1. Bacteriophage cocktails for treatment Burkholderia cenocepacia infections in a Lemna minor model
Madison Kavanaugh
Research Advisor: Dr. Christine Weingart, Ph.D.
Denison University, Granville, Ohio 43023
Burkholderia cenocepacia is an opportunistic pathogen, a pathogen that can cause
acute and chronic infections in hosts with weakened immune systems, such as patients
with cystic fibrosis (5). B. cenocepacia is specifically associated with the development of
“cepacia syndrome” in CF patients, a disease characterized by heightened
transmissibility, a high risk of developing acute respiratory disease and sepsis, and low
survivorship (2). B. cenocepacia is also highly resistant to antibiotics. Consequently,
there is a critical need for alternative strategies to treat B. cenocepacia infections and
block transmission. This study compared the ability of DCMK bacteriophages and
bacteriophage cocktails to reduce B. cenocepacia K56-2 infection in a Lemna minor
infection. All bacteriophages and bacteriophage cocktails were successful in reducing B.
cenocepacia infection. DCMK-1,2,3,4 and DCMK-2,3,4 cocktails rescued duckweed
plants with the highest survivorship. Further investigation will include testing DCMK
bacteriophages on B. cenocepacia-infected human tissue.
Burkholderia cenocepacia is developing increasing antibiotic resistance, which
presents a challenge to the treatment of cepacia syndrome in CF patients.
Bacteriophages, also known as phages, have long been envisioned as a potential
therapy for bacterial infections (2). Bacteriophages are viruses that solely infect and
replicate within bacteria. Phages have the ability to lyse bacterial cells with high
specificity and therefore allow treatment of infections without disrupting the natural host
microflora (5).
Phage cocktails are a relatively novel idea that developed after the mixed success of
monophage treatments of bacterial infections (3). Weiling et. al (2009) found a decrease
in P. aeruginosa biofilms by 99.9% with the use of phage cocktails. Kelly et al. found the
level of bacterial phage resistance to Staphylococcus phage K was 16.1%, while the
level of phage resistance to a cocktail was 2.8%. This recent phage cocktail success
has led our team to explore the effectiveness of phage cocktails with our four
bacteriophages: DCMK-1, DCMK-2, DCMK-3, and DCMK-4.
The monophage assay confirmed that our phages are capable of rescuing duckweed
plants from B. cenocepacia with a high degree of success. DCMK-4 was the most
successful phage with 87.0±6.2% post-infection duckweed survival. DCMK-2 was the
least successful with 71.7±6.5% survival. The phages all caused significantly higher
survivorship than K56-2 controls; however, they all performed with statistically similarity
(FIG 1).
In the phage cocktail rescue assay, DCMK-1,2,3,4 and DCMK-2,3,4 had an average
of 100.0±0.0% survivorship. This indicates that addition of DCMK-1 might not be
important. Additionally, DCMK-2,3 had higher survivorship than DCMK-1,2,3. This
again indicates DCMK-1 might not play a key role in cocktails. The other cocktails
ranged from 95.8±7.2% to 79.2±7.2%. The phage cocktails seem to have performed
better than the individual phages; however, most results were not significant. In fact,
two cocktails (DCMK-3,4 and DCMK-2,4) and DCMK-2 were the least effective at
reducing infection. DCMK-4 produced higher survivorship than DCMK-2,4 and DCMK-
3,4. This suggests cocktails are not a mere addition of anti-bacterial power--phage
behavior changes depending on interaction with other phages.
Overall, all phages and cocktails were successful in reducing B. cenocepacia
infection. This is a promising result for future studies in the Weingart lab and for other
researchers focused on bacteriophages as a possible therapy for infection.
Abstract Discussion
Introduction
Monophage Rescue Assay In order to test the phages’ efficacy in-vivo, 96-well plates
were filled with SHS and one sterile duckweed plant. Twenty microliters of a 100xLD50
dose (10-2
) of B. cenocepacia was added to each well and phage was added. Plates
were stored in a sterile bag and incubated for 96 hours at 30C prior to counting survivors.
Three trials were performed for each phage. Plants were deemed alive if 10% or more of
the plant remained green. Controls included K56-2 only, phage only, and SHS only
treated plants.
Cocktail Rescue Assay To determine if cocktails increased the survivorship of the
duckweed plants post-infection, 96-well plates were filled with SHS and one sterile
duckweed plant. Twenty microliters of a 100xLD50
dose (10-2
) of B. cenocepacia was
added to each well and the plants were placed were incubated for 4 hours to allow for
infection. Then various phage cocktails were added. Plates were stored in a sterile bag
and incubated for 96 hours at 30C prior to counting survivors. Three trials were
performed for each phage cocktail. Plants were deemed alive if 10% or more of the plant
remained green. Controls included K56-2 only, phage cocktail only, and SHS only treated
plants.
