Sticky bacteria and the growth of biofilms on surfaces

Rosalind AllenSchool of Physics and Astronomy,

University of Edinburgh

QLSB II, Como, 20th June 2016

Edinburgh University (Physics)Gavin Melaugh

UT Austin (Physics)Vernita Gordon Jaime Hutchison

U. Copenhagen (Microbiology)Thomas BjarnsholtKasper Kragh

U. Nottingham(Evolutionary Biology)Steve Diggle Yasuhiko IrieAled Roberts

Bacterial biofilms

http://labrat.fieldofscience.com/2011/04/social-evolution-in-bacteria-sgm-series.html

Communities of bacteria growing on surfacesCause chronic infections and industrial biofouling

Also:A beautiful example of multicellular self-assembly

40m Copenhagen and Austin groups

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?

Pseudomonas aeruginosaRod-shaped cells, common in soilCauses 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

Lifecycle of a P. aeruginosa flow-cell biofilm

http://biofilmbook.hypertextbookshop.com

• Individual cells attach• Transient formation of microcolonies• Production of exopolymers• Proliferation• Dispersal

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

Experiments: Track biofilm growth in a flow cell

and Kasper Kragh

Chamber is exposed to slow nutrient flow3D images of biofilm as it growsCan image over several days

Bacteria are represented as spherical particlesFood is represented as a continuum concentration field

Bacteria consume food, grow and divideBacteria push each other out of the wayFood diffuses and is consumed by bacteria

Computer simulations: Model growth of individual cells with iDynoMics

Specific questions

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

ExperimentsSeed flow cells with stationary phase cultureLocate aggregates and track their fate by voxel countingCompare 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 aggregateVary density of surrounding cells, nutrient concentration, etc

Experimental resultsP. 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 KraghCopenhagenJaime HutchisonUT Austin

Simulations: what is the fate of an initial aggregate?

Aggregate has a strong effect on final biofilm structureThis seems to be due to competition for nutrients

Gavin MelaughEdinburgh

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

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

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 competition Low competition

Single cells

Cells in aggregate

Single cells grow faster Cells in aggregate grow faster

•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 behaviourKin selection: less genetic mixing -> more potential for cooperation

ConclusionsBacterial 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?

Edinburgh University (Physics)Gavin Melaugh

UT Austin (Physics)Vernita Gordon Jaime Hutchison

U. Copenhagen (Microbiology)Thomas BjarnsholtKasper Kragh

U. Nottingham(Evolutionary Biology)Steve Diggle Yasuhiko IrieAled Roberts