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.