Susan Springthorpe [sspring@uottawa.ca]

Free-living Protozoa [FLP] and opportunistic bacterial pathogens

in distributed water

FLP presence is a double edged sword

Grazing and digestion reduces biofilm thickness - actively growing and Gram -ve preference, including human pathogens

Bacterial strategies to reduce predation include:

clumping and chemical signals to reduce ingestion

strategies to resist digestion

Trapping during encystment of FLP

Can escape treatment barriers including disinfection if harboured by FLP – opportunistic pathogens

Some bacteria pathogens of FLP replicate and kill FLP – specific

best example of bacteria as FLP pathogen – legionellae

many bacteria can be protected by FLP

Replication? Energy demands. Co-evolution.

Ecto- and endosymbionts. Advantage for FLP.

Amoebal cysts highly resistant to high temperatures, biocide treatments, ...bacteria in

periphery

Amoebal trophozoite – bacteria in food vacuoles

Adverse conditions

Amoebal forms showing bacteria

ACTIVE STAGE RESTING STAGE

What exactly are FLP?

• Small unicellular motile organisms – ubiquitous in wet or damp places – cysts persistent when dried

• Hunt bacterial prey – most encyst (survival) • >15,000 species recognized – many more • 3 basic groups classified by locomotion

– Ciliates many cilia move synchronously, large, create water currents, mostly in soils, sediments

– Flagellates – one or more flagella – biggest open water predators

– Amoebae – creep along surfaces/detritus by extending and withdrawing pseudopodia (false feet) – engulf prey

• Also amoebo-flagellates (e.g. Naegleria fowleri)

Which FLP are found in distributed water? • All groups expected at filter stage of treatment • Active bacterial growth on filter suggests all groups might

colonize filter matrices • Large active protozoa are fragile - destroyed by backwashing • Only encysted protozoa likely to pass filter

– Didn’t excyst on filter – passes filter as cyst – Replicate and re-encyst fast - < length of filter cycle

• Found & isolated in all DS samples regardless of treatment • Distance from treatment plant unimportant

– Amoebae (acanthamoebae, hartmannellae and others), flagellates (cercomonads – most numerous), amoebo-flagellates, ciliates (rarely seen)

• Isolates are small fraction of those present – inhibition observed

• Isolated by culture on bait bacteria, amplified, sub cultured and identified by molecular tools

Warm water, unfiltered, chloramine

Warm water, unfiltered, chloramine

Warm water, filtered , chlorine

Temperate, unfiltered, chlorine

Temperate filtered chloramine

Paracercomonas ambulans strain W80

Acanthamoeba lenticulata

Several Mayor-ella-like isolates

Cercozoan Penicilliun oxalicum

Cercomonas Sphaerodes retispora

Several unidentified trophozoites

Cercozoan Penicillium griseofulvum

Cryptococcus laurentii, Eupenicillium crustaceum

Acanthamoeba hatchetti or A. polyphaga

Almost no cysts after >6 weeks incubation

Protacanth-amoeba bohemica

Vexillifera (trophozoite)

Echinamoeba Platyamoeba (trophozoite), Gymnamoeba cysts

Lobosea sp., Chloroscypha cf. enterochroma

Cercomonadida, Aspergillus sp.

Hartmanella vermiformis clone 9341

Nematode Foraminifera (amoeba with test)

Cercomonas, Echinamoeba sp., Nematode

Warm water, unfil-tered, chloramine

Warm water, unfil-tered, chloramine

Warm water, filtered , chlorine

Temperate, unfiltered, chlorine

Temperate filtered chloramine

Acanthamoeba lenticulata

Paracercomonas ambulans strain W80

Hartmanella vermiformis clone 9341

Cercomonas longicauda

Acanthamoeba sp. (cysts)

Hartmannella vermiformis strain CRIB-19,

Hartmanella vermiformis CRIB-19

Cercomonas

Aspergillus Clone S-10

Paracercomonas minima strain SW2

Tylocephalus auriculatus (Nematode)

Acanthamoeba sp.

Naegleria sp.? Gymnamoeba trophozoite

Penicillium griseofulvum

Acanthamoeba sp (Troph & cyst)

Slime mold? Valkampfia Eimeriidae Testate amoeba

Difflugia (Arcella) Testate

Thaumatomonadida, Uncultured Cercozoan

Mortierella sp. Valkampfia

Vanella

trophozoites

cysts

Hartmannella vermiformis Bullet-shaped amoebo-flagellate – one foot usual

Acanthamoeba spp.

Legionellae and L. pneumophila More that 50 species – several pathogens – some autofluorescent. L. pneumophila most common. Why? Specific association with FLP? L. pneumophila (Lp)-can be hosted by 17 species in vitro but Hartmannella vermiformis appears overwhelmingly most important for Lp amplification in situ Epidemiology suggests FLP most commonly associated with outbreaks is Hartmannella vermiformis Hartmannella commonly isolated from distributed water Acanthamoebae also commonly isolated from distributed water Acanthamoebae can host L. pneumophila in vitro – unclear that it happens much in situ Interest in hartmannellae is less BUT…..

