Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents

Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus

Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response. Introductio

Deep Sea Microbes Probed by Incoherent Neutron Scattering Under High Hydrostatic Pressure

The majority of the biosphere is a high pressure environment. Around 70% of the marine biosphere lies at depths below 1000 m, i.e. at pressures of 100 bars or higher. To survive in these environments, deep-biosphere organisms have adapted to life at high pressure. In vitro studies showed that the activity of certain proteins originating from deep-sea organisms is less affected by high pressure than that of enzymes from surface organisms [1-3]. However, the genetic and structural bases for this increased pressure resistance are still unknown. Elastic incoherent neutron scattering studies, which provide access to information about molecular dynamics, constitute a very promising approach to decipher the structural adaptation in proteins living under high pressure. This approach has been used in the past to investigate the adaptation of biological systems to temperature and salinity and proved to be essential and complementary to structural studies. Here first investigations of high pressure effects on cell dynamics are presented using Thermococcales as models