Joint meeting presentation

April 28, 2020

Factors Influencing Microbial Regrowth and

Occurrence of Opportunistic Pathogens in
Premise Plumbing

Iftita Rahmatika
Water Environment Technology Lab
Department of Urban Engineering
The University of Tokyo
 Research background
Microbial regrowth in drinking water
Premise plumbing
Treatment plant Cl2
Cl2 is maintained during distribution

Microbes is disinfected Chlorine is depleted due to long stagnation

during treatment plant period causing microbial regrowth and
opportunistic pathogens occurrence.
Molecular survey of clinically important opportunistic pathogens in several countries:
Source Microorganisms Number
Household tap water in US1 Legionella pneumophila 9.8 copies/mL
Mycobacterium avium 1.1 copies/mL
Pseudomonas aeruginosa 1.8 copies/mL
Washroom and kitchen tap Legionella pneumophila 3 log copies/mL
water in China2 Pseudomonas aeruginosa 1.04 log copies/mL

Biofilm in shower hoses in Legionella pneumophila 6.6×105 copies/cm2

Europe3 6.6×107 copies/cm2
Mycobacterium avium
Pseudomonas aeruginosa 2.5×104 copies/cm2
Acanthamoeba spp 2.5×104 copies/cm2
To control microbial regrowth and opportunistic pathogens in drinking water, factors
influencing their regrowth needs to be evaluated. 2
 Research background
Factors influencing microbial regrowth in premise plumbing
Problem during water stagnation in premise plumbing:
Depletion of free chlorine due to the contact of
water with pipes, which supports regrowth.

Depletion of Cl2 Organic matter released from

Bacteria from pipes during stagnation.
water distribution BOM in During flushing:
Food
drinking Food • Bacteria growing in water
water BOM • Biofilm
Bacteria in water
Foo Detachment
d from biofilm
Bacteria in biofilm
Some of them can be
pathogen that cause
Biodegradable organic matter supporting
health risk.
regrowth of bacteria in water and in biofilm.

Current method to evaluate the microbiological effect of carbon migration from plastic pipes 4

Method Parameters to determine microbial

W270 (Germany)
Biomass production
potential (Netherland)
BioMig (Eawag)
regrowth
No Information on the composition
of biodegradable organic matter
Volume of surface growth
released from pipes and their
Adenosine triphosphate (ATP)
effect to microbial community.
TCC

3
 Research objectives

Objectives: to evaluate the influence of stagnation to

microbial regrowth and opportunistic pathogen occurrence and
to evaluate the effect of organic matter released from pipes
to microbial regrowth in premise plumbing.

Specific objectives:
1. To investigate microbial regrowth and opportunistic pathogens
occurrence after stagnation in premise plumbing.
2. To evaluate the fate of opportunistic pathogen and other dominant
bacteria growing in premise plumbing in drinking water supply.
3. To reveal the composition of organic matter released from plastic pipe
material during stagnation on microbial community and opportunistic
pathogens using high-resolution Orbitrap MS.

4
 Research framework

Chapter 1 Chapter 2 Chapter 3

Research background Literature review Research methodology

Chapter 4
Microbial regrowth and opportunistic pathogens in premise plumbing after stagnation
1. Influence of stagnation to microbial regrowth.
2. Seasonal and spatial differences on microbial regrowth.
3. Relationship with water quality parameter.

Chapter 5
Fate of opportunistic pathogens and
bacteria growing in premise plumbing
in drinking water supply
1. Changes of microbial community
composition in drinking water supply.
2. Fate of opportunistic pathogens and
other dominant bacteria.
Chapter 6
Composition of organic matter released
from pipes during stagnation influencing
microbial regrowth
1. Changes of DOM compositions in premise
plumbing after stagnation.
2. Effect of organic matter released from pipes to
microbial regrowth.
3. DOM compositions released from pipes
promoting microbial regrowth.

