Date post: | 01-Jan-2016 |
Category: |
Documents |
Upload: | bethany-armstrong |
View: | 214 times |
Download: | 0 times |
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 ([email protected])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)?”
3
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)
5
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
10
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?
13
*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
15
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
17
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
18
*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
19
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?
21