Lecture 23: Biofilms
Today:
Disclaimer: very little is known about biofilms. Not everything in this lecture necessarily applies very widely!
What is the environment of the microbes we’ve studied so far?
Liquid Culture
Gel Pads
What are the key features of these laboratory environments?
Often very nutrient-rich
Often little spatial structure
Often strains bred for growth on these substrates (more on this later)
What are features of the natural microbial environment?
Not always rich nutrient availability
~50 mM dissolved carbon
~40-60 µM dissolved carbon
What are features of the natural microbial environment?
Intricate spatial surface structure
Coral
In the wild, a huge fraction of microbial biomass is associated with surfaces!
plant root
These surface-attached communities are called biofilms!
What is a biofilm?
100 µm
Species 1
Species 2
Warning: a lot of bickering, challenges in manuscript review!
B. subtilis biofilm growth
Where are biofilms? Everywhere.
Biofilms are notorious for forming on medical devices and liquid processing systems
Biofilm-forming strain of opportunistic pathogen Pseudamonas Aeruginosa
Flow-cell with dimensions of a medical device, e.g. catheter
Biofilms are thought to represent a major evolutionary transition toward multicellularity
Often called “planktonic” cells
Often called “biofilms”, “plaques”, etc
-James Shapiro,
Scientific American (1988)
The biofilm life cycle
(image courtesy of George O’Toole, Dartmouth)
1. Swimming cells find a surface
2. Cells adhere to the surface and secrete extracellular matrix
3. Cells form an attached colony
4. Cells form a mature biofilm
5. Motile cells disperse from the biofilm to potentially form a new one elsewhere
Decent amount of research here.
Not much here.
What is a biofilm vs a mere colony?
“Colony”:
“Biofilm”:
Domesticated colony: little spatial structure
Smooth colony-scale structure
Some cell-scale structure
Undomesticated “biofilm” of B. subtilis
Credit to Michael Zulch
Emergent structure over many scales
Domestication
1890s E. coli isolate
Nutrient-rich broth
Cell-dense culture
Nutrient-rich broth
Cell-dense culture
X10,000
1990s E. coli “lab strain”
This process selects for mutants that grow quickly in the nutrient-rich conditions.
Many behaviors quickly disappear in the domestication process, notably biofilm formation.
Earliest biofilm experiments: how does the presence of a surface impact bacterial growth?
2 bacterial cultures:
How does the presence of a surface impact bacterial growth?
2 bacterial cultures:
Let’s quickly plot the data in python!
Open up jupyter
How does the presence of a surface impact bacterial growth?
At “high” nutrient concentration, maybe not at all?
At low nutrient concentration, potentially a lot!
Already ~100x less than standard growth medium
How do biofilms form?
Potentially through a sense of touch! Experiment:
outlet/cell waste
inlet
flow cell 1 (FC1)
flow cell 2 (FC2)
How do biofilms form?
Potentially through a sense of touch!
How do biofilms form?
Potentially through a sense of touch!
Surface-exposed cells, when exposed to a new surface, stick to it more quickly than cells that have not been exposed to a surface.
FC1 cells
FC2 cells
Time since introduction into chamber
Each dot represents one cell that stuck to glass for at least 15 minutes
“Surface-naïve” cells take hours to stick to the glass surface
Surface-exposed cells start to stick immediately
(Ignore y-axes for now)
How do biofilms form?
Surface-exposed cells stick to surfaces for more generations than surface-naïve cells
FC1 cells
FC2 cells
Parent sticky cells
Example family trees of cells that stick to the glass
Cells that detach and swim away
Many generations of cells that stick. There is a memory of encountering the surface that facilitates multiple generations sticking and growing on the surface
How do biofilms form?
Surface exposure triggers intracellular cyclic AMP signaling that engages biofilm genes. Perform the experiment with a fluorescent reporter for cAMP activity
Mutant strain with high cAMP levels
cAMP level
Growth on surface
# generations on surface
How do biofilms form?
Surface sensing and cAMP levels are associated with extracellular pili that encounter the surface!
Type-IV Pilus
P. aeruginosa plausibly touching surfaces with a pilus, activating cAMP signaling sticking to surface, and forming a biofilm!
What are the benefits to being in a biofilm?
We will cover two:
Biofilms are notably more resistant to antibiotics than planktonic cells
medical device, e.g. catheter
fluid flow
Experiment 1
Experiment 2
Same as above but with biofilm.
Planktonic cells dead after 50 µg/ml
Biofilm cells still viable at 1000 µg/ml
“20 times more tolerant of antibiotics”.
Anti-biotic tolerance is not due merely to the inability of antibiotics to penetrate the matrix
P. Aeruginosa strains:
Phase
Membrane stain
Forms normal biofilms
45E7 strain forms robust biofilms indistinguishable from WT, but is killed easily by antibiotics
After tobramycin treatment:
Phase
Cell viability stain
Cell death stain
The ability to secrete matrix and form a biofilm is not sufficient to tolerate antibiotics. Tolerance is conferred by distinct phenotypic states that arise in biofilms or emergent properties!
Biofilm cells divide labor by taking up different phenotypic states
B. subtilis can differentiate into different cell states, much like a developing organism!
Can swim
Can secrete matrix to form biofilm
Become dormant to survive extreme conditions
Can take up DNA
B. subtilis cell types form developmental patterns!
Biofilm section:
B. subtilis cell types form developmental patterns!
Extracellular matrix
Motility
B. subtilis biofilm heterogeneity
Extracellular matrix
Spore
B. subtilis biofilm heterogeneity
Extracellular matrix
Motility
Spore
What have we learned?