RESEARCH PLAN FOR
MANAGEMENT OF EMERGING
PATHOGENS IN DISTRIBUTION
SYSTEMS AND PREMISE
PLUMBING – WATER RESEARCH
FOUNDATION PROJECT #4606
Dr. 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
Five Webinar Project Themes - 1 pm EDT
1) Webinar #1: Emerging Pathogens in Distribution
2)
3)
4)
5)
1
Systems and Premise Plumbing
July 28, Webinar #2: Understanding the Factors
Governing the Proliferation of the Targeted Pathogens
Aug 18, Webinar #3: Develop Best Practices for Risk
Management
Sept 1, Webinar #4: Formulate Effective Communication
Strategies
Sept 24, Webinar #5: Apply Biostability Principles for
Control of Emerging Pathogens
Emerging Pathogens in
Distribution Systems and Premise
Plumbing (DS-PP)
Nicholas Ashbolt (Ashbolt@Ualberta.ca)
Alberta Innovates – Health Solutions
Translational Chair in Water
Office of Research and Development
National Exposure Research
Laboratory
Webinar
#1:
Water Res Foundation #4606, July, 7th 2015
How to address DS-PP pathogens
• Key Question:
“Will the study of N. fowleri, L. pneumophila,
and M. avium complex (MAC) lead us to an
understanding of the mechanisms that
promote the proliferation of emerging
(water-based) pathogens in distribution and
premise plumbing systems (DS-PP)?”
3
Recent (open access) reviews:
• Falkinham III, et al. (2015) Epidemiology and ecology
of opportunistic premise plumbing pathogens:
Legionella pneumophila, Mycobacterium avium, and
Pseudomonas aeruginosa. Environ Health Perspect
DOI: 10.1289/ehp.1408692 (WRF Project #4379)
• Ashbolt (2015) Microbial contamination of drinking
water and human health from community water
systems. Curr Environ Health Rpt. 2(1): 95-106
• Ashbolt (2015) Environmental (saprozoic) pathogens
of engineered water systems: Understanding their
ecology for risk assessment and management.
Pathogens 4(2): 390-405
4
The particular situation for Naegleria fowleri
(~ 8 cases are recognized each year in USA)
• In August 2013, 4 year old died of Primary Amoebic
Meningoencephalitis via US drinking water (New Orleans LA)*
–First reported PAM death associated with culturable
N. fowleri in tap water from a US treated drinking water system
–(yet residual <0.02 mg/L and service line water at 29 °C)
• N. fowleri is a climate-sensitive, thermotolerant (25-40 °C), freeliving amoeba naturally present in warm source freshwater
• Free-living trophozoite stage may grow in DS-PP biofilms, + cysts
• Australian experience from 1970’s shows it is easily controlled by
maintaining a 0.5 mg/L chlorine residual throughout the DS
• Particular issue from warm tap water sources when using neti pot
for sinus irrigation or performance of ritual nasal rinsing
5
*Cope et al. (2015) Clin Infect Dis 60(8): e36-42
Etiologic agents & percentages for 780
drinking water outbreaks, 1971-2006 USA
(28% since 2001)
(85% Norovirus)
(30% Cu, 12% F,
9% NO3- )
Craun et al. (2010)
CMR 23: 507-528
6
6
6
(Many likely to be
viral & parasitic
protozoa, but how
many are nonculturable bacteria?)
