MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor.

Vibrio vulnificus biotype 2 is a polyphyletic group whose virulence for fish relies on a plasmid. This plasmid contains an rtxA gene duplicated in the small chromosome that encodes a MARTX (Multifunctional, Autoprocessing Repeats-in-Toxin) unique within the species in domain structure (MARTX type III). To discover the role of this toxin in the fitness of this biotype in the fish-farming environment, single- and double-knockout mutants were isolated from a zoonotic strain and analysed in a series of in vivo and in vitro experiments with eel, fish cell lines and amoebae isolated from gills. Mice, murine and human cell lines were also assayed for comparative purposes. The results suggest that MARTX type III is involved in the lysis of a wide range of eukaryotic cells, including the amoebae, erythrocytes, epithelial cells and phagocytes after bacterium-cell contact. In fish, MARTX type III may act as a toxin involved in the onset of septic shock, while in mice it may promote bacterial colonization by preventing phagocytosis of bacterial cells. Moreover, this toxin could protect bacteria from predation by amoebae, which would increase bacterial survival outside the host and would explain the fitness of this biotype in the fish-farming environment.

Study of the antifungal potential of novel cellulose/copper composites as absorbent materials for fruit juices.

Cellulose/copper composites with antifungal properties have been synthesized by physical/chemical methods. Physical treatments by heat or by a combination of heat and UV radiation provided composites with metallic copper and excellent interfacial adhesion; in contrast, chemical reduction with borohydride generated small although partially aggregated copper oxide nanoparticles. Copper micro/nano-particles and copper ions (Cu(2+)) were released from the cellulose matrix at an adequate rate to achieve a strong antimicrobial activity against Saccharomyces cerevisiae in in vitro experiments. Moreover, the copper oxide composites showed an excellent antifungal activity in pineapple and melon juice, reducing about 4 log cycles the loads of spoilage-related yeasts and moulds. The metallic copper composites reduced in 4 log cycles the load of yeasts and moulds in pineapple juice, although their antifungal activity was weaker in contact with melon juice. Copper loaded absorbent materials could be selectively implemented during the shelf-life of minimally processed fruits to reduce the number of spoilage-related microorganisms in the drip.

pilF Polymorphism-based PCR to distinguish vibrio vulnificus strains potentially dangerous to public health.

Vibrio vulnificus is a heterogeneous species that comprises strains virulent and avirulent for humans and fish, and it is grouped into three biotypes. In this report, we describe a PCR-based methodology that allows both the species identification and discrimination of those isolates that could be considered dangerous to public health. Discrimination is based on the amplification of a variable region located within the gene pilF, which seems to be associated with potential human pathogenicity, regardless of the biotype of the strain.

Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality.

Vibrio vulnificus biotype 3 has been implicated as the causative pathogen of an ongoing disease outbreak that erupted in Israel in 1996. Recent work based on multi-locus sequence typing (MLST) showed that V. vulnificus biotype 3 is genetically homogeneous. The aim of this study was to investigate the existence of subpopulations within this homogeneous biotype by characterizing the surface antigens and analysing the sequence diversity of selected outer-membrane protein (OMP)-encoding genes. Rabbit antisera were prepared against biotype 1, 2 and 3 strains. The results of the slide-agglutination test, dot-blot assay (using fresh and boiled cells), and immunoblotting of lipopolysaccharides (LPS) and OMPs were evaluated. By slide-agglutination and dot-blot assays all biotype 3 strains agglutinated with the selected biotype 3 strain. This homogeneity was supported by immunoblot analysis of the LPS. Analysis of OMP patterns revealed that all three biotypes share a considerable number of common bands that are antigenically related. Cluster analysis of DNA sequence data from selected OMP-encoding genes showed that biotype 3 strains form a genetically distinct and homogeneous clone. The homogeneity of surface antigens and the lack of any sequence diversity among both housekeeping and OMP-encoding genes reaffirms the highly clonal nature of biotype 3 and suggests that it has only recently descended from the parent population of V. vulnificus.

Comparative assessment of solid-phase extraction clean-up procedures, GC columns and perfluoroacylation reagents for determination of type B trichothecenes in wheat by GC-ECD.

Various solid-phase extraction (SPE) procedures for clean-up, two perfluoroacylation reagents (pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA)) and two chromatographic columns (HP-1701 and HP-5) have been assessed comparatively to achieve the determination of type B trichothecenes (deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol (3- and 15-ADON)) in wheat grain by gas chromatography (GC)-electron-capture detection (ECD). Spiked wheat samples were extracted with acetonitrile-water (84:16, v/v). Tested SPE procedures were MycoSep 225 column, Florisil and different cartridges prepared in the laboratory with mixtures of various sorbents like alumina, Celite 545, C18, silica and charcoal. We propose MycoSep 225 column, and cartridges made with alumina-charcoal-silica and alumina-charcoal-C18 silica mixtures as clean-up procedures on the basis of recovery values (89.6, 87.3 and 86.1% for deoxynivalenol, respectively, at 1.0mg/kg spiking level). The two last procedures are less expensive. Pentafluoropropionic anhydride was more stable against moisture and less expensive, while recoveries were similar to those obtained with heptafluorobutyric anhydride. HP-1701 column can separate 3- and 15-acetyldeoxynivalenol derivatives while HP-5 cannot, although this last column provided lower bleed and better sensitivity.

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates.

In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B1 and fumonisin B2 by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers. The molecular characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B1 and B2 producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.

Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp.

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.