Susan Springthorpe [[email protected]]
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.
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