Phenotypic characterization of enzymatic activity of clinical dermatophyte isolates from animals with and without skin lesions and humans.

AIMS: The pathogenesis of dermatophytoses is associated with the secretion of enzymes degrading the infected tissue components. Although many studies on enzymatic activity of dermatophytes have been conducted over the years, there have been no concrete proposals on the construction of the profile of enzymes characteristic of individual species, genus or ecological types of dermatophytes. The aim of this study was to assess the capability of clinical dermatophyte isolates from both symptomatic and asymptomatic animals and humans to produce different enzymes. METHODS AND RESULTS: Clinical isolates of 234 dermatophyte strains collected during routine examination of animal health were used in this study. The enzymatic production of keratinase, elastase, phospholipase, lipase, protease, DNase and gelatinase as well as the haemolytic activity were evaluated using specific test media. The overall degree of enzymatic activity of the analysed clinical isolates of the dermatophytes was 67%. All tested clinical isolates of different species of dermatophytes showed keratinase activity and 96% additionally exhibited phospholipase activity. The weakest activity among the tested enzymes was demonstrated for elastase and gelatinase. 83% of the isolates of the dermatophytes showed haemolytic activity. CONCLUSION: Our data indicate that clinical isolates of dermatophytes from different species produce enzymes with different levels of activities. SIGNIFICANT AND IMPACT OF THE STUDY: Profile of enzymes characteristic of individual species, genus or ecological types of dermatophytes is possibly dependent upon factors related to the host. The relationship between each enzyme and the occurrence of skin lesions in animals and humans or asymptomatic animal carriers varies on whether the infection is caused by Trichophyton mentagrophytes, Trichophyton verrucosum or Microsporum canis. Interestingly, only keratinase seems to be correlated with the appearance of dermatophyte infections, irrespective of the pathogen species, and elastase is a characteristic enzyme for dermatophyte strains infecting humans. Haemolysis seems to be dependent on host factors and is more common in the case of human dermatophyte isolates.

Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes.

AIMS: Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum. METHODS AND RESULTS: Samples were prepared of each of the DNA extraction methods (phenol-chloroform, CTAB and four different kits) for all of the incubation periods (4, 7 and 10 days) of the cultures on the solid and in the liquid medium. The highest DNA concentrations were obtained using the phenol-chloroform method. The concentration of DNA extracted with the CTAB method accounted for 62·21%, for kits it corresponded from 35·53 to 15·41%. The analysis of the DNA weight yield revealed the highest isolation efficiency of the phenol-chloroform method, 1 mg of mycelium yielded 223·8 µg DNA. Lower DNA yield (by 39·32%) was obtained with the CTAB method; in the case of kits by 68·46-85·32%. In most of the techniques, the DNA yield on the solid medium was higher. CONCLUSION: In summary, the highest DNA yield was noted in the 7-day cultures and extraction with the phenol-chloroform method. Importantly, the type of culture was not relevant for the diagnostic result. SIGNIFICANCE AND IMPACT OF THE STUDY: Most mycoses are caused by fungi that reside in nature. The severity of the infection depends on the pathogenic attributes, socioeconomic factors and local environmental conditions. Recent diagnosis increasingly relies on not only the clinical features. Molecular identifications have determined the need for a rapid and easy method of extracting DNA. Usually two factors have to be considered: maximize the DNA yield and ensure that the extracted DNA is susceptible to enzymatic reactions. These data suggest that phenol-chloroform methods and a 7-day culture period may be useful for validation and constitute the first step of molecular diagnosis of dermatophytes.

Antimicrobial activity of some plant extracts against bacterial pathogens isolated from faeces of red deer (Cervus elaphus).

Antibacterial activity is the most widely studied aspect of plant extracts. Antibiotics extensively produced and consumed in large quantities, have proved to be problematic due to various types of adverse effects. The development of bacterial resistance to currently available antibiotics has necessitated the search for new antibacterial agents. One of the alternative strategies for fighting antibiotic- resistant bacteria is the use of natural antimicrobial substances such as plant extracts. We tested the antimicrobial activity of nine extracts from different plants against pathogenic bacteria isolated from the faeces of red deer (Cervus elaphus). Selected bacteria commonly contaminated the natural environment and constitute a source of infection in other animals and humans. Extracts obtained from the following plants were tested: Hypericum perforatum L., Chamomilla recutita L., Achillea millefolium L., Salvia officinalis L., Thymus vulgaris L., Pinus sylvestris L., Mentha x piperita L., Valeriana officinalis L. and Foeniculum vulgare Mill. The highest degree of antibacterial properties was observed for Mentha x piperita L., narrower spectrum of activity possessed Hypericum perforatum L. Extracts of Achillea millefolium L. had the lowest spectrum of antibacterial activity. Our study confirms that many plant extracts shows in vitro antibacterial activity.

