Transcript Inhibition of Staphylococcus aureus biofilm formation using AIP

Inhibition of Staphylococcus aureus
biofilm formation using AIP
Jordan Shivers
Kevin Kim
Professor Howard Stone
Outline
• Background
– Staphylococcus aureus
– Biofilms and quorum sensing
– agr and AIP
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Goal
Experimental design
Results
Plans
Staphylococcus aureus
• Notorious human pathogen for causing both
hospital-acquired and community-acquired
infections, ranging from minor skin infections
to pneumonia, osteomyelitis, endocarditis,
food poisoning, and infections associated
with indwelling medical devices
• Antibiotic-resistant strains:
• MRSA: Methicillin-resistant Staphylococcus aureus
• VISA: Vancomycin-resistant Staphylococcus aureus
• MRSA: emerged in 1960s, highly virulent, resistant to many antibiotics, associated
with 20000 deaths per year in the US, and a mortality rate of about 20%.
• The infections associated with indwelling medical devices, such as intravenous
catheters, happen because S. aureus is able to colonize within the devices and form
biofilms, which can cause problems within the devices and lead to infection.
Catheter-related bloodstream infections can increase hospital costs by over $50,000
for each patient.
Biofilms and Quorum Sensing
• Biofilms are communities of cells attached to
either an abiotic or a biotic surface, encased
in a self-produced extracellular polymeric
substance (EPS)
– Typically exhibit increased resistance to antibiotic
treatments and host defenses compared to
planktonic cells
– Quorum sensing system play an important role in
biofilm development in S. aureus
• Quorum sensing is the regulation of gene
expression in response to fluctuations in cellpopulation density
– QS bacteria release chemical signal molecules
called autoinducers that increase in concentration
as a function of cell density
– The detection of a threshold concentration of an
autoinducer leads to a change in gene expression
Accessory Gene Regulator system
• QS system that monitors the
extracellular concentration of
autoinducing peptides (AIP) secreted
by S. aureus
• AIP accumulates extracellularly, and
once it reaches a threshold
concentration (at post-exponential
growth stage) the agr QS system
initiates expression of gene for
producing RNA III
• Growth stages:
– Early exponential: OD < about 0.75
– Post-exponential: OD > 1.2
• B. Structures and sequences of the four AIP signals (I–
IV) corresponding to the four S. aureus groups (I–IV)
• RNAIII enables production of surfactant molecule
delta-hemolysin
Goal of our experiment
• Determine the effects of agr gene, and the
effects of inhibitor of agr gene (AIP), on
biofilm formation in the presence of flow in
curved microfluidic channels
Experimental Setup
• Preparation of bacterial solution: First grown overnight in solution of 3% TSB,
3% NaCl, and 0.5% glucose, then diluted 1:100 in the TSB solution and grown
for necessary amount of time to reach desired growth level
• Microfluidic channel coated with 20% human plasma solution about 24 hours
before running each experiment
Our Trials
Without added AIP I molecule:
With added AIP I molecule:
Group II agr wild-type S. aureus, postexponential, no AIP-I
Group II agr wild-type S. aureus, postexponential, AIP-I added during
growth
Results
Group I
Group II
Conclusion
– Biofilms of wild type strains in post exponential
phase (agr gene expressed) will detach quickly due
to RNAIII-regulated surfactant production
– Because AIP-I inhibits group II agr expression,
biofilms of wild type group II in post exponential
phase with extra AIP-I added (during growth) will
not detach easily, due to resulting lack of RNAIII
regulated surfactant production
Future plans
• Complete current experiment and run more
trials to verify that results are reproducible
• Investigate introducing activator AIP during
early-exponential growth to activate agr and
initiate surfactant production before biofilm
attachment actually takes place, in order to
inhibit formation