Antibiotic Resistance of Antibiotic Resistance of Staphylococcus aureus Staphylococcus aureus

BiofilmsBiofilms

Smitha NeerukondaSmitha NeerukondaVCU BBSI Summer 2009VCU BBSI Summer 2009

Mentor: Dr. Kimberly JeffersonMentor: Dr. Kimberly JeffersonAugust 3, 2009August 3, 2009

Staphylococcus aureusStaphylococcus aureus

- Gram-positive bacteria normally colonizing Gram-positive bacteria normally colonizing the nasal passages, skin and mucous the nasal passages, skin and mucous membranesmembranes11

- Opportunistic pathogen Opportunistic pathogen

Gram stain; S. aureus

http://www.labnews.co.uk/laboratory_article.php/2912/5/combating-bacterial-infection

BiofilmsBiofilms Biofilms = community of bacteria covered in Biofilms = community of bacteria covered in

extracellular polymers and attached to a surfaceextracellular polymers and attached to a surface22

Biofilms play an important role in chronic, recurrent Biofilms play an important role in chronic, recurrent infections and in medical device (eg. catheter) -infections and in medical device (eg. catheter) -related infectionsrelated infections

http://www.labnews.co.uk/laboratory_article.php/2912/5/combating-bacterial-infection

Dangers associated with Dangers associated with S. S. aureus aureus BiofilmsBiofilms

Chronic Infections/Diseases:Chronic Infections/Diseases:

Biofilms are resistant to antibiotic levels 10-1,000 times Biofilms are resistant to antibiotic levels 10-1,000 times higher than planktonic bacteriahigher than planktonic bacteria33

– Concentrations of antibiotics required to kill biofilms are Concentrations of antibiotics required to kill biofilms are not therapeutically achievablenot therapeutically achievable

Osteomyelitis Endocarditis

http://www.who.int/buruli/photos/Osteomyelitis_Nigeria_large.jpghttp://www.who.int/buruli/photos/Osteomyelitis_Nigeria_large.jpg

http://www.pathology.vcu.edu/education/cardio/images/2g-a.jpg

HypothesisHypothesis

The gross morphology of the biofilm and The gross morphology of the biofilm and the specific gene expression profile of the specific gene expression profile of biofilm bacteria are involved in increased biofilm bacteria are involved in increased resistance to antibiotics.resistance to antibiotics.

Experimental Questions:Experimental Questions:– Do antibiotics induce changes in gene expression that Do antibiotics induce changes in gene expression that

increase antibiotic resistance? increase antibiotic resistance? – What is the MBCWhat is the MBC9999 for high cell density planktonic for high cell density planktonic

cultures vs biofilms?cultures vs biofilms?– Does biofilm architecture contribute to antibiotic Does biofilm architecture contribute to antibiotic

resistance?resistance?– Does exopolysaccharide contribute to antibiotic Does exopolysaccharide contribute to antibiotic

resistance?resistance?

What is Nafcillin?What is Nafcillin? Beta-lactam antibiotic in the penicillin Beta-lactam antibiotic in the penicillin

group of drugsgroup of drugs44

Bactericidal = kills bacterial cells Bactericidal = kills bacterial cells

Prevents bacterial cell wall synthesisPrevents bacterial cell wall synthesis44

Nafcillin

Cell wall breaks down

Changes in Gene Expression Changes in Gene Expression and Antibiotic Resistance and Antibiotic Resistance

Does exposure to nafcillin induce Does exposure to nafcillin induce changes in gene expression that changes in gene expression that increase antibiotic resistance? increase antibiotic resistance?

Microarray AnalysisMicroarray AnalysisIsolate

mRNA and purify

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http://www.mun.ca/biology/scarr/cDNA_microarray_Principle.jpg

Microarray ResultsMicroarray ResultsGeneGene UpregulatedUpregulated DownregulatedDownregulated

SAS050SAS050 7.88x7.88x

SA2331SA2331 4.06x4.06x

SA1266SA1266 4.03x4.03x

SA0267SA0267 2.66x2.66x

SAR0626SAR0626 2.85x2.85x

SACOL1579SACOL1579 2.92x2.92x

SAS2352SAS2352 3.40x3.40x

SAV0901SAV0901 4.85x4.85x

SA1927SA1927 4.89x4.89x

SACOL1788SACOL1788 9.90x9.90x

Biofilm versus Planktonic Biofilm versus Planktonic Cultures’ Resistance to Cultures’ Resistance to

Nafcillin Nafcillin

– What is the nafcillin MBCWhat is the nafcillin MBC9999 for for high density planktonic high density planktonic cultures?cultures?

