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Opportunistic Pathogens and the Brain-eating Amoeba, Naegleria fowleri, in Reclaimed Water,
Municipal Drinking Water, and Private Well Water
Laurel E. Strom
Thesis submitted to the faculty of Virginia Polytechnic Institute and State University in partial
fulfillment of the requirements for the degree of
Master of Science
in
Environmental Engineering
Amy J. Pruden-Bagchi
Marc A. Edwards
Joseph O. Falkinham, III
August 28, 2017
Blacksburg, Virginia
Keywords: opportunistic pathogens, Naegleria fowleri, reclaimed water, drinking water, private
well water
Opportunistic Pathogens and the Brain-eating Amoeba, Naegleria fowleri, in Reclaimed Water,
Municipal Drinking Water, and Private Well Water
Laurel E. Strom
Abstract
Opportunistic pathogens (OPs) are of special concern for immunocompromised populations and
are known to grow in both drinking water and reclaimed water (i.e., non-potable recycled water)
distribution systems, with aerosol inhalation and other non-ingestion exposures that are not
addressed by existing regulatory frameworks. Factors enabling the growth of OPs in water
distribution and premise (i.e., building) plumbing systems distributing reclaimed and other water
sources systems are poorly understood especially for the emerging OP, Naegleria fowleri (i.e.
brain-eating amoeba). Three phases of investigation were carried out to identify factors that
facilitate the growth of OPs in main distribution and premise plumbing systems, with particular
attention on reclaimed water systems, aging water mains, and private well systems. Phase one
examined the role of biological treatment to remove organic carbon and disinfectant type on the
occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed
water distribution systems were employed to systematically examine the effects of prior granular
activated carbon (GAC) biofiltration of the water; chlorine, chloramines, or no disinfectant, and
water ages ranging up to 5 days. The second and third phases of research explored the role of
nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal
drinking water from a city grappling with aging water infrastructure and untreated private well
water.
Results from the simulated reclaimed water distribution systems suggested that biologically-
active GAC filtration may unintentionally select for specific OPs, contrary to expectations and
experiences with oligotrophic conditions in potable water systems. While GAC biofiltration was
associated with lower total bacteria and Legionella spp. gene markers, there were no apparent
benefits in terms of other OPs analyzed. Similarly, disinfectant treatments successful for
controlling OPs in potable water were either ineffective or associated with increased levels of
OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water examined.
In the potable water study, it was possible to recreate conditions associated with growth of N.
fowleri in the aged main distribution system from where the water for the experiment was
collected; including corroding iron mains, nitrification, and disinfectant decay. While the effects
of nitrification could not be confirmed, there was a clear association of iron corrosion with N.
fowleri proliferation. The role of iron was explored further in what, to the author’s knowledge,
was the first study of N. fowleri in private wells. Analysis of 40 wells found correlations
between N. fowleri and stagnant iron levels, further supporting the hypothesis that iron corrosion
or iron encourages the growth of N. fowleri, and, because wells are not routinely disinfected, not
necessarily as a result of promoting disinfectant decay. As this study took place following a
major flooding event, it provided insight not only into how surface water contamination may
influence private well water microbial communities, but also added to the understanding that
current recommendations for disinfecting private wells are inadequate and standards should be
implemented to aid homeowners in the event of flooding.
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This exploratory research illuminated several factors influencing the OP growth in a range of
water systems. Identifying key variables that control growth is crucial to improving the safety of
these systems.
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Opportunistic Pathogens and the Brain-eating Amoeba, Naegleria fowleri, in Reclaimed Water,
Municipal Drinking Water, and Private Well Water
Laurel E. Strom
General Audience Abstract
Water borne bacteria that effect the immune systems of the sick, known as opportunistic
pathogens (OPs), have become a major heath concern. These organisms are known to grow in
drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems yet
there are no regulations aimed at prevention. There is also limited knowledge on how premise
plumbing and water sources effect the growth, population, and risk of infection by OPs,
especially for Naegleria fowleri (i.e. brain-eating amoeba). An investigation was carried out in
three parts to determine what influences the growth of OPs in water distribution and household
plumbing systems, with particular attention to reclaimed water, municipal drinking water, and
private well systems. Phase one examined the role of biological treatment to remove organic
carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water.
