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.

[Phospholipase and proteinase production by Malassezia pachydermatis isolated in dogs with and without otitis]. / Producción de fosfolipasa y proteinasa en cepas de Malassezia pachydermatis aisladas de perros con otitis y sin otitis.

BACKGROUND: Malassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied. AIMS: In the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis. METHODS: The phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis. RESULTS: In our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%). CONCLUSIONS: Our results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis.

Extracellular phospholipase activity of Malassezia strains isolated from individuals with and without dermatological disease.

BACKGROUND: The Malassezia genus includes mainly lipophilic yeasts belonging to the cutaneous microbiota of man and other mammals. Some Malassezia species have been associated with various dermatological diseases. The factors permitting the transformation of yeasts of the Malassezia genus from a commensal organism to a pathogenic agent are still little known but the production of various enzymes such as lipase, phospholipase and lipoxygenase could contribute to the pathogenic activity of these yeasts. AIMS: Here we have determined and compared the extracellular phospholipase activity of sixty human isolates of Malassezia so as to relate this feature to the species of Malassezia and to the origin (from dermatological diseases or not) of the strains examined. METHODS: Phospholipase production was determined using the semi-quantitative egg-yolk plate method. RESULTS AND CONCLUSIONS: Malassezia obtusa, Malassezia slooffiae, Malassezia globosa, Malassezia restricta had difficulty developing in the chosen culture medium so that it was not possible to measure phospholipasic activity. Malassezia pachydermatis showed the highest phospholipase activity. Twenty-nine Malassezia sympodialis strains produced phospholipase; the isolates from patients with pityriasis versicolor had significantly higher phospholipasic activity than those isolated from healthy individuals. This observation suggests that the phospholipasic activity of Malassezia may play a role in the onset of skin lesions, especially in the case of pityriasis versicolor.

Immune regulation and tolerance to fungi in the lungs and skin.

The balance of pro- and anti-inflammatory signaling is a prerequisite for successful host/fungal interactions and requires the coordinate actions of both innate and adaptive immune systems. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases and limit protective antifungal immune responses. The newly described Th17 develop - mental pathway may play an inflammatory role previously attributed to uncontrolled Th1 responses and serve to accommodate the seemingly paradoxical association of chronic inflammatory responses with fungal persistence in the face of an ongoing inflammation. In this scenario, unrestricted fungal growth could result from the activation of not only pathogenic Th17 cells, but also Th2 cells whose activation is strictly dependent on fungal burden. The capacity of regulatory T cells (Tregs) to inhibit aspects of innate and adaptive antifungal immunity is required for protective tolerance to fungi. Indoleamine 2,3-dioxygenase (IDO) and tryptophan catabolites contribute to such a homeostatic condition by providing the host with immune defense mechanisms adequate for protection, without necessarily eliminating fungal pathogens - which would impair immune memory - or causing an unacceptable level of tissue damage. IDO and tryptophan metabolites may prove to be potent regulators capable of taming overzealous or heightened inflammatory host responses.

[Enzyme activities of dermatophytes isolated from different clinical samples by ApiZYM method]. / Ce,itli klinik orneklerden izole edilen dermatofitlerin enzim aktivitelerinin ApiZYM yöntemiyle tayini.

Dermatophytes' enzymes may have a role in chronic superficial infections. In this study it was planned to investigate the possible relationship between the enzymatic activities of the dermatophytes and acute or chronic course of the infections. Dermatophytes were isolated from 58 (72%) out of 81 patients with superficial infections. The infections were divided into two groups as acute (< or = 6 weeks) or chronic (> 6 weeks). Isolated fungi were identified by the classical methods and ApiZYM method. ApiZYM exhibits 19 different enzymatic activities. From 11 acute cases; five Epidermophyton floccosum, three Trichophyton rubrum, two Microsporum canis and one Trichophyton mentagrophytes strains were isolated. Of the 47 chronic cases, 96% was due to T. rubrum and 4% due to T. mentagrophytes. Production of alkaline phosphatase, leucine arylamidase and beta-glucosidase enzymes were detected in all tested strains. There was no difference between the enzymatic activities of acute and chronic cases. Alpha-mannosidase activity was detected in all of the species isolated from acute cases whereas none of the chronic cases were positive (P < 0.001). The results of this study suggest that alpha-mannosidase activity may play a role in both cutaneous inflammatory response caused by dermatophytes and the chronicity of the lesions.

