Chromatographic and Biological Screening of Chosen Species of Schisandraceae Family: Schisandra chinensis, S. rubriflora, S. sphenanthera, S. henryi and Kadsura japonica.

HPLC and TLC profiling was carried out for leaf and fruit extracts of five Schisandraceae species: Schisandra chinensis, S. rubriflora, S. spehenanthera, S. henryi and Kadsura japonica. HPLC measurements confirmed presence of lignans and phenolic compounds in fruits and leaves of all tested species. The most abundant in lignans was S. chinensis fruit extract in which 15 compounds were detected (e. g.: schisandrol A, schisanhenol, γ-schisandrin, gomisin N). The effect-directed detection, i. e., TLC-direct bioautography against Bacillus subtilis, showed exceptionally high activity for S. chinensis and S. rubriflora fruit extracts. On the other hand, TLC-DB enzyme tests (α-glucosidase, lipase, tyrosinase and acetylcholinesterase (AChE) inhibition assays) showed that all fruit and leaf extracts have ability to inhibit the above-mentioned enzymes (except for the K. japonica fruit). The leaf extracts showed much stronger antioxidant activity than the fruit ones, which were assessed and compared using both TLC-direct bioautography and spectrophotometric measurements based on ABTS, DPPH and FRAP tests.

European Hedgehogs (Erinaceus europaeus L.) as a Reservoir of Dermatophytes in Poland.

The European hedgehog (Erinaceus europaeus Linnaeus) frequently colonises areas located close to human life in cities, as these are more suitable nest sites offering an abundance of food and allowing avoidance of predators. However, urbanisation has a significant impact on the epidemiology of infectious diseases, including dermatophytoses, the primary source of which are wild animals. In this study, we determined the spectrum of dermatophytes isolated from the European hedgehog and assessed their susceptibility profile to antifungal drugs. Symptomatic and asymptomatic dermatophyte infections were observed in 7.7% and 8% of the 182 examined free-living hedgehogs, respectively. In the pool of the isolated dermatophyte strains, Trichophyton erinacei was dominant (29.9%), followed by Trichophyton mentagrophytes (17.9%), Trichophyton benhamiae (13.4%), Nannizzia gypsea (11.9%), Microsporum canis (10.4%), Nannizzia nana (7.5%), Paraphyton cookei (6.0%), and Nannizzia fulva (3.0%). Susceptibility tests revealed the highest activity of luliconazole and the lowest of activity fluconazole among the azole drugs applied. Although terbinafine generally exhibited high efficacy, two Trichophyton mentagrophytes isolates showed resistance to this drug (MIC = 2 µg/ml) resulting from missense mutations in the SQLE gene corresponding to the amino acid substitution Leu393Phe. Summarising, our study has also revealed that such wildlife animals as hedgehogs can be a reservoir of pathogenic human dermatophytes, including harmful strains resistant to commonly used antifungal drugs.

Airborne dermatophyte propagules concentration in cowsheds as an underestimated reservoir of potential zoonoses.

AIMS: Indoor air quality in stables, cowsheds or henhouses has recently become in interest due to the potential risks of zoonotic infections. Cowsheds are commonly known to have high fungal loads, particularly if insufficient attention is paid to the monitoring and control of the indoor microclimate around three elements, that is heating, ventilation and lighting. The aim of this study was to determine the concentrations and spectrum of dermatophyte propagules in the indoor air of cowsheds. METHODS AND RESULTS: Air samples were collected on five farms, and the dermatophyte species were identified using MALDI-TOF MS analysis. The quantitative analysis of the fungal pollutants showed an average of 0.084 dermatophyte propagules (CFU) per m3 of flowing air in spring and 0.0239 CFU/m3 in the summer. Dermatophyte species were identified in case of 64.6% of the obtained colonies. Trichophyton verrucosum as dominant species was isolated on all five farms. In turn, Nannizzia gypsea was isolated on four farms and Trichophyton mentagrophytes as well as Paraphyton cookei were isolated on two farms. CONCLUSIONS: This study demonstrated that indoor aerosol appears to be one of the underestimated risks of dermatophyte infections. Moreover, the risk of zoonotic infections is posed by airborne zoophilic dermatophytes, especially T. verrucosum, whose prevalence of infections has been increasing in recent years. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of dermatophytes to infect animals and humans is thought to be a consequence of not only their adaptation to new ecological niches but also occurring as an aerosol component, which we demonstrate for the first time in this study. The microclimate of the cowshed may be an underestimated reservoir of zoophilic dermatophytes, which pose a zoonotic threat to farmers, animal breeders and veterinarians.