1. Abuladze, T. et al. “Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef
by Escherichia coli O157:H7.” Applied and Environmental Microbiology. 74, 20 (2008): 6230-6238.
2. Carmody, L., Gill, J., Summer, E., Sajjan, U., Gonzalez, C., Young, R., & Lipuma, J. (2010). Efficacy of Bacteriophage Therapy in a Mode
of Pulmonary Infection. The Journal of Infectious Diseases, 264-271. Retrieved January 17, 2015.
3. Chan, Benjamin., Abedon, Stephen., and Catherine Loc-Carrilo. (2013) Phage cocktails and the future of phage therapy, Future
Microbiology. Retrieved January 26, 2015.
4. Drevinek, P. &E. Mahenthiralingam, Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence.
Clinical Microbiology and Infection, 16:7, 821-830. Retrieved January 20, 2015.
5. Ganesan, S., & Sajjan, U. (2012). Host Evasion by Burkholderia cenocepacia. Frontiers in Cellular and Infection Microbiology, 1, 1-9.
Retrieved January 20, 2015
6. Kelly, David., McAuliffe, Olivia., Ross, R.P., O’Mahony, Jim., and Coffey, Aidan. Development of a broad-host-range phage cocktail for
biocontrol, Bioengineered Bugs 2:1, 31-37. Retrieved 28 January 2015.
7. Tomat, D. et al. “Hard surfaces decontamination of enteropathogenic and Shiga toxin-producing Escherichia coli using bacteriophages.”
Food Research International. 57 (2014): 123-129..
8. Semler, Diana, Goudie, A., Finlay, W., Dennis, J. Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory
Infections, Antimicrobial Agents and Chemotherapy, 58(7): 4005-4013. Retrieved January 21, 2015.
9. Weiling, Fu., Forster, Terri., Mayer, Oren., Curtin, John., Lehman, Susan., and Donlan, Rodney. Bacteriophage cocktail for the prevention
of biofilm formation by Pseudomonas aeruginosa on catheters in an In vitro model system, Antimicrobial Agents and Chemotherapy 54 (1):
297-404. Retrieved January 2015.
Results
Methods
The Weingart lab plans to use the duckweed infection model to test various cocktails at
different stages of infection. This will allow insight into how effective cocktails are at
saving plants after long periods of infection. Additional plans also are to test the infection
model on human tissue cultures. The duckweed model showed that DCMK phages and
cocktails significantly reduce infection by B. cenocepacia. A successful human tissue
model will be more useful in showing the future medicinal use of these phages.
TABLE 1 Duckweed survival rates for
bacteriophages DCMK-1, DCMK-2, DCMK-3
and DCMK-4.
TABLE 2 Duckweed survival rates for
bacteriophage cocktails.
Future Directions
FIG 1 Duckweed survival rates for DCMK individual
phages and cocktails. Phage controls not included.
Data points that share the same letter are not
significantly different, and were denoted through a
Tukey HSD test. Error bars indicate a ±1 SE.
I would like to thank the Department of Biology, the Gilpatrick Center for Student Research, and the
Anderson Endowment Program for funding this beginning of this project. I would also like to thank Dr.
Weingart for all her support and guidance and Devon Chosky for her humorous company and
willingness to make our overnight cultures when I forgot.
• DCMK-1, DCMK-2, DCMK-3, and
DCMK-4 rescued the duckweed
with high significance (ANOVA,
p<0.0001); however, they
performed with statistical similarity.
• DCMK-4 had the highest average
survivorship, while DCMK-2
appeared to be the least effective.
• DCMK-2 performed significantly
worse than the SHS and phage
controls, while the other phages
did not.
• All cocktails were successful in
rescuing duckweed plants from
infection when compared non-
treated controls (ANOVA, p<0.
0001).
• DCMK-1,2,3,4 and DCMK-2,3,4
performed significantly better
than DCMK-2,4 and DCMK-3,4
(p=0.0177). All other pairs
performed similarly.
• DCMK-2,4 and DCMK-3,4
performed significantly worse
than the SHS and cocktail
controls (ANOVA, p=0.0177).
• All phages and cocktails
were successful in
reducing B. cenocepacia
infection.
• DCMK-1,2,3,4 and DCMK-
2,3,4 had higher percent
survivals than all other
cocktails or phages.
• DCMK-2,3 had a higher
survival than DCMK-2.
• DCMK-3,4, DCMK-2,4 and
DCMK-2 were the only
phages or cocktails
significantly different than
the SHS controls.
References
Acknowledgements