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

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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

• 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

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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

Intracellular Growth and Vesicle Formation

Shaheen & Ashbolt (2018) Exposure and Health. 10(3), 201-209.

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Vesicle formation

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

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

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

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

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

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

No options Vs Options

W. magna L. pneumophila E. coli MG1655 E. coli MC1061

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Encystation

Hypothetical Model

Thanks

Ashbolt Lab

Thanks

Ramón Areces Foundation

Edmonton

University of Alberta