Cold atmospheric pressure plasma (CAPP) as a new alternative treatment method for onychomycosis caused by Trichophyton verrucosum: in vitro studies.

PURPOSE: Anthropophilic dermatophytes as etiological factors of onychomycoses are more common than zoophilic fungi. In the case of the latter, reverse zoonoses are possible, which poses a threat to the persistence of dermatophytes in the environment. Nevertheless, without treatment, both types of tinea unguium may lead to complete nail plate destruction and secondary mixed infections with fungi and bacteria. One of the zoophilic dermatophytes that cause onychomycosis is Trichophyton verrucosum, whose prevalence has been increasing in recent years. Such infections are usually treated with allylamines and/or azoles, but such a conventional treatment of infections caused by T. verrucosum often fails or is discontinued by patients. METHODS: Herein, we reveal the results of our in vitro studies related to direct application of cold atmospheric pressure plasma (CAPP) on Trichophyton verrucosum growth, germination and adherence to nail as a new alternative treatment method of such types of dermatomycoses. RESULTS: Our in vitro studies showed that, while exposure to CAPP for 10 min delays germination of conidia and clearly impairs the fitness of the fungal structures, 15 min is enough to kill all fungal elements exposed to plasma. Moreover, the SEM images revealed that T. verrucosum cultures exposed to CAPP for 10 and 15 min were not able to invade the nail fragments. CONCLUSION: The results revealed that single exposure to CAPP was able to inhibit T. verrucosum growth and infection capacity. Hence, cold atmospheric pressure plasma should be considered as a promising alternative treatment of onychomycoses.

Therapeutic efficacy of topically used luliconazole vs. terbinafine 1% creams.

OBJECTIVES: Dermatomycoses of zoophilic origin, especially those caused by Trichophyton mentagrophytes, often pose considerable therapeutic problems. This is reflected in the growing number of strains of this species with resistance to terbinafine caused by a mutation in the squalene epoxidase (SQLE) gene. Therefore, it is reasonable to look for alternative therapies to the commonly used terbinafine. The aim of the present study was to assess the in vivo effectiveness of topical therapy with luliconazole or terbinafine 1% cream. METHODS: Therapeutic efficacy was assessed using direct examination in KOH with DMSO, qPCR analysis with pan-dermatophyte primers and culturing. Moreover, in vitro susceptibility tests for luliconazole and terbinafine were performed. RESULTS: The results demonstrated significantly higher antifungal activity of luliconazole than terbinafine against dermatomycoses caused by T. mentagrophytes. The geometric mean of the MIC value for luliconazole against all T. mentagrophytes strains was 0.002 µg/ml, while this value for terbinafine was 0.004 µg/ml. In all studied cases, 28-day local therapy with luliconazole contributed to complete eradication of the aetiological agent of infection. CONCLUSIONS: Given the increasingly frequent reports of difficult-to-treat dermatophytoses caused by zoophilic terbinafine-resistant strains, the 1% luliconazole cream can be alternative solution in topical therapy.

Complementary effect of mechanism of multidrug resistance in Trichophyton mentagrophytes isolated from human dermatophytoses of animal origin.

