The Caenorhabditis elegans cuticle plays an important role against toxicity to bisphenol A and bisphenol S.

Caenorhabditis elegans represents a favorite non-mammalian animal model, which is often used to study the effect of foreign substances on living organisms. Its epidermal barrier is a primary biological barrier that protects nematodes from the toxicity of chemicals. In this study, we investigated the effect of Bisphenol A (BPA), an endocrine disrupting chemical, and its structural analog Bisphenol S (BPS), which is often used as a substitute for BPA in some products, on the behavior of C. elegans wild type (N2) and C. elegans bli-1 mutant strain, which is characterized by the production of abnormal cuticle blisters. We found that exposure of C. elegans wild type (N2), as well as its mutant strain bli-1, to selected concentrations of BPA (0.1, 0.5, 1 and 5 µM) and BPS (0.1, 0.5, 1 and 5 µM) resulted in significant changes in reproduction, habituation behavior, and body length of nematodes. Based on our findings, we can conclude that BPS, which was supposed to be a safer alternative to BPA, caused almost identical detrimental effects on C. elegans behavior. Furthermore, compared to the wild type of C. elegans, these effects were more pronounced in the bli-1 strain, which is characterized by a mutation in an individual collagen gene responsible for proper cuticle formation, underlying the role of the epidermal barrier in bisphenol toxicity. Taken together, our data indicate the potential risks of using BPS as a BPA alternative.

Occurrence, antifungal susceptibility, and virulence factors of opportunistic yeasts isolated from Brazilian beaches.

BACKGROUND: Opportunistic pathogenic yeast species are frequently associated with water habitats that have pollution sources of human or animal origin. Candida albicans has already been suggested as a faecal indicator microorganism for aquatic environments. OBJECTIVES: The goal of this study was to investigate the occurrence of C. albicans and other opportunistic yeasts in sand and seawater samples from beaches in Brazil to assess their correlation with Escherichia coli, and to characterise the pathogenic potential of the yeast isolates. METHODS: Opportunistic species (yeasts that grow at 37ºC) were isolated from sand and seawater samples from eight beaches in Brazil during the summer and the winter. Opportunistic yeast species were evaluated for their susceptibility to antifungal drugs, virulence factors, and the in vitro and in vivo biofilm formation. Strains were selected to carry out virulence tests using BALB/c mice. FINDINGS: Several water samples could be classified as inappropriate for primary contact recreation in relation to E. coli densities. C. albicans was isolated in low densities. Of the 144 opportunistic yeasts evaluated, 61% displayed resistance or dose-dependent sensitivity to at least one tested drug, and 40% produced proteinase. Strains of C. albicans and Kodamaea ohmeri exhibited the highest rates of adhesion to buccal epithelial cells. All the C. albicans strains that were tested were able to undergo morphogenesis and form a biofilm on catheter fragments in both in vitro and in vivo experiments. It was possible to confirm the pathogenic potential of three of these strains during the disseminated infection test. MAIN CONCLUSIONS: The identification of opportunistic yeast species in seawater and sand samples from Brazilian beaches suggest a potential risk to the health of people who use these environments for recreational purposes.

Candidalysin Crucially Contributes to Nlrp3 Inflammasome Activation by Candida albicans Hyphae.

Candida albicans is an opportunistic fungal pathogen that can cause life-threatening infections, particularly in immunocompromised patients. C. albicans induced activation of the Nlrp3 inflammasome, leading to secretion of bioactive interleukin 1ß (IL-1ß) is a crucial myeloid cell immune response needed for antifungal host defense. Being a pleiomorphic fungus, C. albicans can provoke Nlrp3 inflammasome responses only upon morphological transformation to its hyphal appearance. However, the specific hyphal factors that enable C. albicans to activate the Nlrp3 inflammasome in primary macrophages remain to be revealed. Here, we identify candidalysin, a peptide derived from the hypha-specific ECE1 gene, as a fungal trigger for Nlrp3 inflammasome-mediated maturation and secretion of IL-1ß from primary macrophages. Direct peptide administration experiments showed that candidalysin was sufficient for inducing secretion of mature IL-1ß from macrophages in an Nlrp3 inflammasome-dependent manner. Conversely, infection experiments using candidalysin-deficient C. albicans showed that candidalysin crucially contributed to the capacity of this fungus to induce maturation and secretion of IL-1ß from primary macrophages. These complementary observations identify the expression of candidalysin as one of the molecular mechanisms by which hyphal transformation equips C. albicans with its proinflammatory capacity to elicit the release of bioactive IL-1ß from macrophages.IMPORTANCE Candidiasis is a potentially lethal condition that is caused by systemic dissemination of Candida albicans, a common fungal commensal residing mostly on mucosal surfaces. The transition of C. albicans from an innocuous commensal to an opportunistic pathogen goes hand in hand with its morphological transformation from a fungus to a hyphal appearance. On the one hand, the latter manifestation enables C. albicans to penetrate tissues, while on the other hand, the expression of many hypha-specific genes also endows it with the capacity to trigger particular cytokine responses. The Nlrp3 inflammasome is a crucial component of the innate immune system that provokes release of the IL-1ß cytokine from myeloid cells upon encountering C. albicans hyphae. Our study reveals the peptide candidalysin as one of the hypha-derived drivers of Nlrp3 inflammasome responses in primary macrophages and, thus, contributes to better understanding the fungal mechanisms that determine the pathogenicity of C. albicans.

