The great divide: rhamnolipids mediate separation between P. aeruginosa and S. aureus

The interactions between bacterial species during infection can have significant impacts on pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that can co-infect hosts and cause serious illness. The factors that dictate whether one species outcompetes the other or whether the two species coexist are not fully understood. We investigated the role of surfactants in the interactions between these two species on a surface that enables P. aeruginosa to swarm. We found that P. aeruginosa swarms are repelled by colonies of clinical S. aureus isolates, creating physical separation between the two strains. This effect was abolished in mutants of S. aureus that were defective in the production of phenol-soluble modulins (PSMs), which form amyloid fibrils around wild-type S. aureus colonies. We investigated the mechanism that establishes physical separation between the two species using Imaging of Reflected Illuminated Structures (IRIS), which is a non-invasive imaging method that tracks the flow of surfactants produced by P. aeruginosa. We found that PSMs produced by S. aureus deflected the surfactant flow, which in turn, altered the direction of P. aeruginosa swarms. These findings show that rhamnolipids mediate physical separation between P. aeruginosa and S. aureus, which could facilitate coexistence between these species. Additionally, we found that a number of molecules repelled P. aeruginosa swarms, consistent with a surfactant deflection mechanism. These include Bacillus subtilis surfactant, the fatty acids oleic acid and linoleic acid, and the synthetic lubricant polydimethylsiloxane. Lung surfactant repelled P. aeruginosa swarms and inhibited swarm expansion altogether at higher concentration. Our results suggest that surfactant interactions could have major impacts on bacteria-bacteria and bacteria-host relationships. In addition, our findings uncover a mechanism responsible for P. aeruginosa swarm development that does not rely solely on sensing but instead is based on the flow of surfactant.


Figure S2
. Swarm repulsion by clinical isolates of P. aeruginosa.(A) Swarm interaction assays in which wild-type P. aeruginosa was spotted at the center and CIPa strains were spotted at satellite positions with or without tobramycin (TOB).Tobramycin treatment was performed by mixing TOB with bacteria to a final concentration of 0.5 mg/mL and spotting 6 μL of the mixture onto the swarm plate.
Color bars indicate if the strains were isolated from the airway or skin wound.# CIPa-9 was resistant to TOB. (B) Swarm interaction assay in which wild-type P. aeruginosa was spotted at the center and PQS at the indicated concentrations in mM was spotted at satellite positions.Dashed lines indicate the boundaries of initial inoculum spots.Images were acquired 18 to 20 hours following inoculation.aeruginosa that are dynamic.(B) Graphic depicting how the surfactant deflection area was determined, using S. aureus USA300 s1 at the satellite position as an example.The boundary of the surfactant layer near the satellite position was identified (blue line).An arc (red) that connects two of the nearest surfactant layer boundaries that were not deflected was defined as an additional boundary.The surfactant deflection area (pink) was defined as the area enclosed by these boundaries.(C) IRIS images of a swarm interaction assay in which P. aeruginosa was spotted at the center and 6 μL of lung surfactant at the indicated concentrations were spotted at satellite positions.Images were processed to produce the masked IRIS images in Fig. 8B.(D) Schematic depicting how tendril repulsion radius is determined.Test strains or compounds (gray) were spotted along a 5.8 cm-diameter circle (red dashed line) that is concentric with the swarming plate.The repulsion radius (blue line) at each satellite colony was measured as the distance from the center of the satellite position (black dot) to the nearest tendril along a line that is tangent to the circle.If a tendril contacted the boundary of the initial satellite spot, the repulsion radius was recorded as zero.

Figure S3 .
Figure S3.Swarm repulsion by clinical isolates of S. aureus.(A) Swarm interaction assays in which wildtype P. aeruginosa was spotted at the center and clinical isolates of S. aureus (CISa) were spotted at satellite positions with or without tobramycin (TOB).Tobramycin treatment was performed by mixing TOB with bacteria to a final concentration of 0.5 mg/mL and spotting 6 μL of the mixture onto the swarm plate.Color bars indicate if the strains were isolated from the airway or skin wound.# CISa-2 was resistant to TOB.Images were acquired 18 to 20 hours following inoculation.(B) Quantification of surface growth for CISa strains for which the surface growth is not apparent in the TOB-treated colonies in (A).Surface growth intensities were measured within 1 hour and at 12.5 hours following inoculation.Bars represent the surface growth intensities from at least three satellite positions and error bars indicate standard error.T-tests were performed using two-tailed distributions with unequal variance.*** denotes p < 0.001.

Figure S4 .
Figure S4.Transmission electron microscopy (TEM) images in the vicinity of S. aureus colonies.TEM images of areas that were between 1.5 and 3mm from the edge of colonies of (A) wild-type S. aureus strain USA300 s1 and (B) the S. aureus USA300 s1 Δpsmα Δhld mutant.Scale bars indicate 200 nm in (A) and 500 nm in (B).

Figure S5 .
Figure S5.Contact angles and repulsion radii of hydrophobic molecules.(A) Images of hydrophobic molecules on an oleophobic surface acquired by a contact angle goniometer.Triton X-100 and Tween-20 were used at concentrations of 0.2% and 2%, respectively.(B) Tendril repulsion radii by 200 cSt and 1000 cSt viscosities of PDMS.Measurements were performed on images that were acquired 15 hours following inoculation.Red lines indicate average repulsion radius and error bars indicate standard deviation.The t-test was performed as a two-tailed distribution with unequal variance.

Figure S6 .
Figure S6.Surfactant production and surfactant interactions.(A) Schematic of the Imaging using Reflected Illuminated Structures (IRIS) setup.A structured image is projected onto a Petri dish and the image reflected from the surface is captured using a camera.(B) IRIS image (left) and masked IRIS image (right) of P. aeruginosa ΔrhlAB (green) spotted at the center of the plate and imaged after 20 hours of growth.A surfactant boundary was not detected.(C) IRIS images in which wild-type P. aeruginosa was spotted at the center and test strains or compounds were spotted at satellite positions.The images in I, II, and III were processed to produce the masked IRIS images in Figs.5B, 6A, and 7A, respectively.(D) IRIS images in which wild-type P. aeruginosa was spotted at the center and oleic acid or linoleic acid were spotted at satellite positions.The volumes that were spotted and fluidic boundaries are indicated (red dashed lines) in the images to the right.Images were acquired 5 hours following inoculation.

Figure S7 .
Figure S7.Swarm interactions with DMSO and surfactant, and tendril measurement methods.(A) IRIS, masked IRIS, and differential IRIS images of a swarm interaction assay in which P. aeruginosa was spotted at the center and DMSO was spotted at satellite positions.Images were acquired 14 hours following inoculation.Masked IRIS images indicate the surfactant layer (yellow), P. aeruginosa (green), and the initial boundaries of the satellite spots (red).The differential IRIS image indicates components of the surfactant layer and P. aeruginosa that are dynamic.(B) Graphic depicting how the surfactant deflection area was determined, using S. aureus USA300 s1 at the satellite position as an example.The boundary of the surfactant layer near the satellite position was identified (blue line).An arc (red) that connects two of the nearest surfactant layer boundaries that were not deflected was defined as an additional boundary.The surfactant deflection area (pink) was defined as the area enclosed by these boundaries.(C) IRIS images of a swarm interaction assay in which P. aeruginosa was spotted at the center and 6 μL of lung surfactant at the indicated concentrations were spotted at satellite positions.Images were processed to produce the masked IRIS images in Fig.8B.(D) Schematic depicting how tendril repulsion radius is determined.Test strains or compounds (gray) were spotted along a 5.8 cm-diameter circle (red dashed line) that is concentric with the swarming plate.The repulsion radius (blue line) at each satellite colony was measured as the distance from the center of the satellite position (black dot) to the nearest tendril along a line that is tangent to the circle.If a tendril contacted the boundary of the initial satellite spot, the repulsion radius was recorded as zero.