Microbial Interkingdom Biofilms and the Quest for Novel Therapeutic Strategies.

Fungal and bacterial species interact with each other within polymicrobial biofilm communities in various niches of the human body. Interactions between these species can greatly affect human health and disease. Diseases caused by polymicrobial biofilms pose a major challenge in clinical settings because of their enhanced virulence and increased drug tolerance. Therefore, different approaches are being explored to treat fungal-bacterial biofilm infections. This review focuses on the main mechanisms involved in polymicrobial drug tolerance and the implications of the polymicrobial nature for the therapeutic treatment by highlighting clinically relevant fungal-bacterial interactions. Furthermore, innovative treatment strategies which specifically target polymicrobial biofilms are discussed.

Bioluminescence Imaging to Study Mature Biofilm Formation by Candida spp. and Antifungal Activity In Vitro and In Vivo.

The widespread use of indwelling medical devices has increased the number of device-related infections in hospitalized patients. These infections are often associated with the formation of biofilms on the medical implants that are difficult to treat because of their resistance to the classical antifungal drugs. The most common fungi isolated from catheters and other medical devices are Candida species. The Candida genus contains multiple species of which C. albicans and C. glabrata are the two most common pathogenic yeasts in humans. A limited number of animal models is available for investigating host-pathogen interactions and testing novel antifungal drugs in vivo against these species. Fungal load in biofilms in these models is traditionally analyzed postmortem, requiring host sacrifice and enumeration of microorganisms from individual biofilms in order to evaluate the amount of colony forming units and the efficacy of antifungal treatment. Bioluminescence imaging (BLI) made compatible with small animal models for in vivo biofilm formation is a valuable tool to follow biofilm development and its treatment longitudinally. Due to the noninvasive nature of BLI, the imaging procedure can be repeated in the same animal, allowing for follow-up of the biofilm growth in vivo without removing the implanted device or detaching the biofilm from its substrate. Although detecting a quantifiable in vivo BLI signal from biofilms formed on the inside of implanted catheters is challenging, BLI proved to be a practical tool in the study of fungal biofilms. This method describes the use of BLI for in vitro and in vivo follow-up of device-related fungal biofilm formation in mice and rats and antifungal activity testing against both C. albicans and C. glabrata device-associated biofilms. It can further be applied for efficient in vivo screening for interesting genes of the pathogen and the host involved in biofilm formation.

Adhesion of Staphylococcus aureus to Candida albicans During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response.

Interspecies interactions greatly influence the virulence, drug tolerance and ultimately the outcome of polymicrobial biofilm infections. A synergistic interaction is observed between the fungus Candida albicans and the bacterium Staphylococcus aureus. These species are both normal commensals of most healthy humans and co-exist in several niches of the host. However, under certain circumstances, they can cause hospital-acquired infections with high morbidity and mortality rates. Using a mouse model of oral co-infection, we previously showed that an oral infection with C. albicans predisposes to a secondary systemic infection with S. aureus. Here, we unraveled this intriguing mechanism of bacterial dissemination. Using static and dynamic adhesion assays in combination with single-cell force spectroscopy, we identified C. albicans Als1 and Als3 adhesins as the molecular players involved in the interaction with S. aureus and in subsequent bacterial dissemination. Remarkably, we identified the host immune response as a key element required for bacterial dissemination. We found that the level of immunosuppression of the host plays a critical yet paradoxical role in this process. In addition, secretion of candidalysin, the C. albicans peptide responsible for immune activation and cell damage, is required for C. albicans colonization and subsequent bacterial dissemination. The physical interaction with C. albicans enhances bacterial uptake by phagocytic immune cells, thereby enabling an opportunity to disseminate.

Candida albicans and Staphylococcus Species: A Threatening Twosome.

Candida albicans and Staphylococcus species are, respectively, the most common fungal and bacterial agents isolated from bloodstream infections, worldwide. Moreover, it has been shown that 20% of all C. albicans bloodstream infections are polymicrobial in nature, with Staphylococcus epidermidis and Staphylococcus aureus being the first and third most common co-isolated organisms, respectively. These species are part of the commensal microbial flora but can cause hospital-acquired infections with an extreme ability to inhabit diverse host niches, especially in immunocompromised patients. They are well known for their ability to form persistent biofilms in the host or on abiotic surfaces such as indwelling medical devices. Interactions within these biofilm communities can lead to increased virulence, drug tolerance, and immune evasion. This can ultimately impact morbidity and infection outcome, often leading to an increased mortality. Therefore, characterizing the interactions between these species could lead to the development of novel therapeutic approaches that target polymicrobial infections. In this mini review, we briefly highlight the current knowledge and most recent insights into the complex interspecies interactions of C. albicans with Staphylococcus bacteria.

Inhibition of Vesicular Transport Influences Fungal Susceptibility to Fluconazole.

Fungal infections pose a substantial threat to the human population. They can cause either mild and relatively harmless infections or invasive and often lethal diseases in patients with a weakened immune system. The majority of these human fungal infections are caused by Candida species. The limited amount of available therapies, together with the development of resistance against these drugs, strongly emphasizes the need for novel therapeutic strategies. As it is quite time-consuming to introduce completely new drugs to the market, potentiating the efficacy of existing drugs would be a better strategy. Therefore, it is important to identify cellular pathways involved in the development of drug resistance. We found that vesicular transport is involved in fungal susceptibility to the most widely used antifungal drug, fluconazole. We identified specific complexes in the vesicular transport pathway which contribute to fluconazole resistance or tolerance in the model organism Saccharomyces cerevisiae Furthermore, we confirmed our findings in the clinically relevant fungi Candida albicans and Candida glabrata Finally, we show that the combination of fluconazole with a specific inhibitor of the vesicular transport pathway increases the susceptibility of Candida species, indicating the potential of using vesicular transport as a target in combination therapy.

Essential oils and their components are a class of antifungals with potent vapour-phase-mediated anti-Candida activity.

Multi-resistant microorganisms continue to challenge medicine and fuel the search for new antimicrobials. Here we show that essential oils and their components are a promising class of antifungals that can have specific anti-Candida activity via their vapour-phase. We quantify the vapour-phase-mediated antimicrobial activity (VMAA) of 175 essential oils and 37 essential oil components, representing more than a 1,000 unique molecules, against C. albicans and C. glabrata in a novel vapour-phase-mediated susceptibility assay. Approximately half of the tested essential oils and their components show growth-inhibitory VMAA. Moreover, an average greater activity was observed against the intrinsically more resistant C. glabrata, with essential oil component citronellal having a highly significant differential VMAA. In contrast, representatives of each class of antifungals currently used in clinical practice showed no VMAA. The vapour-phase-mediated susceptibility assay presented here thus allows for the simple detection of VMAA and can advance the search for novel (applications of existing) antimicrobials. This study represents the first comprehensive characterisation of essential oils and their components as a unique class of antifungals with antimicrobial properties that differentiate them from existing antifungal classes.