Monitoring of Fluconazole and Caspofungin Activity against In Vivo Candida glabrata Biofilms by Bioluminescence Imaging.

Candida glabrata can attach to various medical implants and forms thick biofilms despite its inability to switch from yeast to hyphae. The current in vivoC. glabrata biofilm models only provide limited information about colonization and infection and usually require animal sacrifice. To gain real-time information from individual BALB/c mice, we developed a noninvasive imaging technique to visualize C. glabrata biofilms in catheter fragments that were subcutaneously implanted on the back of mice. Bioluminescent C. glabrata reporter strains (lucOPT 7/2/4 and lucOPT 8/1/4), free of auxotrophic markers, expressing a codon-optimized firefly luciferase were generated. A murine subcutaneous model was used to follow real-time in vivo biofilm formation in the presence and absence of fluconazole and caspofungin. The fungal load in biofilms was quantified by CFU counts and by bioluminescence imaging (BLI). C. glabrata biofilms formed within the first 24 h, as documented by the increased number of device-associated cells and elevated bioluminescent signal compared with adhesion at the time of implant. The in vivo model allowed monitoring of the antibiofilm activity of caspofungin against C. glabrata biofilms through bioluminescent imaging from day four after the initiation of treatment. Contrarily, signals emitted from biofilms implanted in fluconazole-treated mice were similar to the light emitted from control-treated mice. This study gives insights into the real-time development of C. glabrata biofilms under in vivo conditions. BLI proved to be a dynamic, noninvasive, and sensitive tool to monitor continuous biofilm formation and activity of antifungal agents against C. glabrata biofilms formed on abiotic surfaces in vivo.

An antibiofilm coating of 5-aryl-2-aminoimidazole covalently attached to a titanium surface.

Biofilms, especially those formed by Staphylococcus aureus, play a key role in the development of orthopedic implant infections. Eradication of these infections is challenging due to the elevated tolerance of biofilm cells against antimicrobial agents. In this study, we developed an antibiofilm coating consisting of 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine, designated as LC0024, covalently bound to a titanium implant surface (LC0024-Ti). We showed in vitro that the LC0024-Ti surface reduces biofilm formation of S. aureus in a specific manner without reducing the planktonic cells above the biofilm, as evaluated by plate counting and fluorescence microscopy. The advantage of compounds that only inhibit biofilm formation without affecting the viability of the planktonic cells, is that reduced development of bacterial resistance is expected. To determine the antibiofilm activity of LC0024-Ti surfaces in vivo, a biomaterial-associated murine infection model was used. The results indicated a significant reduction in S. aureus biofilm formation (up to 96%) on the LC0024-Ti substrates compared to pristine titanium controls. Additionally, we found that the LC0024-Ti substrates did not affect the attachment and proliferation of human cells involved in osseointegration and bone repair. In summary, our results emphasize the clinical potential of covalent coatings of LC0024 on titanium implant surfaces to reduce the risk of orthopedic implant infections. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1908-1919, 2019.

