Transcript Effects of Sub-Inhibitory Antibiotic Concentrations on Genes

Effects of Sub-Inhibitory Antibiotic
Concentrations on Genes Associated
with Biofilm Formation in
Mycobacterium avium
Molly Dyan McNab
HHMI Summer 2004
Mentor: Dr. Luiz Bermudez
OSU College of Veterinary Medicine
Department of Biomedical Science
What is Biofilm?
 A multi-cellular aggregate of bacteria that forms on any surface
exposed to the bacteria capable and some amount of water.
 Forms to protect the bacteria from host defenses, harsh
environmental conditions, and antibiotic agents.
 Found almost everywhere in nature, including in rivers, lakes,
soil, water pipes, and even inside the human body.
 Bacterial biofilms are often a cause of infections associated
with medical devices such as catheters and IV lines.
A type of bacterial biofilm,
responsible for plaque.
How is biofilm formed?
Surface
Planktonic bacteria, in
the presence of water,
attach to a surface.
Surface
Bacteria continue to
grow, outer cells provide
a physical barrier to
protect inner cells
Biofilm and antibiotic resistance
Little or no effect
because the bacteria in
biofilm are in a different
phase than most
antibiotics target.
Surface
Mycobacterium avium
 A biofilm-forming
opportunistic human
pathogen found in the
environment
 Several strains have
been isolated from
AIDS patients and
others with
compromised immune
systems
www.med.sc.edu:85/ fox/mycobacteria.htm
Past work
Past work on genes associated with
biofilm formation in M. avium has yielded
approximately 12 genes that are upregulated in biofilm formation.
Several of these genes are important for
glycopeptidolipid (GPL) biosynthesis, while
others play a key role in fatty acid
metabolism or the citric acid cycle.
Current work
Using primers for previously identified
genes associated with biofilm formation,
quantify gene expression M. avium strains
MAC A5, MAC 101, and MAC 104 in the
presence and absence of three different
antibiotics at sub-inhibitory concentrations
using Real-Time PCR.
Antibiotics used
 Macrolide antibiotics- Bacteriostatic antibiotics, Do not
kill the bacteria, instead they prevent the production of proteins by
binding to specific sites on the bacteria’s ribosomes
Claritromycin, fairly low MIC, 2 µg/ml, shown in
previous studies to prevent biofilm formation
when used early in treatment.
Azithromycin, higher MIC, 16 µg/ml
 Fluoroquinolones-
synthetic broad-spectrum bactericidal
drugs
Moxifloxycin, MIC is 2 µg/ml
Hypothesis
Claritromycin at a concentration of 50% its
minimum inhibitory concentration will
selectively limit the expression of genes
typically up-regulated in biofilm formation
Sub-inhibitory concentrations of
Azithromycin and Moxifloxycin will have
little or no effect on those genes.
Genes / Gene Products
Biofilm genes
 Glycosyl Transferase, essential for the
expression of mature GPLs.
 GuaB2 (IMP dehydrogenase), catalyzes the
first reaction in GMP biosynthesis
 PmmB (Phosphomannose mutase), converts
D-Mannose 1-Phosphate TO D-Mannose 6Phosphate
Control gene
 16S RNA, not involved in biofilm formation, to be
used as a control
Mechanism of M. avium infection
M. Avium bacterium is
inhaled and ends up in
the lung
As the bacteria colony
grows, biofilm forms along
the epithelial tissue of the
airway.
Sub-Inhibitory
Antibiotic Treatment
When enough bacteria are
present, infection spreads
across the epithelium.
Procedure
1. Samples of each strain were plated on
Polyvinyl Chloride (PVC) with or without the
presence of 50% of the minimum inhibitory
concentration of each of the three antibiotics
and incubated at 37°C for one week.
Procedure, cont.
2. After one week, the RNA from each
sample was purified, cDNA was made,
and Real-Time PCR was performed with
primers from the previously identified
genes to quantify gene expression in each
sample.
Results
MAC A5
Control
Claritromycin
(1 µg/ml)
Azithromycin
(16 µg/ml)
Moxifloxicin
(0.25 µg/ml)
GuaB2
+++
-
+
++
GTF
+++
-
+
++
PmmB
+++
-
+
++
+++
+++
+++
16S RNA +++
Results
MAC 101
Control
Claritromycin
(1 µg/ml)
Azithromycin
(8 µg/ml)
Moxifloxicin
(1 µg/ml)
GuaB2
+++
-
-
-
GTF
+++
-
++
-
PmmB
+++
-
++
-
+++
+++
+++
16S RNA +++
Results
MAC 104
Control
Claritromycin
(1 µg/ml)
Azithromycin
(8 µg/ml)
Moxifloxicin
(1 µg/ml)
GuaB2
+++
-
No data
GTF
No data
No data
No data
No data
PmmB
No data
No data
No data
No data
+++
No data
No data
16S RNA +++
No data
Discussion / Conclusions
 Claritromycin at a concentration of 1 µg/ml
selectively disrupts expression in genes
associated with biofilm formation in MAC A5
 Azithromycin at a concentration of 16 µg/ml
has a small selective effect on the expression on
genes associated with biofilm formation in MAC
A5
 Moxifloxycin at a concentration of 0.25 µg/ml
has little effect on genes associated with biofilm
formation in MAC A5
Future work
Studying the effects of the treatment on
the remaining genes associated with
biofilm formation.
Extending this treatment to an animal
model.
Developing a protocol for using this
concept to prevent biofilm formation, and
thus infection, in immunocompromised
individuals.
Thank you!
Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Luiz Bermudez
Dr. Yoshitaka Yamazaki
Bermudez Lab Soccer Team