Martin Kierans

Microemulsions are membrane-active, antimicrobial, self-preserving systems

Microemulsions are physically stable oil/water systems that have potential use as delivery systems for many pharmaceuticals which are normally of limited use due to their hydrophobicity, toxicity or inability to access the site of action. It has been suggested that microemulsions are self‐preserving antimicrobials in their own right, although there is little evidence to support this. In this experiment, microemulsions of various compositions were formulated and tested for their stability and antimicrobial action. The physical stability of the different microemulsions was assessed by centrifugation at 4000 g and by storage in a water bath at 37 °C for one month, during which no phase separation was observed. The antimicrobial activity of the microemulsions was tested using the compendial method, observation of the kinetics of killing, and transmission electron microscopy (TEM) of microemulsion‐exposed cultures of Pseudomonas aeruginosa PA01. These latter experiments on Ps. aeruginosa indicated distinct signs of membrane disruption. The results indicated that the microemulsions are self‐preserved, and that their killing of microbial cultures is very rapid and may be the result of membrane activity.

Solubilisation of some naturally occurring metal-bearing minerals, limescale and lead phosphate by Aspergillus niger

The ability of the soil fungus Aspergillus niger to tolerate and solubilise seven naturally occurring metal-bearing minerals, limescale and lead phosphate was investigated. A. niger was able to solubilise four of the test insoluble compounds when incorporated into solid medium: cuprite (CuO2), galena (PbS), rhodochrosite (Mn(CO3)x) and limescale (CaCO3). A. niger was able to grow on all concentrations of all the test compounds, whether solubilisation occurred or not, with no reduction in growth rate from the control. In some cases, stimulation of growth occurred, most marked with the phosphate-containing mineral, apatite. Precipitation of insoluble copper and manganese oxalate crystals under colonies growing on agar amended with cuprite and rhodochrosite was observed after 1-2 days growth at 25 degrees C. This process of oxalate formation represents a reduction in bioavailability of toxic cations, and could represent an important means of toxic metal immobilisation of physiological and environmental significance.

Rock-Building Fungi

Fungi are a major component of the biota in soils and mineral substrates occurring over a wide range of geographical and climatic zones, and are often dominant when compared to other organisms, including bacteria. Due to their filamentous growth habit and ability to excrete organic acids, protons and other metabolites, fungi are perfectly suited as biological weathering agents of natural rocks, minerals, including those used in building materials. Small tunnels discovered inside weatherable minerals in soil were hypothesized to be formed by mineral-solubilizing activities of ectomycorrhizal fungi. Using scanning electron microscopy techniques to visualise in situ fungal weathering of rock, patina formation and secondary mineral precipitation we provide an additional explanation for the occurrence of fungal tunnels inside minerals using carbonates and oxalates as examples. Our findings highlight fungal potential for mineral transformations and their ability to precipitate secondary mycogenic minerals within rock substrata.

Transformation and tolerance of tellurite by filamentous fungi: accumulation, reduction, and volatilization

Accumulation and transformation of tellurite (TeO 3 2- ) by a species of Fusarium and Penicillium citrinum was examined using both solid and liquid Czapek Dox medium. In liquid medium, tellurite partitioned into soluble and insoluble species, and in tellurite-containing (1 mM) liquid medium at pH 6, approx. 60% of added tellurite precipitated after 48 h. Experiments showed that in liquid medium containing 1 mM sodium tellurite, the Fusarium sp. accumulated a maximum of ∼ 0.6 μmol Te (mg D.w.) -1 after 48 h. P. citrinum accumulated a much lower amount of tellurium, ∼ 0.07 μmol (mg D.W.) -1 falling to -1 after 48 h. Both organisms showed marked differences in the pattern of pH change of the medium, with the pH increasing during growth of the Fusarium sp. in 1 mM tellurite to ∼ pH 6.7 after 2 wk. In contrast, the pH decreased during growth of P. citrinum in 1 mM tellurite, to a level of ∼ pH 2.7 after 2 wk. On agar medium, test fungi exhibited tolerance to high levels of tellurite (up to 100 mM Na 2 TeO 3 ) and this was associated with blackening of the growing colonies as well as the surrounding agar. TEM revealed the deposition of large black granules, apparently in vacuoles, which corresponded with the reduction of tellurite to amorphous elemental tellurium. Precipitation of amorphous tellurium on and around the biomass was also observed and confirmed by energy-dispersive X-ray microprobe analysis. In addition to the reductive transformation of tellurite, Fusarium sp. also displayed transformation of tellurite into a volatile form. The production of volatile tellurium by Fusarium sp. occurred over the whole growth period and amounted to an average value of 7.8 μmol Te (∼ 0.16%) from 51 growth medium with an initial concentration of 1 mM Na 2 TeO 3 . Although P. citrinum also transformed tellurite to elemental tellurium, volatilization of tellurium was not detected. It is concluded that different mechanisms of tellurium transformation are species-dependent and can be influenced by physico-chemical changes in the medium, e.g. pH, which can affect tellurium speciation into soluble and insoluble forms and bioaccumulation. In view of the extremely small amounts of Te volatilized by the Fusarium sp., this process cannot be considered to be an important detoxification mechanism.

Silver tolerance and accumulation in yeasts

SummaryDebaryomyces hansenii (NCYC 459 and strain 75-21),Candida albicans (3153A),Saccharomyces cerevisiae (X2180-1B),Rhodotorula rubra (NCYC 797) andAureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO3. Although Ag+ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1–2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag+ to Ag0 resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO3-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag0 although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag+ reduction to Ag0.

