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Development of a Risk
Management Strategy
for Legionella in
Water Systems
Mark W. LeChevallier, Ph.D.
Director, Innovation & Environmental Stewardship
NJAWWA Annual Meeting
Atlantic City. March 18, 2015
WateReuse-12-05 Project Team
American Water
Technical Advisory Group
• Dr. Zia Bukhari
• Dr. Patrick Jjemba
• William Johnson
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Dr. Craig Riley
Dr. Nick Ashbolt
Dr. William Keevil
Dr. Tom Armstrong
Drexel University
• Dr. Charles Haas
Staff / PAC
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Stefani McGregor
Rick Danielson
Terri Slifko
Michael Storey
Bob Vincent
Channah Rock
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Legionella
• Aerobic, non-encapsulated, non-spore forming,
Gram-negative bacterium
• Consists of approximately 50 species
consisting of 70 serogroups
• L. pneumophila has at least 35 serotypes
• Requires cysteine and iron for growth
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Legionella Ecology
• Widely found in diverse water environments - groundwaters,
drinking water systems, seawater and reclaimed water systems
• Engineered systems – hospitals, spas, cooling towers, humidifiers,
ice machines, vegetable misters, cutting oils, etc.
• grow best at temperatures from 20oC to 42oC
• Acid tolerant and thermotolerant- surviving 50oC for several hours
and tolerate temperatures up to 66oC
• Infect free-living amoebae such as Hartmanella sp.
and Acanthamoeba castellanii.
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Legionellosis
• Legionnaire's disease - acute, sometimes
fatal, pneumonia-infection. Nationally notifiable.
• Pontiac fever - self limiting mild illness
• L. pneumophila serogroup 1 is responsible for
about 95% of the Legionnaires' Disease cases
• Causes 8,000 - 18,000 infections/year. Infections peak during
summer months. About 4-5% of all pneumonia. Mortality 5-30%
• At risk individuals are >50 years age, immunocompromised,
smokers, or those with chronic lung disease
• Infection acquired through inhalation
• In the lungs, the organism is engulfed by alveolar macrophages
where they multiply and grow
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57% cases
summer months
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Drinking Water Disease Outbreaks, 1971–2010
Morbidity & Mortality Weekly Report. 62(35):714-720, September 6, 2013.
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Legionella Culture Method
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100 mL filtered through 0.45 µm pore size
Filter placed in 10 mL phosphate-buffered solution
Vortexed for 30 seconds
1mL mixed with an equal amount of acid (HCl-KCl, pH= 2.2 for
15 minutes, then neutralized with KOH-KCl.
0.1 mL spread-plated on BCYE agar supplemented with Lcysteine
Incubated at 36.5°C under 2.5% CO2 with
91% relative humidity for up to 10 days
Typical colony is gray-white with a textured,
cut-glass appearance
Verification by latex agglutination tests to
determine serotype
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Legionella PCR Methods
• Evaluated two targets:
- Macrophage infectivity potentiator (mip) gene
- 23S-5S rRNA gene spacer region
• PCR Sensitivity
700 bp
300 bp
• PCR Specificity
- 99-100% identity with Legionella pneumophila
(Philadelphia-1 strain, ATCC 33152),
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Legionella PCR Strategy
• For water analyses, the mip gene will be the primary target for
identifying Legionella
 Relatively stable gene
 Sufficient interspecies nucleotide variation
 Can differentiate between serotypes
Roche LightCycler 480 System II
• Negative mip PCR samples will be verified
using the 23S-5S target
• Representative isolates will be sequenced
Qiagen QIAamp DNA Mini Kit
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Viable Legionella w/ ethidium monoazide
100%
Percent Viable
75%
R² = 0.9948
50%
25%
0%
0
200000
400000
600000
800000
1000000
1200000
GU / mL
1.20E+06
y = 0.4308x
R² = 0.9857
1.00E+06
GU per mL
8.00E+05
6.00E+05
4.00E+05
Allows PCR to detect only
viable cells
2.00E+05
0.00E+00
0.00E+00
5.00E+05
1.00E+06
1.50E+06
Legionella CFU per mL
2.00E+06
2.50E+06
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Legionella in Reclaimed Water
Characteristics of reclaimed water - growth/risk of Legionella
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Warm water
High level of AOC
No/little disinfectant residual
Little distribution system cleaning/ flushing/ maintenance
Spray irrigation
• 11 of 19 (58%) utilities
positive
• 15 of 38 (39%) samples
positive
• 5 effluents & 10 DS
positive
• 115 CFU/mL in DS, 60
CFU/mL in effluent
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Physical
Temperature ( oC)
Corrosivity (mpy)
Operational
Design
Flow rate (gpm)
Hydraulic pressure (psi)
Biological
Total coliform (cfu/100mL)
Legionella (cfu/100mL)
E. coli (cfu/100mL)
Detention time (days)
Flushing
Reservoir management
Aeration - mixing
Short circuiting
Run-off control
Cleaning and dredging
Distribution system operations
Heterotrophic bacteria (HPC/mL)
Algae
Chlorophyll
AOC
R2 0.625
R2 0.385
300
15000
250
12500
200
10000
AOC (µg / L)
Chlorophyll a (µg/mL)
Dissolved oxygen (mg/L)
Total suspended solids (mg/L)
Turbidity (NTU)
Chemical
pH
Conductivity
Nitrogen (NO 2-N, NO3-N, NH3-N;
mg/L)
AOC (mg/L)
BOD (mg/L)
COD (mg/L)
TDS (mg/L)
Total chlorine (mg/L)
Free chlorine (mg/L)
Phosphorus (mg/L)
Salinity (mg/L)
DPBs (HAA, TTHM; μg/L)
TOC (mg/L)
H2S (mg/L)
Fe3+ (mg/L)
Mn2+ ( mg/L)
150
7500
100
5000
50
2500
0
0
Effluent (n=5)
Distribution (n=10)
Legionella Positive
Effluent (n=14)
Distribution (n=9)
Legionella Negative
Effluent (n=5)
Distribution (n=10)
Legionella Positive
Effluent (n=14)
Distribution (n=9)
Legionella Negative
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Evidence of Regrowth
Legionella
AOC levels
Legionella Species
Species
L. pneumophila
L. oakridgensis
L. moravica
L. longbeachae
L. hackeliae
L. parisiensis
L. steigerwaltii
L. anisa
L. tucsonensis
L.waltersii
L. wadsworthii
L. feeleii
L. spiritensis
L.cincinatiensis
L. lansingensis
L. jordansis
Number
Percent
96
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9
8
8
5
3
3
2
1
1
1
1
1
1
1
63
8
6
5
5
3
2
2
1
1
1
1
1
1
1
1
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Lessons from Real Life:
San Francisco, CA
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53 buildings
Sampled 3 times pre- and post-conversion to chloramines
Sampled hot water heater and four distal sites
Sampled swab and water from distal sites
Surveys collected data on building age, height, type and number of
hot water heaters
pH, temperature, free or total Cl2 residual measured for each sample
Flannery, B. et al. 2006. Reducing Legionella colonization of water systems with
monochloramine. Emerg. Infect. Dis. 12(4): 588-596.
