Biofilm antibiotic resistance in P. aeruginosa
For reasons that are
not understood, bacteria growing in biofilms can become up
to 1000-fold more resistant to antibiotics and other biocides
as compared to their planktonic counterparts. As a result of
this increased resistance, biofilm infections cannot be effectively
treated with conventional antibiotic therapy. This biofilm
resistance is distinct from commonly known resistance mechanisms
such as plasmid-borne resistance markers or resistance conferred
by mutation.
Biofilm antibiotic resistance
is thought to occur due to changes in gene expression or physiology
as a consequence of transitioning to a surface-attached existence.
Our lab has used a genetic approach to isolate mutants capable
of biofilm formation, but which do not develop normal biofilm
antibiotic resistance. The long-term goal of these studies is
to develop new strategies to disable biofilm resistance, thus
rendering these communities sensitive to conventional antibiotic
therapy.
We performed a screen to isolate
mutants of Pseudomonas aeruginosa capable of forming a biofilm,
but unable to develop full biofilm resistance to the antibiotic
tobramycin (Tb). We chose Tb because this is one of the antibiotics
used to treat patients with Cystic Fibrosis (CF). Some current
studies from other groups suggest that P. aeruginosa grows as
a biofilm in the CF lung.
One of the strains isolated carried
a mutation in the ndvB gene. We grew the wt and the ndvB mutant
in a flow cell for 24 hrs followed by treating the biofilm with
Tb for 24hrs. The biofilms were stained with
the BacLight viability stain and images were acquired with phase-contrast
microscopy as well as epifluorescence in green and red channels.
The wild type showed more green stain (indicating live cells)
than red stain (indicating dead cells). The reverse is true
for the ndvB mutant. The phase-contrast images (left-most panels)
shows that there is no difference in the structure of this biofilm.
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The proposed localization of the NdvB protein to the inner membrane
is shown to the right. NdvB is thought to catalyze the synthesis
of circular glucose molecules known as cyclic glucans. These
molecules are transported to the periplasm and are also exported
outside the cell, possibly via the action of the the NdvA protein.
Shown below is our current model for the role of cyclic glucans
in biofilm resistance. The ndvB gene is preferentially expressed
in biofilm cells likely resulting in an increase in the NdvB
protein (yellow). NdvB catalyzes the synthesis of cyclic glucans
in the periplasm (yellow circles). The red stars represent the
fact that the antibiotic Tb can penetrate the biofilm. However,
based on a demonstrated interaction between the periplasmic
glucans and Tb, we propose that the diffusion of Tb into the
cytoplasm may be slowed by the glucans. The decreased diffusion
of Tb may allow the biofilm cells additional time to adapt to
and resist the action of the antibiotic.
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These studies are continuing
to be pursued by Dr. Thien-Fah “Thienny” Mah, a former post-doc
in the O’Toole lab. She now has her own group at
the University of Ottawa. Check out her website.
Related publications
Mah
TF, O'Toole GA. 2001. Mechanisms of biofilm resistance to antimicrobial
agents. Trends Microbiol. 9:34-9.
Mah
TF, Pitts B, Pellock B, Walker GC, Stewart PS, O'Toole GA. 2003.
A genetic basis for Pseudomonas aeruginosa biofilm antibiotic
resistance. Nature. 426:306-10.