Determination of resistance and virulence genes in Enterococcus faecalis and E. faecium strains isolated from poultry and their genotypic characterization by ADSRRS-fingerprinting.

The aim of this study was to determine the antimicrobial resistance of E. faecalis and E. faecium strains isolated from poultry and to carry out genotypic characterization thereof with the ADSRRS-fingerprinting method (amplification of DNA fragments surrounding rare restriction sites) and analysis of the genetic relatedness between the isolates with different resistance and virulence determinants. Samples were collected from 70 4-week-old chickens and tested for Enterococcus. Minimum inhibitory concentrations of 11 antimicrobials were determined using the broth microdilution method. Detection of antibiotic resistance and virulence genes was performed using PCR, and molecular analysis was carried out using the ADSRRS-fingerprinting method. The highest percentage of strains was resistant to tetracycline (60.5%) and erythromycin (54.4%), and a large number exhibited high-level resistance to both kanamycin (42.1%) and streptomycin (34.2%). Among 8 genes encoding AME, the tested strains showed mainly the presence of [aph(3΄)-IIIa], [ant(6)-Ia], [aac(6΄)-Ie-aph(2΄΄)-Ia], and [ant(9)-Ia] genes. Phenotypic resistance to erythromycin was encoded in 98.4% strains by the ermB gene. Genotypic resistance to tetracycline in E. faecium was associated with the presence of tetM and tetL (respectively, in 95.5 and 57.7% of the isolates); in contrast, E. faecalis strains were characterized mainly by the presence of tetO (83.3%). The virulence profile was homogenous for all E. faecium strains and included only efaAfm and ccf genes. All E. faecalis strains exhibited efaAfs, gelE, and genes encoding sex pheromones. The strains tested exhibited 34 genotypic profiles. Comparative analysis of phenotypic and genotypic resistance and virulence profiles and confrontation thereof with the genotypes of the strains tested showed that strains assigned to a particular genotype have an identical phenotypic resistance profile and a panel of resistance and virulence genes. The results of this study confirm that poultry can be a reservoir of resistant E. faecium and E. faecalis strains with multiple combinations of resistance and virulence genes, whose specific panel determines not only phenotypic characteristics but also has a strong correlation with the genotypic profiles of the strains.

Structure, physicochemical and biological properties of new complex salt of aqua-(nitrilotriacetato-N,O,O',O")-oxidovanadium(IV) anion with 1,10-phenanthrolinium cation.

The crystal structure of new 1,10-phenathrolin-1-ium aqua-(nitrilotriacetato-N,O,O',O")-oxidovanadium(IV) semihydrate of molecular formula (phenH)[VO(NTA)(H2O)]∙1/2H2O was determined. This is the first example of structurally characterized compound that comprises a distinctly separated, monomeric [VO(NTA)(H2O)](-) coordination entity. The crystallographic measurements have subsequently been complemented by the IR spectroscopic characterization and thermal analysis. Furthermore, the electrochemical (cyclic voltammetry) as well as spectrophotometric (UV-vis) studies revealed that the compound is capable of scavenging the superoxide free radicals (O2(-)) as well as stable organic radicals i.e. 2,2'-azinobis(3-ethylbenzothiazoline-6 sulfonic acid) cation radical (ABTS(+)) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), but its reactivity towards radicals is lower than that of VOSO4. Finally, biological properties of the complex in the range of 1-100 µM were investigated in relation to its cytoprotective activity against the oxidative damage generated exogenously by using hydrogen peroxide in the hippocampal neuronal HT22 cell line (the MTT and LDH tests). It has been established that in contrast to VOSO4 the title compound protects the HT22 from the oxidative damage. The paper presents a new perspective for oxidovanadium(IV) complexes as candidates for novel, low-molecular mass cytoprotective agents.

Experimental studies of microbial populations and incidence of zoonotic pathogens in the faeces of red deer (Cervus elaphus).

