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.

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.

Susceptibility testing of Aspergillus niger strains isolated from poultry to antifungal drugs--a comparative study of the disk diffusion, broth microdilution (M 38-A) and Etest methods.

The aim of this study was to determine the sensitivity of Aspergillus niger strains isolated from birds to available antifungal drugs using different in vitro assays--classical disk diffusion, Etest and broth microdilution NCCLS/CLSI M 38-A. The study material consisted of about 2.000 swabs and samples from different species of birds. A. niger (n=10) was accounted for 6.81% of the total pool of strains isolated. Determinations were made for 13 antifungal drugs using the disk diffusion method. The A. niger exhibited high susceptibility to enilconazole, terbinafine, voriconazole, tioconazole and ketoconazole, low susceptibility to clotrimazole, miconazole and nystatin, and resistance to amphotericin B, itraconazole, pimaricin, fluconazole and 5-fluorocytosine. Minimum inhibitory concentration (MIC) was determined for 9 antifungal drugs using the micromethod of duplicate serial dilutions in a liquid medium. A. niger strains were most susceptible to enilconazole and voriconazole. MIC ranged from 0.0625 to 0.5 microg/ml for enilconazole, with MIC90-0.5 microg/ml and MIC50-0.125 microg/ml. The corresponding values for voriconazole were 0.25-1 microg/ml, 1 microg/ml and 0.5 microg/ml. MIC for amphotericin B and terbinafine ranged from 0.5 to 4 microg/ml, while the values for the remaining drugs were highly varied. MIC was measured by the gradient diffusion method using Etest for 5 antifungal drugs: amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. By far the highest susceptibility was obtained in the case of voriconazole, with MIC ranging from 0.0625 to 1 microg/ml. MIC for amphotericin B ranged from 0.25 to 4 microg/ml, for itraconazole and ketoconazole ranging from 0.5 to 16 microg/ml. Methods available for this purpose are not always applicable in field conditions. The present results indicate that the Etest technique, due to its high percentage of agreement with the M 38-A microdilution method, should find application in medical and veterinary practice.

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.

[Micromorphology of Trichophyton verrucosum]. / Mikromorfologia Trichophyton verrucosum.

Using 6 samples of pathologically altered epidermis of guinea pigs and cattle, chosen strains Trichophyton verrucosum (6 strains) and the two layer method of inoculation on a classic Sabouraud's medium, arthropogenesis of the fungus was investigated. The authors described successive stages of the developmental cycle of T. verrucosum and they found that in all cases the process of arthropogenesis was very similar irrespective of the source of the material used for inoculation. Using various modifications of Sabouraud's medium and a rice medium microscopic examinations revealed in the examined strains of T. verrucosum the presence of chlamydospores (Sabouraud's medium without sugar) macroconidia (rice medium) and spiral forms (Sabouraud's medium with gluconic acid or natrium sulphite) analogical to the forms noted in Trichophyton mentagrophytes.

An evaluation of the resistance to Microsporum canis on the basis of the guinea pig.

The purpose of the work was to assess the immune response of guinea pigs after the experimental infection with Microsporum canis, and after immunization with a specific live vaccine. The guinea pigs after the recovery from infection showed a delayed type of hypersensitivity and in addition, 30 per cent of the animals were characterized by the presence of immediate hypersensitivity reactions. All the animals were resistant to reinfection with M. canis and to some extent to Trichophyton verrucosum, however, no cross-immunity against T. mentagrophytes was found. The vaccinated animals were characterized mainly by delayed type of hypersensitivity and the resistance to approximately 1000 infectious doses of M. canis.

Immunological properties of selected strains of Trichophyton genus.

The investigations demonstrated that the strains under study, either virulent or in the form of inactivated vaccines, induced a delayed type allergy. The response depended upon the affiliation to a determined species and individual properties of a strain. The strain T. mentagrophytes 1 displayed the highest biological activity, since it was a good inducer of allergy and a manufacturer of active trichophytin in tests performed both in vivo and in vitro.

Lipid composition of the mycelial and spore forms of Trichophyton verrucosum.

Neutral lipid composition and that of phospholipids of mycelial and spore forms of Trichophyton verrucosum were examined. It was found that arthrospores had more than twice as high content of lipids (99.3 mg/g) than the corresponding mycelial form (44.7 mg/g). Differences were also found in the qualitative composition: almost two times more neutral lipids (58.5 mg/g) and three times more phospholipids (40.8 mg/g) occurred in the spores than in the mycelial form 30.6 and 14.1 mg/g, respectively). Analysis of the neutral lipid fraction composition of both examined forms of T. verrucosum showed that the basic component were triglycerides, constituting about 70% in the spores and 44% in the mycelium of all the lipids. In the case of phospholipids no significant differences were observed between the spore and mycelial forms, phosphatidylcholine and phosphatidylethanolamine were predominating in both forms. A much higher content of lipids in the infectious form of the fungus, the arthrospores, suggest a possibility of participation of this fraction in the pathogenicity of the fungus.