Modulation of the Substitution Pattern of 5-Aryl-2-Aminoimidazoles Allows Fine-Tuning of Their Antibiofilm Activity Spectrum and Toxicity.

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N1- and 2N-substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2N-substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N1 and 2N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N1-,2N-disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N1-,2N-disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N1 and 2N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

OBJECTIVES: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Synergistic activity of the tyrocidines, antimicrobial cyclodecapeptides from Bacillus aneurinolyticus, with amphotericin B and caspofungin against Candida albicans biofilms.

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.

Structure-activity relationship study of the plant-derived decapeptide OSIP108 inhibiting Candida albicans biofilm formation.

We performed a structure-activity relationship study of the antibiofilm plant-derived decapeptide OSIP108. Introduction of positively charged amino acids R, H, and K resulted in an up-to-5-fold-increased antibiofilm activity against Candida albicans compared to native OSIP108, whereas replacement of R9 resulted in complete abolishment of its antibiofilm activity. By combining the most promising amino acid substitutions, we found that the double-substituted OSIP108 analogue Q6R/G7K had an 8-fold-increased antibiofilm activity.

Oral administration of the broad-spectrum antibiofilm compound toremifene inhibits Candida albicans and Staphylococcus aureus biofilm formation in vivo.

We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans, Candida glabrata, Candida dubliniensis, Candida krusei, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.

Plant-derived decapeptide OSIP108 interferes with Candida albicans biofilm formation without affecting cell viability.

We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation.

Derivatives of the mouse cathelicidin-related antimicrobial peptide (CRAMP) inhibit fungal and bacterial biofilm formation.

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 µM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 µM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.

Repurposing as a means to increase the activity of amphotericin B and caspofungin against Candida albicans biofilms.

OBJECTIVES: Biofilms of Candida species, often formed on medical devices, are generally resistant to currently available antifungal drugs. The aim of this study was to identify compounds that increase the activity of amphotericin B and caspofungin, commonly used antifungal agents, against Candida biofilms. METHODS: A library containing off-patent drugs was screened for compounds, termed enhancers, that increase the in vitro activity of amphotericin B against Candida albicans biofilms. Biofilms were grown in 96-well plates and growth was determined by the cell titre blue assay. Synergy between identified enhancers and antifungal agents was further characterized in vitro using fractional inhibitory concentration index (FICI) values and in vivo using a worm biofilm infection model. In light of the application of these enhancers onto implants, their possible effect on the growth potential of MG63 osteoblast-like cells was assessed. RESULTS: Pre-incubation of C. albicans biofilms with subinhibitory concentrations of the enhancers drospirenone, perhexiline maleate or toremifene citrate significantly increased the activity of amphotericin B or caspofungin (FICI  < 0.5) against C. albicans and Candida glabrata biofilms. Moreover, these enhancers did not affect the growth potential of osteoblasts. Interestingly, toremifene citrate also enhanced the in vitro activity of caspofungin in a mixed biofilm consisting of C. albicans and Staphylococcus epidermidis. Furthermore, we demonstrate synergy between toremifene citrate and caspofungin in an in vivo worm C. albicans biofilm infection model. CONCLUSIONS: Our data demonstrate an in vitro and in vivo enhancement of the antibiofilm activity of caspofungin by toremifene citrate. Furthermore, our results pave the way for implant-related applications of the identified enhancers.

Evaluation of the toxicity of 5-aryl-2-aminoimidazole-based biofilm inhibitors against eukaryotic cell lines, bone cells and the nematode Caenorhabditis elegans.

Previously, we have synthesized several series of compounds based on the 5-aryl-2-aminoimidazole scaffold, which showed a preventive activity against microbial biofilms. We here studied the cytotoxicity of the most active compounds of each series. First, the cytostatic activity was investigated against a number of tumor cell lines (L1210, CEM and HeLa). A subset of monosubstituted 5-aryl-2-aminoimidazoles showed a moderate safety window, with therapeutic indices (TIs) ranging between 3 and 20. Whereas introduction of a (cyclo-)alkyl chain at the N1-position strongly reduced the TI, introduction of a (cyclo-)alkyl chain or a triazole moiety at the 2N-position increased the TI up to 370. Since a promising application of preventive anti-biofilm agents is their use in anti-biofilm coatings for orthopedic implants, their effects on cell viability and functional behavior of human osteoblasts and bone marrow derived mesenchymal stem cells were tested. The 2N-substituted 5-aryl-2-aminoimidazoles consistently showed the lowest toxicity and allowed survival of the bone cells for up to 4 weeks. Moreover they did not negatively affect the osteogenic differentiation potential of the bone cells. Finally, we examined the effect of the compounds on the survival of Caenorhabditis elegans, which confirmed the higher safety window of 2N-substituted 5-aryl-2-aminoimidazoles.

Identification of fungicidal 2,6-disubstituted quinolines with activity against Candida biofilms.

