Respective roles of culturable and viable-but-nonculturable cells in the heterogeneity of Salmonella enterica serovar typhimurium invasiveness

Abstract

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.

FIG. 1.

Distribution of the cells in a Radioselectan density gradient after ultracentrifugation of a 10-, 24-, or 48-h culture of S. Typhimurium. Fifty successive samples of 200 μl have been collected from the gradient from top to bottom of the ultracentrifuge tube. Bars represent the OD₆₀₀s of the individual samples expressed as percentages of the cumulated OD₆₀₀ of the 50 samples. In the graphs, cell density increases from their upper part to their lower part. LD, MD, and HD cell subpopulations are indicated.

FIG. 2.

Cytometric analyses of the S. Typhimurium total cell population (T) and of the LD, MD, and HD cell subpopulations obtained after a Radioselectan density gradient centrifugation of a 48-h culture. Cells were stained using a Live/Dead BacLight bacterial-viability kit (Molecular Probes). The abscissa indicates the green fluorescence intensity (FL 1) of cells stained with SYTO 9. The ordinate indicates the red fluorescence intensity (FL 3) of cells stained with propidium iodide.

FIG. 3.

β-Galactosidase activity of lacZ transcriptional fusions of three invasion genes (white bars, hilA; gray bars, invF; black bars, orgA) in S. Typhimurium subpopulations. The LD, MD, and HD cell subpopulations were obtained using Radioselectan density gradient ultracentrifugation after 10, 24, and 48 h of culture. The standard deviations for the triplicate β-galactosidase assays are so small that error bars are not visible. Results are representative of three independent experiments.

FIG. 4.

Effect of the addition of heat-killed cells on the invasion rate of S. Typhimurium culturable cells on HeLa cells. Heat-killed (121°C, 15 min) S. Typhimurium cells were added to a 100% culturable cell inoculum. No heat-killed cells (white bars), 10 heat-killed cells (gray bars), or 100 heat-killed cells (black bars) were added for each culturable cell. Error bars represent standard deviations for triplicate invasion assays.

FIG. 5.

Invasion rates of S. Typhimurium on HeLa cells after 10, 24, and 48 h of culture expressed in the total (T) cell population and in the LD, MD, and HD cell subpopulations obtained using Radioselectan density gradient centrifugation. The invasion rate represents the percentage of invasive cells expressed with respect to the number of culturable cells inoculated (10⁶ for each inoculum). Error bars represent the standard deviation of sextuplicate β-galactosidase assays. Results are representative of three independent experiments.