Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype

Abstract

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25 degrees C after 3 days of incubation. Incubation at 37 degrees C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25 degrees C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.

FIG. 1

Morphology of rugose (Rv/25) (A and C) and smooth (Rv/37) (B and D) colonies of S. enterica serovar Typhimurium DT104 grown for 4 days on TSA at 25 and 37°C, respectively. Scale bars, 5 mm (A and B) and 2 mm (C and D).

FIG. 2

Progression of rugose formation at day 2 through day 4 at 25°C on TSA. Panels A to C show Rv/25 colonies at day 2, 3, and 4, while panels D to F show scanning electron micrographs of cells from the respective colonies. Expression of ES is apparent in cells starting at day 3 when the colonies just become wrinkled. Scale bars, 5 mm (A to C) and 1 μm (D to F).

FIG. 3

Pellicle formation was produced by Rv/25 (right) but not by Stv (left) in TSB after 7 days of incubation at 25°C.

FIG. 4

Scanning electron micrograph of pellicle cells. Note the presence of ES in both slime and the fibrillar structure. Scale bar, 1 μm.

FIG. 5

Transmission electron micrographs of cells from the pellicle (A) and from broth under the pellicle (B) grown in TSB for 7 days, showing the presence of capsular material and a thin layer of ES, respectively, that binds to cationic ferritin. The fibrillar material was also bound to cationic ferritin (arrow in panel A). Scale bars, 0.5 μm.

FIG. 6

Formation of biofilm matrix on the inner surface of glass flasks. The cultures of Rv/25 (A) and Stv (B) in TSB were incubated with shaking (50 rpm) at 25°C for 7 days with the TSB changed every 24 h. The resulting biofilms were stained with crystal violet for immediate observation.

FIG. 7

Survival of S. enterica serovar Typhimurium DT104 cells from 4-day-old Rv/25 (■) and Stv (▴) upon a 90-min exposure to 10 mM hydrogen peroxide at 37°C (—) or pH 3 at 25°C (- - - ). Each data point represents the mean with standard deviation for three separate experiments.