BACKGROUND: Dermatophytoses have gained interest worldwide due to the increased resistance to terbinafine and azoles and difficulty in management of these refractory diseases. OBJECTIVES: In this study, we identified and analysed Trichophyton mentagrophytes clinical isolates obtained from humans with infections of animal origin. METHODS: We used quantitative real-time PCR (qRT-PCR) to examine the transcriptional modulation of three MDR genes (PDR1, MDR2 and MDR4) and analysed squalene epoxidase (SQLE) gene sequences from multidrug-resistant Trichophyton mentagrophytes isolates. RESULTS: The expression profile revealed a 2- to 12-fold increase in mRNA accumulation in the presence of any of the antifungals, compared to cells incubated without drugs. A statistically significant relationship between the isolates exposed to itraconazole and increased expression of the tested genes was revealed. Substantially lower transcription levels were noted for cells exposed to luliconazole, that is, a third-generation azole. Additionally, in the case of 50% of terbinafine-resistant strains, Leu397Phe substitution in the SQLE gene was detected. Furthermore, the reduced susceptibility to itraconazole and voriconazole was overcome by milbemycin oxime. CONCLUSIONS: In conclusion, our study shed more light on the role of the ABC transporter family in T. mentagrophytes, which, if overexpressed, can confer resistance to single azole drugs and even cross-resistance. Finally, milbemycin oxime could be an interesting compound supporting treatment with azole drugs in the case of refractory dermatomycoses.

Genetic Predisposition and its Heredity in the Context of Increased Prevalence of Dermatophytoses.

Dermatophytosis is a widespread disease with high prevalence and a substantial economic burden associated with costs of treatment. The pattern of this infectious disease covers a wide spectrum from exposed individuals without symptoms to those with acutely inflammatory or non-inflammatory, chronic to invasive, and life-threatening symptoms. Moreover, the prevalence of cutaneous fungal infections is not as high as might be expected. This curious disparity in the dermatophyte infection patterns may suggest that there are individual factors that predispose to infection, with genetics as an increasingly well-known determinant. In this review, we describe recent findings about the genetic predisposition to dermatophyte infections, with focus on inheritance in families with a high frequency of dermatophyte infections and specific host-pathogen interactions. The results of studies indicating a hereditary predisposition to dermatophytoses have been challenged by many skeptics suggesting that the varied degree of pathogenicity and the ecological diversity of this group of fungi are more important in increasing sensitivity. Nonetheless, a retrospective analysis of the hereditary propensity to dermatophytoses revealed at least several proven genetic relationships such as races, CARD9 deficiency, HLA-DR4 and HLA-DR8 type and responsible genes encoding interleukin-22, ß-defensin 2 and 4 as well as genetic defects in dectin-1, which increased the prevalence of the disease in families and were involved in the inheritance of the proneness in their members. In future, the Human Genome Diversity Project can contribute to elucidation of the genetic predisposition to dermatophytoses and provide more information.

Population differentiation, antifungal susceptibility, and host range of Trichophyton mentagrophytes isolates causing recalcitrant infections in humans and animals.

The major problems in determining the causative factors of the high prevalence of dermatophytoses include the lack of a well-standardized antifungal susceptibility testing method, the low consistency of in vitro and clinical minimal inhibitory concentration values, the high genomic diversity of the population, and the unclear mechanism of pathogenicity. These factors are of particular importance when the disease is recalcitrant and relapses. Herein, we identified and characterized Trichophyton mentagrophytes isolates obtained from therapy-resistant cases in humans and animals. We used genomic diversity analysis of 17 human and 27 animal clinical isolates with the MP-PCR technique, determined their phenotypic enzymatic activity and host range, and performed antifungal susceptibility testing to currently available antifungal drugs from various chemical groups. Genomic diversity values of 35.3% and 33.3% were obtained for clinical isolates from humans and animals, respectively, yet without any relationship to the host species or antifungal drug to which resistance in therapy was revealed. The highest activity of keratinase enzymes was recorded for fox, guinea pig, and human hairs. These hosts can be considered as the main species in the host range of these isolates. A phenyl morpholine derivative, i.e. amorolfine, exhibited superior activity against strains obtained from both humans and animals with the lowest MIC50. Interestingly, high compliance of terbinafine in vitro resistance with clinical problems in the treatment with this substance was shown as well. The high resistance of dermatophytes to drugs is the main cause of the recalcitrance of the infection, whereas the other features of the fungus are less important.