Chapter 7 Conclusions and recommendations

5
 1. Influence of stagnation to microbial regrowth
Sampling methodologies:
• Sample: drinking water sample from different faucets (pre- and post- stagnation)
• Sampling location: Engineering building 14, The University of Tokyo.
• Sampling time: May 2018 – April 2019

Sample code Building Water source Sampling time

F1 Buidling A Surface water treated Summer, Autumn, Winter, Spring
F2 (Eng. 14) by advanced drinking Summer
water treatment plant
F3 Summer, Autumn, Winter, Spring
(Ozonation –BAC)
F4 Summer, Autumn, Winter, Spring
F5 Summer, Autumn, Winter, Spring
F6 Summer
F7 Summer, Autumn, Winter, Spring
F8 Summer, Autumn, Winter, Spring

6
 1. Influence of stagnation to microbial regrowth

1) Pre-stagnation Sample Measurement in 10 L

sample:
1. Free chlorine and temperature
2. Dissolved organic carbon
(DOC)
3. Carboxylic acids
4. Total cell counts (TCC)
5. HPC
Open and run the Sample collection: 6. DNA extraction for qPCR and
faucet for 5 min 10 L sequencing

Close the faucet and let it stagnant for 24 h

2) Post-stagnation
Composite of the first 1L
1 2 3 4 5 6 7 8 9 10  DNA extraction for
qPCR and sequencing
Sample collection: every 100 mL in the first 1 L
Open the faucets
and collect
sample Sample measurement in each 100 mL samples
immediately 1. Free chlorine and Temperature
2. Total cell counts (TCC)
3. HPC (only first 100 mL)
7
 1. Influence of stagnation to microbial regrowth
List of parameters:
Parameter Method Sample
Dissolved organic carbon TOC-L Pre-stagnation
(DOC)
Carboxylic acids Ion chromatography Pre-stagnation
Free chlorine HACH chlorine pocket colorimeter Pre- and post-stagnation
Temperature Pre- and post-stagnation
Total cell counts (TCC) Flow cytometer Pre- and post-stagnation
Heterotrophic plate count Standard method 9215 C Pre- and post-stagnation
(first 100 mL)
Opportunistic pathogen qPCR Pre-and post- stagnation
 Legionella spp. (first 1 L)
 L. pneumophila
 Mycobacterium spp
 M. avium
 P. aeruginosa
 Achantamoeba spp.
Total bacteria (16S rRNA qPCR Pre-and post- stagnation
gene) (first 1 L)
Microbial community Illumina miseq of 16S rRNA gene Pre-and post- stagnation
structure and Nanopore sequencing (first 1 L)

8
 1. Influence of stagnation to microbial regrowth
Influence of stagnation to cell concentrations
• Free chlorine and TCC pre- and post- stagnation in F1 (summer) :
F re e c h lo rin e

0.35
• Microbial regrowth
(m g /L )

0.3

0.25 Free chlorine gradually occurred when chlorine

increased after flushing.
0.4
0.2 is depleted, especially in
0.3
24 0.15
h Free chlorine the first 100 mL after
0.2 0.1 depleted to stagnation.
0.05 < 0.02 mg/L
0.1
Free chlorine • Stagnation and
0
0
0.4 0.6 0.8 1 0 100 200 300 400 500 600 700 800 900 1000 flushing influences the
(c e lls /m L × 1 0 5 )

Open plot shows value < LOD

8.0.E+04 cell concentration  a
Total cell counts
7.0.E+04
TCC brief flushing prior the
6.0.E+04
TCC

120000
5.0.E+04
significantly consumption can be a
100000 increased
4.0.E+04 mitigation to remove
80000
24 h
60000
3.0.E+04
TCC gradually decreased after
bacteria.
2.0.E+04
40000
1.0.E+04 flushing.
20000
0.0.E+00
0
0.4 0.6 0.8 1 0 100 200 300 400 500 600 700 800 900 1000
Flushed volume (mL)
9
 1. Influence of stagnation to microbial regrowth
Influence of stagnation to cell concentrations
• Free chlorine, TCC and HPC pre- and post- stagnation (first 100 mL) in all faucets:
F re e c h lo rin e (m g /L )

TCC (cells/m L)
10
1E+06
6

105
0.4 Decay to < 0.02 Significant
mg/L 104
1E+05 increase of TCC (4-
0.3 220 folds)
103
0.2
1E+04
102
0.1
101
Free chlorine p= 10 -23 Total cell counts p= 10 -7
0 1E+03
10 0
Pre-stagnation (n= 26) Post-stagnation (n= 26) Pre-stagnation (n= 26) Post-stagnation (n= 26)
HPC (cfu/mL)

