Loss of flagellum-based motility by Listeria monocytogenes results in formation of hyperbiofilms

Abstract

Biofilm formation by the gram-positive, motile, food-borne pathogen Listeria monocytogenes was demonstrated to occur by an ordered series of stages. Biofilm development involves flagellum-based motility, which when blocked decreases initial bacterial surface attachment but subsequently leads to the formation of hyperbiofilms, surface-attached communities reaching high density.

FIG. 1.

Bright-field microscopy of flow cells inoculated with L. monocytogenes 10403s. The flow cell at ambient temperature was inoculated and allowed to remain static for 1 h. Medium flow was then restored, and images of surface-attached cells were captured at a magnification of either ×1,000 to image individual surface-attached cells or ×62.5 to visualize masses of surface-attached cells (dark regions against light background). The time points and level of magnification for each image are indicted in the upper left and right, respectively. Stage 1, surface attachment of individual cells; stage 2, microcolony formation; stage 3, biofilm maturation; stage 4, community dissociation (60 h) followed by regeneration (72 h).

FIG. 2.

Loss of flagellar motility alters the dynamics of L. monocytogenes biofilm development. (A) Quantification of crystal violet staining (optical density at 595 nm [OD₅₉₅]) to indirectly measure the amount of surface-attached biomass for selected strains following 24 h of cultivation in static 96-well polyvinyl chloride microtiter plates at 30°C. Each strain examined is indicated along with the relevant genotype. Each assay was completed in triplicate. The data were statistically analyzed by a two-tailed t test to compare each mutant to the wild-type strain. Significant differences are indicated by an asterisk when P is <0.05. (B) Examination of motility mutants cultured in a flow cell at ambient temperature. Mutants display an initial lag in surface attachment (compare at 0 h). Subsequently, the mutants progressively form surface-attached communities to a greater extent than the wild-type strain. The flow cells were treated as described in the legend of Fig. 1. The time points and level of magnification for each image are indicted in the upper left and right, respectively. (C) Motility mutants form HBs, an accumulation of surface-attached cells that impede the flow of medium to the flow cell. Bacterial strains were cultivated as described in the legend of Fig. 1 but were allowed to grow for 60 h. The image was captured with a standard digital camera to show a macroscopic view. Each chamber of the flow cell shown was inoculated with a different L. monocytogenes strain as indicated.

FIG. 3.

Microscopic examination of selected motility mutants and the wild-type strain. Each strain was cultivated in liquid medium as static planktonic cultures. Bacteria were stained as previously described (8). As expected, GMY1447, GMYL1448, GMY1432, and GMY1380 did not produce flagella. The wild-type (WT) strain 10403s and GMYL1455 produced flagella and were motile. Strain characteristics are indicated on the figure; plasmid pGMY21 is a flaA⁺ derivative of the integration vector pPL2 (10). Strains GMYL1434 and GMYL1385 produced flagella but were not motile, which is consistent with loss of motor function resulting in paralyzed flagella.

FIG. 4.

Deletion of flagellar genes affects biofilm development. The experimental conditions are as described in the legend of Fig. 1. Each chamber of the flow cell was inoculated with a different L. monocytogenes strain, as indicated above the images.