Laboratory-scale simulated reclaimed water distribution systems were used to examine the
effects of granular activated carbon (GAC) biofiltration of the water, disinfectants (chlorine,
chloramines, or no disinfectant), and water ages ranging zero to five days. The second and third
phases of research explored the role of nitrification, iron corrosion, and disinfectant on the
growth of N. fowleri both in municipal drinking water from a city with aging water infrastructure
and untreated private well water. Results from the simulated reclaimed water distribution
systems suggested that biologically-active GAC filtration may allow for the growth of specific
OPs. While GAC biofiltration was associated with lower total bacteria and Legionella spp.,
there were no apparent benefits in reducing the presence of other OPs. Similarly, common
disinfectant treatments for preventing OPs in drinking water were either ineffective or increased
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levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water. In
the drinking water study, conditions were introduced to grow N. fowleri in aged drinking water
distribution systems with the additions of corroding iron, nitrification (using nitrifying bacteria),
and disinfectant. While the effects of nitrification could not be confirmed, there was a clear
relationship between iron corrosion and N. fowleri growth. The role of iron was explored further
in what, to the author’s knowledge, was the first study of N. fowleri in private wells. Forty wells
were examined and the relationships between N. fowleri and stagnant iron levels supported the
hypothesis that iron corrosion or iron increases the growth of N. fowleri. As this study took place
following a major flooding event, it provided data not only into how surface water contamination
may influence private well water microbial communities, but also added to the understanding
that current recommendations for disinfecting private wells are inadequate and standards should
be implemented to aid homeowners in the event of flooding.
This exploratory research highlighted several variables that may allow for the growth of OPs in a
range of water systems. Identifying key variables that control growth is crucial to improving the
safety of these systems.
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Acknowledgements
This thesis would not have been possible without the constant encouragement, patience, and
guidance of Dr. Amy Pruden, as well as the invaluable input, support, and encouragement of Dr.
Marc Edwards. Their thoughtfulness and kindness will always be appreciated. I would also like
to thank Dr. Joseph Falkinham III for his support as a part of this committee.
This work was completed due to the contributions of Ni (Joyce) Zhu, Dr. Dongjuan Dai, Dr.
Kelsey Pieper, and Dr. Adrienne Katner, whom I would like to thank for their hard work and
assistance. I also would like to thank Robert Bielitz and Kandace Donaldson for their hard work
maintaining and analyzing data from the reclaimed water rigs. I would like to thank Jacob Bond
and Connor Brown for their help with water changes for the batch reactors. I must also thank
Owen Strom for his assistance in all the projects, endless patience, and for reviewing my work.
I would like to thank Corona Environmental Consulting, for their support of my work on
Naegleria fowleri in drinking water. Specifically, I must thank Dr. Sheldon Masters for
supporting and guiding my research, Randi McCuin for introducing me to amoeba research and
providing necessary cultures, and Jake Causey for collecting water from Louisiana at a moment’s
notice.
Lastly, I would like to thank the rest of the Pruden/Edwards lab (namely, Dr. William Rhoads,
Emily Garner, Pan Ji, and Dr. Jeff Parks) for their constant guidance, assistance, and patience.
None of this work would have been possible without them and I hope they have gained from this
experience as well.