Intrinsic resistance to terbinafine among human and animal isolates of Trichophyton mentagrophytes related to amino acid substitution in the squalene epoxidase.

BACKGROUND: Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. PURPOSE: Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. METHODS: We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. RESULTS: In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. CONCLUSION: Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

Unusual dermatomycoses caused by Nannizzia nana: the geophilic origin of human infections.

BACKGROUND: Fungal infections of the skin, hair, and nails are the largest and most widespread group of all mycoses. Nannizzia nana is a relatively rare etiological factor of dermatomycosis in humans, as it usually affects animals, e.g. pigs and boars. In addition to the zoophilic nature, there are also reports of the geophilic reservoir of this dermatophyte species. OBJECTIVE: In this study, we present symptomatic infections with N. nana aetiology in humans reported recently in Poland. Interestingly, these cases had a non-specific clinical picture and occurred as skin lesions on the neck and foot as well as onychomycosis of the toenails. From the medical history, the patients had no contact with pigs. METHODS: Diagnostics of these infections was performed with a combination of classical phenotypic and molecular genomic methods. The genomic diversity of the isolates was determined using the MP-PCR method. In vitro antifungal susceptibility tests against itraconazole, ketoconazole, terbinafine and naftifine hydrochloride were also performed. RESULTS: Nannizzia nana has been identified as an etiological factor of dermatomycosis. Moreover, heterogeneity of the genomes was revealed for the obtained strains. In vitro activities of antifungal agents showed that isolates were susceptible to all tested drugs. The patients were treated with oral terbinafine and topical ketoconazole cream, which led to a complete recovery. CONCLUSIONS: In conclusion, the cases studied by us may indicate that the infrequency of N. nana infections may not necessarily be related to the low infectivity of this fungal agent, but they are rather associated with misdiagnosis. Furthermore, N. nana reservoirs should also be sought in soil.

Comparison of in vitro activities of 11 antifungal agents against Trichophyton verrucosum isolates associated with a variety hosts and geographical origin.

The high prevalence of dermatophytosis in animals is usually associated with extra expenditure on prevention, diagnosis and long-term treatment. Humans are usually infected from animals, also from asymptomatic carriers, through direct contact or indirectly via fungus-bearing hair, scales and fomites. Despite the medical importance of Trichophyton verrucosum infections, there are limited in vitro data on the fungal susceptibility to antifungal drugs, including new-generation triazoles, imidazoles and allyloamines. The aim of the current study was to evaluate comprehensively the in vitro activity of 11 antifungal drugs against a large collection of T. verrucosum isolates obtained in Poland, Latvia, Lithuania and Slovakia from humans and animals using a microdilution assay. In vitro susceptibility testing of 11 antifungal drugs was performed according to the Clinical and Laboratory Standards Institute (CLSI) document M38. The MICs of clotrimazole, ciclopirox, enilconazole, miconazole, naftifine and terbinafine against all T. verrucosum isolates were below 1 µg/mL, whereas those of fluconazole, griseofulvin, itraconazole, ketoconazole and voriconazole were above 1 µg/mL. Ciclopirox was demonstrated to have superior activity against all strains in comparison with the other drugs, whereas fluconazole exerted the weakest in vitro effect and exhibited the highest MIC values. Our study has shown that drugs of different chemical origin have satisfactory antifungal activity and can be promising candidates for the treatment of T. verrucosum dermatophytosis. Moreover, no significant disparity in drug sensitivity between isolates obtained from different hosts and geographical regions have been demonstrated.

A case of anti- pityriasis versicolor therapy that preserves healthy mycobiome.

BACKGROUND: The impact of Malassezia yeasts on skin mycobiome and health has received considerable attention recently. Pityriasis versicolor (PV), a common dermatosis caused by Malassezia genus worldwide, is a manifestation of dysbiosis. PV can be associated with hyper- and/or hypopigmented skin lesions. This disease entity is characterized by high percentage of relapses, which demands a proper antifungal therapy that is based on unambiguous species identification and drug susceptibility testing. CASE PRESENTATION: Comprehensive analysis of PV case in man presenting simultaneously hyper- and hypopigmented skin lesions was performed. Conventional and molecular diagnostic procedures revealed Malassezia furfur and Malassezia sympodialis, respectively as etiological agents of skin lesions observed. Susceptibility tests showed significantly lowered sensitivity of M. furfur cells to fluconazole. Based on susceptibility profiles local antifungal therapy with drugs characterized by entirely different mechanism of action was included. CONCLUSIONS: Our study indicates that cases of PV represented by two types of skin lesions in one patient may be associated with distinct Malassezia species. Moreover, as observed in this case, each of the isolated etiological agents of PV may differ significantly in susceptibility to antifungals. This can significantly complicate the treatment of dermatosis, which by definition is associated with a significant percentage of relapses. In the presented case localized topical treatment was sufficient and successful while allowing maintaining the physiological mycobiome.

Dermatophytosis with concurrent Trichophyton verrucosum and T. benhamiae in calves after long-term transport.

BACKGROUND: Dermatophytosis is a common problem in cattle. The aetiological factors associated with this disease are filamentous fungi with the ability to digest and grow on keratinized substrates. In cattle, and less frequently in other domestic animals and people, the dermatophyte Trichophyton verrucosum is most commonly isolated from skin lesions. The dermatophyte Trichophyton benhamiae is an important zoonotic pathogen, and the main sources of transmission are guinea pigs and other small rodents. OBJECTIVES: In this report, we show multispecies infection in calves (Bos taurus) after long-term transport and vaccination against trichophytosis. ANIMALS: Sixty animals were imported of which 32 were observed to be affected with superficial infection nine to 12 days after vaccination for dermatophytosis. METHODS AND MATERIALS: Diagnosis was made correlating the clinical signs with a micro- and macroscopic examination of cultured fungi. Molecular differentiation was used to confirm the species affiliation. RESULTS: Eight of the calves were infected with T. verrucosum alone, and 24 calves with both T. verrucosum and T. benhamiae. We suggest that the cause of this large outbreak was immunosuppression of the animals resulting from the stress of transport and administration of vaccine. CONCLUSION: Both T. verrucosum and T. benhamiae can be seen concurrently in cattle.

Contamination of the urban environment with excrements of companion animals as an underestimated source of Staphylococcus species posing a threat to public health.

The aim of the study was to assess the incidence, resistance, virulence, and genotypic characteristics of Staphylococcus spp. residing in the gastrointestinal tract of dogs and cats, as a group of animals causing potential contamination of the urban space. A high percentage of strains resistant to penicillin (58%), oxacillin (9%) and tetracycline (60%) were found. All isolates resistant to penicillin, kanamycin, or chloramphenicol carried genes responsible for individual resistance (blaZ, aph(3')-IIIa, and cat (pC194)/cat (pC223), respectively. The mecA gene was detected in 45% of the oxacillin-resistant Staphylococcus pseudintermedius strains. The amplification of DNA fragments surrounding rare restriction sites analysis demonstrated high heterogeneity of genotypic profiles correlating with phenotypic resistance profiles. Multilocus sequence typing analysis classified the methicillin-resistant S. pseudintermedius strains as ST71, ST890, and the totally new ST1047. The presence of a high level of resistance among Staphylococcus strains may suggest a potential risk of transfer of these bacteria between companion animals and humans.

Multiple-strain Trichophyton mentagrophytes infection in a silver fox (Vulpes vulpes) from a breeding farm.

