RAPID DETECTION OF ENTEROBACTERIACEAE IN URINE BY FLUORESCENT 16S RIBOSOMAL-RNA IN-SITU HYBRIDIZATION ON MEMBRANE FILTERS

The detection and enumeration of putatively pathogenic bacteria in uri ne is the single-most important diagnostic criterion for the diagnosis of urinary tract infections (UTI). We have developed a fluorescent in situ hybridization (FISH) assay which utilizes a fluorescently-tagged 16S rDNA oligonucleotide probe specific for 16S rRNA of the Enterobac teriaceae family. The technique involves fixation of a urine specimen, filtration through a 0.2 mu m polycarbonate membrane, staining with t he nucleic acid dye, 4',6-diamidino-2-phenylindole (DAPI), hybridizati on with a fluorescently tagged nucleic acid probe, and examination und er epifluorescence microscopy. The technique was found to be sensitive and specific, recovering less than or equal to 10(3) Escherichia coli /ml within 4 h, both in spiked PBS and in filter-sterilized urine. Two non-Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aur eus, did not react with the probe but were visualized via DAPI stainin g. Eighty-three urine specimens were randomly selected from the clinic al laboratory and assayed using this new method. A total of 10 specime ns were identified by the hospital laboratory as containing members of the Enterobacteriaceae family, including E. coli and Proteus mirabili s. All 10 of these specimens were also positive by the membrane-based FISH assay. Of those specimens characterized as either 'no growth' or 'mixed organisms' by the hospital laboratory, 24 were positive using t he membrane-based FISH assay. This FISH assay for bacteriuria shows pr omise as a rapid technique for use in clinical diagnosis of urinary tr act infections. (C) 1997 Elsevier Science B.V.