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Table of Contents
Abstract .......................................................................................................................................... ii
General Audience Abstract ........................................................................................................ vii
Acknowledgements ..................................................................................................................... vii
Table of Contents ....................................................................................................................... viii
Table of Tables ............................................................................................................................. xi
Table of Figures........................................................................................................................... xii
Chapter 1: Introduction ............................................................................................................... 1
1.1 Opportunistic Pathogens ......................................................................................................................... 1
1.1.1 Consequences & Significance in Reclaimed Water ............................................................................. 1
1.2 Naegleria fowleri .................................................................................................................................... 3
1.2.1 Consequences and Significance in Drinking Water ........................................................................ 5
1.2.2 Potential for OP Occurrence in Private Well Water Settings ......................................................... 6
1.3 Thesis Overview ..................................................................................................................................... 7
1.4 Attributions ............................................................................................................................................. 9
1.5 References ............................................................................................................................................. 10
Chapter 2. Effect of biofiltration and disinfectant type on occurrence of opportunistic
pathogens in simulated reclaimed water distribution systems ............................................... 18
2.1 Introduction ........................................................................................................................................... 18
2.2 Materials and Methods .......................................................................................................................... 23
2.2.1 Simulated Reclaimed Water Distribution Systems (SRWDSs) ....................................................... 23
2.2.2 Influent Water Treatment and Water Changes .............................................................................. 23
2.2.3 Sample Collection .......................................................................................................................... 26
2.3.4 DNA extraction & qPCR ............................................................................................................... 26
2.2.5 Water Chemistry Parameters ........................................................................................................ 27
2.2.6 Statistical analysis ......................................................................................................................... 28
2.3 Results & Discussion ............................................................................................................................ 28
2.3.1 Water Chemistry data .................................................................................................................... 28
2.3.2 Relationships between Targeted Gene Markers for OPs ............................................................... 31
2.3.3 Effect of AOC on Targeted Gene Markers for OPs ....................................................................... 34
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2.3.4 Effect of Disinfectant on Targeted OP Gene Markers ................................................................... 37
2.3.5 Effect of Water Age on Targeted Gene Markers for OPs .............................................................. 39
2.4 Conclusions ........................................................................................................................................... 40
2.5 Acknowledgements ............................................................................................................................... 42
2.6 References ............................................................................................................................................. 43
Appendix A ................................................................................................................................................. 50
Chapter 3: Effects of pipe material on the growth and survival of Naegleria fowleri in
drinking water ............................................................................................................................. 53
3.1 Introduction ........................................................................................................................................... 53
3.2 Materials & Methods ............................................................................................................................ 56
3.2.1 Simulated warm water batch reactors (SWWBR) .......................................................................... 56
3.2.2. Influent water and water changes................................................................................................. 56
3.2.3 Water Chemistry Analyses ............................................................................................................. 58
3.2.4 Microbiological Analyses .............................................................................................................. 59
3.2.5 Statistical Analysis ......................................................................................................................... 60
3.3 Results & Discussion ............................................................................................................................ 60
3.3.1 Nitrification ................................................................................................................................... 60
3.3.2. Effect of iron ................................................................................................................................. 61
3.3.3. Disinfectant conditions ................................................................................................................. 62
3.3.4. Inhibition Troubleshooting ........................................................................................................... 64
3.4 Conclusions ........................................................................................................................................... 65
3.5 Acknowledgements ............................................................................................................................... 66
3.6 References ............................................................................................................................................. 67
Chapter 4: Naegleria fowleri in private well water after major flooding .............................. 71
4.1 Introduction ........................................................................................................................................... 71
4.2 Methods................................................................................................................................................. 75
4.2.1 Study Design .................................................................................................................................. 75
4.2.2 Sample Analysis ............................................................................................................................. 76
4.2.3 DNA extraction & qPCR ............................................................................................................... 77
4.2.4 Statistical Analysis ......................................................................................................................... 77
4.3 Results & Discussion ............................................................................................................................ 77
4.3.1. Correlations among bacteria ........................................................................................................ 77
4.3.2 Naegleria fowleri & iron ............................................................................................................... 80
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4.3.3 Survey data correlations ................................................................................................................ 82
Table 4.2. Homeowner reported maintenance before and after flooding. Error! Bookmark not
defined.