(403,000 cases from a single outbreak of
Cryptosporidium hominis in Milwaukee (WI) April
1993, but only 9% of outbreaks vs. Giardia 86%)
Alternative approach: Hospital
claims: US drinking water cases
• CDC estimate drinking water disease costs > $970 m/y
–Less so enterics, largely Legionnaires’ disease, otitis externa
(Pseudomonas aeruginosa) & non-tuberculous mycobacteria
causing >40 000 hospitalizations/year with costs identified as:
Disease
Annual costs
Cryptosporidiosis
Giardiasis
Legionnaires’ disease
NTM infection/Pulmonary
7
$46M
$34M
$434M
$426M/ $195M
Collier et al. (2012) Epi Inf 140: 2003-2013
Saprozoic/Opportunistic pathogen niche:
Biofilms/sediments in DS-PP Shared features
• Non-culturable states
• Free-living in biofilm
• + Intracellular in host
cells (e.g. free-living
protozoa, amoebae)
8
Lau & Ashbolt (2009) J Appl Microbiol 107(3): 368–378
Water-based microbial pathogens of DS-PP
Microbial
Group
Recognized
Potential
Viral
None
Mimivirus, Mamavirus of amoebae*
Bacterial
Legionella spp.,
non-tuberculous
mycobacteria (NTM),
P. aeruginosa
Acinetobacter baumannii
Aeromonas hydrophila
ARB* (Afipia, Bosea, Parachlamydia)
E. coli (toxigenic strains), Listeria
monocytogenes, Staphylococcus aureus,
Stenotrophomonas maltophilia
Protozoan
Acanthamoeba T4
Acanthamoeba, Vahlkampfia, Vannella spp.,
Balamuthia mandrillaris** Vermamoeba vermiformis
Naegleria fowleri
Aspergillus fumigatus, A. Candida albicans, C. parapsilosis
terreus (nosocomial)
Exophiala dermatitidis (grows at ~40 °C)
*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
9
Fungal
Ashbolt (2015) Curr Environ Health Rpt. 2(1): 95-106
Antimicrobial-resistance (AMR) genes
in DS-PP pathogens
• Class 1 integrons are capable of integrating gene cassettes
containing AMR genes into native bacteria and pathogens
–to date there are over 130 gene cassettes conferring a range of
antibiotic-resistant phenotypes*
–thus the presence of a class 1 integron (and other
determinants) enables resistant to antibiotics and maybe a
useful proxy for antibiotic resistance in DS-PP pathogens*
• 3rd gen cephalosporin- resistant E. coli & MRSA predicted deaths
3.3 per 100,000 in EU in 2015**
• Globally 700,000 AMR-deaths, some 10 million by 2050***
• Unclear fraction of AMR bacteria exposure via drinking water
10
*Amos et al. (2015) ISME J 10.1038/ismej.2014.237
**Ashbolt et al. (2013) Env Health Perspect 121(9): 993-1001
***Hoffman et al. (2015) Bull WHO 3(2): 66
Non-tuberculous mycobacteria
~16,000 hospitalization/y NTM reported in US*
• Various species of Mycobacterium including:
–M. avium complex (MAC) that contains M. intracellulare that
are ubiquitous atypical mycobacteria (i.e. non-TB) found in the
environment, best known as infect patients with HIV and low
CD4 cell counts via inhalation or ingestion; some also include
M. avium subspecies paratuberculosis (MAP) in this group
–MAC causes disseminated disease in up to 40% of patients
with human immunodeficiency virus (HIV) in US, producing
fever, sweats, weight loss, and anemia and clear link to DW
–Other species (e.g. rapid growers [M. abscessus, M. chelonae, M.
fortuitum] & slow growers [M. gordonae, M. kansasii, M. immunogenum,
M. ulcerans] less clearly identified via DW, yet cause hypersensitivity
pneumonitis, other respiratory problems & wound infections at work/home
11
*Collier et al. (2012) Epi Inf 140: 2003-2013
Rising incidence of NTM infections
• Due to a number of factors, including:
–Host Effects: Aging population, particularly among the
slender elderly woman, decrease in TB-cross immunity, use
of immunosuppressive agents, increasing survival of risk
groups (e.g. cystic fibrosis patients)
–Pathogen traits: hydrophobic (mycolic acid rich) outer
membrane protects cells from disinfectants, so both slow and
rapid growing NTM selected for by a residual disinfectant
• Compared to E. coli, the CT99.9% for water-adapted cells of
L. pneumophila is 1050-fold & M. avium 2000 higher
12
Falkinham III (2008) J Wat Health 6: 209-213
(2009) J Appl Microbiol 107: 356-367
(2010) J Med Microbiol 59: 1198-1202
Falkinham III et al. (2015) Pathogens 4: 373-386
Legionellosis (only via aerosols)
~14,000 hospitalization/y reported in US*
Characteristic
Legionnaires’ Disease
Pontiac Fever
Incubation period
2 – 10 days
5 h – 3 days
Weeks
2 – 5 days
Depends on susceptibility
Nosocomial cases 40-80%
No deaths
Attack rate
0.1 – 5% of the general population
0.4 – 14% in hospitals
Up to 95%
Symptoms
Anorexia, malaise, fever, chills,
lethargy, vomiting, diarrhea
Fever, chills, vomiting,
diarrhea
Duration
Case-Fatality rate
13
Legionella and the prevention of Legionellosis, WHO (2007)
*Collier et al. (2012) Epi Inf 140:2003-2013
Legionella Virulence
>70 species/serogroups of Legionella
L. pneumophila serogroup 1 causes > 85% cases of disease
20-50% of environmental isolates are Lp1
MAb2(+) strains cause >80% cases of disease
< 20% environmental isolates are MAb2(+)
~1700
I STs
170 are known to
cause disease in
the US
16 STs cause > 80% of
Source: Claressa Lucas
disease in the US
CDC
14
ST – Sequence Type; typing from seven gene targets (flaA, pilE, asd,
mip, momp, proA, and neuA)
Legionellosis
• Cases of Legionellosis and spread attributed to inhalation of
aerosols from domestic pluming systems, hots tubs, indoor
fountains, humidifiers and cooling towers
• causative agent of Legionnaires’ disease: Legionella
pneumophila serogroup 1 then sg2, then other species:
(L. micdadei > L. bozemanii > L. dumoffii > L. longbeachae)
15
Role of Free-living Protozoa in Biofilms
• L. pneumophila are able to persist and remain viable for about
15 days within artificial biofilms (VBNC >90 d in Cu-pipe biofilms)
• Addition of Vermamoeba vermiformis, Acanthamoeba spp. or ?