MBC AssayMBC Assay

MBC = minimal bactericidal MBC = minimal bactericidal concentrationconcentration

Count colony forming units (CFUs)

Results from MBC AssayResults from MBC Assay

Biofilm Architecture and Biofilm Architecture and Antibiotic Resistance Antibiotic Resistance

– Does biofilm architecture (gross Does biofilm architecture (gross morphological structure) morphological structure) contribute to antibiotic contribute to antibiotic resistance?resistance?

Confocal MicroscopyConfocal Microscopy Method to visualize Method to visualize viability of cells in viability of cells in biofilm after treatment biofilm after treatment with nafcillinwith nafcillin

LIVE/DEAD ViabilityLIVE/DEAD Viability/Cytotoxicity Kit: two-color /Cytotoxicity Kit: two-color assayassay55

– RedRed indicates dead cells indicates dead cells– GreenGreen indicates viable, indicates viable, alive cellsalive cells

http://www.olympusconfocal.com/theory/images/theoryheader.jpg

Confocal Images

Control Nafcillin-treatedNafcillin-treated

Confocal Images

Control Nafcillin-treatedNafcillin-treated

MBC AssaysMBC Assays

Exopolysaccharide Matrix and Exopolysaccharide Matrix and Antibiotic Resistance Antibiotic Resistance

– Does exopolysaccharide Does exopolysaccharide contribute to antibiotic contribute to antibiotic resistance?resistance?

Exopolysaccharide Matrix and Exopolysaccharide Matrix and Antibiotic ResistanceAntibiotic Resistance

Biofilm matrix can Biofilm matrix can selectively hinder antibiotic selectively hinder antibiotic penetration through the penetration through the biofilm (depends on size biofilm (depends on size and charge)and charge)33

PNAG = PNAG = ββ-1-6-linked -1-6-linked NN--acetylglucosamine = acetylglucosamine = polysaccharide polysaccharide encapsulating biofilmencapsulating biofilm33

PNAG is produced by PNAG is produced by icaADBCicaADBC gene products gene products

Does PNAG contribute to Does PNAG contribute to antibiotic resistance? antibiotic resistance? http://www3.niaid.nih.gov/NR/rdonlyres/263D4EDB-3C96-4AC6-8C58-B7AF8F6CF2C5/0/staphylococcus_epidermidis.jpg

PNAG-negative PNAG-negative S. aureus S. aureus MutantsMutants

Approach: Compare resistance of a Approach: Compare resistance of a wildtype strain to a PNAG-negative wildtype strain to a PNAG-negative strain (SA113 vs SA113strain (SA113 vs SA113ica)ica)..

If PNAG contributes to biofilm If PNAG contributes to biofilm formation, then how do we get biofilms formation, then how do we get biofilms from SA113from SA113icaica??

ResultsResults

Optimal Media for Biofilm FormationOptimal Media for Biofilm Formation– Tryptic Soy Broth (TSB) + 3.5% NaClTryptic Soy Broth (TSB) + 3.5% NaCl

MBC AssayMBC Assay– No difference in viability of SA113 and No difference in viability of SA113 and

SA113SA113ΔΔicaica in the presence of 100 in the presence of 100g/mL g/mL nafcillinnafcillin

ConclusionsConclusions

Increased antibiotic resistance of Increased antibiotic resistance of S. S. aureus aureus biofilms:biofilms:– Gene expression is altered Gene expression is altered – Biofilms are more resistant to nafcillin Biofilms are more resistant to nafcillin

thanthanplanktonic culturesplanktonic cultures

– Biofilm architecture does not contribute Biofilm architecture does not contribute substantially substantially

– PNAG does not contribute substantiallyPNAG does not contribute substantially

What are persister cells?What are persister cells?

Nondividing, dormant cellsNondividing, dormant cells Will neither grow nor die in the Will neither grow nor die in the

presence of antibiotic stresspresence of antibiotic stress

Experimental QuestionsExperimental Questions

Which genes in the Which genes in the S. aureus S. aureus genome contribute to the persister genome contribute to the persister phenotype? phenotype?

How many persister cells are present How many persister cells are present in biofilm and planktonic (free-in biofilm and planktonic (free-floating bacteria) populations after floating bacteria) populations after antibiotic treatment?antibiotic treatment?

The genotypic basis of The genotypic basis of persister cellspersister cells

Which genes in the Which genes in the S. aureus S. aureus genome contribute to the persister genome contribute to the persister phenotype?phenotype?

Genomic Expression LibraryGenomic Expression Library

•Digested fragments separated via gel electrophoresis

•Transformed into E. coli cells

•Transformants selected on ampicillin-infused media

Gel ElectrophoresisGel Electrophoresis

Restriction Enzymes to Cut Restriction Enzymes to Cut Genomic DNAGenomic DNA

S. aureus S. aureus strain 10833strain 10833 genomic DNAgenomic DNA– DNase I + DNA polymerase IDNase I + DNA polymerase I– XbaI/SpeI XbaI/SpeI

XbaI: XbaI: T C T A G A becomes T C T A G A becomes T + C T A G A 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 SpeI: A C T A G T becomes A + C T A G T 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

– Sau3AISau3AI AGATCG becomes AGATCG becomes A + GATCG A + GATCG TCTAGCTCTAGC TCTAG CTCTAG C

Restriction Enzymes to Cut Restriction Enzymes to Cut PlasmidPlasmid

Plasmid pCl15Plasmid pCl15– SmaISmaI– BamHI BamHI

Results: Digestion of Genomic Results: Digestion of Genomic DNADNA

XbaI/SpeI:XbaI/SpeI:

MiniprepsMinipreps

Digestion and gel electrophoresis

Visualize fragments

Quantification of Persister Quantification of Persister CellsCells

How many persister cells are present How many persister cells are present in biofilm and planktonic (free-in biofilm and planktonic (free-floating bacteria) populations after floating bacteria) populations after antibiotic treatment?antibiotic treatment?

CiprofloxacinCiprofloxacin

Quinolone antibiotic Quinolone antibiotic Prevents supercoiling Prevents supercoiling

of DNA by inhibiting of DNA by inhibiting

DNA gyraseDNA gyrase Bactericidal = kills Bactericidal = kills

bacteriabacteria

DNA gyrase

ReferencesReferences1. Götz, F., T. Bannerman, and K.-H. Schleifer. 2006. The Genera 1. Götz, F., T. Bannerman, and K.-H. Schleifer. 2006. The Genera

Staphylococcus Staphylococcus and and Macrococcus. Macrococcus. In: In: The Prokaryotes: A Handbook on the The Prokaryotes: A Handbook on the Biology of Bacteria: Firmicutes: Firmicutes with Low GC Content of DNA Biology of Bacteria: Firmicutes: Firmicutes with Low GC Content of DNA (Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, H.-K., Stackebrandt, E., (Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, H.-K., Stackebrandt, E., eds.). eds.). Springer New York, NY, pp. 11-59Springer New York, NY, pp. 11-59

2. 2. Monroe, D. 2007. Looking for Chinks in the Armor of Bacterial Biofilms. Monroe, D. 2007. Looking for Chinks in the Armor of Bacterial Biofilms. PLoS PLoS BiologyBiology. 5(11): 2458.. 5(11): 2458.

3. Jefferson K. K., D. A. Goldmann, and G. B. Pier. (2005). Use of confocal 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 microscopy to analyze the rate of vancomycin-binding in Staphylococcus aureus biofilms. Antimicrob. Agents Chemother. 49(6): 2467-2473.aureus biofilms. Antimicrob. Agents Chemother. 49(6): 2467-2473.

4. Cerner Multum, Inc. Nafcillin. November 1, 2006. July 30, 2008 4. Cerner Multum, Inc. Nafcillin. November 1, 2006. July 30, 2008 http://www.drugs.com/mtm/nafcillin.htmlhttp://www.drugs.com/mtm/nafcillin.html

5. Invitrogen. LIVE/DEAD® 5. Invitrogen. LIVE/DEAD® BacBacLight™ Bacterial Viability Kit. 2008. July 30, Light™ Bacterial Viability Kit. 2008. July 30, 2008 <http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?2008 <http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=L7007&messageTycmd=catProductDetail&entryPoint=adirect&productID=L7007&messageType=catProductDetail>pe=catProductDetail>