In vitro susceptibility of Aspergillus spp. clinical isolates to albendazole.

The in vitro antifungal activity of albendazole, a benzimidazole widely used as an antihelmintic drug in humans, was investigated and assessed for its activity against Aspergillus spp. Forty-eight isolates, representing the most frequent species found in human pathology [Aspergillus fumigatus (n = 27), Aspergillus flavus (n = 10), Aspergillus terreus (n = 7), Aspergillus nidulans (n = 3) and Aspergillus niger (n = 1)], and one quality control strain (A. niger ATCC 9804 83435) were tested according to the NCCLS M38-P methodology for moulds. All the strains were susceptible to albendazole, with homogeneous MICs for each species; three strains were resistant to itraconazole.

Inter-strain variability of insertion/deletion events in the Encephalitozoon cuniculi genome: a comparative KARD-PFGE analysis.

We applied a two-dimensional pulsed-field gel electrophoresis procedure to the genomes of two karyotype variants assigned to two different strains of the microsporidian Encephalitozoon cuniculi, termed D (strain III) and F (strain II). Data obtained for BssHII and MluI restriction fragment length polymorphisms in each chromosome are compiled and compared to the reference strain I variant A. Six Insertion/Deletion (InDels) are found in subterminal position, some of these being characteristic of either D or F. Like in strain 1, the terminal fragments extending between each telomere and rDNA locus are conserved in length for each chromosome. They are however smaller than in reference variant. This size reduction is estimated to be 2.5 kbp for the strain III isolate and 3.5 kbp for the strain II isolate. We hypothesize that for the three E. cuniculi strains, all chromosome extremities are prone to a constant process of sequence homogenization through mitotic recombination between conserved regions.

Occurence of subtelomeric rearrangements in the genome of the microsporidian parasite Encephalitozoon cuniculi, as revealed by a new fingerprinting procedure based on two-dimensional pulsed field gel electrophoresis.

In Microsporidia, mitochondria-lacking eukaryotic intracellular parasites, genomic comparisons were so far based on molecular karyotyping. The mammal-infecting species Encephalitozoon cuniculi is characterized by a very low haploid genome size (approximately 2.8 Mbp) and rather high karyotype variability. Recently, we developed a two-dimensional pulsed field gel electrophoresis (2-D PFGE) fingerprinting technique useful for constructing a restriction map fo the genome of a mouse E. cuniculi isolate (karyotype variant A). The so-called karyotype and restriction display 2-D PFGE (KARD-PFGE) protocol involved 1-D chromosome separation, digestion with a rare cutter, Klenow radiolabeling of genomic DNA and 2-D separation of restriction fragments followed by autoradiography. In order to assess its suitability for detecting polymorphic loci in E. cuniculi, we applied KARD-PFGE with either BssHII or Mlul digestion to genome analysis of two rabbit isolates representative of two different karyotype variants (A and C). The 2-D spot pattern of the rabbit isolate variant A is identical to the reference mouse isolate but differs greatly from the rabbit isolate variant C. Chromosomal restriction fragment length polymorphisms (RFLPs) provide strong evidence for homologous chromosomes and frequent DNA rearrangements within subtelomeric regions just upstream of the dispersed rDNA units closely associated with each chromosomal end.

Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes.

A restriction map of the 2.8-Mb genome of the unicellular eukaryote Encephalitozoon cuniculi (phylum Microspora), a mammal-infecting intracellular parasite, has been constructed using two restriction enzymes with 6 bp recognition sites (Bss HII and Mlu I). The fragments resulting from either single digestions of the whole molecular karyotype or double digestions of 11 individual chromosomes have been separated by two-dimensional pulsed field gel electrophoresis (2D-PFGE) procedures. The average distance between successive restriction sites is approximately 19 kb. The terminal regions of the chromosomes show a common pattern covering approximately 15 kb and including one 16S-23S rDNA unit. Results of hybridisation and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies on each chromosome. We have also located 67 DNA markers (clones from a partial E. cuniculi genomic library) by hybridisation to restriction fragments. Partial or complete sequencing has revealed homologies with known protein-coding genes for 32 of these clones. Evidence for two homologous chromosomes III, with a size difference (3 kb) related to a subtelomeric deletion/insertion event, argues for diploidy of E.cuniculi. The physical map should be useful for both the whole genome sequencing project and studies on genome plasticity of this widespread parasite.

In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes.

A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [alpha-(32)P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics.