Alessandro Calo, Janne Thöming, Brian Conlon, Kim Lewis
Undergraduate/Graduate Category:Physical and Life Sciences Degree Level: Undergraduate Abstract ID# 807
Novel Method for Analysis of Antibiotic Activity Against Biofilm Alessandro Calo, Janne Thöming, Brian Conlon, Kim Lewis Antimicrobial Discovery Center, Department of Biology
A biofilm is any bacterial population that forms a sessile aggregate that adheres to a surface and to other cells by producing extracellular polymeric substance (EPS). Individual cells initially attach to a surface and once they have established an irreversible attachment, the cells can proliferate and excrete EPS. Cells will then disperse to continue colonization. Biofilm is formed by many different bacteria. It can include single species but is often comprised of a myriad of bacterial species. Biofilms are not very susceptible to antibiotics making biofilm infections hard to treat. They contribute to many types of in a number of infections such as osteomylitis, endocarditis, chronic infections, and infections of implanted devices.
Dynamic
Figure 1: A The traditional static model of biofilm formation. Cells are incubated in microtiter wells and incubated statically. Biofilm forms on well base. B The dynamic model. Cells produce biofilm on glass beads in constant motion.
Rich media (BHI)
Glass beads
A
B
8 7
6.00
6
Log(CFU)
5 SH1000 WT SH1000 ∆clpP
4
5.00 SH1000 Control SH1000 Rif 4.00
SHΔClpP Control SHΔClpP Rif
3 3.00
2 1 control
Figure 2: Survival of Stapphyloccus aureus strain SH1000 and clpP mutant biofilm treated with rifampicin. (A) Experiment using the traditional static method in a 96-well format. (B) Experiment using our dynamic method.
Rifampicin
0
8
1 Time (days)
2
3 2 1
Pros: • The constant motion of the beads produces an environment similar to the movement of a surface when inside of an animal • Multiple beads allow for measuring population from the same experiment over a series of time points • Movement assures all of the biofilm have equal exposure to oxygen and antibiotic • One time addition of antibiotic to media • Protocol is quick and simple Cons: • Not as high-throughput as 96-well plate model.
7.00
6
4
Cons: • Biofilm lays on bottom of well giving restricted access to oxygen • Antibiotic may not disperse • One population can contribute to one experiment making it necessary to use a separate population for each time point
8.00
7
5
Pros: • 96-well format allows for small samples and high throughput experimentation • Small amount of sample and antibiotic are needed
2.00
B
A
Static Model:
Dynamic Model:
7.00
6.00 Log(CFU)
Background:
B Shaker table
Log (cfu/well)
Biofilm is a population of bacteria attached to a surface and embedded in an exopolymerix matrix or 'slime'. In this state they are difficult to treat with antibiotics and are implicated in a number of infections. In order to find new antibiotic therapies to kill biofilm, we need a suitable assay for examination of novel compound against biofilm associated cells. Currently, the most common protocol involves growing bacteria in a biofilm attached to the base of a 96well plate and adding fresh media with antibiotic. Although useful, this method has important limitations. Firstly, the assay is static and therefore cells may get an uneven distribution of antibiotic. Secondly, many of the antibiotics being tested are hydrophobic, causing insolubility or they binding to plastic surfaces. An alternative to this static environment is the use of a flow cell, whereby media containing antibiotic can be continuously flowed over biofilm adhered to a capillary tube. Bacteria are difficult to remove from the capillary and quantification is not easily achievable. Also, experimental antibiotics often have limited quantities. As a drug is flowing continuously, large quantities are required. Here I investigate the use of a novel method for the analysis of antibiotic activity against biofilm. I grow biofilm on glass beads suspended in rich media on a rotating table. This allows for a flowing system similar to a natural environment. Here I compare results using the traditional 96-well plate method and the new glass bead protocol.
Static
Log (c.f.u. well-1)
Abstract:
A
Control
5.00
ADEP Rif
4.00
ADEP+Rif
3.00 2.00 0
1
2 Time (days)
3
Figure 3: Survival of Stapphyloccus aureus strain UAMS-1 biofilm treated with ADEP and rifampicin. (A) Experiment using the traditional static method in a 96-well format. (B) Experiment using our dynamic method.
Future Experiments: • Examine the feasibility of this method to measure differences in biofilm formation in various strains. • Observe the nature of the biofilm formed on the bead using microscopy. • Determine if both proteinaceous and polysaccharide biofilm can form on the glass beads. • Examine different antibiotics against biofilm in this model to determine the best antibiotics for biofilm infection treatment. • Use a bioluminescent strain to produce the biofilm and determine if luminescence can be used to measure viablility over time.
