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 membranes 1 - Opportunistic pathogen Gram stain; S. aureus

Biofilms Biofilms = community of bacteria covered in extracellular polymers and attached to a surface 2 Biofilms = community of bacteria covered in extracellular polymers and attached to a surface 2 Biofilms play an important role in chronic, recurrent infections and in medical device (eg. catheter) -related infections 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: Chronic Infections/Diseases: Biofilms are resistant to antibiotic levels 10-1,000 times higher than planktonic bacteria 3 Biofilms are resistant to antibiotic levels 10-1,000 times higher than planktonic bacteria 3 –Concentrations of antibiotics required to kill biofilms are not therapeutically achievable OsteomyelitisEndocarditis

Hypothesis The gross morphology of the biofilm and the specific gene expression profile of biofilm bacteria are involved in increased resistance to antibiotics. The gross morphology of the biofilm and the specific gene expression profile of biofilm bacteria are involved in increased resistance to antibiotics. Experimental Questions: Experimental Questions: –Do antibiotics induce changes in gene expression that increase antibiotic resistance? –What is the MBC 99 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 drugs 4 Beta-lactam antibiotic in the penicillin group of drugs 4 Bactericidal = kills bacterial cells Bactericidal = kills bacterial cells Prevents bacterial cell wall synthesis 4 Prevents bacterial cell wall synthesis 4 Nafcillin Cell wall breaks down

Changes in Gene Expression and Antibiotic Resistance Does exposure to nafcillin induce changes in gene expression that increase antibiotic resistance? Does exposure to nafcillin induce changes in gene expression that increase antibiotic resistance?

Microarray Analysis Isolate mRNA and purify

Microarray Results GeneUpregulatedDownregulated SAS x SA x SA x SA x SAR x SACOL x SAS x SAV x SA x SACOL x

Biofilm versus Planktonic Cultures’ Resistance to Nafcillin –What is the nafcillin MBC 99 for high density planktonic cultures?

MBC Assay MBC = minimal bactericidal concentration 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 Method to visualize viability of cells in biofilm after treatment with nafcillin LIVE/DEAD Viability LIVE/DEAD Viability /Cytotoxicity Kit: two-color assay 5 –Red indicates dead cells –Green indicates viable, alive cells

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 Biofilm matrix can selectively hinder antibiotic penetration through the biofilm (depends on size and charge) 3 PNAG = β -1-6-linked N- acetylglucosamine = polysaccharide encapsulating biofilm 3 PNAG = β -1-6-linked N- acetylglucosamine = polysaccharide encapsulating biofilm 3 PNAG is produced by icaADBC gene products PNAG is produced by icaADBC gene products Does PNAG contribute to antibiotic resistance? Does PNAG contribute to antibiotic resistance? staphylococcus_epidermidis.jpg

PNAG-negative S. aureus Mutants Approach: Compare resistance of a wildtype strain to a PNAG-negative strain (SA113 vs SA113  ica). Approach: Compare resistance of a wildtype strain to a PNAG-negative strain (SA113 vs SA113  ica). If PNAG contributes to biofilm formation, then how do we get biofilms from SA113  ica? If PNAG contributes to biofilm formation, then how do we get biofilms from SA113  ica?

Results Optimal Media for Biofilm Formation Optimal Media for Biofilm Formation –Tryptic Soy Broth (TSB) + 3.5% NaCl MBC Assay MBC Assay –No difference in viability of SA113 and SA113 Δ ica in the presence of 100  g/mL nafcillin

Conclusions Increased antibiotic resistance of S. aureus biofilms: 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 Nondividing, dormant cells Will neither grow nor die in the presence of antibiotic stress 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? 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? 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? 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 genomic DNA S. aureus strain genomic DNA –DNase I + DNA polymerase I –XbaI/SpeI  XbaI: T C T A G A becomes  T + C T A G A T C T A G A becomes  T + C T A G A A G A T C T A G A T C T A G A T C T A G A T C T  SpeI: A C T A G T becomes  A + C T A G T T G A T C A T G A T C A T G A T C A T G A T C A –Sau3AI  AGATCG becomes  A + GATCG TCTAGCTCTAG C TCTAGCTCTAG C

Restriction Enzymes to Cut Plasmid Plasmid pCl15 Plasmid pCl15 –SmaI –BamHI

Results: Digestion of Genomic DNA XbaI/SpeI: 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? How many persister cells are present in biofilm and planktonic (free-floating bacteria) populations after antibiotic treatment?

Ciprofloxacin Quinolone antibiotic Quinolone antibiotic Prevents supercoiling Prevents supercoiling of DNA by inhibiting DNA gyrase Bactericidal = kills Bactericidal = killsbacteria DNA gyrase

References 1. Götz, F., T. Bannerman, and K.-H. Schleifer 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 Monroe, D Looking for Chinks in the Armor of Bacterial Biofilms. PLoS Biology. 5(11): 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): Cerner Multum, Inc. Nafcillin. November 1, July 30, Invitrogen. LIVE/DEAD® BacLight™ Bacterial Viability Kit July 30, Invitrogen. LIVE/DEAD® BacLight™ Bacterial Viability Kit July 30, 2008