Yvonne Gräser

IDENTIFICATION AND DETERMINATION OF THE RELATIONSHIPS OF SPECIES AND STRAINS WITHIN THE GENUS LEISHMANIA USING SINGLE PRIMERS IN THE POLYMERASE CHAIN REACTION

DNA polymorphisms were assessed in different species and strains within the genus Leishmania by amplifying genomic DNA with single non-specific primers. This polymerase chain reaction (PCR) method employed non-random primers which anneal to mini- and microsatellite DNA sequences like the M13 core sequence and the simple repeat sequences (GTG)5 and (GACA)4, and the T3B primer derived from an intergenic spacer for tRNA genes. Distinctive and reproducible sets of amplified DNA fragments were obtained for all Leishmania isolates tested. The number and size of amplification products were found to be characteristic for a given taxon. Highly similar PCR profiles were observed when genomic DNA of representatives of the L. donovani, L. mexicana or L. braziliensis complexes was amplified. By comparing PCR patterns of unidentified Leishmania isolates with those obtained from reference strains it was possible to identify these isolates at the species level. The information of the amplification patterns was used for the construction of phylogenetic trees to measure the genetic relatedness within the genus Leishmania.

First isolation of Trichophyton raubitschekii (syn. T. rubrum) in Europe

Summary. Trichophyton raubitschekii is a rare dermatophyte which belongs to the Trichophyton rubrum species complex. Since 1981, only a few cases of dermatophytosis due to this anthropophilic causative agent were published. In this paper the authors report the first cases of Tinea corporis caused by Trichophyton raubitschekii (syn. T. rubrum) in Europe. The patients, one immigrant from Ghana and three from Cameroon, had typical lesions of tinea corporis. Four strains were isolated and characterized by conventional and molecular methods. On morphological and physiological grounds the isolates were identified as T. raubitschekii by the following phenotypical features: (1) velvety colony texture; (2) brown pigment; (3) abundant macroconidia and (4) positive urease activity. Molecular diagnostics were performed by single strand conformation polymorphism (SSCP) and sequence analysis of the ATPase9 intron of the mitochondrial (mt) DNA and the internal transcribed spacer (ITS) region of the rDNA, respectively. The ITS sequences and SSCP patterns of the ATPase9 intron were found to be identical among the four strains and also when compared to reference strains of T. rubrum. As shown in the present paper, T. raubitschekii is genetically identical to T. rubrum but differs in some phenotypical characteristics. Since misidentification with other dark‐coloured dermatophyte variants is possible, medical mycologists should bear in mind the special morphological characteristics of T. raubitschekii (syn. T. rubrum) for future identifications.

Determination of S Fimbriae Among Escherichia coli Strains from Extraintestinal Infections by Colony Hybridization and Dot Enzyme Immunoassay

449 E. coli strains obtained from septic infections (124 isolates), urinary tract infections (246) and water (79) were surveyed for S/F1C fimbrial DNA by DNA hybridization and for expression of S fimbriae by enzyme immunoassay. S/F1C fimbrial DNA was detected with greater frequency in septic (35%) and urinary (43%) isolates than in water isolates and was often associated with the O2, O4, O6, O18, O83, and O156 serogroups. Most O45 strains did not possess such sequences. Only 12% and 28%, respectively, of the septic and urinary strains possessing S/F1C fimbrial DNA expressed S fimbriae.

Monoseptic growth of fungal lipase producers under minimized sterile conditions: Cultivation of Phialemonium curvatum in 350 L scale

A disadvantage of most microbial production processes is the need for sterile techniques. The objective of this study was the development of a robust fungal system allowing monoseptic growth with a minimum of sterile technique in plastic barrels. Selective growth conditions were achieved by mineral salts medium, known for the cultivation of Botrytis cinerea, but containing rapeseed oil instead of glucose as the sole source of carbon and energy. Furthermore, pH 3 was adjusted. A screening of fungi suitable for that system revealed Phialemonium curvatum AW02 isolated from compost. P. curvatum AW02 was superior in comparison with four further fungal isolates because high titers of hydrophilic spores were found in submerged production. Second, a biofilm formation on plastic segments or moving beds made harvesting of the biomass comfortable. Cultivations with volumes of 100 or 350 L showed no contaminations by bacteria when all conditions were controlled. Two independent approaches showed the dependance of growth on lipases in the cultivation system. A B. cinerea strain knocked out in lip1 showed reduced growth in comparison to the wild type because the first catabolic step is the triglyceride hydrolysis. P. curvatum AW02 lipase activity was detected. More than 90% was found to be cell wall associated. Solid shear stress liberated two active proteins showing IEPs of 4.7 or 5.6.