1E+04
104
• TCC and HPC increased significantly (p<
1E+03
103 Japan’s water quality 0.05) and 6 samples were above the HPC
guideline guideline.
1E+02
102 • Stagnation greatly affects chlorine decay and
Significant microbial regrowth  may come from the
1E+01
101 increase of HPC
growth of bacteria in bulk water or biofilm
HPC p= 10 -4
10 0
1E+00
detachment5.
Pre-stagnation (n= 26) Post-stagnation (n= 26)

10
 1. Influence of stagnation to microbial regrowth
Influence of stagnation to microbial community
• Microbial community of pre-stagnation samples in all faucets:
100%

• Microbial
Pseudomonas spp.
80% community of
pre-stagnation
Relative abundance (%)

60% samples in winter

Phreatobacter. spp was different.
40%

20%

0%

Others (<5%) Betaproteobacteria-DQ395705 Sediminibacterium

Dechloromonas Acinetobacter Planctomycetaceae
Unclassified betaproteobacteria Deltaproteobacteria-FM253606 Uclassified comamonadaceae
Cyanobacteria-AY957901_s Cyanobacteria-HM445329 Reyranella
Cyanobacteria-JN616139 Methylobacterium Pseudomonas
Cyanobacteria-PAC000053 Sphingomonas Cyanobacteria-AY945890
Phreatobacter

11
 1. Influence of stagnation to microbial regrowth
Influence of stagnation to microbial community
• Microbial community of post-stagnation samples in all faucets
100%
• Microbial community of
Pseudomonas spp. post-stagnation was
80%
different with pre-
Relative abundance (%)

stagnation samples,
60%
especially in summer,
autumn and spring.
40%
• Stagnation influences
the shift of microbial
20% community.

0%

Others (<5%) Unclassified Babela

Peredibacter
Unlcassified Acidiferrobacteraceae
Unclassified Cenarchaeaceae
Dechloromonas
Mycobacterium
Cyanobacteria-PAC000053_g
Immundisolibacter
Unlassified Rhodospirillaceae
Terrimonas
Unclassified Comamonadaceae
Sphingomonadaceae-AY957890
Porphyrobacter

12
 1. Influence of stagnation to microbial regrowth
Influence of stagnation to microbial community
• Top bacteria that regrow after stagnation in each faucet (based on absolute changes):
Copies/mL of bacteria= % of bacteria × copies/mL of total bacteria (16S rRNA gene)

Top bacteria
Winter Pseudomonas
Other seasons Sphingomonas
Sediminibacterium,
Comamonadaceae,
Dechloromonas,
Cyanobacteria

• Some species of Pseudomonas spp. can

proliferate at temperature as low as 4°C6.
• Sphingomonas spp. dominated in F1, F4
and F5 (faucets with high-use-frequency that
is frequently exposed to free chlorine) 
Sphingomonas is resistant to chlorine7
• Dechloromonas spp. was dominated in F7
(faucets with low-use-frequency which might
1E+01 1E+020 1E+03 1E+04 have low oxygen)  they could grow under
10 101 10 2
103 104
anaerobic conditions8.
Changes in copies/mL Microbial regrowth is very site-specific.
13
 1. Influence of stagnation to microbial regrowth
Occurrence of clinically important opportunistic pathogens
• Clinically important opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa and
Acanthamoeba spp.) were below the LOQ (5×10-3 copies/mL) for all samples pre- and
post- stagnation  health risk is low.
• However, Legionella and Mycobacterium spp. might contain other opportunistic pathogens,
and thus their abundance was also quantified.
Mycobacterium and Legionella’s copies number pre- and post stagnation:
1E+02 1E+01
Copies/mL

10 3
10 2

102 101
1E+01

Copies/mL
101
100
100 Significant increase
1E+00 Significant increase of
of Mycobacterium Legionella
10-1
10-1
Mycobacterium spp. p= 0.005 Legionella spp. p= 0.014
10-2
1E-01 10-2
1E+00
Pre-stagnation (n= 26) Post-stagnation (n= 26) Pre-stagnation (n= 26) Post-stagnation (n= 26)

• Mycobacterium and Legionella spp. significantly increased in the first 1 L after 24 h

stagnation (p< 0.05) regrowth in stagnant water, detachment from biofilms or the release
from free-living amoeba1. 14
 1. Influence of stagnation to microbial regrowth
Composition of Mycobacterium and legionella spp. after stagnation
• Nanopore sequencing was able to reveal the species of Mycobacterium and
Legionella spp., and pathogenic species associated with those genera was
detected after stagnation.
Occurrence of of pathogenic Mycobacterium:
Name of species No. of Relative abundance Potential health risk
detected to Mycobacterium
samples spp. (%)
M. gordonae 4/14 3.1 Pulmonary infection to immunocompetent patient 9
M. haemophilum 1/14 0.2 Skin infection and arthritis to immunocompromised
patient10
Occurrence of of pathogenic Legionella:
Name of species No. of Relative abundance to Potential health risk
detected Legionella spp. (%)
samples
L. feelei 13/14 47 Legionnaires’ disease11
L. waltersii 4/14 12 Penumonia
L. maceachernii 3/14 3.5 Pneumonia to immunocompromised patient 12
L. feelei was frequently detected in most of the faucets after
L. micdadei 1/14 0.3 Legionnaires’ disease11
stagnation  health risk should be considered.
15
2. DOM compositions released from pipes promoting microbial regrowth

Experimental methods:
1. Pipe migration assay to screen DOM released from pipes
Pipe pieces
Pipe materials:
Incubation 1. New cross-linked polyethylene (PE)
24 h, 25oC
2. New high density polyethylene: (HDPE)
3. New polyvinyl chloride (PVC)
4. Negative control (w/o pipes)
Milli-Q with Post-migration
and without Cl2 samples Pipes were disinfected with NaClO for 1-2 h.
(n= 3) (n= 3)
DOM composition analysis:
Peaks newly
1. SPE with Bond Elut PPL :
released after
DOM composition Analysis: incubation concentration from 750 mL10 mL
DOM molecules in Methanol
Peak intensity

Peak intensity

migrated from 2. Orbitrap MS

pipes • Column separation with
InertSustain® AQ-C18
• ESI negative ionization.
m/z m/z • Molecular formulae assignment:
Milli-Q Post-migration samples Compound discoverer 3.0 software.
16
2. DOM compositions released from pipes promoting microbial regrowth

2. Regrowth potential assay to screen BOM candidates promoting regrowth

Regrowth potential assay (post-migration and autoclaved drinking water)

Inoculated assay (I) Non-inoculated assay (NI) as control

Inoculum (bacteria from

faucet)+inorganic media Inorganic media

Incubation Incubation
3 d, 25oC 3 d, 25oC

Cl2 = 0
Cl2 = 0
mg/L
mg/L

Post-migration samples Post-migration samples

(n= 2) (n= 1)

BOM screening analysis:

DOM decreased their peak intensity > 10%
Peak intensity

Peak intensity

in inoculated assay, but stable in non-

inoculated assay  BOM candidate

m/z m/z
Post-migration samples 17
2. DOM compositions released from pipes promoting microbial regrowth

Chlorine decay and DOC migration from pipes

Free chlorine pre- and post-migration DOC in post-migration:
(in chlorinated assay):

D O C m ig r a tio n (μ g /c m 2 )
Without Cl With Cl
0.4
PE- with Cl
released higher
0.3 DOC

0.2
F r e e c h lo rin e (m g /L )

Pre-migration Post-migration 0.1

<LOQ (0.1 mg/L DOC)

0
PE HDPE PVC Neg

• Free chlorine decay in all pipes> Negative control (p< 0.05) contact of water with pipes
accelerate the chlorine decay.
• DOC released from PE > HDPE and PVC. The release of DOC was dependent on pipe
material.
• PE–with Cl > PE-without Cl (p< 0.05) chlorine accelerates the release of DOC.

18
2. DOM compositions released from pipes promoting microbial regrowth

DOM compositions released from pipes

N u m b e r o f a s s ig n e d fo r m u la e

Number of molecular formulae of DOM released from pipes:

Cl-containing CHOS CHONS CHON CHO

600

500

400

300

200

100
NA NA
0

• DOM compositions of PE and PVC was different.