Novel Method for Analysis of Antibiotic Activity Against Biofilm Alessandro Calo, Janne Thöming, Brian Conlon, Kim Lewis Antimicrobial Discovery Center, Department of Biology
A biofilm is any bacterial population that forms a sessile aggregate that adheres to a surface and to other cells by producing extracellular polymeric substance (EPS). Individual cells initially attach to a surface and once they have established an irreversible attachment, the cells can proliferate and excrete EPS. Cells will then disperse to continue colonization. Biofilm is formed by many different bacteria. It can include single species but is often comprised of a myriad of bacterial species. Biofilms are not very susceptible to antibiotics making biofilm infections hard to treat. They contribute to many types of in a number of infections such as osteomylitis, endocarditis, chronic infections, and infections of implanted devices.
Dynamic
Figure 1: A The traditional static model of biofilm formation. Cells are incubated in microtiter wells and incubated statically. Biofilm forms on well base. B The dynamic model. Cells produce biofilm on glass beads in constant motion.
Rich media (BHI)
Glass beads
A
B
8 7
6.00
6
Log(CFU)
5 SH1000 WT SH1000 ∆clpP
4
5.00 SH1000 Control SH1000 Rif 4.00
SHΔClpP Control SHΔClpP Rif
3 3.00
2 1 control
Figure 2: Survival of Stapphyloccus aureus strain SH1000 and clpP mutant biofilm treated with rifampicin. (A) Experiment using the traditional static method in a 96-well format. (B) Experiment using our dynamic method.
Rifampicin
0
8
1 Time (days)
2
3 2 1
Pros: • The constant motion of the beads produces an environment similar to the movement of a surface when inside of an animal • Multiple beads allow for measuring population from the same experiment over a series of time points • Movement assures all of the biofilm have equal exposure to oxygen and antibiotic • One time addition of antibiotic to media • Protocol is quick and simple Cons: • Not as high-throughput as 96-well plate model.
7.00
6
4
Cons: • Biofilm lays on bottom of well giving restricted access to oxygen • Antibiotic may not disperse • One population can contribute to one experiment making it necessary to use a separate population for each time point
8.00
7
5
Pros: • 96-well format allows for small samples and high throughput experimentation • Small amount of sample and antibiotic are needed
2.00
B
A
Static Model:
Dynamic Model:
7.00
6.00 Log(CFU)
Background:
B Shaker table
Log (cfu/well)
Biofilm is a population of bacteria attached to a surface and embedded in an exopolymerix matrix or 'slime'. In this state they are difficult to treat with antibiotics and are implicated in a number of infections. In order to find new antibiotic therapies to kill biofilm, we need a suitable assay for examination of novel compound against biofilm associated cells. Currently, the most common protocol involves growing bacteria in a biofilm attached to the base of a 96well plate and adding fresh media with antibiotic. Although useful, this method has important limitations. Firstly, the assay is static and therefore cells may get an uneven distribution of antibiotic. Secondly, many of the antibiotics being tested are hydrophobic, causing insolubility or they binding to plastic surfaces. An alternative to this static environment is the use of a flow cell, whereby media containing antibiotic can be continuously flowed over biofilm adhered to a capillary tube. Bacteria are difficult to remove from the capillary and quantification is not easily achievable. Also, experimental antibiotics often have limited quantities. As a drug is flowing continuously, large quantities are required. Here I investigate the use of a novel method for the analysis of antibiotic activity against biofilm. I grow biofilm on glass beads suspended in rich media on a rotating table. This allows for a flowing system similar to a natural environment. Here I compare results using the traditional 96-well plate method and the new glass bead protocol.
Static
Log (c.f.u. well-1)
Abstract:
A
Control
5.00
ADEP Rif
4.00
ADEP+Rif
3.00 2.00 0
1
2 Time (days)
3
Figure 3: Survival of Stapphyloccus aureus strain UAMS-1 biofilm treated with ADEP and rifampicin. (A) Experiment using the traditional static method in a 96-well format. (B) Experiment using our dynamic method.
Future Experiments: • Examine the feasibility of this method to measure differences in biofilm formation in various strains. • Observe the nature of the biofilm formed on the bead using microscopy. • Determine if both proteinaceous and polysaccharide biofilm can form on the glass beads. • Examine different antibiotics against biofilm in this model to determine the best antibiotics for biofilm infection treatment. • Use a bioluminescent strain to produce the biofilm and determine if luminescence can be used to measure viablility over time.
