Cell density and mobility protect swarming bacteria against antibiotics

Abstract

Swarming bacteria move in multicellular groups and exhibit adaptive resistance to multiple antibiotics. Analysis of this phenomenon has revealed the protective power of high cell densities to withstand exposure to otherwise lethal antibiotic concentrations. We find that high densities promote bacterial survival, even in a nonswarming state, but that the ability to move, as well as the speed of movement, confers an added advantage, making swarming an effective strategy for prevailing against antimicrobials. We find no evidence of induced resistance pathways or quorum-sensing mechanisms controlling this group resistance, which occurs at a cost to cells directly exposed to the antibiotic. This work has relevance to the adaptive antibiotic resistance of bacterial biofilms.

Fig. 1.

Antibiotic response and cell densities of bacteria moving within soft agar (swim) or over the surface of medium agar (swarm). (A) E-test strips containing a gradient of indicated antibiotics (decreasing from top to bottom) were placed in the center of swim (0.3%) or swarm (0.6%) agar plates. Bacteria (Salmonella) were inoculated at a point indicated by the asterisk and allowed to migrate outward. Plates were incubated at 37°C overnight and photographed against a black background so that zones of bacterial colonization appear white and uncolonized agar appears black. Cipro, ciprofloxacin; Kan, kanamycin; Polymyx, polymyxin. (B) Relative local cell densities of indicated Salmonella cultures determined by controlled sampling using the flat end of a cylindrical culture stick (see Methods). The cell numbers represent cells per stick sampled (cfus). Measurements of swim and swarm edges were taken ~6 h after the initial inoculation (swarming motility initiates at ~3 h). The accuracy range of this method was tested using broth-grown cells concentrated to various degrees, as shown in Fig. S1.

Fig. 2.

Nonswarmers show cell density-dependent antibiotic resistance. (A) Phase-contrast images (100× magnification) showing the density distribution of cells during 0–2 h of growth on the surface of swarm and nonswarm hard-agar plates. The pour-and-drain method of inoculation from a broth culture at an OD₆₀₀ of ~0.7 was used to get an initially uniform distribution of cells on the agar surface (6). At 0 h, the cell density is low and cells do not touch each other. Cell density increases continuously with time, and growth rates on both sets of plates are similar (see figure 4 of ref. 6). Cells tend to grow in aggregates on the hard agar, likely because the surface of hard agar appears not to be as smooth as that of the swarm agar. Cell density becomes confluent by 2 h; some clear pockets remain on hard agar, likely attributable to the initial uneven distribution of cells. Motility initiates on swarm plates between 2 and 2.5 h. (B) Ciprofloxacin E-test strips were applied to the surface of plates shown in A at indicated times after the plates had been evenly inoculated. Plates were photographed 3 h after E-test strip application. Similar results were obtained with kanamycin and polymyxin. The opaque halo around the clear zones surrounding the E-strips on 0–1-h swarm plates likely results from accumulating dead cells that migrate into this region (Fig. 3C).

Fig. 3.

Border-crossing assay, adaptive resistance, and cell death in Salmonella. (A) Cells were inoculated in the left no-antibiotic chamber and allowed to migrate to the right antibiotic-containing chamber (Methods). Numbers refer to μg/mL of indicated antibiotic. Plates were incubated at 37 °C for 16 h, which is the time it took for bacteria in the no-antibiotic control plates to colonize the entire right chamber. (B) Antibiotic sensitivity of swarmer cells that crossed the border on ciprofloxacin (Cip) and kanamycin (K) plates. Cells just behind the edge of the moving front were transferred by the flat end of a cylindrical toothpick to fresh swarm plates containing the same antibiotic concentration from which cells were picked (Fig. S3). Controls (Ctrl) included cells from the no-antibiotic side. The control no-antibiotic plates were solidified with 1.5% (w/v) agar to prevent swarming. (C) Swarmer cells from indicated plates stained with the live/dead stain. The red cell fraction was 6% on the control plates, 38% on kanamycin (Kan) 20, and 30% on ciprofloxacin (Cipro) 0.25 (Methods).

Fig. 4.

Faster migration enables higher adaptive resistance. (A) Cross-border swarm plates were inoculated with Salmonella as described in Fig. 3A. The 37 °C and 30 °C plates were incubated for 16 and 26 h, respectively, the time at which control plates were fully colonized. Further incubation did not promote additional migration. Sensitivity of corresponding broth cultures (OD₆₀₀ ~0.7) spotted on indicated (μg/mL) antibiotic plates is shown on the right. (B) Plates were inoculated with either Salmonella or Serratia and incubated at 30 °C. The experiment was stopped at 12 h when Serratia colonized the entire right side on control plates. Salmonella had just arrived at the border in 12 h and did not cross the border significantly on the antibiotic plates, even when incubated for longer times. We note that experiments in A and B were performed on different days; thus, the 30 °C Salmonella plates cannot strictly be compared between the two panels. The strips on the right show antibiotic sensitivity of corresponding broth-grown cultures as described in A. The dark color of Serratia is attributable to a red pigment that accumulates during overnight growth. Cipro, ciprofloxacin; Kan, kanamycin.

Fig. 5.

Behavior of Bacillus in the cross-border assay. (A) Cross-border migration of B. subtilis over increasing polymyxin concentrations. Numbers refer to μg/mL. Plates were incubated at 37 °C for 16 h. (B) Increasing cell death in the monolayer swarms on polymyxin plates stained with live/dead stain. The fraction of red cells was 8% on the control plates, 15% on polymyxin 5, 20% on polymyxin 20, and 80% on polymyxin 50 in monolayer samples from plates shown in A. (C) As in A, except that cells were allowed to build up cell density in the left chamber (16 h at 37 °C) before pouring antibiotic media into the right chamber. The plates were incubated for another 16 h.