We have identified two subseries of 2,6-disubstituted quinolines, consisting of 6-amide and 6-urea derivatives, which are characterized by fungicidal activity against Candida albicans with minimal fungicidal concentration (MFC) values < 15 µM. The 6-amide derivatives displayed the highest fungicidal activity against C. albicans, in particular compounds 1, 5 and 6 characterized by MFC values of 6.25-12.5 µM. Compounds 1 and 5 of this series displayed fungicidal activity against the emerging pathogen Candida glabrata (MFC < 50 µM). The 6-amide derivatives 1, 2, 5, and 6 and the 6-urea derivatives 10, 12, 13 and 15 could also eradicate C. albicans biofilms. We found that the 6-urea derivatives 10, 13, and 15 induced accumulation of endogenous reactive oxygen species in Candida albicans biofilms.

Antimicrobial Peptides as a Strategy to Combat Fungal Biofilms.

Invasive fungal infections caused by opportunistic fungal pathogens are associated with high mortality rates, mainly due to the occurrence of genotypic and/or phenotypic resistance. One of the causes of phenotypic resistance is the preferred growth of various fungal pathogens as biofilms, which are tolerant or resistant to most classes of antifungal agents. Moreover, increasing evidence points to biofilm formation as a general prerequisite for the development of systemic infections. Therefore, new antibiofilm agents are urgently needed to reduce the incidence of biofilm-associated infections. Nowadays, antimicrobial peptides (AMPs) are considered as valuable alternatives for or complements to the classical antifungal agents to combat fungal infections. Many review reports describe activity of AMPs against free-living planktonic fungal pathogens. In contrast, this review summarizes the antibiofilm properties of natural or synthetic AMPs against fungal biofilms and their potential to enhance the antibiofilm activity of existing antifungal agents.

Alternating Current Electrophoretic Deposition for the Immobilization of Antimicrobial Agents on Titanium Implant Surfaces.

One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen C. albicans as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities.

Combinatorial drug approaches to tackle Candida albicans biofilms.

The human fungal opportunistic pathogen Candida albicans resides in the human gut, genitourinary tract and on the skin. The majority of infections caused by C. albicans are biofilm-related. In the first part of this review, we discuss new insights into C. albicans biofilm characteristics, concentrating on the extracellular matrix, phenotypic switching, efflux pumps and persister cells. It is widely accepted that this multicellular lifestyle is more resistant to traditional antifungal treatment compared to free-living cells. Therefore, much effort is put in the search for combinations of drugs leading to synergistic interactions against microbial biofilms to achieve lower effective doses of the drugs. In the second part of this manuscript, we review all recently identified compounds that act synergistically with azoles, echinocandins and/or polyenes against C. albicans biofilms.

Novel anti-infective implant substrates: controlled release of antibiofilm compounds from mesoporous silica-containing macroporous titanium.

Bone implants with open porosity enable fast osseointegration, but also present an increased risk of biofilm-associated infections. We design a novel implant material consisting of a mesoporous SiO2 diffusion barrier (pore diameter: 6.4 nm) with controlled drug release functionality integrated in a macroporous Ti load-bearing structure (fully interconnected open porosity: 30%; pore window size: 0.5-2.0 µm). Using an in vitro tool consisting of Ti/SiO2 disks in an insert set-up, through which molecules can diffuse from feed side to release side, a continuous release without initial burst effect of the antibiofilm compound toremifene is sustained for at least 9 days, while release concentrations (up to 17 µM daily) increase with feed concentrations (up to 4mM). Toremifene diffusivity through the SiO2 phase into H2O is estimated around 10(-13)m(2)/s, suggesting configurational diffusion through mesopores. Candida albicans biofilm growth on the toremifene-release side is significantly inhibited, establishing a proof-of-concept for the drug delivery functionality of mesoporous SiO2 incorporated into a high-strength macroporous Ti carrier. Next-generation implants made of this composite material and equipped with an internal reservoir (feed side) can yield long-term controlled release of antibiofilm compounds, effectively treating infections on the implant surface (release side) over a prolonged time.

Reactive oxygen species-inducing antifungal agents and their activity against fungal biofilms.

Invasive fungal infections are associated with very high mortality rates ranging from 20-90% for opportunistic fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Fungal resistance to antimycotic treatment can be genotypic (due to resistant strains) as well as phenotypic (due to more resistant fungal lifestyles, such as biofilms). With regard to the latter, biofilms are considered to be critical in the development of invasive fungal infections. However, there are only very few antimycotics, such as miconazole (azoles), echinocandins and liposomal formulations of amphotericin B (polyenes), which are also effective against fungal biofilms. Interestingly, these antimycotics all induce reactive oxygen species (ROS) in fungal (biofilm) cells. This review provides an overview of the different classes of antimycotics and novel antifungal compounds that induce ROS in fungal planktonic and biofilm cells. Moreover, different strategies to further enhance the antibiofilm activity of such ROS-inducing antimycotics will be discussed.

VEGF mediates commissural axon chemoattraction through its receptor Flk1.

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.