1. Combined Treatment of
Pseudomonas aeruginosa Biofilms
with Bacteriophages & Chlorine
Yanyan Zhang, Zhiqiang Hu
Journal: Biotechnology & Bioengineering
Presented By-
Miss Mugdha Pramod Padhye
M. Sc. II Biotechnology
8th
February 2013
2. A Line of Approach...
Introduction
Study Design Overview
Materials & Methods
Results & Concluding Remarks
Advantages & Limitations
References
Discussion
4. What is Bacterial Biofilm?
A biofilm is an aggregate of microorganisms in which
cells adhere to each other on a surface. These adherent
cells are frequently embedded within a self-produced
matrix of extracellular polymeric substance(EPS).
5. Why to remove Biofilms?
Biofilms are a source of infection and contamination in
drinking water supply, medical devices, and food
processing environments.
6. Methods of Biofilm Removal
Traditional Methods-
1. Flushing
2. Chlorination
3. UV Disinfection
4. Use of antibiotics
New Methods-
1. Quorum Sensing Inhibition
2. Nitric Oxide Removal
3. Enzymatic Disruption
4. Phage Therapy
8. Objectives of Study
To study the effect of combined treatment of Phages &
Chlorine on-
1. Inhibition of P. aeruginosa Biofilm Formation
2. Removal of Pre-existing P. aeruginosa Biofilms
To compare combined effect of Phages + Cl treatment
with that of Cl & Phage treatments separately.
9. Why did they choose
P. aeruginosa as a test organism?
Exhibits multiple mechanisms of antimicrobial
resistance.
Highly active in biofilm formation as it secretes cis-2-
decenoic acid (Quorum sensing).
Thrives on most surfaces, uses a wide range of organic
material for food.
Thrives not only in normal atmospheres, but also in
hypoxic atmosphere.
10. Why did they use chlorine along with
Phages?
Besides being cheap and easily available, it kills
pathogens such as bacteria and viruses by breaking the
chemical bonds in their molecules.
12. Bacteriophage Isolation, Enrichment &
Visualization
Isolation of lytic phages from municipal waste
Filtration
Phage enrichment with exponentially growing cultures of
P. aeruginosa (ATCC 39018)
Enrichment by DAL method
Centrifugation
First round of enrichment
Collect the Filtrate
Standard double agar layer (DAL) method
Second round of enrichment
13. Use of Supernatant as inoculum
Supernatant phage concentration was determined by the
DAL method (typically at about 109
PFU/ml)
Filtration
Storage at 4o
C before use
Preparation of desired phage concentrations in PBS
by 1:10 serial dilutions
Detection of morphology of phages by negative staining
with uranyl acetate, TEM & Nucleic Acid analysis
14. Evaluation of effect of Phage concentration
on biofilm formation
Aliquots of overnight P. aeruginosa culture in 8 replicates
Addition of LB medium + aliquot of phages of known
concentration
Final phage concentration in each microwell varied from
0 to 4×107
PFU/ml
Harvesting & Evaluation by crystal violet assay
In polystyrene microplate wells
Incubation for 70 h
15. Evaluation of use of phages in removal of
existing P. aeruginosa biofilms
Aliquots of overnight P. aeruginosa culture
Addition of LB medium
Sustaining biofilm growth
Addition of phage stock aliquots to reach final phage
concentrations ranging from 60 to 6×107
PFU/ml
Harvesting & Evaluation by crystal violet assay
In polystyrene microplate wells
Incubation at 37o
C, 72 h without mixing
Incubation for 72 h, at room temp.
16. A Combined use of Phages + Chlorine to
control P. aeruginosa biofilm formation
Aliquots of overnight P. aeruginosa culture in 8 replicates
Addition of LB medium + aliquot of phages
Addition of NaClO
Harvesting & Evaluation by crystal violet assay
To remove pre-existing P. aeruginosa biofilms by phage
and chlorination treatment, after 72 h of treatment at
room temperature, the amount of remaining biofilm was
quantified by crystal violet staining.
In polystyrene microplate wells
Incubation for 50h, at room temp.
