Quantitative and Qualitative Analyses of the Cell Death Process in Candida albicans Treated by Antifungal Agents

The death process of Candida albicans was investigated after treatment with the antifungal agents flucytosine and amphotericin B by assessing morphological and biophysical properties associated with cell death. C. albicans was treated varying time periods (from 6 to 48 hours) and examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM and AFM images clearly showed changes in morphology and biophysical properties. After drug treatment, the membrane of C. albicans was perforated, deformed, and shrunken. Compared to the control, C. albicans treated with flucytosine was softer and initially showed a greater adhesive force. Conversely, C. albicans treated with amphotericin B was harder and had a lower adhesive force. In both cases, the surface roughness increased as the treatment time increased. The relationships between morphological changes and the drugs were observed by AFM clearly; the surface of C. albicans treated with flucytosine underwent membrane collapse, expansion of holes, and shrinkage, while the membranes of cells treated with amphotericin B peeled off. According to these observations, the death process of C. albicans was divided into 4 phases, CDP0, CDP1, CDP2, and CDP4, which were determined based on morphological changes. Our results could be employed to further investigate the antifungal activity of compounds derived from natural sources

Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells

Peptidoglycans provide bacterial cell walls with mechanical strength. The spatial organization of peptidoglycan has previously been difficult to study. Here, atomic force microscopy, together with cells carrying mutations in cell-wall polysaccharides, has allowed an in-depth study of these molecules

Adhesion of Azospirillum brasilense: Role of proteins at the cell-support interface

The adhesion of the soil bacterium Azospirillum brasilense to polystyrene has been investigated using a parallel-plate chamber in conditions allowing cell transport to the support by sedimentation and rinsing under controlled hydrodynamic conditions. The adhesion pattern, which was heterogeneous, and the density of adhering cells were determined by cell aggregation at the support surface and detachment of aggregates upon rinsing. Determination of the support surface composition by X-ray photoelectron spectroscopy after detachment of adhering cells and analysis of the supernatant of cell suspensions revealed that, during the course of adhesion, extracellular proteins are released progressively into the aqueous phase and adsorb at the support surface. Support preconditioning by contact with a cell suspension promoted adhesion after a short contact time (2 h) due to protein adsorption at the support surface. Cell ageing prior to the adhesion test also enhanced adhesion after 2 h due to protein accumulation at the cell surface. Moreover, when supports were preconditioned and when cells were aged prior to the test, adhesion was still dependent on cell-support contact time, which pointed to the influence of in situ secretion of proteins by the adhering cells. It was therefore concluded that the role played by proteins at the cell-support interface is twofold. In the first stage, proteins accumulate at the cell surface, are liberated into the solution and adsorb at the support surface; the increase of the protein concentration at the interface promotes initial adhesion. In the next stage, in situ secretion of proteins during the prolonged contact between the cells and the support strengthens adhesion

Adhesion of Lactococcus lactis to model substrata: direct study of the interface

The adhesion of Lactococcus lactis harvested in the exponential and stationary growth phases was examined on glass and on polystyrene with cells resuspended in water. A low adhesion density was observed with exponential cells whatever the substratum. Stationary cells adhered in large amount on polystyrene and not on glass. This adhesion behavior cannot be explained simply in terms of physicochemical properties of the surfaces. The attention is drawn on particular aspects which are not taken into consideration in classical approaches of cell adhesion. Atomic force microscopy showed that, after a forced contact, the bacterial surface sticks to the retracting AFM probe, indicating that the force applied when the cell comes in contact with the substratum may influence its adhesion. This may affect the relevance of adhesion tests in laminar conditions with respect to real situations. The X-ray photoelectron spectroscopy analysis of the substratum surface after detachment of adhering cells, on the one hand, and of evaporated cell supernatants, on the other hand, revealed that extracellular substances were released by L. lactis in the aqueous phase and at cell-substratum interface. They may have a critical effect on cell adhesion by increasing the ionic strength of the solution confined between the cells and the substrata and by bringing macromolecules at the interface. (C) 2001 Elsevier Science B.V. All rights reserved

Modification of the aggregation behaviour of the environmental Ralstonia eutropha-like strain AE815 is reflected by both surface hydrophobicity and amplified fragment length polymorphism (AFLP) patterns

After inoculation of the plasmid-free non-aggregative Ralstonia eutropha-like strain AE815 in activated sludge, followed by reisolation on a selective medium, a mutant strain A3 was obtained, which was characterized by an autoaggregative behaviour. Strain A3 had also acquired an IncP1 plasmid, pLME1, co-aggregated with yeast cells when co-cultured, and stained better with Congo red than did the AE815 strain. Contact angle measurements showed that the mutant strain was considerably more hydrophobic than the parent strain AE815, and scanning electron microscopy (SEM) revealed the production of an extracellular substance. A similar hydrophobic mutant (AE176R) could be isolated from the AE815-isogenic R, eutropha-like strain AE176, With the DNA fingerprinting technique repetitive extragenic palindromic-polymerase chain reaction (REP-PCR), no differences between these four strains, AE815, A3, AE176 and AE176R, could be revealed. However, using the amplified fragment length polymorphism (AFLP) DNA fingerprinting technique with three different primer combinations, small but clear reproducible differences between the banding patterns of the autoaggregative mutants and their non-autoaggregative parent strains were observed for each primer set. These studies demonstrate that, upon introduction of a strain in an activated sludge microbial community, minor genetic changes readily occur, which can nevertheless have major consequences for the phenotype of the strain and its aggregation behaviour