In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus

Abstract

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4,6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the cold-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5°C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.

Profiling bacterial survival through a water treatment process and subsequent distribution system

“…nutrient limitation and chlorination) resulting in intracellular rRNA levels below that detectable by oligonucleotide hybridization for subsequent EFM visualization. Such a decrease in rRNA below that for detection by FISH has been demonstrated previously in cold-induced VBNC cells of the human pathogenic bacterium Vibrio vulnificus (Weichart et al 1997).…”

Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment

“…Membrane-impermeative fluorescent probes that can passively diffuse through the cell wall of a bacterium can act as an indicator of a loss in membrane integrity, which, in turn, can often act as an indicator of cell viability (3,10). Indeed, the loss of membrane integrity results in membrane permeabilization and thus, in the degradation of nucleic acids whose maintenance is at least essential to retain viability (4,17,18). However, such degradation may occur with a delay after the loss of membrane integrity which may be dependent on both environmental conditions and species.…”

“…To better understand this decrease in the number of dead cells as determined by SYTOX Green staining, we analyzed the temporal evolution of the nucleic acid content of starved cells by staining the cells with SYBR-I nucleic acid dye. The degradation of nucleic acids is sometimes considered an alternative method for assessing the death of bacterial cells (5,17). FCM analysis of the nucleic acid content of SYBR-I-stained cells revealed important changes in the course of starvation (Fig.…”

Behavior of sulfate reducing bacteria under oligotrophic conditions and oxygen stress in particle-free systems related to drinking water

“…In situ hybridization was first introduced in 1989 by DeLong et al [17] as a tool for the identification of whole bacterial cells and for the quantification of the ribosome content of growing cells. In the past, FISH signal intensities of bacterial cells have been used as measurement for the metabolic activity or potential of slow growing [18] or starving bacteria [37,38]. Nevertheless, ribosomes are known to undergo various changes, e.g.…”