Swarming Motility Assays in Salmonella

Abstract

Salmonella enterica has six subspecies, of which the subspecies enterica is the most important for human health. The dispersal and infectivity of this species are dependent upon flagella-driven motility. Two kinds of flagella-mediated movements have been described-swimming individually in bulk liquid and swarming collectively over a surface substrate. During swarming, the bacteria acquire a distinct physiology, the most significant consequence of which is acquisition of adaptive resistance to antibiotics. Described here are protocols to cultivate, verify, and study swimming and swarming motility in S. enterica, and an additional "border-crossing" assay, where cells "primed" to swarm are presented with an environmental challenge such as antibiotics to assess their propensity to handle the challenge.

Keywords: Flagella; Motility; Surface motility; Swarming; Swimming.

Fig. 1

Observing swimming and swarming bacteria. (a) Salmonella swimming motility on a soft agar (0.3% [w/v] agar) plate, after 8 h incubation at 30 °C. (b) Salmonella swarming motility on a swarm agar (0.6% [w/v] agar, 0.5% [w/v] glucose) plate, after 6 h incubation at 30 °C. (c) Salmonella swarming motility across a border-crossing plate (0.6% w/v agar, 0.5% w/v glucose), after 8 h incubation at 30 °C. In this example, kanamycin (20 μg/mL) is added to the right-side compartment. (d) Visualization of an expanding Salmonella swarm front propagated as in (a), imaged from above using an Olympus microscope with a 40× objective

Fig. 2

Border-crossing swarm plates. A schematic showing the various stages involved in preparing for Salmonella border-crossing assays. From left to right, take a dual-compartment petri dish, pour ~30 mL of 0.6% [w/v] swarm agar (with 0.5% [w/v] glucose, and any desired challenge supplementation, e.g., antibiotic) to the right chamber until the chamber is level with the divider, and allow to set. Fill the left chamber with the same swarm agar, omitting the supplementation, until flush with the divider. Use a sterile pipette tip to gently drag the molten agar across the divider, thereby connecting the two sides with a ~1 mm tall bridge. Allow the completed plate to dry at room temperature overnight for use in the following day. For a representative image of Salmonella colonizing a border-crossing plate, see Fig. 1d