17. Phage + Chlorine treatment
in a continuous flow system
Overnight P. aeruginosa was introduced in
each channel of drip flow biofilm reactor
Biofilms were grown by continuously feeding with 10 g/L
LB (flow rate=0.423 ml/min) for about 5 days
A combined treatment with phages and chlorination
Live/Dead viability test
Visualization of biofilm structure & morphology by TEM
19. Microscopic Observations
TEM images depicted presence of 2 types of phages-
1. Rod shaped phages (Length: 50 nm)
2. Spherical phages (Mean spherical diameter: 40 nm)
Nucleic acid analyses followed by gel electrophoresis
& enzyme digestion indicated that all of them were
RNA phages.
20. Effect of Bacteriophage Concentration on-
Inhibition of P. aeruginosa
Biofilm Formation
Pre-existing Biofilm Removal
21. Phage Concentration Inhibition of
Biofilm Formation
Removal of Pre-
existing Biofilm
40 PFU/ml No Significant Effect --
400 to 4.0×107
PFU/ml 45±15% to 73±8% --
6000 to 6.0×107
PFU/ml -- 45±9% to 75±5%
Chlorine Concentration Inhibition of
Biofilm Formation
Removal of Pre-
existing Biofilm
21 mg/l 53±11% No Significant Effect
210 mg/l 86±3% No Significant Effect
Phage Treatment
Chlorine Treatment
22. A combined treatment with
phages and chlorine to-
Inhibition of P. aeruginosa
biofilm formation Removal of pre-existing biofilms
23. Concentration
of Chlorine
Concentration
of Phage
Effect on
Inhibition of
Biofilm
Formation
Effect on
Removal of Pre-
existing Biofilm
21 mg/l 3.0×107
PFU/ml 92±1% 60±19%
210 mg/l 3.0×107
PFU/ml 94±1% 88±6%
Phage + Chlorine Treatment
24. P. aeruginosa biofilm structure changes after treatments with
P. aeruginosa phages. Shiny surfaces indicate clean areas with no
biofilm attachment.
25. Continuous Flow System After Phage + Cl Treatment
Concentrations of- Removal of Pre-
existing Biofilm in
96 h
Phages 89±1%
Chlorine (Cl) 40±5%
Phage + Cl 97±1%
Continuous Drip System After Phage + Cl Treatment
Chlorine
Treatment
Time (h)
Phage
Concentration
Removal of Pre-
existing Biofilm
12 3.8×105
PFU/ml 96±1% (in 36 h)
26. Live/Dead Viability Test
A comparison of biofilm bacterial viability determined by the live
(green)/dead (red) assay after phage and chlorine treatment.
Control Chlorine
Phage Phage+Cl
29. Limitations
Low concentration of bacterial host
Low nutrient availability
Narrow host range of phage
Loss of phage infectivity
Lack of Safety of phage preparations in humans.
Presence of residual phages in water after treatment
Additional disinfection steps needed to inactivate the
residual phages
Bacterial cells in the lysogenic state may become
resistant to infection.
30. Advantages
More practical in use.
Mixture of phages can delay the appearance of phage
resistant bacteria.
Number of phages produced is directly related to the
available host bacteria.
Additional phage inactivation steps-
1. Addition of Chlorine dioxide at a concentration of
0.02mg/l inactivates phage MS2.
2. Use of lower phage concentrations & shorter Cl
treatment time.
32. A combination of phages (3×107
PFU/ml) and chlorine at
210 mg/l concentration reduced biofilm growth by
94±2% and removed 88±6% of existing biofilms.
In a continuous flow system with continued biofilm
growth, a combination of phages with chlorine
removed 97±1% of biofilms after Day 5.
Laser scanning confocal microscopy supplemented with
electron microscopy indicated that the combination
treatment resulted in biofilms with lowest cell density
and viability.
Thus, results suggest that the combination treatment of
phages and chlorine is a promising method to control and
remove bacterial biofilms from various surfaces.
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removal of infectious biofilms. Curr Pharm Biotechnol
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formation, control and novel strategies for eradication.
Science against microbial pathogens: communicating
current research and technological advances. 2011.
Krylov V*; Olga S; Krylov S and Pleteneva E. A Genetic
Approach to the Development of New Therapeutic
Phages to Fight Pseudomonas Aeruginosa in Wound
Infections. Viruses, 5, 15-53. 2013.
http://www.phage-therapy.org/