in these systems necessary for L. pneumophila replication
Buse et al. (2014) FEMS Microbiol Ecol 88: 280-295
• Some 30-40% of biofilms samples isolated from various hospital
water supply sources, dental units and taps are 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 presence
16
Trojan horse: amoebae in water
Source
Samples +ve
(+ve/total #)
Amoebae/L
# Genera
River water
Post treatment
Groundwater
26/26
6/6
9/11
200 – 90,000
2 – 4,000
0 – 3,000
6 – 17
1–8
0–5
Drinking water
7/21
0 – 100
0–4
Hoffmann & Michel (2001) Int. J. Hyg. Environ. Health 203: 215-219
Up to 104 Legionella/L DW sediments, some 8% L. pneumophila*
70-2900 amoebae-cilliates.cm-2 biofilm in 23-33 mg.L-1 AOC water**
17
17
*Loret & Greub (2011) Int. J. Hyg. Environ. Health 213: 167-75
*Lu et al. (2015) J Appl Microbiol 119: 278-288
**Långmark et al. (2007) Water Research 41: 3327-3336
QMRA for critical Legionella densities –
used in Dutch & German regs, ASHRAE
Critical # in DW
106 – 108 CFU L-1
based on QMRA model
Needs hosts to reach that
Biofilm colonization
and detachment
18
Aerosolization
Critical # 35 – 3,500 CFU m-3
based on QMRA model
Inhalation
Deposition
1-1,000 CFU in lung
for potential illness
Schoen & Ashbolt (2011) Water Research 45(18): 5826-5836
Biotic and abiotic factors influencing
Legionella growth – control 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)
• Soluble ions increase with water stagnation in pipes (hot & cold)
19
19
*Lu et al. (2015) J Appl Microbiol 119: 278-288
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 increases Acanthamoebidae presence
–VBNC Legionella forms, so culture ID not totally inclusive
–Increased resistance to disinfectants
• Cu/Ag treatment also may increase VBNC
• Cu pipes select for biofilms supportive of Legionella
compared to PVC pipes – more in Webinar #5
20
Buse et al. (2014a) FEMS Microbiol Ecol 88(2): 280-295
Buse et al. (2014b) Int J Hyg Environ Health 217(1): 219-225
Lu et al. (2013) Appl Environ Microbiol 79(8): 2713-2720
Water-based microbial pathogens of DS-PP
Microbial
Group
Recognized
Potential
Viral
None
Mimivirus, Mamavirus of amoebae*
Bacterial
Legionella spp.,
non-tuberculous
mycobacteria (NTM),
P. aeruginosa
Acinetobacter baumannii
Aeromonas hydrophila
ARB* (Afipia, Bosea, Parachlamydia)
E. coli (toxigenic strains), Listeria
monocytogenes, Staphylococcus aureus,
Stenotrophomonas maltophilia
Protozoan
Acanthamoeba T4
Acanthamoeba, Vahlkampfia, Vannella spp.,
Balamuthia mandrillaris** Vermamoeba vermiformis
Naegleria fowleri
Aspergillus fumigatus,
Candida albicans, C. parapsilosis
A. terreus (nosocomial)
Exophiala dermatitidis (grows at ~40 °C)
*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 of the skin
21
Fungal
Ashbolt (2015) Curr Environ Health Rpt. 2(1): 95-106
Are P. aeruginosa & other DS-PP
pathogens needing an additional index?
• Do these opportunistic pathogens also grow more in
protozoan hosts or just freely in drinking water or
associated biofilms?
• Total Legionella i.e. other than pathogenic Legionella
strains, may grow freely in biofilm, but will they index the
above?
• Many non-pathogenic NTM grow freely in biofilms, but will
they index health risks from the above group?
• Noting that, limited general biofilm growth may not be a
problem but a solution (Webinar #5 discussion)
22
Discussion –
Not DBP but WBP!
(WBP: Water-based pathogens)
• Once you manage enteric pathogens, water-
based pathogens likely to cause most health
burden, via respiratory & wound infections
–Non-tuberculous mycobacterial (NTM) infections
now > TB, & infection only via environment
23
–Similarly Legionella pneumophila only via aerosols
and mostly grow in biofilm amoebae