms, have already showed susceptibility to APDT. Therefore, this treatment might consist in an alternative for the management of fungal infections that is mainly caused by biofilms, since they respond poorly to conventional antibiotics and may play a role in persistent infections. Biofilms are the leading cause of microbial infections in humans, thus representing a serious problem in health care. Candida albicans is the main type of fungi able to form biofilms, which cause superficial skin and mucous membrane infections as well as deep-seated mycoses, particularly in immunocompromised patients. In these patients, invasive infections are often associated with high morbidity and mortality. Furthermore, the increase in antifungal resistance has decreased the efficacy of conventional therapies. Treatments are time-consuming and thus demanding on health care budgets. Additionally, current antifungal drugs only have a limited spectrum of action and toxicity. The use of APDT as an antimicrobial topical agent against superficial and cutaneous diseases represents an effective method for eliminating microorganisms.     

Cryptococcus laurentii Biofilms: Structure, Development and Antifungal Drug Resistance

A great number of fungal infections are related to biofilm formation on inert or biological surfaces, which are recalcitrant to most treatments and cause human mortality. Cryptococcus laurentii has been diagnosed as the aetiological pathogen able to cause human infections mainly in immunosuppressed patients and the spectrum of clinical manifestations ranges from skin lesions to fungaemia. The effect of temperature, pH and surface preconditioning on C. laurentii biofilm formation was determined by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay. Scanning electron microscopic (SEM) analysis of C. laurentii biofilms demonstrated surface topographies of profuse growth and dense colonization with extensive polymeric substances around the cells. In this study, we determined the activity of amphotericin B, itraconazole and fluconazole against C. laurentii free-living cells and biofilms. The activity of antifungals tested was greater against free-living cells, but sessile cells fell into the resistant range for these antifungal agents. Extracellular polymeric substances (EPS), comprising the matrix of C. laurentii biofilms, were isolated by ultrasonication. Fourier transform infrared spectroscopy (FT-IR) was performed with ethanol-precipitated and dried samples. Also, the multielement analysis of the EPS was performed by inductively coupled plasma optical emission spectroscopy (ICP-OES).     

How to build a biofilm: a fungal perspective

Biofilms are differentiated masses of microbes that form on surfaces and are surrounded by an extracellular matrix. Fungal biofilms, especially those of the pathogen Candida albicans, are a cause of infections associated with medical devices. Such infections are particularly serious because biofilm cells are relatively resistant to many common antifungal agents. Several in vitro models have been used to elucidate the developmental stages and processes required for C. albicans biofilm formation, and recent studies have begun to define biofilm genetic control. It is clear that cell-substrate and cell-cell interactions, hyphal differentiation and extracellular matrix production are key steps in biofilm development. Drug resistance is acquired early in biofilm formation, and appears to be governed by different mechanisms in early and late biofilms. Quorum sensing might be an important factor in dispersal of biofilm cells. The past two years have seen the emergence of several genomic strategies to uncover global events in biofilm formation and directed studies to understand more specific events, such as hyphal formation, in the biofilm setting.     

The role of bacterial biofilms in ocular infections

There is increasing evidence that bacterial biofilms play a role in a variety of ocular infections. Bacterial growth is characterized as a biofilm when bacteria attach to a surface and/or to each other. This is distinguished from a planktonic or free-living mode of bacterial growth where these interactions are not present. Biofilm formation is a genetically controlled process in the life cycle of bacteria resulting in numerous changes in the cellular physiology of the organism, often including increased antibiotic resistance compared to growth under planktonic conditions. The presence of bacterial biofilms has been demonstrated on many medical devices including intravenous catheters, as well as materials relevant to the eye such as contact lenses, scleral buckles, suture material, and intraocular lenses. Many ocular infections often occur when such prosthetic devices come in contact with or are implanted in the eye. For instance, 56% of corneal ulcers in the United States are associated with contact lens wear. Bacterial biofilms may participate in ocular infections by allowing bacteria to persist on abiotic surfaces that come in contact with, or are implanted in the eye, and by direct biofilm formation on the biotic surfaces of the eye. An understanding of the role of bacterial biofilm formation in ocular infections may aid in the development of future antimicrobial strategies in ophthalmology. We review the current literature and concepts relating to biofilm formation and infections of the eye.     

Dipeptide cis-cyclo(Leucyl-Tyrosyl) produced by sponge associated Penicillium sp. F37 inhibits biofilm formation of the pathogenic Staphylococcus epidermidis

Infections associated to microbial biofilms are involved in 80% of human infections and became a challenge concerning public health. Infections related to Staphylococcus epidermidis biofilms are presently commonly associated to medical devices, increasing treatment costs for this type of infection. Alternatives to eliminate this kind of disease have been employed in screening programs using diverse marine-derived fungi source of bioactive compounds capable to combat biofilm formation. In this work was isolated the dipeptide cis-cyclo(Leucyl-Tyrosyl) from a sponge associated Penicillium sp. possessing a remarkable inhibition up to 85% of biofilm formation without interfering with bacterial growth, confirmed by scanning electron microscopy. This is the first demonstration that cis-cyclo(Leucyl-Tyrosyl) is able to specifically inhibit biofilm formation adding another aspect to the broad spectrum of bioactivities of cyclic dipeptides.     

Biofilm of Candida albicans: formation,regulation and resistance

Candida albicans is the most common human fungal pathogen, causing infections that range from mucous membranes to systemic infections. The present article provides an overview of C. albicans, with the production of biofilms produced by this fungus, as well as reporting the classes of antifungals used to fight such infections, together with the resistance mechanisms to these drugs. Candida albicans is highly adaptable, enabling the transition from commensal to pathogen due to a repertoire of virulence factors. Specifically, the ability to change morphology and form biofilms is central to the pathogenesis of C. albicans. Indeed, most infections by this pathogen are associated with the formation of biofilms on surfaces of hosts or medical devices, causing high morbidity and mortality. Significantly, biofilms formed by C. albicans are inherently tolerant to antimicrobial therapy, so the susceptibility of C. albicans biofilms to current therapeutic agents remains low. Therefore, it is difficult to predict which molecules will emerge as new clinical antifungals. The biofilm formation of C. albicans has been causing impacts on susceptibility to antifungals, leading to resistance, which demonstrates the importance of research aimed at the prevention and control of these clinical microbial communities.     

Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments

The biofilm formation on abiotic surfaces in food and medical sectors constitutes a great public health concerns. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodborne and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food and medical area, seem also to enhance the biofilm formation and their resistance to disinfectant agents. In this regard, this review highlights the effect of environmental conditions on bacterial adhesion and biofilm formation on abiotic surfaces in the context of food and medical environment. It also describes the current and emergent strategies used to study the biofilm formation and its eradication. The mechanisms of biofilm resistance to commercialized disinfectants are also discussed, since this phenomenon remains unclear to date.     

Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.