Free living amoebae -bacteria interactions shift the ... · Shaheen & Ashbolt (2018) Exposure and...
Transcript of Free living amoebae -bacteria interactions shift the ... · Shaheen & Ashbolt (2018) Exposure and...
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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
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Exposure to Legionella and Aquatic Ecology
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Pipe-Biofilms and Exposure to Legionella
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(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
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Water-Flow System and Biofilms
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• 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
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• 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
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W. magna-L. pneumophila in Tap Water
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W. magna-L. pneumophila in Tap Water
Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.
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Intracellular Growth Vesicle formation
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Intracellular Growth and Vesicle Formation
Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.
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Vesicle formation
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Vesicles and Infectious Dose
Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.
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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
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Attachment and Colonization
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• Introduction of L. pneumophila, A. polyphaga and W. magna trophozoites • Observed colonization of L. pneumophila to predeveloped water-biofilms
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Amoebae-L. pneumophila interaction in Flow-Cell
On 20th d of inoculation
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• No immediate feeding of L. pneumophila by the FLA trophozoites • Observed a time delay in feeding on Legionella by the FLA trophozoites
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Amoebae-L. pneumophila interaction in Flow-Cell
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Persistence of Legionella within Amoeba Trophozoites
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• No nutrient feed • Closed-loop system • Temperature: 22±1 °C • Flow: 30 ml/h • Study period: 850 days
After 2 years After 1 years
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Re-Isolation of Amoebae
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Persistence of Legionella in Bulk Water
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W. magna with Bacteria at 30 min
L. pneumophila
E. coli MG1655
E. coli MC1061
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W. magna with Bacteria at 24h
E. coli MC1061 E. coli MG1655
L. pneumophila
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W. magna with Bacteria at 96h
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L. pneumophila E. coli MG1655
E. coli MC1061
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Internalization of Bacteria
E. coli MC1061 at 15 min E. coli MG1655 at 24h
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W. magna with Bacteria
20 Total E. coli MC1061 E. coli MG1655 L. pneumophila
30 min 24 h
48 h 96 h
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No options Vs Options
W. magna L. pneumophila E. coli MG1655 E. coli MC1061
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Encystation
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Hypothetical Model
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Thanks
Ashbolt Lab
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Thanks
Ramón Areces Foundation
Edmonton
University of Alberta