Identification and characterisation of human pathogenic filamentous fungi and susceptibility to Thymus schimperi essential oil Rita Pagiotti, 1 Paola Angelini, 1 Andrea Rubini, 2 Bruno Tirillini, 3 Bruno Granetti 1 and Roberto Venanzoni 1 1 Department of Applied Biology, University of Perugia, Perugia, Italy, 2 CNR – Institute of Plant Genetics, Perugia Division, Perugia, Italy and 3 Institute of Botany, University of Urbino, Urbino, Italy Summary Twenty-eight clinical fungal isolates were characterised by morphological (macro- and micro-features and growth response at 25, 30 and 37 °C) and molecular (nuclear rDNA-internal transcriber spacer, calmodulin, cytochrome c oxidase 1 and the largest subunit of RNA polymerase II) analyses. The clinical fungal isolates were ascribed to the following taxa: Penicillium chrysogenum, Verticillium sp., Aspergillus tubingensis, Aspergillus minutus, Beauveria bassiana and Microsporum gypseum. In addition, in vitro susceptibility testing of the isolates to conventional antifungal agents and to two chemically well-defined chemotypes of Thymus schimperi essential oil was performed. Most of the isolates were resistant to amphotericin B (except A. minutus), and itraconazole, while terbinafine was quite active on these fungi. T. schimperi essential oil showed antifungal activity against all of the tested fungal isolates with minimal inhibitory concentration values similar or lower than those of terbinafine. Transmission electron microscopy analyses revealed that fungal growth inhibition by essential oil was accompanied by marked morphological and cytological changes. Key words: Antifungal agents, culture medium, molecular typing, morphotypes, transmission electron microscopy. Introduction The phyla ascomycota and basidiomycota are com- prised of a wide range of fungal species that form a heterogeneous group in terms of clinical occurrence, phenotypic and genetic complexity and association with diseases. 1 Many fungi behave as facultative pathogens in the immunocompromised host, and uncommon or newly described fungal species are currently thought to be the cause of numerous diseases. 2 Factors responsible for the increased number of cases include AIDS, haematological malignancies, organ transplantation, use of corticosteroids, antineoplastic drug treatments, complex surgical procedures and long-term indwelling vascular catheterisation. 3 Clinical microbiology labora- tories rely heavily on morphology-based methods to identify fungal species wherein diagnostic criteria include the recognition of asexual or sexual structures and their characteristics such as shape, size, colour and ornamentation. Unfortunately, there are numerous difficulties in such a phenotype-based scheme largely because these characteristics are unstable, and clinical fungi are sometimes atypical with slow sporulation and aberrant conidiophores formation. Correct identification of clinical fungal isolates is essential for optimal clinical management, detailed epidemiological studies and pre- vention and containment of outbreaks. The most useful information for selecting an appropriate antifungal treatment is still often obtained from a reliable identi- fication of the fungus at the species level. 4 Recent advances in molecular biology have impacted the field of mycology, and many new genetically distinct species that are not easily distinguishable by phenotypic char- acteristics have been described. The increase in fungal resistance to classical drugs and the fact that most available antifungal drugs have Correspondence: Paola Angelini, Department of Applied Biology, University of Perugia, Borgo XX Giugno, 74 – 06121 Perugia, Italy. Tel.: +39 075 585 6423. Fax.: +39 075 585 6404. E-mail: paola@vnr.unipg.it Accepted for publication 10 May 2010 Original article Ó 2010 Blackwell Verlag GmbH doi:10.1111/j.1439-0507.2010.01926.x mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases