RESEARCH PLAN FOR MANAGEMENT OF EMERGING PATHOGENS IN DISTRIBUTION SYSTEMS AND PREMISE PLUMBING – WATER RESEARCH FOUNDATION PROJECT #4606CALL IN NUMBER: 1-866 528 2256CODE: 32 33 772 # PLEASE MUTE *6Dr. Mark LeChevallier, Dr. Zia Bukhari, American Water, and

Dr. Nicholas Ashbolt, University of Alberta, leading this project

Series of five webinar presentations and interactive discussions as part of participants sharing their ideas on each project theme

Office of Research and DevelopmentNational Exposure Research Laboratory

Apply Biostability Principles for Control of Emerging PathogensNicholas Ashbolt (Ashbolt@Ualberta.ca)Alberta Innovates – Health Solutions Translational Chair in Water

Webinar #5: Water Res Foundation #4606, September 24th 2015

How to address DS-PP pathogens

•Key Question:

“Can biostability / biocontrol approaches minimize the growth of N. fowleri, L. pneumophila, M. avium complex (MAC) and other emerging (water-based) pathogens in distribution and premise plumbing systems (DS-PP)?”  

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What is meant by biostability?

• Ideally biologically stable water does not support the growth of any microorganism• In practice means limiting growth so no undesirable:

– Tastes and odors;– Visual turbidity; and most relevant here– No growth of water-based pathogens

•Control of microbial growth is normally achieved by:– Limiting biologically available org carbon (maybe P)– Using residual disinfectant (e.g. Cl2 , ClO2 or NH2Cl)

4 Key reference: Hammes et al. (2010) AQUA 59(1): 31-40

Biostability in practice - 1•Some European countries (e.g. Netherlands, Germany, Austria & Switzerland) distribute high quality drinking water without a residual disinfectant–By using biologically-filtered / advance oxidation-treated water (to minimize growth substrates)

–And using pipe materials that limit biofilm growth•Biological stability (biostability) is also termed ‘microbial stability’, ‘microbiological stability’ or ‘no regrowth potential’• Instability seen as an increase in biomass and utilization of substrate (generally organic carbon limits growth)

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Biostability in practice - 2• In Zurich, biostability seen with drinking water with:

– total cell count (TCC) of 9 x 104 cells mL-1

– dissolved organic carbon (DOC) of 0.78 mg.L-1 – assimilable organic carbon (AOC) of 32 mg.L-1

–No sig change throughout the distribution system*•How to measure this biostability?

–HPC provide a relative index, but vastly underestimate total viable count of bacterial cells present

–Hence, Swiss* pioneered use of TCC by flow cytometry & test materials with Dutch biofilm formation rate (BFR) assay

6 *Hammes et al. (2010) AQUA 59(1): 31-40, AOC test-inoculum comprised autochthonous bacteria from the treatment plant

Biostability in summary• To produce biologically stable water in the North American context is problematic and costly–Need to reduce surface water DOC/AOC substantially, e.g. by use of BAC & O3 oxidation-like treatment steps

–Water temp < 15 °C preferred to aid stability in DWDS–Pipe materials with low biofilm growth potential, i.e. not PVC and caste iron (stainless steel not Cu in hospitals)

•Even then, drinking water biofilms release bacteria well in access of those assayed by HPC/culture-based methods–Which may include various water-based pathogens, potentially further selected for by a residual chlorine

–Requires ecological understanding to control pathogens7

Water-based microbial pathogens of DS-PPMicrobial Group

Recognized Potential

Viral None Mimivirus, Mamavirus of amoebae*

Bacterial Legionella spp., non-tuberculous mycobacteria (NTM, including MAC) and P. aeruginosa

Acinetobacter baumanniiAeromonas hydrophilaARB* (Afipia, Bosea, Parachlamydia)E. coli (toxigenic strains), Listeria monocytogenes, Staphylococcus aureus, Stenotrophomonas maltophilia