http://www.cdc.gov/ncidod/EID/vol12no04/05-1101.htm.
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Round 1
Heater
#2
#3
#4
#5
#6
#7
#8
Round 2
#9 Heater
#2
#3
#4
#5
#6
#7
#8
#9
Round 3
Heater
#2
#3
#4
#5
#6
#7
#8
#9
Round4
Heater
#2
#3
#4
#5
#6
#7
#8
Round5
#9 Heater
#2
#3
#4
#5
#6
#7
#8
Round 6
#9 Heater
#2
#3
#4
#5
#6
#7
#8
#9
93% reduction
in Legionella
occurrence
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Impact of Disinfection on Legionella and Amoebae
• Detection of Legionella spp. significantly (p = 0.01) associated with
detection of amoebae - 61 (36%) of 169 samples versus 291 (24%)
of 1,236 samples without amoebae
• After conversion to monochloramine, Legionella were found in 1% of
samples with or without amoebae
• Avg. disinfectant residual in hot water heaters; 0.13 (0-0.86) vs 1.10
(0-2.20) mg/L
• Legionella concentration was higher in samples containing amoebae
(median 9.0 CFU/mL, range 0.1–25.0) compared with those without
amoebae (median 1.5 CFU/mL, range 0.05–25.0, p<0.001).
• The prevalence of amoebae decreased from 169 (12%) of 1,405
samples when free chlorine was the residual disinfectant to 78 (8%)
of 944 samples after conversion to monochloramine (p = 0.006).
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Legionella and Amoebae
• Intracellular Legionella in: Acanthamoeba, Amoeba,
Comandonia, Echinamoeba, Filamoeba,
Hartmannella, Naegleria, Paratetramitus, Vahlkamfia,
Tetrahymena, Dictyostelium
• Legionella survive for months, resistant to 50 mg/L
free chlorine for 18 hr
Trophozoite
• Coated with amoebal proteins
• Increases virulence, replication
• Legionella-containing vacuoles expelled prior to
encystation
Cyst
• Trophozoite stage sensitive to disinfectants
(CT99.9 = 1.5 mg-min/L)
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Trophozoite and Cyst Differentiation
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Conceptual Risk Model
A. Legionella in biofilm or
amoebae
B. Detachment
C. Aerosolized
D. Inhaled
E. Deposited in lungs
Critical range: 3.5 103 - 3.5 105 cfu/mL
Schoen and Ashbolt. Water Research 45(18): 5826–5836, 2011
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Managing Legionella Risk
• Determining Legionella densities, species and serotypes
– Methods for culture and PCR
AOC (ug acetate C/L)
Legionella/mL
• Emphasis on maintaining a disinfectant residual
– Some advantage for a chloramine residual
7000
- More persistent
6000
5000
- Impact on encycstation?
4000
3000
– Investigation of other biocides
2000
1000
- peracetic acid?
0
M
AOC results
A -1ft.
B - 50 ft.
C - 100 ft.
D - 150 ft.
2000
1500
1000
500
0
0
2
Control
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FreeChlorine
ChlorineChloramine
From Jjemba et. al. 2010, WRF – 05 – 002
• Distribution System BMPs
– recommendations from project WRRF-11-03
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Summary
• Legionella is an emerging public health that water utilities
should be aware of
• Existing data demonstrates that Legionella spp. are present in
reclaimed/water distribution systems
• There is a gap in information on the ecology of free living
amoebae in reclaimed water systems
• Currently risk models are incomplete
• There is a lack of best management practices for Legionella
and free living amoebae
• Water utilities can focus on maintaining a disinfectant
residual, regular flushing/cleaning
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Contact Information:
Mark W. LeChevallier, Ph.D.
Director, Innovation & Environmental
Stewardship
American Water
1025 Laurel Oak Road
Voorhees, NJ 08043 USA
phone: (856) 727-6106
fax:
(856) 727-6199
e-mail: [email protected]
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