4.4 Conclusions ........................................................................................................................................... 84
4.5 Acknowledgements ............................................................................................................................... 85
4.6 References ............................................................................................................................................. 86
Appendix B ................................................................................................................................................. 93
Chapter 5: Conclusions and Final Thoughts ............................................................................ 95
5.1 Conclusions and Contributions ............................................................................................................. 95
5.1.1 Reclaimed Water ............................................................................................................................ 95
5.1.2 Naegleria fowleri ........................................................................................................................... 96
5.2 Future Work .......................................................................................................................................... 97
5.2.1 Reclaimed Water ............................................................................................................................ 97
5.2.2 N. fowleri in drinking water .......................................................................................................... 98
5.2.2 N. fowleri in private well water ..................................................................................................... 98
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Table of Tables
Table 2.1. Logistics of experiment. (a) Conditions of each SRWDS. (b) Timeline of experiment
including sampling dates....................................................................................................... 25
Table 2.2. Water Chemistry parameters assessed with references to APHA Standard Method or
published method. ................................................................................................................. 27
Table 2.3. Spearman rank correlation (⍴) values for each of the highest quantified genes assessed
and associated p values. ........................................................................................................ 33
Table 2.4. Percentage of samples positive for a gene from each SRWDS conditions.. ............... 34
Table 2.5. qPCR primers, probes, and assay conditions used in this study. ................................ 50
Table 3.1. Water Chemistry parameters assessed with references to APHA Standard Method or
other published standard method. ......................................................................................... 59
Table 3.2. Comparison of 24 replicate PVC only and PVC with iron reactors. .......................... 62
Table 3.3. Experimental conditions for second phase of experiment. ......................................... 63
Table 3.4. Summary of positive detections via qPCR in the iron reactors following shifting them
to the indicated conditions for the time period indicated.. .................................................... 64
Table 4.1. Positive detection of bacteria and OP DNA in three sample types. ............................ 78
Table 4.2. Homeowner reported maintenance before and after flooding ..................................... 84
Table 4.3. Maintenance, treatment, or action reported before and after flooding.. ...................... 84
Table 4.4. qPCR primers, probes, and assay conditions used in this study. ................................ 93
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Table of Figures
Figure 1.1. Life cycle of N. fowleri and route of infection. ........................................................... 3
Figure 2.1. (a) Schematic of simulated reclaimed water distribution system (SRWDS) (b) Photo
of actual SRWDS set up with all six SRWDSs in temperature-controlled room. ................ 24
Figure 2.2 (a) Dissolved Oxygen (mg/liter) over time in all SRWDSs (b) Chlorine residuals
(mg/liter) in chlorine-fed (free chlorine residual) and chloramine-fed (total chlorine
residual) SRWDSs over time. ............................................................................................... 29
Figure 2.3. Assimilable organic carbon (AOC) over time for each SRWDS. ............................. 31
Figure 2.4. Four most abundant gene targets quantified by qPCR in this study, plotted as a
function of water age in the. SRWDS bulk water and biofilm are plotted in the top and
bottom, respectively, and Low and High AOC on the left and right, respectively, of each
tile. (a) Total Bacteria (16S rRNA) (b) Legionella spp. (c) Mycobacterium spp. (d)
Acanthamoeba spp. ............................................................................................................... 36
Figure 3.1. 125 mL simulated warm water batch reactors containing PVC only (left) and PVC
with iron coupons (right). ..................................................................................................... 56
Figure 4.1. Comparison of log10(16S rRNA) and log10(N. fowleri+1) gene copies. No
correlation was found between total bacterial DNA and N. fowleri (Spearman’s ρ=0.29,
p>0.05). ................................................................................................................................. 80
Figure 4.2. Comparison of log10(N. fowleri+1) gene copies versus first draw (cold stagnant)
total iron concentrations.. ...................................................................................................... 81
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Chapter 1: Introduction
1.1 Opportunistic Pathogens
Opportunistic pathogens (OPs), in the drinking water context, are those microorganisms that can
reside in the low nutrient conditions characteristic of tap water and cause potentially life-
threatening disease in those exposed, particularly those with compromised immune systems (J.