Dermatophyte infections are extremely frequent worldwide, and their epidemiological features and distribution make them one of the most frequent infections all over the world. We identified and analysed multiform T. mentagrophytes strains isolated from a silver fox (Vulpes vulpes) kept on a breeding farm. Identification of dermatophyte strains was carried out traditionally by correlating both the clinical manifestations of the infection with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. DNA was isolated from the dermatophytes with the phenol-chloroform method. The reaction of chitin synthase 1 (chs1) amplification was carried out to confirm the dermatophytes. The phylogenetic analysis was based on the ITS sequences. The polymerase chain reaction melting profile (PCR-MP) procedure was used for differentiation of dermatophyte genomes. Direct analysis of the material sampled from the clinical lesions revealed the presence of arthrospores in the samples collected from all animals with skin lesions. The macromorphology of the colonies obtained from material sampled from the same individual was not homogeneous. The PCR-MP electrophoregram indicated high variability of their genomes. Although the dermatophytes were isolated from one infected fox, no two identical genomic profiles were obtained. The PCR-MP result corresponds with the phenotypic diversity of the isolates. The findings about the multiple dermatophyte infection in one individual complicate any future epidemiology work and other clinical investigation. Previously, using only morphological characteristics, it had been assumed that one fungal isolate per patient could be diagnosed. The novel findings encourage application of the newly developed molecular typing methods in the diagnosis of dermatophytosis.

Determination of chlorogenic acid, polyphenols and antioxidants in green coffee by thin-layer chromatography, effect-directed analysis and dot blot - comparison to HPLC and spectrophotometry methods.

The great prevalence of thin-layer chromatography over high-performance liquid chromatography is connected with the possibility of analyzing many samples in parallel. Therefore, the method is often used in screening and/or effect directed analysis to compare composition and chemical/biological properties of many samples in one run. It was already proved, that high performance thin-layer chromatography, in many cases, can replace high-performance liquid chromatography for quantitative analysis. The main aim of the paper is to show that simple thin-layer chromatography can also be used as a quantitative or at least as a semi-quantitative method, even when it concerns effect directed analysis e.g. direct bioautography. Chlorogenic acid content was measured in four methanol extracts of various green coffees and in one extract of black coffee using thin-layer chromatography with ultraviolet detection and thin-layer chromatography with effect directed detection. High-performance liquid chromatography was used as a reference method. Additionally, total contents of polyphenols and antioxidants were estimated using thin-layer chromatography or dot-blot on chromatography plates. These results were compared to spectrophotometric methods. It was proved that thin-layer chromatography can be used as a quantitative (using densitometry) or semi-quantitative method (using other detection methods including effect directed detection) as well as for estimating total antioxidants or polyphenols content.

The host range of dermatophytes, it is at all possible? Phenotypic evaluation of the keratinolytic activity of Trichophyton verrucosum clinical isolates.

Dermatophytes are fungi that have an ability to invade keratinised structures. Enzymes secreted by dermatophytes can underlie fungal survival on the host and development of infection. It is possible that the range of activity of keratinases from various dermatophytes is limited to specific species of animals and groups of people. The aim of this study was to carry out phenotypic analysis of the degree of keratinolytic activity of Trichophyton verrucosum strains using hairs of humans and various animal species as substrates. Our results indicated that the activity of keratinases is substrate-induced. The host range of T. verrucosum can be defined as wide. The highest activity of keratinases was recorded in media containing keratin from cow (Bos taurus) and sheep (Ovis aries) hairs in comparison with that from other tested species. The production of keratin-degrading enzymes is a function of time, with the peak of their activity occurring on day 15 of incubation. The role of keratin-degrading enzymes in the pathogenesis of dermatophytosis is becoming increasingly clearer. Given the conceptual understanding that keratin breakdown may require more than just one enzyme, the use of phenotypic methods is an optimal approach to in vitro study of the decomposition of species-specific keratin.

Tinea corporis by Microsporum canis in mycological laboratory staff: Unexpected results of epidemiological investigation.

