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Antibiotic Resistance of Staphylococcus aureus Biofilms Smitha Neerukonda VCU BBSI Summer 2009 Mentor: Dr. Kimberly Jefferson August 3, 2009 Staphylococcus aureus - Gram-positive bacteria normally colonizing the nasal passages, skin and mucous membranes1 - Opportunistic pathogen Gram stain; S. aureus http://www.labnews.co.uk/laboratory_article.php/2912/5/combating-bacterial-infection Biofilms Biofilms = community of bacteria covered in extracellular polymers and attached to a surface2 http://www.labnews.co.uk/laboratory_article.php/2912/5/combating-bacterial-infection Biofilms play an important role in chronic, recurrent infections and in medical device (eg. catheter) -related infections Dangers associated with S. aureus Biofilms Chronic Infections/Diseases: Osteomyelitis Endocarditis Biofilms are resistant to antibiotic levels 10-1,000 times higher than planktonic bacteria3 – Concentrations of antibiotics required to kill biofilms are not therapeutically achievable http://www.who.int/buruli/photos/Osteomyelitis_Nigeria_large.jpg http://www.pathology.vcu.edu/education/cardio/images/2g-a.jpg Hypothesis The gross morphology of the biofilm and the specific gene expression profile of biofilm bacteria are involved in increased resistance to antibiotics. Experimental Questions: – Do antibiotics induce changes in gene expression that increase antibiotic resistance? – What is the MBC99 for high cell density planktonic cultures vs biofilms? – Does biofilm architecture contribute to antibiotic resistance? – Does exopolysaccharide contribute to antibiotic resistance? What is Nafcillin? Beta-lactam antibiotic in the penicillin group of drugs4 Nafcillin Cell wall breaks down Bactericidal = kills bacterial cells Prevents bacterial cell wall synthesis4 Changes in Gene Expression and Antibiotic Resistance Does exposure to nafcillin induce changes in gene expression that increase antibiotic resistance? Microarray Analysis Isolate mRNA and purify http://www.mun.ca/biology/scarr/cDNA_microarray_Principle.jpg http://www.intervet.co.nz/binaries/90_79841.jpg Microarray Results Gene SAS050 SA2331 SA1266 SA0267 SAR0626 SACOL1579 SAS2352 SAV0901 SA1927 SACOL1788 Upregulated 7.88x 4.06x 4.03x 2.66x Downregulated 2.85x 2.92x 3.40x 4.85x 4.89x 9.90x Biofilm versus Planktonic Cultures’ Resistance to Nafcillin – What is the nafcillin MBC99 for high density planktonic cultures? MBC Assay MBC = minimal bactericidal concentration Count colony forming units (CFUs) Results from MBC Assay Biofilm Architecture and Antibiotic Resistance – Does biofilm architecture (gross morphological structure) contribute to antibiotic resistance? Confocal Microscopy Method to visualize viability of cells in biofilm after treatment with nafcillin LIVE/DEAD Viability /Cytotoxicity Kit: two-color assay5 – Red indicates dead cells – Green indicates viable, alive cells http://www.olympusconfocal.com/theory/images/theoryheader.jpg Confocal Images Control Nafcillin-treated Confocal Images Control Nafcillin-treated MBC Assays Exopolysaccharide Matrix and Antibiotic Resistance – Does exopolysaccharide contribute to antibiotic resistance? Exopolysaccharide Matrix and Antibiotic Resistance Biofilm matrix can selectively hinder antibiotic penetration through the biofilm (depends on size and charge)3 PNAG = β-1-6-linked Nacetylglucosamine = polysaccharide encapsulating biofilm3 PNAG is produced by icaADBC gene products Does PNAG contribute to antibiotic resistance? http://www3.niaid.nih.gov/NR/rdonlyres/263D4EDB-3C96-4AC6-8C58-B7AF8F6CF2C5/0/ staphylococcus_epidermidis.jpg PNAG-negative S. aureus Mutants Approach: Compare resistance of a wildtype strain to a PNAG-negative strain (SA113 vs SA113Dica). If PNAG contributes to biofilm formation, then how do we get biofilms from SA113Dica? Results Optimal Media for Biofilm Formation – Tryptic Soy Broth (TSB) + 3.5% NaCl MBC Assay – No difference in viability of SA113 and SA113Δica in the presence of 100g/mL nafcillin Conclusions Increased antibiotic resistance of S. aureus biofilms: – Gene expression is altered – Biofilms are more resistant to nafcillin than planktonic cultures – Biofilm architecture does not contribute substantially – PNAG does not contribute substantially What are persister cells? Nondividing, dormant cells Will neither grow nor die in the presence of antibiotic stress Experimental Questions Which genes in the S. aureus genome contribute to the persister phenotype? How many persister cells are present in biofilm and planktonic (free-floating bacteria) populations after antibiotic treatment? The genotypic basis of persister cells Which genes in the S. aureus genome contribute to the persister phenotype? Genomic Expression Library •Digested fragments separated via gel electrophoresis •Transformed into E. coli cells •Transformants selected on ampicillin-infused media Gel Electrophoresis Restriction Enzymes to Cut Genomic DNA S. aureus strain 10833 genomic DNA – DNase I + DNA polymerase I – XbaI/SpeI XbaI: T C T A G A becomes T + CTAGA AGATCT AGATC T SpeI: A C T A G T becomes A + CTAGT TGATCA TGATC A – Sau3AI AGATCG becomes A + TCTAGC TCTAG GATCG C Restriction Enzymes to Cut Plasmid Plasmid pCl15 – SmaI – BamHI Results: Digestion of Genomic DNA XbaI/SpeI: Minipreps Digestion and gel electrophoresis Visualize fragments Quantification of Persister Cells How many persister cells are present in biofilm and planktonic (free-floating bacteria) populations after antibiotic treatment? Ciprofloxacin Quinolone antibiotic Prevents supercoiling of DNA by inhibiting DNA gyrase Bactericidal = kills bacteria DNA gyrase References 1. Götz, F., T. Bannerman, and K.-H. Schleifer. 2006. The Genera Staphylococcus and Macrococcus. In: The Prokaryotes: A Handbook on the Biology of Bacteria: Firmicutes: Firmicutes with Low GC Content of DNA (Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, H.-K., Stackebrandt, E., eds.). Springer New York, NY, pp. 1159 2. Monroe, D. 2007. Looking for Chinks in the Armor of Bacterial Biofilms. PLoS Biology. 5(11): 2458. 3. Jefferson K. K., D. A. Goldmann, and G. B. Pier. (2005). Use of confocal microscopy to analyze the rate of vancomycin-binding in Staphylococcus aureus biofilms. Antimicrob. Agents Chemother. 49(6): 2467-2473. 4. Cerner Multum, Inc. Nafcillin. November 1, 2006. July 30, 2008 http://www.drugs.com/mtm/nafcillin.html 5. Invitrogen. LIVE/DEAD® BacLight™ Bacterial Viability Kit. 2008. July 30, 2008 <http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetai l&entryPoint=adirect&productID=L7007&messageType=catProductDetail>