Pablo Bravo
PhD Candidate in Quantitative Biosciences at GeorgiaTech. I am interested in biofilms, and how their topography evolves and what we could learn about their development by looking at them
Colony size and antibiotic tolerance:
the role of spatial dynamics
Pablo Bravo
Yunker Lab, Georgia Tech
“Experiment is the only means of knowledge at our disposal. Everything else is poetry, imagination.”
-
all the way down
Gradients
This is just thinking how things are changing, both in space and time!
\vec{\nabla} u
\frac{\partial u}{\partial x}
\frac{\partial u}{\partial t}
\dot{u}
Horizontal Growth
Vertical
Growth
Growth and accumulation
- How do aggregates grow?
- What are the underlying processes?
- Simple quantitative model?
Extracellular matrix formed of polysaccharides, DNA, and proteins
Surface
Interface
Cells
Biofilms are 3D structures
complex surface-attached
cycle
The (expanded) biofilm
Sauer, K., et al. Nature Reviews Microbiology (2019)
in the biofilm surface
\(1 cm\)
\(1 cm\)
Dietrich, L., et al. Journal of Bacteriology (2013)
Structures
inside the colony
Nutrient dynamics
\(z\)
\frac{\partial c}{\partial t} = D \cdot \frac{\partial^2 c}{\partial z^2} - \lambda \cdot \frac{c}{k+c}
c(0, t) = c_{0} \quad \quad \quad \dot{c}(h, t) = 0
Concentration in the substrate
No flux in the top
Region where cells can grow is finite
\int_0^h \frac{c(z)}{k+c(z)}dz \approx \text{min}(h, L)
Total growth of a colony saturates once they reach a critical length \(L\)
Diffusion constant
Consumption rate
Monod constant
Local and death
growth
random
Adding time, we can see the actual dynamics
- Colonies would grow forever!
- Adding a death term, proportional to the population, dynamics reach an equilibrium
No death
Random death
- Multiple light sources in the instrument
- Super-resolution measurements
- Non-invasive
- No preparation needed
\( 0.5 mm\)
0
2
4
6
8
10
\(\Delta z\) (\( \mu m\))
Central region of a vibrio cholerae biofilm
Surface topography + intensity!
White-light interferometry
Using to measure
interferometry
biofilm growth
Homeland
Agar
Coffee Ring
Vertical growth dynamics ( )
Experiments across a large cohort of microbes
- Bacteria and fungi
- Gram + and -
- Different shapes
- Different EPS production
VGD
Interferometry at the single-cell level
Surface topography and intensity from Interferometry
S. cerevisiae, 48 hours of growth.
Bravo 2022
Things didn't go quite well the first ~10 attempts
Hours
0
48
Not the whole story, but !
close enough
Kannan et al. Biorxiv (2023)
"All models are wrong, but some are useful"
- George E. P. Box
The metabolite gradients are more tricky than in the 1D story.
What would happen when we introduce ?
antibiotics
Source of antibiotics is from the same interface as the nutrients. How will the interactions change dynamics?
No death
Random death
- Zones could overlap
- Or they could not...
No Growth
Growth
What would happen when we introduce ?
antibiotics
Source of antibiotics is from the same interface as the nutrients. How will the interactions change dynamics?
- Zones could overlap
- Or they could not...
No Growth
Growth
What would happen when we introduce ?
antibiotics
Source of antibiotics is from the same interface as the nutrients. How will the interactions change dynamics?
No death
Random death
- Zones could overlap
- Or they could not...
No Growth
Growth
Antibiotics
What would happen when we introduce ?
antibiotics
Source of antibiotics is from the same interface as the nutrients. How will the interactions change dynamics?
No death
Random death
No Growth
Growth
Antibiotics
Will the zone of antibiotics then propagate, or remain fixed?
Could it be that growth zones manages to just escape?
- Zones could overlap
- Or they could not...
of antibiotic tolerance in biofilms
Mechanisms
There is many different factors related to tolerance in biofilms!