Microsporum canis is a zoophilic dermatophyte, which is very contagious, especially to cats and dogs. Asymptomatic animal carriers of M. canis are regarded a critical factor in the epidemiology of the disease. The aim of this study was to investigate the epidemiological origin of M. canis isolates using morphological traits in combination with molecular analysis. Identification of dermatophyte strains was carried out by correlating the clinical manifestation of the infection with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. A positive result of the culture examination was obtained from the samples with arthrospores in the direct analysis, that is, from a symptomatic cat and humans, and from a cat without any signs of infection. The microsatellite-primed PCR fingerprinting (MSP-PCR) electro-profiles were identical for all analysed strains. The melting profile-PCR (MP-PCR) electrophoregram indicated variability of the genomes of the strains. The search for the source of the infection indicated one cat that did not have any signs of dermatophytosis. PCR-fingerprinting techniques are useful tools for epidemiological investigation of the origin of dermatophyte infection. These methods can also be used in many cases for species identification of dermatophytes and clarification of the relationships among varieties of a species.

Infection of Trichophyton verrucosum in cattle breeders, Poland: A 40-year retrospective study on the genomic variability of strains.

Trichophyton verrucosum is a zoophilic fungus that is the most frequent aetiological agent of dermatophytosis in cattle. During the last few years, the number of cases of T. verrucosum from humans has been increasing constantly, which is correlated with the presence of cattle-rearing farms. We identified and analysed T. verrucosum strains isolated from humans and cattle. Identification was carried out traditionally by correlating both the clinical manifestations with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. Direct analysis revealed the presence of arthrospores. The macro- and micromorphology of the isolates obtained from material sampled was homogeneous, and characterised for T. verrucosum. The phylogenetic analysis based on the ITS sequences demonstrated that the strains formed a monophyletic group with T. verrucosum ATCC10 695 with a support of 99%. The MP-PCR analysis indicates high invariability of genomes of strains from humans and animals. MSP-fingerprinting analysis gives the same results as the MP-PCR analysis. To sum up, the rDNA ITS sequence analysis in combination with macro- and micro-morphology only facilitated T. verrucosum species identification without the possibility of intraspecific differentiation. Finding and testing methods, especially molecular technique, with sufficient discriminatory power, is the present challenge for mycologists.

Multilocus sequence analysis supports the taxonomic position of Astragalus glycyphyllos symbionts based on DNA-DNA hybridization.

In this study, the phylogenetic relationship and taxonomic status of six strains, representing different phenons and genomic groups of Astragalus glycyphyllos symbionts, originating from Poland, were established by comparative analysis of five concatenated housekeeping gene sequences (atpD, dnaK, glnA, recA and rpoB), DNA-DNA hybridization and total DNA G+C content. Maximum-likelihood phylogenetic analysis of combined atpD, dnaK, glnA, recA and rpoB sequence data placed the studied bacteria into the clade comprising the genus Mesorhizobium. In the core gene phylograms, four A. glycyphyllos nodule isolates (AG1, AG7, AG15 and AG27) formed a cluster common with Mesorhizobium ciceri, whereas the two other A. glycyphyllos symbionts (AG17 and AG22) were grouped together with Mesorhizobium amorphae and M. septentrionale. The species position of the studied bacteria was clarified by DNA-DNA hybridization. The DNA-DNA relatedness between isolates AG1, AG7, AG15 and AG27 and reference strain M. ciceri USDA 3383T was 76.4-84.2%, and all these A. glycyphyllos nodulators were defined as members of the genomospecies M. ciceri. DNA-DNA relatedness for isolates AG17 and AG22 and the reference strain M. amorphae ICMP 15022T was 77.5 and 80.1%, respectively. We propose that the nodule isolates AG17 and AG22 belong to the genomic species M. amorphae. Additionally, it was found that the total DNA G+C content of the six test A. glycyphyllos symbionts was 59.4-62.1 mol%, within the range for species of the genus Mesorhizobium.