UNLABELLED: Wild animals can serve as hosts, amplifiers or reservoirs for various zoonotic diseases. Most species of deer in highly fragmented agricultural landscapes, search out maximum cover from intrusive human activity. Hence, the likelihood of zoonosis transmission is likely to increase the more humans and wildlife interact. In our study, we conducted a comparative analysis of bacteria isolated from the faeces of red deer (Cervus elaphus) living in their natural environment in south-western Poland and brought in from Hungary and Slovakia under a species reintroduction programme. The faecal bacterial flora from 120 specimens of deer were examined, with particular attention to potentially pathogenic agents. We isolated 458 micro-organisms, of which 13 (2·84%) were identified as EHEC (Enterohaemorrhagic Escherichia coli) strains, and of these one strain, produced the Shiga toxin. No strain was identified as having ESBL (Extended-Spectrum Beta-Lactamase) resistance. Other bacteria that are important in terms of the health of humans and animals included Yersinia enterocolitica (4, 0·67%) and Staphylococcus aureus (4, 0·67%), but without methicillin resistance, and Listeria monocytogenes (8, 1·75%). Of all the micro-organisms 138 (30·13%) were bacteria of the genus Enterococcus, including 12 (2·62%) of the species Enterococcus faecium. The results of the study indicate that red deer may play an important role in the environmental maintenance of zoonotic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: A particularly important factor in the epidemiology of bacterial infections is the introduction of pathogens posing a risk to other animals and humans into the soil, plants and especially water, as contaminants together with faeces. Our study presents screening of potentially pathogenic bacteria in different populations of deer that were displaced under reintroduction programmes. Based on our own research and the literature data, it seems that wild ruminants play an important role in the maintenance of zoonotic pathogens and information about zoonoses from red deer will become increasingly important as deer populations continue to grow, especially in Europe.

Morphology and chemical composition of Ag/Sn/Ag interconnections.

The main goal of the present contribution was to describe morphology and chemical composition of the intermetallic phases, which were formed during diffusion soldering process of the silver using tin. The Ag(3)Sn intermetallics is the main constituent of the joint after diffusion soldering at 235 degrees C and 265 degrees C. A closer inspection of the Ag/Ag(3)Sn interface revealed also the small crystallites of the second intermetallic phase, Ag(5)Sn, which was not previously observed using scanning electron microscope. Both phases are characterized by high melting temperatures: 480 degrees C and 724 degrees C, respectively. Therefore, their presence guarantees high thermal stability of the interconnection, which can be even three times higher than the temperature used for soldering.

Absence of lower extremity ossification centers in term infants with congenital syphilis.

Radiologic evaluation is valuable for confirmation of congenital syphilis. Bony abnormalities are thought to be secondary to active disease or a growth response of bone to systemic illness. Anteroposterior lower extremity radiographs of 85 euthyroid term infants (> or = 38 weeks' gestation; mean weight, 3244 gm; range, 1940-4380 gm) with presumptive congenital syphilis were evaluated for the presence of the distal femoral and proximal tibial secondary ossification centers and other bony abnormalities. Both proximal tibial and distal femoral ossification centers were absent in only 9 of 85 infants with syphilis; the proximal tibial center was absent in 26 of 85 (total 30.6%; 95% confidence limits 0.2 to 0.4). This observation is significant at the p < 0.01 level by chi-square analysis with use of historical controls. Periosteal new bone formation was seen in 2 of 85 infants. Metaphyseal lucent bands were present in 21 of 85 infants, 8 of whom also had absent lower extremity ossification centers. For comparison we reviewed lower extremity radiographs from 12 term infants without congenital syphilis. Ossification centers were absent in one growth-retarded infant, and metaphyseal lucent bands were found in one infant. Although standard texts report nearly universal ossification of the distal femoral epiphysis at term, we observed delayed skeletal maturation in 30.1% of term infants with congenital syphilis. We speculate that this delay may be reflective of systemic infection.

Positioning and stability of mitotic spindle orientation in the neuroepithelial cell.

To verify non-random positioning and to define the stability of the mitotic spindle orientation in neuroepithelial cells of mouse foetuses, computer - assisted morphometric analysis at the light microscopy level was performed. It was confirmed that the mitotic spindle axis is positioned non-randomly in relation to the cell polarity axis and could be displaced only within a narrow range. This orientation was found to be attained at metaphase and it does not change until telophase is completed. However, in relation to the long axis of the neural tube the mitotic spindle axis was found to be positioned randomly. In the light of these findings centrosome movement and positioning are discussed.

Spatial orientation of the mitotic apparatus and its stability in a polarized epithelial cell. A computer-assisted morphometric analysis.

The spatial and temporal orientation of the mitotic apparatus in reference to the polarized structure of an epithelial cell, as well as the relationship between mitotic spindle positioning and the direction of epithelial cell migration, was investigated. As an experimental model the epithelium of the mouse small intestine was chosen. Computer-assisted morphometric analysis applied in this study disclosed that the position of the mitotic spindle axis is restricted to a plane which is perpendicular to the axis of cell polarity. On this plane the spindle axis may occupy a random position. The latter finding indicates that there exists no correlation between the orientation of the mitotic spindle and the direction of epithelial cell migration. The plane of the spindle axis is established at metaphase, and no repositioning at further stages of mitosis has been observed. It is suggested that cell polarity plays a role in determination and stabilization of the mitotic spindle orientation in epithelial cells.