Ciofu et al. Nat Rev Microbiology (2022)
dynamics in biofilm
Macroscopic
The outcome of treating a biofilm with antibiotics will be at least determined by some macroscopic factors:
- Antibiotic concentration
- Exposure time
tolerance
Antibiotics, Stress time
Survival
Preliminary Results - Growth and stress in MH
- \(h_0\) of 90 and 120 μm
- Growth seems to saturate at ~220μm
- Saturation height is consistent with and without transfer
- Chloramphenicol seems to have little effect in growth
- Carbenicillin is increasing height significantly
behind tolerance and resistance
Mechanisms
dynamics in biofilm
Macroscopic
tolerance
Biofilm size
Survival
The outcome of treating a biofilm with antibiotics will be at least determined by some macroscopic factors:
- Antibiotic concentration
- Exposure time
- Biofilm size
, the good and the not so good!
Homeland
Experiments
Goal: test the role of biofilm size under different antibiotic stresses
Prelim Exp #1: wild type
Aeromonas veronii (wt)
48 hours of stress
Colonies grow up to 80% of the ones without antibiotics!
: No changes
M9
: growth after ~30 μm
Chloramphenicol
: growth after ~5 μm
Carbenicillin
biofilm-former
Prelim Exp #2: strain
Day 1
Day 2
Day 3
Eschericia coli (lc)
48 hours of stress
: No apparent changes
M9
: some growth
after ~10 μm?
Carbenicillin
: expected VGD
MH
Colonies did not grow, but carbenicillin is doing something to large colonies...
Each point is a different colony
M9
Carb
MH
low "complexity"
Improvements:
- 108 colonies over 10 hours
- 3 measurements instead of 2
Antibiotics change biofilm
M9
Carbenicillin
Small
Large
Day 1
Day 2
Day 3
Let's look at individual profiles:
- Small colonies remain mostly unchanged
- Coffee Ring
- Large colonies get disrupted!
Is there a biological response? Or is it post-lysis physics?
structure
Assessing the final
population
Looking at the height is an OK proxy, but how do we actually quantify survival?
- We could look at the unstructured population, either by OD or CFU's
- The structured population keeps a macroscopic/multicellular perspective
Planning a better experiment
Help!
Bug choice
- Wild type v. cholerae
- EPS- QS-: \( \Delta hapR \Delta vpsR \)
- EPS-: \(\Delta vpsR\)
Stress plates
- 1x respective MIC
- 10x MIC
"Revival" step
- CFU/OD
- Back to growth plates
Stress time?
- 4-12 hours?
- 48 hours may be too much
Same or different story?
Different/more antibiotics?
Dr. Peter Yunker
Dr. Thomas Day
Dr. Miles Wetherington
Dr. Aawaz Pokhrel
Dr. Adam Krueger
Emma Bingham
Raymond Copeland
Maryam Hejri
Dr. Brian Hammer
Dr. Siu Lung Ng
Kathryn MacGillvray
Chris Zhang
has an important role in , since gradients set the underlying spatial dynamics.
Thanks!
Colony size
antibiotic tolerance
Results from 2024-11-13
- BH1514, Vibrio cholerae (wt)
- 78 different colonies
- 13 growth times
- 2 treatments (10x, 100x)
- 3 replicates
- Measurements
- Before transfer (clean)
- After 24h of stress
- After 48h of stress
*Using \(\langle h \rangle \)
Transition seems to be the same for 10x and 100x!
Behaviors across the same colony
- Large colonies grow without a problem
- Small colonies do not grow
- Medium sized colonies grow a bit?
How should we classify/quantify growth?
How do we get numbers from the plot?
24h to 48h - Relevant dynamics?
There seems to be little change between 24 and 48 hours
Coffee Ring - Homeland Split
| 10x | 100x | |
|---|---|---|
| Average | ||
| Homeland | ||
| CR |
- Homeland removes points in the y-axis during transition
- CR taller than Homeland (XY)
- Clean transition in y-axis too
Why don't the x-axis transitions match?
Current explanation (there is a rotation...)
Height threshold
Lactamase threshold
Disperse homeland
Connected homeland
Coffee Ring
How do we connect the heights to the metabolic threshold?
We can measure!
Real?
Or how to characterize the dependency in a simple experimental way?
CMDI 2024
By Pablo Bravo