Occurrence, antifungal susceptibility, and virulence factors of opportunistic yeasts isolated from Brazilian beaches

BACKGROUND Opportunistic pathogenic yeast species are frequently associated with water habitats that have pollution sources of human or animal origin. Candida albicans has already been suggested as a faecal indicator microorganism for aquatic environments. OBJECTIVES The goal of this study was to investigate the occurrence of C. albicans and other opportunistic yeasts in sand and seawater samples from beaches in Brazil to assess their correlation with Escherichia coli, and to characterise the pathogenic potential of the yeast isolates. METHODS Opportunistic species (yeasts that grow at 37ºC) were isolated from sand and seawater samples from eight beaches in Brazil during the summer and the winter. Opportunistic yeast species were evaluated for their susceptibility to antifungal drugs, virulence factors, and the in vitro and in vivo biofilm formation. Strains were selected to carry out virulence tests using BALB/c mice. FINDINGS Several water samples could be classified as inappropriate for primary contact recreation in relation to E. coli densities. C. albicans was isolated in low densities. Of the 144 opportunistic yeasts evaluated, 61% displayed resistance or dose-dependent sensitivity to at least one tested drug, and 40% produced proteinase. Strains of C. albicans and Kodamaea ohmeri exhibited the highest rates of adhesion to buccal epithelial cells. All the C. albicans strains that were tested were able to undergo morphogenesis and form a biofilm on catheter fragments in both in vitro and in vivo experiments. It was possible to confirm the pathogenic potential of three of these strains during the disseminated infection test. MAIN CONCLUSIONS The identification of opportunistic yeast species in seawater and sand samples from Brazilian beaches suggest a potential risk to the health of people who use these environments for recreational purposes.

Anidulafungin increases the antibacterial activity of tigecycline in polymicrobial Candida albicans/Staphylococcus aureus biofilms on intraperitoneally implanted foreign bodies.

Objectives: We aimed to establish a novel murine intra-abdominal foreign body infection model to study the activity of anidulafungin and tigecycline against dual species Candida albicans/Staphylococcus aureus biofilms. Methods: In vitro and in vivo single and dual species biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well plates or implanted intraperitoneally in BALB/c mice. The effect of tigecycline and anidulafungin alone and in combination was tested using clinically relevant concentrations. Scanning electron microscopy was used to visualize the mature biofilm structure developed intraperitoneally. Flow cytometry was used to determine the immunological response upon infection. Immunoblot analysis allowed us to determine the effect of anidulafungin on poly-ß-(1,6)-N-acetylglucosamine in in vitro-grown S. aureus biofilms. Results: We determined the MIC, MBC and in vitro susceptibility profile for anidulafungin and tigecycline against C. albicans and S. aureus in mixed and single species biofilms. We demonstrated that anidulafungin acts synergistically when combined with tigecycline against in vivo intra-abdominal biofilms. Moreover, we reveal that anidulafungin reduces the abundance of S. aureus poly-ß-(1,6)-N-acetylglucosamine. The influx of neutrophils is much increased when infected with mixed biofilms compared with single species biofilms. Conclusions: Currently, treatment of intra-abdominal infections, in particular polymicrobial catheter-associated peritonitis, is ineffective. To the best of our knowledge, this is the first study that provides insight into new possible options for treatment of C. albicans/S. aureus biofilms present in the abdominal cavity.

Bioluminescence imaging increases in vivo screening efficiency for antifungal activity against device-associated Candida albicans biofilms.

Fungal infections are a major problem for a growing number of mostly immunocompromised patients. Candida albicans is an important human fungal pathogen causing mucosal and deep tissue infections, of which the majority are associated with biofilm formation on medical implants. Animal models that are currently in use to test antifungal drugs are limited to ex vivo analyses, requiring host sacrifice that excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. As a solution, we introduce non-invasive, dynamic imaging and quantification of C. albicans biofilm formation in vivo and subsequent evaluation of treatment efficacy against these biofilms using bioluminescent C. albicans in a catheter-associated mouse model. Bioluminescence imaging (BLI) allowed us to evaluate baseline biofilm load before the start of therapy, which is necessary for correct evaluation and interpretation of antibiofilm efficacy in vivo. Moreover, we demonstrate that this BLI approach monitors the antibiofilm activity of different antifungal agents efficiently in vitro and in vivo. In this study, BLI revealed superior antibiofilm activity for echinocandins compared with amphotericin B and fluconazole. In vitro, anidulafungin showed the highest antibiofilm activity, followed by micafungin and caspofungin. In vivo, caspofungin significantly decreased the biofilm fungal load, as documented by the lower BLI signal and confirmed by CFU counts. In conclusion, this BLI approach increases the power and efficiency of screening and validation of antimycotics both under in vitro and in vivo conditions, thereby refining pre-clinical therapy studies.

Antifungal Activity of Oleylphosphocholine on In Vitro and In Vivo Candida albicans Biofilms.

