Antifungal-Loaded Calcium Sulfate Beads as a Potential Therapeutic in Combating Candida auris.

Candida auris provides a substantial global nosocomial threat clinically. With the recent emergence that the organism can readily colonize skin niches, it will likely continue to pose a risk in health care units, particularly to patients undergoing surgery. The purpose of this study was to investigate the efficacy of antifungal-loaded calcium sulfate (CS) beads in combatting C. auris infection. We demonstrate that the CS-packed beads have the potential to interfere with planktonic and sessile C. auris.

Small molecule based anti-virulence approaches against Candida albicans infections.

Fungi are considered "silent killers" due to the difficulty of, and delays in diagnosis of infections and lack of effective antifungals. This challenge is compounded by the fact that being eukaryotes, fungi share several similarities with human cellular targets, creating obstacles to drug discovery. Candida albicans, a ubiquitous microbe in the human body is well-known for its role as an opportunistic pathogen in immunosuppressed people. Significantly, C. albicans is resistant to all the three classes of antifungals that are currently clinically available. Over the past few years, a paradigm shift has been recommended in the management of C. albicans infections, wherein anti-virulence strategies are considered an alternative to the discovery of new antimycotics. Small molecules, with a molecular weight <900 Daltons, can easily permeate the cell membrane and modulate the signal transduction pathways to elicit desired virulence inhibitory actions against pathogens. This review dissects in-depth, the discoveries that have been made with small-molecule anti-virulence approaches to tackle C. albicans infections.

Assessing the Bioactive Profile of Antifungal-Loaded Calcium Sulfate against Fungal Biofilms.

Calcium sulfate (CS) has been used clinically as a bone- or void-filling biomaterial, and its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm-inhibitory doses. A panel of different fungal species (n = 15) were selected for planktonic broth microdilution testing with fluconazole (FLZ), amphotericin B (AMB), and caspofungin (CSP). After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n = 5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semidry material, to mimic active infection in vivo In general, antifungals released from loaded CS beads were all effective at inhibiting the pathogenic fungi over 7 days within standard MIC ranges for these fungi. We observed a significant reduction of pregrown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal-loaded CS beads produce a sustained antimicrobial effect that inhibits and kills clinically relevant fungal species in vitro as planktonic and biofilm cells.

Candida albicans biofilms and polymicrobial interactions.

Candida albicans is a common fungus of the human microbiota. While generally a harmless commensal in healthy individuals, several factors can lead to its overgrowth and cause a range of complications within the host, from localized superficial infections to systemic life-threatening disseminated candidiasis. A major virulence factor of C. albicans is its ability to form biofilms, a closely packed community of cells that can grow on both abiotic and biotic substrates, including implanted medical devices and mucosal surfaces. These biofilms are extremely hard to eradicate, are resistant to conventional antifungal treatment and are associated with high morbidity and mortality rates, making biofilm-associated infections a major clinical challenge. Here, we review the current knowledge of the processes involved in C. albicans biofilm formation and development, including the central processes of adhesion, extracellular matrix production and the transcriptional network that regulates biofilm development. We also consider the advantages of the biofilm lifestyle and explore polymicrobial interactions within multispecies biofilms that are formed by C. albicans and selected microbial species.

Non-typeable Haemophilus influenzae chronic colonization in chronic obstructive pulmonary disease (COPD).

Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes to the pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.

Chitosan Ameliorates Candida auris Virulence in a Galleria mellonella Infection Model.

Candida auris has emerged as a multidrug-resistant nosocomial pathogen over the last decade. Outbreaks of the organism in health care facilities have resulted in life-threatening invasive candidiasis in over 40 countries worldwide. Resistance by C. auris to conventional antifungal drugs such as fluconazole and amphotericin B means that alternative therapeutics must be explored. As such, this study served to investigate the efficacy of a naturally derived polysaccharide called chitosan against aggregative (Agg) and nonaggregative (non-Agg) isolates of C. aurisin vitro and in vivo. In vitro results indicated that chitosan was effective against planktonic and sessile forms of Agg and non-Agg C. auris In a Galleria mellonella model to assess C. auris virulence, chitosan treatment was shown to ameliorate killing effects of both C. auris phenotypes (NCPF 8973 and NCPF 8978, respectively) in vivo Specifically, chitosan reduced the fungal load and increased survival rates of infected Galleria, while treatment alone was nontoxic to the larvae. Finally, chitosan treatment appeared to induce a stress-like gene expression response in NCPF 8973 in the larvae likely arising from a protective response by the organism to resist antifungal activity of the compound. Taken together, results from this study demonstrate that naturally derived compounds such as chitosan may be useful alternatives to conventional antifungals against C. auris.

