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ORIGINAL ARTICLE 10.1111/j.1469-0691.2005.01356.x
Rapid identification of clinical Trichoderma longibrachiatum isolates by cellulose-acetate electrophoresis-mediated isoenzyme analysis A. Szekeres1,M.La´day2, L. Kredics3, J. Varga1, Z. Antal3, L. Hatvani1, L. Manczinger1,C.Va´gvo¨lgyi1 and E. Nagy3,4
1Department of Microbiology, University of Szeged, Szeged, 2Plant Protection Institute, Hungarian Academy of Sciences, Budapest, 3Microbiological Research Group, Hungarian Academy of Sciences and University of Szeged and 4Department of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary
ABSTRACT Cellulose-acetate electrophoresis was used to investigate isoenzyme polymorphism among ten clinical and 11 non-clinical isolates of Trichoderma. Initial testing of 13 enzyme systems for activity and resolution of bands showed that seven were appropriate for identifying the different species. Each of the enzyme systems investigated (glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, peptidases A, B and D, and phosphoglucomutase) was diagnostic for at least one species. On the basis of the results of isoenzyme analysis, several isolates identified originally as Trichoderma pseudokoningii, T. koningii or T. citrinoviride were re-identified as T. longibra- chiatum, in agreement with sequence analysis data for the internal transcribed spacer region of the isolates. The availability of a quick, inexpensive and reliable diagnostic tool for the identification of T. longibrachiatum isolates is important, as most clinical Trichoderma isolates belong to T. longibrachiatum. Furthermore, as many different enzyme systems are available, the method may also be suitable for the identification of other clinically relevant fungal species. Keywords Cellulose-acetate electrophoresis, diagnosis, identification of fungi, isoenzyme analysis, Trichoderma spp. Original Submission: 10 January 2005; Revised Submission: 19 July 2005; Accepted: 13 September 2005 Clin Microbiol Infect 2006; 12: 369–375
has been published previously [3]. The number INTRODUCTION of reported cases has grown from year to year, Species of the imperfect filamentous fungal possibly because of the expanding population of genus Trichoderma with teleomorphs belonging immunocompromised hosts. Four further cases to the Hypocreales order of the Ascomycota are mentioned in a study concerning extracellu- division are of great economic importance as lar protease production by clinical isolates of sources of enzymes and antibiotics, as plant T. longibrachiatum [4], and an additional report growth promoters, as decomposers of xenobiot- describes the infection of a cerebrospinal fluid ics, and as commercial biofungicides [1,2]. shunt device in a non-immunocompromised However, this genus is also on the growing list patient [5]. of emerging fungal pathogens, and has been Data concerning the antifungal susceptibilities reported in an increasing number of cases as an of clinical Trichoderma isolates indicate high aetiological agent in human infections. A review levels of resistance to the antifungal drugs used of 40 cases of infection associated with Tricho- routinely, which may cause difficulties in the derma spp., mostly Trichoderma longibrachiatum, treatment of infected patients [6]. Antifungal susceptibility data often correlate with the taxo- nomic position of isolates; therefore, accurate Corresponding author and reprint requests: A. Szekeres, identification of opportunistic fungi is crucial Department of Microbiology, University of Szeged, PO Box 533, H-6701 Szeged, Hungary for adequate therapeutic interventions. As most E-mail: [email protected] clinical Trichoderma isolates are T. longibrachia-