Transcript Antibiotic Resistance of Staphylococcus aureus Biofilms

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 100g/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>