Microemulsions are highly effective anti-biofilm agents

Aims: The demonstration of the antibiofilm effects of pharmaceutical microemulsions.

Failure of centrosome migration causes a loss of motile cilia in talpid3 mutants

Background: Loss of function mutations in the centrosomal protein TALPID3 (KIAA0586) cause a failure of primary cilia formation in animal models and are associated with defective Hedgehog signalling. It is unclear, however, if TALPID3 is required only for primary cilia formation or if it is essential for all ciliogenesis, including that of motile cilia in multiciliate cells. Results: FOXJ1, a key regulator of multiciliate cell fate, is expressed in the dorsal neuroectoderm of the chicken forebrain and hindbrain at stage 20HH, in areas that will give rise to choroid plexuses in both wt and talpid3 embryos. Wt ependymal cells of the prosencephalic choroid plexuses subsequently transition from exhibiting single short cilia to multiple long motile cilia at 29HH (E8). Primary cilia and long motile cilia were only rarely observed on talpid3 ependymal cells. Electron microscopy determined that talpid3 ependymal cells do develop multiple centrosomes in accordance with FOXJ1 expression, but these fail to migrate to the apical surface of ependymal cells although axoneme formation was sometimes observed. Conclusions: TALPID3, which normally localises to the proximal centrosome, is essential for centrosomal migration prior to ciliogenesis but is not directly required for de novo centriologenesis, multiciliated fate, or axoneme formation. Developmental Dynamics 242:923–931, 2013.

A Model Sheet Mineral System to Study Fungal Bioweathering of Mica

The general objective of this research was to examine fungal interactions with silicate minerals within the context of their roles in bioweathering. To achieve this, we used muscovite, a phyllosilicate mineral (KAl2[(OH)2|AlSi3O10]), in the form of a mineral sheet model system for ease of experimental manipulation and microscopic examination. It was found that test fungal species successfully colonized and degraded the surface of muscovite sheets in both laboratory and field experiments. After colonization by the common soil fungus Aspergillus niger, a network of hyphae covered the surface of the muscovite, and mineral dissolution or degradation was clearly evidenced by a network of fungal “footprints” that reflected coverage by the mycelium. For natural soil incubations, microorganisms associated with muscovite sheet material included biofilms of fungi and bacteria on the surface, while mineral encrustation or adhesion to microbial structures was also observed. Our results show that muscovite sheet is a good model mineral system for examination of microbial colonization and degradation, and this was demonstrated using laboratory and field systems, providing more evidence for the bioweathering significance of fungal activities in the context of silicate degradation and soil formation and development. The approach is also clearly applicable to other rock and mineral-based substrates and a variety of free-living and symbiotic microbial systems.

Phosphatase‐mediated bioprecipitation of lead by soil fungi

Geoactive soil fungi were examined for their ability to release inorganic phosphate (Pi ) and mediate lead bioprecipitation during growth on organic phosphate substrates. Aspergillus niger and Paecilomyces javanicus grew in 5 mM Pb(NO3)2-containing media amended with glycerol 2-phosphate (G2P) or phytic acid (PyA) as sole P sources, and liberated Pi into the medium. This resulted in almost complete removal of Pb from solution and extensive precipitation of lead-containing minerals around the biomass, confirming the importance of the mycelium as a reactive network for biomineralization. The minerals were identified as pyromorphite (Pb5(PO4)3Cl), only produced by P. javanicus, and lead oxalate (PbC2O4), produced by A. niger and P. javanicus. Geochemical modelling of lead and lead mineral speciation as a function of pH and oxalate closely correlated with experimental conditions and data. Two main lead biomineralization mechanisms were therefore distinguished: pyromorphite formation depending on organic phosphate hydrolysis and lead oxalate formation depending on oxalate excretion. This also indicated species specificity in biomineralization depending on nutrition and physiology. Our findings provide further understanding of lead geomycology and organic phosphates as a biomineralization substrate, and are also relevant to metal immobilization biotechnologies for bioremediation, metal and P biorecovery, and utilization of waste organic phosphates.

Studies on the kinetics of killing and the proposed mechanism of action of microemulsions against fungi

Microemulsions are physically stable oil/water clear dispersions, spontaneously formed and thermodynamically stable. They are composed in most cases of water, oil, surfactant and cosurfactant. Microemulsions are stable, self-preserving antimicrobial agents in their own right. The observed levels of antimicrobial activity associated with microemulsions may be due to the direct effect of the microemulsions themselves on the bacterial cytoplasmic membrane. The aim of this work is to study the growth behaviour of different microbes in presence of certain prepared physically stable microemulsion formulae over extended periods of time. An experiment was designed to study the kinetics of killing of a microemulsion preparation (17.3% Tween-80, 8.5% n-pentanol, 5% isopropyl myristate and 69.2% sterile distilled water) against selected test microorganisms (Candida albicans, Aspergillus niger, Schizosaccharomyces pombe and Rhodotorula spp.). Secondly, an experiment was designed to study the effects of the microemulsion preparation on the cytoplasmic membrane structure and function of selected fungal species by observation of 260 nm component leakage. Finally, the effects of the microemulsion on the fungal membrane structure and function using S. pombe were studied using transmission electron microscopy. The results showed that the prepared microemulsions are stable, effective antimicrobial systems with effective killing rates against C. albicans, A. niger, S. pombe and Rhodotorula spp. The results indicate a proposed mechanism of action of significant anti-membrane activity, resulting in the gross disturbance and dysfunction of the cytoplasmic membrane structure which is followed by cell wall modifications, cytoplasmic coagulation, disruption of intracellular metabolism and cell death.