Falkinham, 2015; Pruden, Edwards, & Falkinham III, 2013; Yoder et al., 2008). OPs are natural
residents of drinking water distribution systems, not contaminants like fecal organisms, and,
therefore, can be found in drinking water systems worldwide (J. Falkinham, 2015; Pruden et al.,
2013). OPs of key concern in the United States include, Legionella pneumophila,
Mycobacterium avium, Pseudomonas aeruginosa, and the free-living amoebae (FLA),
Acanthamoeba spp., and Naegleria fowleri, among others. While drinking water regulations are
generally designed to address fecal pathogens, a significant route of infection for the bacterial
OPs mentioned above (L. pneumophila, M. avium, P. aeruginosa), is inhalation through
aerosolization, which can occur through a variety of uses of water including showering and
washing dishes (Addiss et al., 1989; J. O. Falkinham, Norton, & Mark, 2001; Gensberger et al.,
2014; Lee et al., 2011; Leoni et al., 2005).
1.1.1 Consequences & Significance in Reclaimed Water
As freshwater resources continue to be depleted, drought-stricken and other water-strapped areas
around the world have been turning with increasing frequency towards reclaimed water as an
alternate potable and non-potable water source. As it is not intended for human consumption,
reclaimed water can be direct effluent from wastewater treatment or be subject to additional
treatments, often with additional disinfection a requirement, as per state regulations, prior to use
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in agriculture (i.e. irrigation), recreation (i.e. watering lawns, golf courses, making snow, etc.), or
other uses such as flushing toilets (Garner, Zhu, Strom, Edwards, & Pruden, 2016), among other
purposes (Bartrand, Causey, & Clancy, 2014; US EPA, 2012). Jjemba et al. (2010) defines
reclaimed water as “effluents that have undergone a combination of physical, chemical, and
biological treatments in engineered systems that utilize wastewater treatment technologies to
remove suspended solids, dissolved solids, organic matter, nutrients, metals, and pathogens.”
Therefore, reclaimed water quality and corresponding human health risk is highly dependent on
those treatments used upstream during wastewater treatment.
As a result of having lower treatment standards than drinking water, conditions in reclaimed
water distribution systems make it more suitable for the growth of bacteria and, potentially,
opportunistic pathogens (Jjemba et al. 2010b). OPs in drinking water are known to present a
substantial risk when conditions are suitable for regrowth in drinking water distribution systems,
with about 6000 cases of Legionnaire’s disease per year (reported in 2015) and 19,600 cases of
pulmonary disease per year due to M. avium (2010) in the U.S. (CDC, 2016; Prevots et al.,
2010). Thus it is important to understand if conditions unique to distributing reclaimed water
could exacerbate risk of illness related to opportunistic pathogens.
Disinfectant residual and assimilable organic carbon (AOC) levels have been identified as key
factors that can limit regrowth of opportunistic pathogens and other microbes (Jjemba et al.
2010a). These two variables go hand-in-hand particularly when it comes to treatment of waters
with high organic carbon, such as reclaimed water. For one, while disinfectant is meant to
inactivate microorganisms, inadequate disinfection residuals can select for OPs against fecal
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bacteria and can promote chlorine resistance, where organisms are able to survive under
conditions with low disinfectant residuals (De Jonckheere & Van De Voorde, 1976; García,
Jones, Pelaz, Millar, & Abu Kwaik, 2007; Francine Marciano-Cabral, Jamerson, & Kaneshiro,
2010). AOC, by definition, is a readily-available nutrient source for microorganisms, but,
interestingly, application of disinfectant can actually tend to increase AOC in water (Falkinham
et al. 2001; Jjemba et al. 2010b). This is due to increased destruction of cells, breaking down
complex organic matter into simper forms and providing new sources of AOC. Studies have
shown inverse correlations between AOC levels and microbial activity due to the consumption of
nutrients by present microbes (Thayanukul, Kurisu, Kasuga, & Furumai, 2013; Weinrich,
Jjemba, Giraldo, & Lechevallier, 2010).