[The enzymatic activity of fungi strains isolated from the skin and skin appendages of humans returning from the tropics]. / Aktywnosc enzymatyczna szczepów grzybów wyizolowanych ze skóry i jej przydatków od osób powracajacych z tropiku.

The aim of the work was to assess the enzymatic activity of 11 fungi strains isolated from the skin of 10 Lódz residents who had visited the tropics over 1998-1999. The strains were cultured by Jeske and Lupa of The Voivodeship Outpatient Clinic of Infectious, Parasitic and Tropical Diseases and Fungal Infections in Lódz. They were as follows: Trichophyton rubrum and Acremonium kiliense from Zambia, Myriodontium keratinophilum, Beauveria bassiana, Cladosporium herbarum, Candida famata and Trichophyton yaoundei from Sudan, Trichophyton tonsurans from Ethiopia, Trichophyton phaseliforme from Egypt, Acremonium strictum from Zimbabwe and Microsporum racemosus from Tanzania. The enzymatic activity was determined with the use of API ZYM of bioMérieux enabling 19 hydrolases to be revealed. In general, all tropical strains were characterised by a weak hydrolytic activity.

The in vitro and in vivo production of a 31.5-kD keratinolytic subtilase from Microsporum canis and the clinical status in naturally infected cats.

BACKGROUND: Microsporum-canis-infected cats, especially the asymptomatic infected ones, are mainly responsible for the zoonotic disease. The important variability of the clinical signs in cats is poorly understood. Recently, a 31.5-kD keratinolytic subtilase was found to be a putative virulence factor. OBJECTIVE: To investigate the possible relationship between the clinical status of dermatophytic cats and the production of the keratinase. METHODS: Seven M. canis strains isolated either from clinically affected, asymptomatic infected or mechanical carrier cats were tested for the in vitro production of the enzyme. The immunohistochemical detection of the enzyme was also assessed in skin biopsies of 4 symptomatic and 7 asymptomatic naturally infected cats. RESULTS: All the strains produced in vitro a 31.5-kD keratinolytic subtilase. The enzyme was present in all but 1 of the infected cats. CONCLUSION: The production of the keratinase is not a factor directly responsible for the clinical picture seen in M.-canis-infected cats.

Detection of keratinolytic proteinase in skin tissues from guinea pigs infected with Microsporum canis by an immunoperoxidase technique.

An immunoperoxidase technique was performed to detect keratinolytic proteinase (KPase) in sections of the skin taken from guinea pigs infected with Microsporum canis and in cultured M. canis using polyclonal antisera to purified KPase. Of tissue samples from guinea pigs infected with M. canis, all sections of erythematous lesions showed positive staining mainly in the horny layer and the hair follicles. Positive depositions were seen only at the level of the outer and inner root sheaths of the hair follicles in sections of skin lesions showing scales and crusts. However, sections from areas of alopecia following desquamation of the crust showed no depositions of bright red reaction products. The patterns of deposition of KPase according to the clinical course of experimental dermatophytosis were consistent with the existence of organisms observed by PAS and methenamine silver stains. These results suggest that KPase may be produced during infection with M. canis after the development of erythema and before desquamation of the crust.

Tolerability and efficacy of bifonazole in dermatomycoses.

1-[(4-Biphenylyl)-phenylmethyl]-1-H-imidazole (bifonazole, Bay h 4502, Mycospor) is a broad spectrum antifungal agent for the topical treatment of dermatomycoses. In addition to tolerability studies, a report is given on the results of 43 clinical studies of efficacy and tolerability involving 1129 patients in various countries. These were placebo-controlled double-blind studies, randomised comparative studies with reference preparations and open studies. They were carried out with bifonazole cream 1%, solution 1%, gel 1% and powder 1%. 58.6% of the patients tested suffered from dermatophytoses, 21.9% cutaneous candidoses, 14.3% from pityriasis versicolor and 5% from other skin infections. The therapeutic efficacy of bifonazole, assessed according to mycological and clinical findings proved good for the indications studied and administered once daily for a treatment period of 2 or 3 weeks. Tolerability - local and systemic - was good. There was no evidence of substance-related side effects.