Biofilms

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Biofilms

• By Tony Urbina

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What is a biofilm?
Biofilms can be defined as communities of
bacteria attached to a surface.
BIOFILM LIFE CYCLE (Video)
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Where can we find biofilms?

• Our teeth
• Slippery coating on river stones
• Clogging in water pipes
• Infected wounds

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Why form biofilm?

• Resistance to antibiotics, biocides
• Synergism between species and metabolisms
• By pooling their biochemical resources, several
  species of bacteria, each armed with different
  enzymes, can break down food supplies that no
  single species could digest alone.

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Why form biofilm?

• Evolutionary strategy to enhance the ability of a
  community to adapt to quick changes in
  environmental selective pressures

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Two Different Lifestyles

• Planktonic (free-swimming)
• Nomadic bacteria
• Surface-attached bacteria
• Form sessile communities called biofilm

Figure 1a, Nature
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Two Different Lifestyles

• How is lifestyle switched?
• cyclic dimeric guanosine monophosphate
  (c-di-GMP)

Low c-di-GMP found in free-swimming
cells High c-di-GMP found in sedentary cells
(in biofilms)
Figure 2, Nature
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Two Different Lifestyles

• How is lifestyle switched?
• cyclic dimeric guanosine monophosphate
  (c-di-GMP)

Low c-di-GMP found in free-swimming
cells High c-di-GMP found in sedentary cells
(in biofilms)
Otoole Lab, Dartmouth Med.
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Overview of Biofilm Formation
Otoole Lab, Dartmouth Med.

• Biofilm formation is a developmental process
  that involves the transition between planktonic
  (free-swimming) and surface-attached bacteria.
• Transition occurs in response to a variety of
  environmental cues including the nutritional
  status of the environment

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Two Different Lifestyles
Figure 1b, Nature
Figure 1c, Nature
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Sticking Together

• How do bacteria in a biofilm stick together?
• Cells secrete a matrix to hold themselves in
  place and to provide a buffer against the
  environment.
• Matrix components extracellular polysacharrides
  (EPS), proteins, and nucleic acids

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Sticking Together

• In many strains of P. aeruginosa, 3 types of
  extracellular polysacharride has been found
• Alginate
• Pel
• Psl

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The Involvement of Cell-to-Cell Signals in
theDevelopment of a Bacterial Biofilm

• Davies et al. 1998.

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Quorum sensing Biofilm formation

• Pseudomonas aeruginosa has 2 acyl-HSL signals.
• 3OC12-HSL produced by LasI
• LasI makes an AHL that binds to LasR, which turns
  on translation of the Rhl gene.
• C4-HSL produced by RhlI

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Quorum sensing Biofilm formation
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Hypothesis

• Because quorum sensing requires a sufficient
  density of bacteria, neither of these signals
  would be expected to participate in the initial
  stages of biofilm formation, attachment, and
  proliferation.
• HOWEVER, these signals may be involved in biofilm
  differentiation.

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Two Different Lifestyles
Figure 1b, Nature
Figure 1c, Nature
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How did they test this?
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Monitoring Biofilm Formation

• WT vs. lasI-rhlI (no quorum-sensing signals)
• Both strains formed a biofilm but the mutant was
  thin and cells more densely packed.
• WT formed characteristic microcolonies made up of
  groups of cells separated by water channels

Figure 1A
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lasI, rhlI, or both?
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Monitoring Biofilm Formation

• rhlI mutant formed biofilms similar to WT
• lasI mutant formed biofilms similar to the double
  mutant

Figure 1A
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Monitoring Biofilm Formation

• These results were consistent with their
  hypothesis.
• One of the quorum-sensing systems participates in
  the subsequent biofilm differentiation process.

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Figure 1B

• Comparing WT and lasI mutant biofilms (used GFP
  and scanning confocal microscopy)
• Scanning confocal microscopy used to produce a
  side view of WT mutant biofilms
• Why is the mutant showing abnormal biofilm
  formation?

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So how did they figure out that abnormal biofilm
formation in the lasI mutant was due to absence
of3OC12-HSL?
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Figure 1B

• Added quorum-sensing signal (3OC12-HSL) to
  mutant biofilm
• In the presence of the compound/autoinducer, the
  lasI mutant formed biofilms similar to that of WT
  (in terms of thickness and cell density).
• Development of clusters of relatively loosely
  packed cells

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Monitoring Biofilm Formation

• They concluded that 3OC12-HSL is required for
  normal biofilm differentiation
• Also, gradients of the signal do not appear to be
  necessary of this differentiation

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Monitoring EPS levels in biofilms
Figure 2

• No significant differences between WT and lasI
  mutant in terms of total carbohydrates and uronic
  acids (component of alginate EPS of P.
  aeruginosa).
• What does this tell us? Did we expect these
  results?

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Monitoring EPS levels in biofilms

• Normal P. aeruginosa biofilm and planktonic cells
  produce similar amounts of EPS
• HOWEVER, the distribution of the EPS is different
• Biofilm cells cemented to each other by EPS
  matrix
• Planktonic cells have a compressed, incomplete
  EPS
• The lasI mutant biofilms may have an EPS similar
  to that of planktonic cells
• This results in tight packing of mutant biofilm

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How did they prove that abnormal,
undifferentiated biofilm formed by lasI mutant is
similar to biofilm of planktonic cells?
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Figure 3
Examined whether the abnormal mutant biofilm is
sensitive to biocides that normally do not
disrupt WT biofilms ? Added sodium dodecyl
sulfate (SDS 0.2) to WT and lasI biofilm
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Figure 3

• ? WT SDS no effect
• lasI SDS detachment/dispersal
• lasI AI SDS
• no effect

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CONCLUSIONS

• Cell-to-cell signal required in quorum sensing
  is also required for biofilm differentiation of
  individual cells of Pseudomonas aeruginosa.
• Mutation blocking generation of signal molecule
  hinders differentiation?results in abnormal
  biofilm sensitive to SDS

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THE END!