Nanomechanical sensors for single microbial cell growth monitoring.

A nanomechanical technique for rapid real time detection and monitoring of microorganism growth will significantly reduce costs and diagnosis times in industrial and clinical settings. Owing to their label free detection mechanism and unprecedented sensitivity to the mass and elastic modulus of biological structures, dynamically operated cantilever arrays provide an opportunity to rapidly detect and track the evolution of microbial growth. Here we report the monitoring of the growth of single Aspergillus niger spores via the multimode response of microcantilevers. The fungal hyphal structure affects the cantilevers' nanomechanical properties as it propagates along the sensor. We demonstrate, for the first time, the mapping of cellular events with great accuracy using a cantilever frequency response. Imaging of growth conditions on the cantilever, which is performed in parallel, allows for verification of these results. Theoretical comparison and finite element modelling confirm experimental findings and allow for determination of the hyphal elastic modulus.

Antifungal activity of statins and their interaction with amphotericin B against clinically important Zygomycetes.

The in vitro antifungal activity of different statins and the combinations of the two most effective ones (fluvastatin and rosuvastatin) with amphotericin B were investigated in this study on 6 fungal isolates representing 4 clinically important genera, namely Absidia, Rhizomucor, Rhizopus and Syncephalastrum . The antifungal effects of statins revealed substantial differences. The synthetic statins proved to be more effective than the fungal metabolites. All investigated strains proved to be sensitive to fluvastatin. Fluvastatin and rosuvastatin acted synergistically and additively with amphotericin B in inhibiting the fungal growth in clinically available concentration ranges. Results suggest that statins combined with amphotericin B have a therapeutic potential against fungal infections caused by Zygomycetes species.

In vitro interactions between primycin and different statins in their effects against some clinically important fungi.

The in vitro antifungal activities of primycin (PN) and various statins against some opportunistic pathogenic fungi were investigated. PN completely inhibited the growth of Candida albicans (MIC 64 microg ml(-1)) and Candida glabrata (MIC 32 microg ml(-1)), and was very effective against Paecilomyces variotii (MIC 2 microg ml(-1)), but had little effect on Aspergillus fumigatus, Aspergillus flavus or Rhizopus oryzae (MICs >64 microg ml(-1)). The fungi exhibited different degrees of sensitivity to the statins; fluvastatin (FLV) and simvastatin (SIM) exerted potent antifungal activities against a wide variety of clinically important fungal pathogens. Atorvastatin, rosuvastatin and lovastatin (LOV) had a slight effect against all fungal isolates tested, whereas pravastatin was completely ineffective. The in vitro interactions between PN and the different statins were investigated using a standard chequerboard titration method. When PN was combined with FLV, LOV or SIM, both synergistic and additive effects were observed. The extent of inhibition was higher when these compounds were applied together, and the concentrations of PN and the given statin needed to block fungal growth completely could be decreased by several dilution steps. Similar interactions were observed when the variability of the within-species sensitivities was investigated.

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides.

In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor hmgR did not affect the carotenoid production of M. circinelloides.

Interactions between statins and Penicillium chrysogenum antifungal protein (PAF) to inhibit the germination of sporangiospores of different sensitive Zygomycetes.

This study reports on the antifungal activities of statins combined with an antifungal compound secreted by Penicillium chrysogenum, PAF. Several species belonging in the class Zygomycetes are considered to be agents of human or animal mycoses; other species have significance as postharvest plant pathogens. In the present work, four species (Rhizopus stolonifer, Mortierella wolfii, Syncephalastrum racemosum and Mycotypha africana) that exhibited different sensitivities to lovastatin and PAF in previous experiments were investigated. The efficiencies with which four statins (lovastatin, simvastatin, rosuvastatin and atorvastatin) inhibited sporangiospore germination in the absence or in the presence of a constant concentration of PAF were studied. PAF and lovastatin acted synergistically on the sporangiospore germination of Mycotypha africana, and similar effects of the combinations PAF-rosuvastatin and PAF-atorvastatin were observed on S. racemosum.

Differentiation of Rhizomucor species on the basis of their different sensitivities to lovastatin.

The opportunistic pathogens Rhizomucor pusillus and Rhizomucor miehei may be agents of frequently fatal mycotic diseases. In the present study, the susceptibilities of 27 clinical and environmental isolates of R. miehei and R. pusillus to lovastatin under different culturing conditions were investigated. Most of the R. miehei strains grew at lovastatin concentrations as high as 64 to 128 microg/ml. In contrast, the inhibitory effect of lovastatin on all of the R. pusillus strains was evident at lovastatin concentrations as low as 1 to 2 microg/ml. A simple and reliable method for species-level differentiation, based on the significantly higher sensitivity of R. pusillus to lovastatin than that of R. miehei, was elaborated. According this, on malt extract agar containing 6 mug of lovastatin/ml, R. pusillus is not able to produce colonies, while R. miehei will form compact colonies.

Cloning and sequence analysis of Mucor circinelloides glyceraldehyde-3-phosphate dehydrogenase gene.

A genomic library of Mucor circinelloides ATCC 1216b has been constructed in Lambda Fix II vector. The library has an average insert site of 10 kb and covers the genome 12 times. The M. circinelloides gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The promoter region, containing a consensus TATA and CAAT box and a 298 nucleotid long termination region were also determined.