Anwendung molekularbiologischer Methoden für Diagnostik und Epidemiologie humaner Pilzinfektionen

Zusammenfassung. Für molekularbiologische Methoden in der mykologischen Diagnostik existieren zwei prospektive Anwendungsgebiete: der Direktnachweis des Erregers ohne vorherige Anzucht und die molekulare Identifizierung von Spezies und Subspezies. Für den Nachweis von Infektionserregern werden vor allem spezifische DNA‐Sonden und/oder die Polymeraseketten‐reaktion (PCR) genutzt, wobei nur die PCR eine ausreichende Empfindlichkeit für den Direktnachweis der Erreger im biologischen Material erreicht. Die in der Literatur beschriebenen Anwendungen der PCR für die Detektion humanpathogener Pilze werden hinsichtlich ihrer Möglichkeiten und Grenzen kritisch beleuchtet.

Tinea barbae caused by a zoophilic strain of Trichophyton interdigitale.

Summary A deep absceding infection is reported of the inframandibular part of the face of a 22‐year‐old male student due to a zoophilic strain of Trichophyton interdigitale. The fungus was probably acquired from the cat of the patient. Initial therapy by a general practitioner was with topical glucocorticoids and oral antihistaminica. The patient developed a severe phlegmoneous inflammation of the bearded part of the face. Later, the patient was successfully treated by a combination of itraconazole and fluconazole. Identification of the species was confirmed by light and scanning microscopy as well as sequence analysis of the internal transcribed spacer region of the ribosomal DNA.

ZUR VARIABILITAT VON TRICHOPHYTON VERRUCOSUM-ISOLATEN AUS IMPFBESTANDEN MIT RINDERTRICHOPHYTIE

Zusammenfassung. 27 Trichophyton verrucosum‐Stämme aus 14 Rinderbeständen in Thüringen und Mecklenburg‐Vorpommern wurden unter Einbeziehung von sechs T. verrucosum‐Referenzstämmen, unter anderem auch die sogenannten album‐, discoides‐ und ochraceum‐Varietäten, kulturmorphologisch und molekularbiologisch (PCR‐Fingerprinting, AFLP‐Analyse sowie Sequenzieung der ITS‐Region der rDNA) geprüft. Trotz großer kulturmorphologischer Variabilität der lsolate und ihrer möglichen Zuordnung zu vier verschiedenen Kolonietypen waren alle geprüften T. verrucosum‐Stämme genotypisch identisch. Auch die zwei mit Gelbpigment wachsenden Feldisolate, die möghcherweise als ochraceum‐Varietät angesehen werden könnten, sowie der ochrceum‐Referenzstamm konnten mittels der drei genannten molekularbiologischen Methoden nicht differenziert werden. Somit ergeben sich weder Hinweise für die eigenständige taxonomische Stellung der drei T. verrucosum‐Varietäten noch konnte die vermutete Infektion mit ochraceum‐Stämmen in Rinderbeständen als Ursache für das Versagen der Impfprophylaxe mit verschiedenen T. verrucosum‐Impfstoffen und für das gehäufte Auftreten sogenannter Impfversager bestätigt werden. Es wird vielmehr vermutet, daß neben der unzureichenden Impfstammpflege vor allem Anwendungsfehler, insbesondere in der lückenlosen, systematischen Immunprophylaxe in der Rinderherde, ursächlich für das gehäufte Auftreten erkrankter Rinder in gegen Trichophytie geimpften Beständen verantwortlich sind.

Identification and characterization of medically importantCandida species by using PCR-fingerprinting

in the same reaction vial together with the template, amplified copy numbers can be quantified.We have constructed a standard for the PCR-detection of the insertion element IS 6110 of Mycobacterium tuberculosis. The PCR-MIMIC construction kit of Clontech was used. In this reaction the same primers amplify a template sequence of 123 bp and the standard of 360 bp. The standard is added in 3 different concentrations (6, 60, 600 molecules per sample), the probe is amplified and the products are separated, blotted and detected with enhanced chemiluminiscence. The results show that the lower detection limit is not constant. In some experiments only 600 molecules of the standard were detectable. This varying efficiency may be caused by inhibitors within the individual sample. Especially in the case of negative results, one has to consider this phenomenon. The use of internal standards gives information concerning the lower detection limit and prevents false negative results.