Uropathogenic Escherichia coli 536 (06:K15:H31) carries two unstable DNA regions, which were shown to be responsible for virulence. These regions, on which the genes for hemolysin production (hly) and P-related fimbriae (prj) are located, are termed pathogenicity islands (PAI) I and II, and were mapped to positions 82 and 97, respectively, on the E. coli K-12 linkage map. Sequence analysis of the PAI region junction sites revealed sequences of the leuX and selC loci specific for leucine and selenocysteine tRNAs. The tRNA loci function as the targets for excision events. Northern (RNA) blot analysis revealed that the sites of excision are transcriptionally active in the wild-type strain and that no tRNA-specific transcripts were found in the deletion mutant. The analysis of deletion mutants revealed that the excision of these regions is specific and involves direct repeats of 16 and 18 nucleotides, respectively, on both sides of the deletions. By using DNA long-range mapping techniques, the size of PAI I, located at position 82, was calculated to be 70 kb, while PA II, mapped at position 97, comprises 190 kb. The excision events described here reflect the dynamics of the E. cohi chromosome.

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Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in a mouse infected with the closely related species C. parapsilosis. Conversely, the C. parapsilosis-specific probe detected fungal cells in the deep organs of a mouse infected with C. parapsilosis but not in the deep organs of a C. albicans-infected mouse. In addition, the C. albicans-specific probe was used to detect this species in human blood spiked with yeast cells by a lysis-filtration assay and subsequent fluorescent in situ hybridization. By this assay, as few as three yeast cells per 0.5 ml of blood were consistently detected. Our results demonstrate that fluorescent in situ hybridization with species-specific rRNA-targeted oligonucleotide probes provides a novel, culture-independent method for the sensitive detection and identification of Candida species in clinically relevant material.

Thirty Candida isolates obtained from the oropharynxes of three AIDS patients were genotypically characterized. In vitro fluconazole MIC determination revealed increasing fluconazole resistances during treatment, thereby confirming the in vivo situation. Pulsed-field gel electrophoresis karyotyping, randomly amplified polymorphic DNA analysis, and hybridizations with Candida albicans repetitive element 2 were used to determine possible genotypic changes. The isolates from two patients showed genetic homogeneity, suggesting the selection for resistant variants. One patient experienced a strain switch to Candida krusei. Horizontal spread of identical strains between the patients could be excluded. However, the molecular methods used might not be sufficient to detect the underlying genetic basis of resistance to fluconazole.

To analyse the function of the equid herpesvirus 4 (EHV-4) glycoprotein M homologue (gM), two different mutated viruses (E4DgM-GFP and E4DgM-w) were generated. Both gM-negative EHV-4-mutants were characterized on complementing and on non-complementing cells and compared with E4RgM, a virus where gM-expression had been repaired. It was demonstrated in virus growth kinetics that deleting gM had a more dramatic influence on EHV-4 replication than expected. Extracellular infectivity was detected 9-12 h later than in EHV-4-infected Vero cells and titres were reduced up to 2000-fold. In addition, mean maximal diameters of plaques were less than 20 % of diameters of wild-type plaques. These results are in contrast to most other alphaherpesviruses, including the closely related equid herpesvirus type 1, where deletion of gM only marginally influences the ability of viruses to replicate in cell culture. Nevertheless, analysis of infected cells by electron microscopy did not reveal a specific defect for deleting gM. It was concluded that EHV-4 gM is important for more than one step in virus replication in cell culture, influencing both efficient virus egress and cell-to-cell spread.

A method is presented for the standardization of Candida albicans DNA fingerprinting, which is based on Southern hybridization of EcoRI-digested chromosomal DNA with the moderately repetitive DNA element CARE-2 and the subsequent rehybridization of the blots with a molecular size marker also included in each DNA sample. This method resulted in extremely precise alignment of all strain-specific CARE-2 hybridization patterns, even when analysed on different gels, and will enhance the accuracy of genetic relationship determinations in epidemiological studies including large numbers of strains.

A total of 277 Candida isolates from various body sites of 149 AIDS and cancer patients treated in four different university clinics in Würzburg, Germany were collected over a period of 27 months and phenotypically and genotypically characterized. The fingerprinting patterns of 194 Candida albicans isolates obtained with the moderately repetitive, C. albicans-specific DNA fragment CARE-2 were digitized and retrospectively compared with a highly accurate computer-assisted standardization method. A total of 168 different genotypic patterns (< 100% identity) could be differentiated using this technique. Although comparative analysis of C. albicans subsets revealed a pronounced tendency of C. albicans isolates from HIV patients to form clusters, the mean genetic variability in HIV and cancer patient isolates was virtually identical. Patients with a specific disease condition or in a certain age group were not found to harbour C. albicans isolates displaying a characteristic "signature genotype". Micro-evolutionary changes were detected by CARE-2 fingerprinting in temporal successive isolates of one patient, but nosocomial transmission of identical isolates between unrelated patients was never seen. Genotyping showed that patient isolates can replace one another; occasionally also species switches were observed. Secreted aspartic protease (SAP) production was not correlated with a specific C. albicans banding pattern; isolates obtained from HIV patients and from an internal control group secreted comparable amounts of SAP. Candida dubliniensis isolates in this study showed an elevated level of SAP production. When used under standardized conditions, CARE-2 fingerprinting is an efficient, reproducible and sensitive technique to characterize C. albicans isolates.