Protozoan Acanthamoeba T4Balamuthia mandrillaris**Naegleria fowleri

Acanthamoeba, Vahlkampfia, Vannella spp., Vermamoeba vermiformis

Fungal Aspergillus fumigatus, A. terreus (nosocomial)

Candida albicans, C. parapsilosisExophiala dermatitidis (grows at ~40 °C)

8From: Ashbolt (2015) Curr Environ Health Rpt. 2(1): 95-106

*Acanthamoeba polyphaga mimivirus (APMV) may cause respiratory disease and unknown health effects from Mamavirus; ARB – amoeba-resisting bacterial pathogens** causes granulomatous amoebic encephalitis (GAE) via skin lesions to blood to brain or may cause amoebic keratitis

What is meant by biocontrol?•Biocontrol is the deliberate manipulation of microorganism to provide a beneficial microbiome, by:– Use of selective chemical (materials, nutrients, biocides) & physical conditions and/or

– Addition of specific microorganisms, the so called probiotic approach* that select for a desirable microbiome

•Hence need to understand the chemical, physical and biological factors governing water-based pathogens in DWDS & premise plumbing (i.e. their ecology) to utilize biocontrol as part of BMP

9*Wang et al. (2013) Environ Sci Technol 47: 10117-28

Anti-biofilm agents & considerationsChung et al. (2014) Pathog Dis 70(3): 231-9

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Attachment inhibitors

Attachment Mature biofilm

Biofilm disruptionSignal transduction & Quorum sensing inhibitors

Dispersion of cellsMicrocolony formation

e.g. biotic and abiotic factors influencing Legionella growth – biocontrol points?

•Biotic (at treatment, storage, distribution, premise)–Biofilm environment (pipe microbiome)• Free-living amoebae (FLA): e.g. Acanthamoeba & Vermamoeba

–Hosts increase on GAC/sand filters & reservoir sediments*• Competitive & predatory bacteria (Lysobacter) and FLP (Cercomonas)

•Abiotic (at treatment, storage, distribution, premise)–Disinfectant residual (free Cl2 vs NH2Cl vs none)

–Water temperature (increasing growth > 25 °C to 42 °C)–Pipe materials for growth/virulence (Fe, Cu, Mn, Zn ions/corrosion)• Soluble ions increase with water stagnation in pipes (hot & cold)

1111 *Lu et al. (2015) J Appl Microbiol 119: 278-288

Wang et al. (2013) Environ Sci Technol 47: 10117-28

Fate of pathogenic Legionella in DS-PP

Adapted from: Lau & Ashbolt (2009) J Appl Microbiol 107(3): 368–37812

Biocontrol in practice - 1•Once attached to the biofilm*, biotic factors include:

–Predator (digestion, replication with hosts, vesicle out)–Lytic phage attach (poorly understood for biofilm pathogens)

–Free biofilm growth and competition/antagonism, incl.•Sloughing factors (nitrous oxide, enzymes, quorum molecules)

–DOC/AOC?

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*Shen et al. (2015) Environ Sci Technol 49: 4274–82

Role of FL Protozoa in biofilms & pipe material• L. pneumophila are able to persist and remain viable for about 15 days-months in artificial biofilms (VBNC >90 d in Cu-pipe biofilms) • Addition of Vermamoeba vermiformis, Acanthamoeba spp. etc. in DW biofilms seem necessary for L. pneumophila &? MAC growth–Legionella in amoebal cysts & vesicles may last years (Cl2 proof)

–Other Legionella spp. may grow freely in biofilms?

Buse et al. (2014) FEMS Microbiol Ecol 88: 280-295

Schwake et al. (2015) Pathogens 4: 269-282• Some 30-40% of biofilms samples isolated from various hospital water sources, dental units & taps positive for Acanthamoeba spp.