1.2 Naegleria fowleri
N. fowleri is one particular OP of emerging concern in water distribution systems and premise
plumbing. Known as “the brain-eating amoeba,” N. fowleri is most often found in warm surface
waters and is traditionally a concern for recreational exposures, such as head-dunking, in hot
springs. Being an ameboflagellate, it is able to take the form of a cyst, trophozoite, or flagellate,
and causes the fatal disease primary amebic meningoencephalitis (PAM) when water in forced
FLAGELLATEDFORMCYST
TROPHOZOITE1
2 3
Mitosis
Figure 1.1. Life cycle of N. fowleri and route of infection.
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up the nasal cavity, facilitating nasal aspiration of the amoeba (Figure 1.1; John 1982).
Fortunately, PAM infections are very rare, with only 143 documented cases between 1962-2016,
but only four known survived individuals (>97% mortality). However, there is concern that
exposure risk is on the rise (Capewell et al., 2015; CDC, 2017). One cause of this concern is that
cases of PAM continue to occur in cooler locations in the United States with the northernmost
known case occurring in Minnesota in 2010 (Kemble et al., 2012). This has caused apprehension
as warming climate conditions could yield further cases in the coming years as average summer
temperatures climb. Of the 143 documented cases of PAM, seven have been confirmed to have
been caused by exposure to contaminated drinking water with other cases thought to also be
connected with drinking water, but not confirmed.
In the past, methods for detection of N. fowleri have involved culture with microscopy and
confirmation using a flagellation test. While providing a high level of accuracy, these methods
are time intensive, costly, and require specific skills to identify the amoeba. Recent
developments of molecular methods have provided faster and more accurate molecular detection
of N. fowleri DNA, allowing for confirmation tests in environmental samples and in suspected
PAM patients (Madarová, Trnková, Feiková, Klement, & Obernauerová, 2010; F. Marciano-
Cabral, MacLean, Mensah, & LaPat-Polasko, 2003; Mull, Narayanan, & Hill, 2013; Puzon,
Lancaster, Wylie, & Plumb, 2009). However, despite molecular methods improving the speed of
DNA confirmation, there are no rapid tests for enumerating viable N. fowleri and rigorous
culture methods are still required to confirm viable amoeba.
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1.2.1 Consequences and Significance in Drinking Water
A variety of OPs have been well-documented in municipal drinking water distribution systems
and premise plumbing. Factors such as water source, treatment methods, disinfection strategy,
pipe material, water temperature, distribution system length and age, and other physicochemical
variables all can influence the organisms that colonize drinking water systems.
N. fowleri is one OP that has gained increased attention due to recent detections in drinking
water, but most recent studies have focused on rapid detection of N. fowleri and disinfecting
colonized systems, rather than understanding the influences of physicochemical factors that
might have led to its growth or persistence in drinking water distribution systems (Bright,
Marciano-Cabral, & Gerba, 2009; Miller et al., 2015, 2016; Mull et al., 2013; Puzon et al., 2009).
In the past four years, several utilities in the state of Louisiana have confirmed the presence of N.
fowleri in their drinking water distribution system resulting in multiple cases of PAM (Louisiana
Department of Health, 2013a, 2013b, 2017). While cases are still rare, the presence of the
amoeba is alarming and should not be minimized.
It has been difficult to establish monitoring and management strategies to ensure the killing of N.
fowleri from distribution systems and premise plumbing, as its presence is sporadic and there are
no specific indicators of its occurrence. Previous studies are inconsistent in terms of the factors
identified that correlate with N. fowleri growth and occurrence. For example, Esterman et al.
