Oligonucleotide probe for the visualization of Escherichia coli/Escherichia fergusonii cells by in situ hybridization: specificity and potential applications.

There are several occasions when enumeration of Escherichia coli cells is needed. These include examination of urine specimens and water or food samples. Present methods rely on growth in more or less selective media (colony-forming units on agar or the most probable number method using liquid media). Unfortunately, no really selective medium with 100% efficiency of plating is available for E. coli. A 24-mer oligonucleotide probe (Colinsitu), complementary to a piece of 16S ribosomal ribonucleic acid, has been tested for specifically visualizing E. coli cells by in situ hybridization and epifluorescence microscopy. The fluorescent dye-labeled probe was able to stain cells of E. coli, Shigella spp. and E. fergusonii. Shigella spp. are known to belong to the E. coli genomospecies and E. fergusonii is the nomenspecies closest to E. coli by DNA-DNA hybridization. The probe did not stain any strain of 169 other genomospecies of the family Enterobacteriaceae or of a few other species frequently encountered in the environment. Revivification without cell division allowed the visualization of E. coli cells in contaminated water. In situ hybridization using the Colinsitu probe is a potential tool for the confirmation of (atypical) E. coli in reference centers and the rapid (3-6 h) detection and enumeration of E. coli in urine specimens, contaminated water and food. More work is needed to include in situ hybridization in laboratory routine.

Problems associated with the direct viable count procedure applied to gram-positive bacteria.

Despite the numerous advantages of fluorescent in situ hybridization (FISH) for identifying a single bacterial cell with 16S rRNA probes, problems are encountered with starving bacteria in natural samples. The original direct viable count procedure (DVC) includes a revivification step in the presence of an antibiotic inhibiting cell division. Cells elongate and accumulate ribosomes. This results in a natural amplification of 16S rRNA molecules (target of FISH). However, it is limited to gram-negative bacteria which are sensitive to nalidixic acid. The objective of this study was to develop a procedure for estimating the number of metabolically active gram-positive Staphylococcus aureus and Enterococcus faecalis cells by the use of a method which combines the number of substrate-responsive cells and their identification by FISH. It was observed that no single published DVC method could apply to taxonomically different gram-positive bacteria. Since cells were not counted, the revivification step in presence of nalidixic acid will be referred to as revivification without cell division. For each species, different low-nutrient media and complex media, different fluoroquinolones and beta-lactam antibiotics, concentrations of antibiotics, combinations of antibiotics, temperature and time were evaluated using bacteria in different physiological states and in natural samples. Enumeration of bacteria by plate counts and direct FISH were compared. The improved procedure should yield information about the physiological state, the taxonomic identity, and the enumeration of viable gram-positive bacteria. The application of DVC to an entire ecosystem is presently still a challenge.

Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells.

A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.