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Free living amoebae-bacteria interactions shift the drinking water biofilm community towards
opportunistic water-based pathogens
Mohamed Shaheen University of Alberta February 14, 2019
Exposure to Legionella and Aquatic Ecology
Pipe-Biofilms and Exposure to Legionella
3
(1-100 cfu)
(3.5 x 106-8 cfu/L)
Lau & Ashbolt (2009) J Appl Microbiol 107(3):368–378 Schoen & Ashbolt (2011). Water research, 45(18), 5826-5836.
Action limit for Legionella 103 cfu/L Heterotrophic plate count 105 cfu/L
Water-Flow System and Biofilms
4
• Natural biofilms under oligotrophic condition for 6 months
• Introduction of L. pneumophila 105/ml and Amoeba trophozoites (50:1) to pre-developed biofilms
• Closed-loop with no additional nutrient for 850 days
• Observed attachment/ colonization under stagnation and flow condition
• Temp: 22±1 °C
• Microscopy/ Culture and Molecular Techniques • Bacterial Strains:
• Legionella pneumophila (ATCC 33152) • Escherichia coli MG1655 • E. coli MC1061
• Free-living amoeba Strains • Willaertia magna (ATCC 50035) (closely related to Naegleria) • Acanthamoeba polyphaga (ATCC 30461) • Vermamoeba vermiformis (ATCC 50237)
Bacteria and Free-living-amoebae
W. magna-L. pneumophila in Tap Water
16
W. magna-L. pneumophila in Tap Water
Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.
16
Intracellular Growth Vesicle formation
Intracellular Growth and Vesicle Formation
Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.
16
Vesicle formation
Vesicles and Infectious Dose
Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.
16
0
300
600
900
1200
1500
20 30 40
No.
of V
esic
les
Incubation Temperature °C
Number of Vesicles Produced Under Different Incubation Temperature
Total vesicles
Respirable-size vesicles
Particle size (D in µm)
No. of Bacteria
5 1.09 x102
10 8.7 x102
20 7.0 x103
50 1.09 x105
3.5 x 106-8 cfu/L
Attachment and Colonization
10
• Introduction of L. pneumophila, A. polyphaga and W. magna trophozoites • Observed colonization of L. pneumophila to predeveloped water-biofilms
Amoebae-L. pneumophila interaction in Flow-Cell
On 20th d of inoculation
11
• No immediate feeding of L. pneumophila by the FLA trophozoites • Observed a time delay in feeding on Legionella by the FLA trophozoites
Amoebae-L. pneumophila interaction in Flow-Cell
12
Persistence of Legionella within Amoeba Trophozoites
13
• No nutrient feed • Closed-loop system • Temperature: 22±1 °C • Flow: 30 ml/h • Study period: 850 days
After 2 years After 1 years
Re-Isolation of Amoebae
14
Persistence of Legionella in Bulk Water
15
W. magna with Bacteria at 30 min
L. pneumophila
E. coli MG1655
E. coli MC1061
16
W. magna with Bacteria at 24h
E. coli MC1061 E. coli MG1655
L. pneumophila
17
W. magna with Bacteria at 96h
18
L. pneumophila E. coli MG1655
E. coli MC1061
Internalization of Bacteria
E. coli MC1061 at 15 min E. coli MG1655 at 24h
19
W. magna with Bacteria
20 Total E. coli MC1061 E. coli MG1655 L. pneumophila
30 min 24 h
48 h 96 h
No options Vs Options
W. magna L. pneumophila E. coli MG1655 E. coli MC1061
21
Encystation
Hypothetical Model
Thanks
Ashbolt Lab
Thanks
Ramón Areces Foundation
Edmonton
University of Alberta
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