Swarming motility, secretion of type 3 effectors and biofilm formation phenotypes exhibited within a large cohort of Pseudomonas aeruginosa clinical isolates

Abstract

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of acutely infecting or persistently colonizing susceptible hosts. P. aeruginosa colonizes surfaces in vitro by either biofilm formation or swarming motility. The choice of behaviour is influenced by the physical properties of the surface and specific nutrient availability, and subject to regulatory networks that also govern type 2 and type 3 protein secretion. Biofilm formation by clinical isolates has been well-studied. However, the swarming behaviour of human isolates has not been extensively analysed. We collected isolates from 237 hospitalized patients without cystic fibrosis and analysed motility and secretion phenotypes of each isolate. We found biofilm formation and swarming to be negatively associated, while swarming was positively associated with the secretion of both proteases and type 3 exoenzymes. Most isolates were capable of type 3 secretion and biofilm formation, even though these traits are considered to favour distinct modes of pathogenesis. Our data demonstrate that while clinical isolates display diverse motility, biofilm and secretion phenotypes, many of the predicted relationships between swarming motility and other phenotypes observed in laboratory strains also hold true for bacteria isolated from human patients.

Fig. 1.

Clinical isolates display variable swarming phenotypes. Swarming plates (0.5 % agar M8) were incubated overnight at 30 °C and then at room temperature for 24–48 h. The colony morphology of clinical isolates varied, even among strains with identical twitching and swimming phenotypes. The laboratory strain PAO1 is at the bottom of each plate. (a) Swim-positive/twitch-negative; (b) swim-negative/twitch-positive; (c) swim-positive/twitch-positive; (d) swim-negative/twitch-negative. Enlarged views of colonies with small swarming diameters are provided in insets. Bars, 2 cm.

Fig. 2.

Increased swimming motility correlates with increased swarming motility among swim-positive/swarm-positive clinical isolates. Swarming and swimming diameters are normalized to PAO1. Median normalized swarm diameters are indicated for each group and are significantly different (P=0.008, Kruskal–Wallis).

Fig. 3.

Most swarm-negative isolates do not produce wetting material. Wild-type PAO1 was inoculated in the centre of the swarming plate. The black arrow shows a swarm-negative clinical isolate that does not produce wetting material and does not repel PAO1, while the white arrow shows PAO1 avoiding a swarm-negative clinical isolate due to the production of wetting material. Bar, 2 cm.

Fig. 4.

Swarming motility on LB plates. Two clinical isolates displayed colony spread consistent with swarming when assayed on 0.3 % agar LB plates. The first isolate (a) displayed no swimming, while the second (b) displayed both a swimming zone and a swarming morphology on the agar surface. Bars, 1 cm. (c) PAO1 does not swarm on 0.5 % agar LB (48 h post-inoculation), while the clinical isolate pictured in (b) shows extensive swarming on 0.5 % agar LB at this time (d).

Fig. 5.

Relationship between swarming, protease activity and type 3 secretion. (a) Swarming is positively correlated with in vitro protease activity (P <0.001, Kruskal–Wallis). (b) Isolates with the largest swarming zones secrete more PopD than isolates with decreased or absent swarming in an in vitro assay for T3SS (P <0.001, Kruskal–Wallis). (c) Isolates that secrete the highest amount of PopD display more active protease activity (P=0.06, Kruskal–Wallis). The median is shown for each group of isolates; values are expressed as percentages of the PA103 control.