In this study, we investigated the potential antifungal activity of the alkylphospholipid oleylphosphocholine (OlPC), a structural analogue of miltefosine, on in vitro and in vivoCandida albicans biofilm formation. The effect of OlPC on in vitro and in vivoC. albicans biofilms inside triple-lumen polyurethane catheters was studied. In vivo biofilms were developed subcutaneously after catheter implantation on the lower back of Sprague-Dawley rats. Animals were treated orally with OlPC (20 mg/kg of body weight/day) for 7 days. The effect of OlPC on biofilms that developed on the mucosal surface was studied in an ex vivo model of oral candidiasis. The role of OlPC in C. albicans morphogenesis was investigated by using hypha-inducing media, namely, Lee, Spider, and RPMI 1640 media. OlPC displayed activity against both planktonic cells and in vitroC. albicans biofilms. To completely abolish preformed, 24-h-old biofilms, higher concentrations (8, 10, and 13 mg/liter) were needed. Moreover, OlPC was able to reduce C. albicans biofilms formed by caspofungin-resistant clinical isolates and acted synergistically when combined with caspofungin. The daily oral administration of OlPC significantly reduced in vivoC. albicans biofilms that developed subcutaneously. In addition, OlPC decreased biofilm formation on mucosal surfaces. Interestingly, the application of subinhibitory concentrations of OlPC already inhibited the yeast-to-hypha transition, a crucial virulence factor of C. albicans We document, for the first time, the effects of OlPC on C. albicans cells and suggest the potential use of OlPC for the treatment of C. albicans biofilm-associated infections.

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization.

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP's). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP's is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1's internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms.

Public health problems are associated with device-associated biofilm infections, with Candida albicans being the major fungal pathogen. We previously identified potent antibiofilm combination treatment in which the antifungal plant defensin HsAFP1 is co-administered with caspofungin, the preferred antimycotic to treat such infections. In this study, we identified the smallest linear HsAFP1-derived peptide that acts synergistically with caspofungin or anidulafungin against C. albicans as HsLin06_18, a 19-mer peptide derived from the C-terminal part of HsAFP1. The [caspofungin + HsLin06_18] combination significantly reduced in vitro biofilm formation of Candida glabrata and C. albicans on catheters, as well as biofilm formation of a caspofungin-resistant C. albicans strain. The [caspofungin + HsLin06_18] combination was not cytotoxic and reduced biofilm formation of C. albicans in vivo using a subcutaneous rat catheter model, as compared to control treatment. Mode of action research on the [caspofungin + HsLin06_18] combination pointed to caspofungin-facilitated HsLin06_18 internalization and immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin.

Modulation of Staphylococcus aureus Response to Antimicrobials by the Candida albicans Quorum Sensing Molecule Farnesol.

In microbial biofilms, microorganisms utilize secreted signaling chemical molecules to coordinate their collective behavior. Farnesol is a quorum sensing molecule secreted by the fungal species Candida albicans and shown to play a central physiological role during fungal biofilm growth. Our pervious in vitro and in vivo studies characterized an intricate interaction between C. albicans and the bacterial pathogen Staphylococcus aureus, as these species coexist in biofilm. In this study, we aimed to investigate the impact of farnesol on S. aureus survival, biofilm formation, and response to antimicrobials. The results demonstrated that in the presence of exogenously supplemented farnesol or farnesol secreted by C. albicans in biofilm, S. aureus exhibited significantly enhanced tolerance to antimicrobials. By using gene expression studies, S. aureus mutant strains, and chemical inhibitors, the mechanism for the enhanced tolerance was attributed to upregulation of drug efflux pumps. Importantly, we showed that sequential exposure of S. aureus to farnesol generated a phenotype of high resistance to antimicrobials. Based on the presence of intracellular reactive oxygen species upon farnesol exposure, we hypothesize that antimicrobial tolerance in S. aureus may be mediated by farnesol-induced oxidative stress triggering the upregulation of efflux pumps, as part of a general stress response system. Hence, in mixed biofilms, C. albicans may influence the pathogenicity of S. aureus through acquisition of a drug-tolerant phenotype, with important therapeutic implications. Understanding interspecies signaling in polymicrobial biofilms and the specific drug resistance responses to secreted molecules may lead to the identification of novel targets for drug development.

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase.

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with ß-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections.

Commensal Protection of Staphylococcus aureus against Antimicrobials by Candida albicans Biofilm Matrix.

Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus specifically are considered leading opportunistic fungal and bacterial pathogens, respectively, mainly due to their ability to form biofilms on catheters and indwelling medical devices. However, the impact of mixed-species biofilm growth on therapy remains largely understudied. In this study, we investigated the influence of C. albicans secreted cell wall polysaccharides on the response of S. aureus to antibacterial agents in biofilm. Results demonstrated significantly enhanced tolerance for S. aureus to drugs in the presence of C. albicans or its secreted cell wall polysaccharide material. Fluorescence confocal time-lapse microscopy revealed impairment of drug diffusion through the mixed biofilm matrix. Using C. albicans mutant strains with modulated cell wall polysaccharide expression, exogenous supplementation, and enzymatic degradation, the C. albicans-secreted ß-1,3-glucan cell wall component was identified as the key matrix constituent providing the bacteria with enhanced drug tolerance. Further, antibody labeling demonstrated rapid coating of the bacteria by the C. albicans matrix material. Importantly, via its effect on the fungal biofilm matrix, the antifungal caspofungin sensitized the bacteria to the drugs. Understanding such symbiotic interactions with clinical relevance between microbial species in biofilms will greatly aid in overcoming the limitations of current therapies and in defining potential new targets for treating polymicrobial infections. IMPORTANCE: The fungus Candida albicans and the bacterium Staphylococcus aureus are important microbial pathogens responsible for the majority of infections in hospitalized patients and are often coisolated from a host. In this study, we demonstrated that when grown together, the fungus provides the bacterium with enhanced tolerance to antimicrobial drugs. This process was mediated by polysaccharides secreted by the fungal cell into the environment. The biofilm matrix formed by these polysaccharides prevented penetration by the drugs and provided the bacteria with protection. Importantly, we show that by inhibiting the production of the fungal polysaccharides, a specific antifungal agent indirectly sensitized the bacteria to antimicrobials. Understanding the therapeutic implications of the interactions between these two diverse microbial species will aid in overcoming the limitations of current therapies and in defining new targets for treating complex polymicrobial infections.

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.

Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Sticky Matrix: Adhesion Mechanism of the Staphylococcal Polysaccharide Intercellular Adhesin.

The development of bacterial biofilms on surfaces leads to hospital-acquired infections that are difficult to fight. In Staphylococci, the cationic polysaccharide intercellular adhesin (PIA) forms an extracellular matrix that connects the cells together during biofilm formation, but the molecular forces involved are unknown. Here, we use advanced force nanoscopy techniques to unravel the mechanism of PIA-mediated adhesion in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain. Nanoscale multiparametric imaging of the structure, adhesion, and elasticity of bacteria expressing PIA shows that the cells are surrounded by a soft and adhesive matrix of extracellular polymers. Cell surface softness and adhesion are dramatically reduced in mutant cells deficient for the synthesis of PIA or under unfavorable growth conditions. Single-cell force spectroscopy demonstrates that PIA promotes cell-cell adhesion via the multivalent electrostatic interaction with polyanionic teichoic acids on the S. aureus cell surface. This binding mechanism rationalizes, at the nanoscale, the well-known ability of PIA to strengthen intercellular adhesion in staphylococcal biofilms. Force nanoscopy offers promising prospects for understanding the fundamental forces in antibiotic-resistant biofilms and for designing anti-adhesion compounds targeting matrix polymers.

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.

Candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Force nanoscopy of hydrophobic interactions in the fungal pathogen Candida glabrata.

Candida glabrata is an opportunistic human fungal pathogen which binds to surfaces mainly through the Epa family of cell adhesion proteins. While some Epa proteins mediate specific lectin-like interactions with human epithelial cells, others promote adhesion and biofilm formation on plastic surfaces via nonspecific interactions that are not yet elucidated. We report the measurement of hydrophobic forces engaged in Epa6-mediated cell adhesion by means of atomic force microscopy (AFM). Using single-cell force spectroscopy, we found that C. glabrata wild-type (WT) cells attach to hydrophobic surfaces via strongly adhesive macromolecular bonds, while mutant cells impaired in Epa6 expression are weakly adhesive. Nanoscale mapping of yeast cells using AFM tips functionalized with hydrophobic groups shows that Epa6 is massively exposed on WT cells and conveys strong hydrophobic properties to the cell surface. Our results demonstrate that Epa6 mediates strong hydrophobic interactions, thereby providing a molecular basis for the ability of this adhesin to drive biofilm formation on abiotic surfaces.