Rapid detection of Enterobacteriaceae in urine by fluorescent 16S rRNA in situ hybridization on membrane filters

The detection and enumeration of putatively pathogenic bacteria in urine 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 Enterobacteriaceae family. The technique involves fixation of a urine specimen, filtration through a 0.2 mu m polycarbonate membrane, staining with the nucleic acid dye, 4',6-diamidino-2-phenylindole (DAPI), hybridization with a fluorescently tagged nucleic acid probe, and examination under 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 aureus, did not react with the probe but were visualized via DAPI staining. Eighty-three urine specimens were randomly selected from the clinical laboratory and assayed using this new method. A total of 10 specimens were identified by the hospital laboratory as containing members of the Enterobacteriaceae family, including E. coli and Proteus mirabilis. 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 the membrane-based FISH assay. This FISH assay for bacteriuria shows promise as a rapid technique for use in clinical diagnosis of urinary tract infections. (C) 1997 Elsevier Science B.V.