Motility of Filamentous Cells of Salmonella enterica Serovar Enteritidis E40

Introduction:  Previous work from our laboratories has shown that exposure to increased salt concentration (7% w/v) results in substantial filament formation by Salmonella Enteritidis E40. These filaments have the ability to invade Caco-2 intestinal epithelial cells in vitroand are virulent upon intragastric inoculation into mice. 

Purpose:  During the course of our experiments, we noted that filamentous cells of Salmonella Enteritidis E40 were motile. The purpose of this study was to compare the motility of filamentous and non-filamentous forms of SalmonellaEnteritidis E40, and to assess the effects of medium and time of incubation on motility.

Methods:  Salmonella Enteritidis E40 was grown on trypticase soy agar (TSA) (control, non-filamentous) and TSA + 7% NaCl (filamentous) at 30°C for 4 days. The bacterial cells were harvested and resuspended in Caco-2 tissue culture medium (DMEM with supplements) with 10 % fetal bovine serum. The cells were then incubated in the same medium for up to 6 hr at 30°C in a CO₂incubator. Every 30 min, a sample was removed and plated on swimming agar (0.25% final concentration). The plates were then incubated at 37°C for 7 hr. At that time the diameter of the bacterial lawn (i.e., motility) was measured and expressed in cm. Samples removed from the Caco-2 medium at the same time points were also used to estimate colony forming units, by dilution in saline and plating on sheep blood agar.

Results: Filamentous cells of Salmonella Enteritidis E40 exhibited substantially greater motility than non-filamentous cells. For example, filamentous cells incubated in DMEM with 10% FBS for 150 min migrated approximately 8 cm on swimming agar, whereas non-filamentous cells migrated approximately 4.4 cm. During a 4-hour incubation in Caco-2 medium, an initial inoculum of 40 µg/ ml wet weight non-filamentous cells increased a little more than one log (1.67 x 10⁷ to 2.7 x 10⁸ CFU) whereas in the same time frame the CFU of filamentous cells increased from 2.7 x 10⁵ to 1.4 x 10⁸CFU.  Microscopic examination revealed that the greater increase in CFU for the filamentous cells reflects, in part, fragmentation of the filaments into smaller cells. Whether or not the medium contained fetal bovine serum seemed to have little effect on filamentous cell motility.  However, there was greater motility of non-filamentous cells as the concentration of FBS increased. By 4 hours, most filamentous cells were broken up into smaller rods, resembling control cells, and swam faster than the control cells as evident by their large initial diameter in the swimming agar.

Significance:  Filamentous cells of Salmonella Enteritidis E40 are highly motile, and can break up into many individual cells. This could facilitate spread of filamentous cells of Salmonella Enteritidis E40 in a contaminated environment.