Carlesso et al. (2007) Rev Soc Bras Med Trop 40: 316-320 Lu et al. (2015) J Appl Microbiol 119: 278-288

Key link: free-living protozoa and DS-PP pathogen presence14

Cu pipes: even more challenging•Most buildings use Cu-pipes for hot/cold water•CuOs form on all these Cu-pipes, which we shown may induce genes involved in:–Phagocytosis by amoebae within biofilms•Noting Cu favors bacteria that support Acanthamoebidae

–VBNC Legionella forms, so culture ID not totally inclusive–Increased resistance to disinfectants•Cu/Ag treatment also may increase VBNC

•Cu pipes may select for biofilms supportive of Legionella compared to PVC pipes & impact downstream biofilms

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Buse et al. (2014a) FEMS Microbiol Ecol 88(2): 280-295Buse et al. (2014b) Int J Hyg Environ Health 217(1): 219-225

Lu et al. (2013) Appl Environ Microbiol 79(8): 2713-2720

Molecular ecology approaches•Current 16 & 18 S rRNA & metagenomic sequencing is identifying major and some minor biofilm/DW members–Such as Mycobacterium dominance in NH2Cl DW*

–But does not indicate ecological interactions nor strains of potential health concern

• Limited transcriptome data to date, but has the potential to target gene regulation in situ**– More likely to aid in identifying mRNA qPCR targets to follow efficacy of an intervention (e.g. biofilm biocide) **

• ‘Deep’ metagenomic whole genome analysis needed in combination with physical location of interacting cells

16 *Gomez-Alvarez et al. (2015) J Wat Health 13(1): 140–151**Lu et al. (2013) Appl Environ Microbiol 79(8): 2713-2720

Phage or microbial/product control?

• Lytic phages know to infect Legionella, Mycobacteria etc.–But unknown if phage addition would be effective control–Or simply follow a bloom of the target group?

• Generally maintenance of a chlorine residual reduces OPPP

–Unclear fraction of Cl2-selected mycobacteria are pathogens

–Hence, given the ubiquitous nature of mycobacteria and resistance to disinfectants and growth with low DOC/AOC

–Mycobacterium biocontrol, while counter intuitive, may work

• Agents like nitrous oxide, quorum sensing molecules could be added to DW or released from premise plumbing materials to facilitate low biofilm biomass & reduce microbial risk niche

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Biocontrol with Probiotics*•As it is possible to influence the downstream drinking water microbiome by manipulating the filter microbial community & subsequent DWDS**•Potential to add a ‘microbial selector’ upstream of a health care facility or building to influence PP microbiota–Could be augmented by addition of additional supplements (microbes/bacteriocins + chemicals)•McHatton et al. Patent US 2015/0225272. Preventing and reducing biofilm formation and planktonic proliferation. Aug 13, 2015

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*Wang et al. (2013) Environ Sci Technol 47: 10117-28**Buse et al. (2014b) Int J Hyg Environ Health 217(1): 219-225

**Lu et al. (2013) Appl Environ Microbiol 79(8): 2713-2720

Commercially available amoeba biocide

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Summary•Reducing AOC/DOC, certain pipe materials,

temp & stagnation control or disinfectant

alone may not control water-based pathogens

–Need to understand the ecological interactions to

harness biocontrol strategies, e.g.

•Go beyond 16/18S rRNA gene sequencing

• In situ proliferation niche needs to be studied from a

multi-factor perspective to suggest options, such as

•Use of selector materials prior to DW building entry?20

Many outstanding questions…• How does controlling Legionella with disinfectants impact on selection for MAC of concern?• Safety with biological control agents, e.g. key amoebae?• Is adaptation to counter any control strategy likely in the long term?•What subset of Legionella, NTM & MAC in particular are potential pathogens versus potentially useful members of biofilms to suppress ecological space for pathogenic members?• How is P. aeruginosa in PP impacted by Legionella/MAC control options?• Do we need to consider further opportunistic pathogens when considering biocontrol strategies?

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