PE: CHO and Cl-containing formulae; PVC: CHOS formulae.
• N- and S- compounds might be derived from antioxidants and heat stabilizers 13,14.
• Cl-containing formulae was dominant in PE with chlorine  Chlorine affects the
DOM compositions released from PE.
19
2. DOM compositions released from pipes promoting microbial regrowth

DOM compositions released from pipes

• Venn diagram of the number of DOM molecular formulae released from pipes:
PE PE PVC PVC
Without Cl With Cl Without Cl With Cl • Several DOM component
released due to the
addition of chlorine.
65 238 209 68 306 131

• Top 5 DOM molecular formulae released from pipes:

PE PVC
• The top DOM component
of PE with and without
Without Cl With Cl Without Cl With Cl
chlorine was different,
C18H26O3 C19H37O5NCl2 C11H16O5S3 C11H16O5S3
while PVC was identical in
C18H24O3 C18H26O4 C10H12O5S2 C10H12O5S2
two conditions.
C21H37O4N C19H28O6SCl2 C10H14O6S3 C10H14O6S3 • Chlorine alters DOM
C13H10O2 C18H27O4Cl C15H24O5S2 C15H24O5S2 compositions released from
C14H30O5S C17H34O4NCl C18H18O3S3 C18H18O3S3 PE.
20
2. DOM compositions released from pipes promoting microbial regrowth

Effect of DOM released from pipes to regrowth potential

TCC pre- and post- incubation of regrowth potential assay:
Pre-incubation Post-incubation
1400000

1200000
TCC increased in the water contained organic matter
1000000
released from pipes  DOM released from pipes could
800000
promote microbial regrowth.
m
C
C

)
×
T

L
c
e

1
0
ll
(

600000

400000

200000
Control Control Control Control Control Control Control
0

• TCC in two conditions (with and without chlorine) was not different  DOM released from
pipes due to Cl2 might not be biodegradable.
• Based on the growth yield of 107 cells/g C15, BOM consumed for regrowth: 90, 18 and 39 μg
C/L in PE, HDPE and PVC, respectively.

21
2. DOM compositions released from pipes promoting microbial regrowth
N u m b e r o f a s s i g n m o l e c u l a r fo r m u l a e

BOM candidates promoting microbial regrowth

N u m b e r o f a s s ig n e d f o r m u la e
Total number of BOM candidates from PE and PVC: Elemental compositions of BOM candidates:

BOM candidates: peak intensity

after incubation decreased > 10%
CHO CHON CHONS
Non-BOM candidates BOM candidates 120 CHOS Cl-containing
600
100
500
80
400
60
300
40
200
20
100 NA NA NA
0
NA NA NA
0
1 2 3 4

• 15 – 40% of number of DOM formulae released from pipes can be biodegradable.

• BOM compositions of PE and PVC was different, while BOM compositions of PVC in
two conditions was identical.
• Pipe material affects BOM composition, while chlorine addition might not influence
the composition.
22
 Conclusions and Future plan

Important finding

• Significant increase of TCC and shifts of microbial community compositions after

stagnation was observed, indicating that stagnation greatly influences
microbial regrowth. Microbial regrowth and microbial community compositions
are very site-specific.
• Clinically important opportunistic pathogens (L. pneumophila, M. avium, P.
aeruginosa and Acanthamoeba spp.) were not detected in all samples,
suggesting that the health risk might be low.
• Contact of water with pipe material during stagnation accelerates the chlorine
decay and DOC migration. Some fractions of DOC migrated from pipes were
found to be biodegradable as it could promote microbial regrowth.

Future plan

• To evaluate the effect of organic matter released from PE, PVC and HDPE to
microbial community composition, by applying NGS.
23
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 Appendix

To semiquantitavely compare the peak intensity before and after incubation, the
reduction of intensity with decreasing concentration of samples was evaluated.
Peak intensity reduction in non-diluted (100) and diluted samples
Relative peak intensity to non-diluuted sample (%)
120

100

80

60

40

20

-
100 90 80 70 60 50 40 30 20 10
Concentration

Using t-test analysis, it was revealed that the peak intensity in non-diluted samples
was significantly difference with 10%-diluted samples (p< 0.05), and thus 10%
was set as criteria to determine the changes of peak intensity after incubation.
25