Multi-omics tools for studying microbial biofilms: current perspectives and future directions.

The advent of omics technologies has greatly improved our understanding of microbial biology, particularly in the last two decades. The field of microbial biofilms is, however, relatively new, consolidated in the 1980s. The morphogenic switching by microbes from planktonic to biofilm phenotype confers numerous survival advantages such as resistance to desiccation, antibiotics, biocides, ultraviolet radiation, and host immune responses, thereby complicating treatment strategies for pathogenic microorganisms. Hence, understanding the mechanisms governing the biofilm phenotype can result in efficient treatment strategies directed specifically against molecular markers mediating this process. The application of omics technologies for studying microbial biofilms is relatively less explored and holds great promise in furthering our understanding of biofilm biology. In this review, we provide an overview of the application of omics tools such as transcriptomics, proteomics, and metabolomics as well as multi-omics approaches for studying microbial biofilms in the current literature. We also highlight how the use of omics tools directed at various stages of the biological information flow, from genes to metabolites, can be integrated via multi-omics platforms to provide a holistic view of biofilm biology. Following this, we propose a future artificial intelligence-based multi-omics platform that can predict the pathways associated with different biofilm phenotypes.

The systemic inflammatory response following hand instrumentation versus ultrasonic instrumentation-A randomized controlled trial.

OBJECTIVE: This study sought to investigate whether the immediate systemic inflammatory response following full-mouth debridement differs following use of hand compared with ultrasonic instruments. METHODS: Thirty-nine periodontitis patients were randomized to treatment with full-mouth debridement using either hand or ultrasonic instrumentation completed within 24 hr. Serum and periodontal clinical parameters were collected at baseline, day 1, day 7 and day 90 post-treatment. Differences in systemic inflammatory markers were assessed using general linear models at each timepoint, corrected for age, gender, smoking status, body mass index and baseline levels of each marker. RESULTS: Across all patients, serum C-reactive protein increased at day 1, with no differences between hand and ultrasonic groups (p(adjusted) = .22). There was no difference between groups in interleukin-6 (p(adjusted) = .29) or tumour necrosis factor α (p(adjusted) = .53) at day 1. Inflammatory markers returned to baseline levels by day 7. Treatment resulted in equal and marked improvements in clinical parameters in both groups; however, total treatment time was on average shorter for ultrasonic instruments (p(adjusted) = .002). CONCLUSIONS: Ultrasonic instrumentation resulted in shorter treatment time with comparable clinical outcomes. Levels of serum C-reactive protein at day 1 were similar following debridement with hand or ultrasonic instruments.

Development of a high throughput and low cost model for the study of semi-dry biofilms.

The persistence of microorganisms as biofilms on dry surfaces resistant to the usual terminal cleaning methods may pose an additional risk of transmission of infections. In this study, the Centre for Disease Control (CDC) dry biofilm model (DBM) was adapted into a microtiter plate format (Model 1) and replicated to create a novel in vitro model that replicates conditions commonly encountered in the healthcare environment (Model 2). Biofilms of Staphylococcus aureus grown in the two models were comparable to the biofilms of the CDC DBM in terms of recovered log10 CFU well-1. Assessment of the antimicrobial tolerance of biofilms grown in the two models showed Model 2 a better model for biofilm formation. Confirmation of the biofilms' phenotype with an extracellular matrix deficient S. aureus suggested stress tolerance through a non-matrix defined mechanism in microorganisms. This study highlights the importance of conditions maintained in bacterial growth as they affect biofilm phenotype and behaviour.

Nanoimprinting of biomedical polymers reduces candidal physical adhesion.

Management of fungal biofilms represents a significant challenge to healthcare. As a preventive approach, minimizing adhesion between indwelling medical devices and microorganisms would be an important step forward. This study investigated the anti-fouling capacity of engineered nanoscale topographies to the pathogenic yeast Candida albicans. Highly ordered arrays of nano-pit topographies were shown to significantly reduce the physical adherence capacity of C. albicans. This study shows a potential of nanoscale patterns to inhibit and prevent pathogenic biofilm formation on biomedical substrates.

Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida auris.

The emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare-associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.

Implications of Antimicrobial Combinations in Complex Wound Biofilms Containing Fungi.

Diabetic foot ulcer treatment currently focuses on targeting bacterial biofilms, while dismissing fungi. To investigate this, we used an in vitro biofilm model containing bacteria and fungi, reflective of the wound environment, to test the impact of antimicrobials. Here we showed that while monotreatment approaches influenced biofilm composition, this had no discernible effect on overall quantity. Only by combining bacterium- and fungus-specific antibiotics were we able to decrease the biofilm bioburden, irrespective of composition.

Biofilms Formed by Isolates from Recurrent Vulvovaginal Candidiasis Patients Are Heterogeneous and Insensitive to Fluconazole.

Vulvovaginal candidiasis (VVC) is a global health problem affecting ∼75% of women at least once in their lifetime. Here we examined the epidemiology of VVC in a patient cohort to identify the causative organisms associated with VVC. Biofilm-forming capacity and antifungal sensitivity profiles were also assessed. We report a shifting prevalence of Candida species with heterogeneous biofilm-forming capacity, which is associated with altered antifungal drug sensitivity.

High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and ß-1,3-glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus.

One step closer to understanding the role of bacteria in diabetic foot ulcers: characterising the microbiome of ulcers.

BACKGROUND: The aim of this study was to characterise the microbiome of new and recurrent diabetic foot ulcers using 16S amplicon sequencing (16S AS), allowing the identification of a wider range of bacterial species that may be important in the development of chronicity in these debilitating wounds. Twenty patients not receiving antibiotics for the past three months were selected, with swabs taken from each individual for culture and 16S AS. DNA was isolated using a combination of bead beating and kit extraction. Samples were sequenced on the Illumina Hiseq 2500 platform. RESULTS: Conventional laboratory culture showed positive growth from only 55 % of the patients, whereas 16S AS was positive for 75 % of the patients (41 unique genera, representing 82 different operational taxonomic units (OTU's). S. aureus was isolated in 72 % of culture-positive samples, whereas the most commonly detected bacteria in all ulcers were Peptoniphilus spp., Anaerococcus spp. and Corynebacterium spp., with the addition of Staphylococcus spp. in new ulcers. The majority of OTU's residing in both new and recurrent ulcers (over 67 %) were identified as facultative or strict anaerobic Gram-positive organisms. Principal component analysis (PCA) showed no difference in clustering between the two groups (new and recurrent ulcers). CONCLUSIONS: The abundance of anaerobic bacteria has important implications for treatment as it suggests that the microbiome of each ulcer "starts afresh" and that, although diverse, are not distinctly different from one another with respect to new or recurrent ulcers. Therefore, when considering antibiotic therapy the duration of current ulceration may be a more important consideration than a history of healed ulcer.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

Development and characterisation of a novel three-dimensional inter-kingdom wound biofilm model.

Chronic diabetic foot ulcers are frequently colonised and infected by polymicrobial biofilms that ultimately prevent healing. This study aimed to create a novel in vitro inter-kingdom wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical wound treatments. Inter-kingdom triadic biofilms composed of Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus were shown to be quantitatively greater in this model compared to a simple substratum when assessed by conventional culture, metabolic dye and live dead qPCR. These biofilms were both structurally complex and compositionally dynamic in response to topical therapy, so when treated with either chlorhexidine or povidone iodine, principal component analysis revealed that the 3-D cellulose model was minimally impacted compared to the simple substratum model. This study highlights the importance of biofilm substratum and inclusion of relevant polymicrobial and inter-kingdom components, as these impact penetration and efficacy of topical antiseptics.

Aspergillus Biofilms in Human Disease.

The biofilm phenotype of Aspergillus species is an important and accepted clinical entity. While industrially these biofilms have been used extensively in important biofermentations, their role in clinical infection is less well defined. A recent flurry of activity has demonstrated that these interesting filamentous moulds have the capacity to form biofilms both in vitro and in vivo, and through various investigations have shown that these are exquisitely resistant to antifungal therapies through a range of adaptive resistance mechanisms independent of defined genetic changes. This review will explore the clinical importance of these biofilms and provide contemporary information with respect to their clinical management.