H. vermiformis replicates very rapidly, easily within filter cycle Could amplify Lp on filter Could also be carried into distribution system, settle on tap biofilms and amplify Lp Chloramine reduces Lp colonization

Hartmanellae feeding frenzy

Mass of amoebae (Hartmannella sp.) consuming the E.coli bait bacteria. Arrows show direction of movement of

hartmanellae ahead of newly formed Hartmanella cysts.

40 X

100 x

Interactions of FLP with mycobacteria

• Mycobacteria tough waxy cell wall – persist & grow slowly

• Experiments with acanthamoebae or other amoebae, conducted by us and others, mycobacterial species often avoided as food – especially by new FLP isolates – prey preference experiments showed preferences for other bacteria

• Often contact or ingestion by FLP resulted in encystation.

• Might be adaptation to mycobacteria if only food source

• Using heat killed cells suggest a signal might prevent mycobacteria-acanthamoeba interaction.

• Laboratory interactions observed might not be reproduced in the field

• Overall, field interactions with mycobacteria and any amplification uncertain for the predators examined

Subculture plate - Acanthamoeba trophozoites feeding

Acanthamoeba trophozoites (molecular id) on non-nutrient agar / E. coli lawn

Subculture plate – presumptive Naegleria feeding on E. coli

Naegleria trophozoites with varied sized cysts-left; Naegleria plasmodium on non-nutrient agar / E. coli lawn - right

Biflagellate cercomonads

Direct microscopic examination of sample Following culture

Extremely numerous in all environments – nothing known about predation habits or interaction with bacteria in drinking water

Disinfection of FLP • Several lines of evidence that monochloramine provides

more control of Legionella pneumophila than free chlorine

• Field observations of a switch from chlorine to monochloramine at Pinellas County Utilities – Switch from legionellae to mycobacteria as

predominant DGGE band in DS biofilms • CDC data – decrease in Lp at showerheads (29% to 6%);

of the 6% , 78% were Lp – 8 spp cultured in chlorine, only 3 in chloramine

• Obvious question about disinfection of FLP – especially acanthamoebae and hartmannellae

• If hartmannellae dominant in maintaining Lp – expect them to be senitive to chloramine

Disinfection Reactors • Simple flow-through reactors – examine

disinfection in DS • Real disinfectant levels delivered by

treated water from plant or in DS • Dialysis sacs holding 10 mL amoeba

saline, challenge level of amoeba and glass beads coated with E. coli biofilm to permit active feeding

• Chlorine or chloramine at real levels and pH 6-9 – neutralized or non-disinfected controls . Modification of pH if required used water from large tanks of ~300 L

• Experiments up to 24 h • Compared effects on H. vermiformis

(mainly cysts) and Acanthamoeba polyphaga (mainly trophozoites, some cysts)

Disinfection summary for FLP

plant DS plant DS DS DS DS DS

chloramine chloramine chloramine chloramine chloramine chloramine chlorine chlorine

H. vermiformis

mg/L 1.57-1.67 1.49-1.54 0.98-1.00 neutralized neutralized 1.00

0.2 1.00

0.2

pH 8.70-9.40 8.60-8.70 6.00 9.00 6.00 8.90-9.00 6.10

log reduction 1 5.30 5.60 nd 5.50 0.03 0.00 0.57 0.00

log reduction 2 5.60 4.70 nd 5.50 0.06 0.07 0.97 0.00

plant DS DS DS DS DS

A. polyphaga chloramine chloramine chloramine chloramine chloramine chloramine chlorine chlorine

mg/L 1.54 1.50-1.53 1.30-1.40 0.98-1.00 neutralized neutralized 0.99-1.09 1.00

0.2

pH 8.60-8.70 8.70-8.80 7.40-7.60 6.00 9.00 6.00 8.97-9.00 6.10

log reduction 1 2.81 -0.03 0.44 0.30 -0.07 0.00 0.27 0.04

log reduction 2 3.10 2.66 1.86 0.71 0.08 0.05 0.56 0.69

Detection of survivors

• Serial dilutions and inoculation onto lawns of E. coli in 24-well dishes

• Plates observed microscopically for days to weeks to look for excysted and feeding amoebae

• Because counts done microscopically, quantitation not highly accurate - very laborious work

• Data obtained by microscopy – no cyst disruption - is inactivation permanent?

• Even in apparently killed but intact cysts bacteria such as Lp might be protected from disinfection

Discussion • Rapid amplification of H. vermiformis and cyst formation –

occurs within filter cycle time

• Chloramine - much greater effect on H. vermiformis even at low disinfectant residuals

• May have significant effects on Lp numbers

• Those Lp in cysts might stay intact and unaffected since cyst integrity retained

• Potentially released later following cyst degradation

• Acanthamoebae likely pass filters as cysts

• Previous experiments examining disinfection in flow-through mode cause little to no inactivation of mycobacteria

• No evidence for FLP-mycobacteria interaction in situ but using M. avium as bait, it can clearly be digested by some FLP.