Inhibition of Staphylococcus aureus biofilm formation using AIP

Jordan ShiversKevin Kim

Professor Howard Stone

Outline

• Background– Staphylococcus aureus– Biofilms and quorum sensing– agr and AIP

• 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 cell-population 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

With added AIP I molecule:

Without added AIP I molecule:

Our Trials

Group II agr wild-type S. aureus, post-exponential, no AIP-I

Group II agr wild-type S. aureus, post-exponential, 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