This document summarizes a study on how pre-formed bacterial aggregates affect biofilm development. Experiments tracking Pseudomonas aeruginosa biofilms in a flow cell showed that initial aggregates drastically change the final biofilm structure depending on competition levels. Computer simulations similarly found that aggregates can outcompete surrounding single cells at high competition due to better nutrient access. Being in an aggregate is disadvantageous at low competition due to nutrient limitations. The findings have implications for biofilm shape, evolution, and the potential for cooperative behavior within aggregates.

1. Sticky bacteria and the growth of
biofilms on surfaces
Rosalind Allen
School of Physics and Astronomy,
University of Edinburgh
QLSB II, Como, 20th
June 2016

2. Edinburgh University (Physics)
Gavin Melaugh
UT Austin (Physics)
Vernita Gordon
Jaime Hutchison
U. Copenhagen (Microbiology)
Thomas Bjarnsholt
Kasper Kragh
U. Nottingham
(Evolutionary Biology)
Steve Diggle
Yasuhiko Irie
Aled Roberts

3. Bacterial biofilms
http://labrat.fieldofscience.com/2011/04/social-evolution-
in-bacteria-sgm-series.html
Communities of bacteria growing on surfaces
Cause chronic infections and industrial biofouling
Also:
A beautiful example of
multicellular self-assembly
40µm
Copenhagen and Austin groups

4. Fundamental questions:
•How do bacteria interact in biofilms?
physical forces, metabolic interactions, cooperative interactions
•What controls biofilm spatial and genetic structure?
rough versus smooth surface, clonal versus genetically diverse
•How does evolution happen in biofilms?
•Can we learn about multicellularity more generally?

5. Pseudomonas aeruginosa
Rod-shaped cells, common in soil
Causes hospital-acquired infections and
chronic infection in cystic fibrosis patients
E. Banin et al, PNAS 102: 11076 (2005)
Produces `sticky’ extracellular polymers
Engages in many cooperative behaviours
Model organism for biofilm formation

6. Lifecycle of a P. aeruginosa flow-cell biofilm
http://biofilmbook.hypertextbookshop.com
• Individual cells attach
• Transient formation of microcolonies
• Production of exopolymers
• Proliferation
• Dispersal

7. But P. aeruginosa can aggregate even in
liquid
Alhede et al, PLOS ONE (2011)
Aggregates < 105
cells in stationary phase
cultures of P. aeruginosa
Also seen in vivo at sites of chronic infection
Lung tissue from cystic fibrosis
patient, courtesy of K. Kragh
How does the picture of biofilm
development change if growth
starts from preformed
aggregates?
Our approach:
Combine flow cell microscopy with computer simulations

8. Experiments:
Track biofilm growth in a flow cell
and Kasper Kragh
Chamber is exposed to slow nutrient flow
3D images of biofilm as it grows
Can image over several days

9. Bacteria are represented as spherical particles
Food is represented as a continuum concentration field
Bacteria consume food, grow and divide
Bacteria push each other out of the way
Food diffuses and is consumed by bacteria
Computer simulations:
Model growth of individual cells with iDynoMics

10. Specific questions
• Do initial aggregates affect final biofilm spatial structure?
• Do cells in aggregates outcompete isolated cells?
• What are the mechanisms involved?

11. Experiments
Seed flow cells with stationary phase culture
Locate aggregates and track their fate by voxel
counting
Compare growth of aggregate to that of cells far
from an aggregate
Simulations
Quantifying the fate of cell aggregates
Start with circular aggregate, surrounded by “single cells”
Track fate of aggregate
Vary density of surrounding cells, nutrient concentration, etc

12. Experimental results
P. aeruginosa PA01 GFP in M9 + phosphate buffer + 0.3mM glucose
Do initial aggregates affect final biofilm structure?
24 h 24 h
Yes
they
do
Kasper Kragh
Copenhagen
Jaime Hutchison
UT Austin

13. Simulations: what is the fate of an initial aggregate?
Aggregate has a strong effect on final biofilm structure
This seems to be due to competition for nutrients
Gavin Melaugh
Edinburgh

14. Cells in aggregate can outcompete single cells at high competition
Average
progeny from
aggregate /
average
progeny from
single cell
Density of surrounding cells (per micron)
Cells in aggregate
outcompete single cells
Simulations:
fate of aggregate depends on
competition from surrounding cells

15. Key mechanism is competition for nutrients
But cells at top of aggregate
have better access to
nutrients
-> being in an aggregate can
be advantageous at high
competition
Cells in aggregate have a
fitness cost because nutrients
are limited in centre
-> being in an aggregate is
disadvantageous at low
competition

16. Experimental results:
Fate of aggregate depends on level of
competition
Aggregate can outcompete surrounding cells but only at high competition
Medium competition
Cells in
aggregate
Single
cells
Cells in
aggregate
Single
cells
High competitionLow competition
Single
cells
Cells in
aggregate
Single cells grow faster Cells in aggregate grow
faster

17. •Pre-formed aggregates can drastically affect biofilm development
•This depends on level of competition
K. Kragh, et al. mBio 7, e00237-16 (2016)
G. Melaugh, et al. PloS One 11, e0149683 (2016)
Why does this matter?
•Biofilm shape
rougher biofilms easier to penetrate with antimicrobials?
rougher biofilms evolve slower? current work, Gavin Melaugh, Edinburgh
• Evolution of cooperative behaviour
Kin selection: less genetic mixing -> more potential for cooperation

18. Conclusions
Bacterial biofilms are a beautiful example of multicellular self-assembly
Pre-formed aggregates can change our picture of biofilm development
Stickiness caused by polymer is crucial in aggregate formation
Density-dependent potential can be a way to simulate polymer production
Ongoing questions
What are the pathways to aggregate formation?
Are aggregates a first step in evolution of multicellularity?

19. Edinburgh University (Physics)
Gavin Melaugh
UT Austin (Physics)
Vernita Gordon
Jaime Hutchison
U. Copenhagen (Microbiology)
Thomas Bjarnsholt
Kasper Kragh
U. Nottingham
(Evolutionary Biology)
Steve Diggle
Yasuhiko Irie
Aled Roberts