(1984) and Tyndall et al. (1989) report correlations between water temperature (positive), air
temperature (positive), chlorine disinfectant residual (negative), and bacterial colony counts at 35
°C (via culture, positive). However, Sykora et al. (1983), Brown et al. (1983), and Bright et al.
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(2009) reported a lack of correlations between factors such as temperature, pH, and bacterial
counts, using similar methods. Therefore, it is unclear what indicators may be useful to describe
systems that are vulnerable to N. fowleri colonization. In their review, Bartrand et al. (2014)
suggest that a correlation between nitrification in distribution systems could contribute to N.
fowleri viability, particularly when utilities utilize chloramine disinfectant, but no study has yet
confirmed this hypothesis.
Strategies to eliminate N. fowleri in drinking water distribution systems currently involve
maintaining adequate chlorine residuals, with one study recommending a contact time (CT) of 30
mg/L-min (Trolio, Bath, Gordon, Walker, & Wyber, 2008). However, other studies show that a
higher CT is required (Sarkar & Gerba, 2016). These discrepancies make it difficult to set
standards for N. fowleri control and, as a result, utilities tend to vary in their responses to positive
detection of the amoeba in their distribution system.
1.2.2 Potential for OP Occurrence in Private Well Water Settings
Of all drinking water sources, private wells are by far the most under investigated for a variety of
reasons. As private well water quality is not regulated by EPA or any other authority, care and
maintenance is the responsibility of homeowners, who often do not have the resources or
education required to properly maintain their water quality (US EPA, 2017). Private well water
is also a challenging topic of study, as quality can vary depending on climate, geographical
location, aquifer type, well type, well owner actions, and surrounding sources of contamination,
among others. Therefore, it is difficult to identify the complex range of well water conditions
and combinations of conditions that are key to influencing the health of those exposed. It is
7
clear, however, that microbial contamination in private wells poses a significant health risk to
those who rely on private well water as their drinking water source (Allevi et al. 2013; Defelice
et al. 2016; Won et al. 2013).
Multiple studies have investigated total coliforms and E. coli in private well water (Allevi et al.,
2013; DeFelice, Johnston, & Gibson, 2016; Won, Gill, & LeJeune, 2013), as these are
standardized tests for regulatory monitoring intended to indicate fecal pollution and are widely
applied to recreational water and municipal drinking water. Each of these studies yielded a
similar conclusion: unacceptably high levels of coliforms and E. coli are often present in private
wells and these are leading to increased prevalence of gastrointestinal illnesses in residents.
However, fecal indicator organisms only represent a single facet of the microbial populations
that actually reside in private well water systems. Knowledge of OP occurrence in private well
water is extremely scarce and those that have investigated well water in any capacity (private or
high-volume use) have found that these waters can support the growth of OPs (Blair, Sarkar,
Bright, Marciano-Cabral, & Gerba, 2008; Richards et al., 2015). The fact that more work has not
been published investigating OPs in private well water presents a considerable knowledge gap
that merits research attention.
1.3 Thesis Overview
The goal of this research was to deepen the understanding of the role that the various factors
characterizing the microbial environments of different water sources and distribution systems
play in stimulating proliferation of OPs, with a particular focus on the vastly understudied N.
fowleri. Chapter 2 introduces OPs in a general framework and applies controlled, laboratory-
scale pipe rigs to systematically examine the effects of varying water chemistries on OP
8
occurrence in reclaimed water distribution systems. Chapter 3 dives deeper into key factors that
could influence the growth and viability of N. fowleri in municipal drinking water systems,
particularly those grappling with aging infrastructure, through a bench-scale experiment.
Chapter 4 takes this further by surveying N. fowleri in private well water, with emphasis on
private wells in warm southern regions of the United States.
The specific objectives of the research summarized in this thesis were to:
• Improve understanding of how OPs behave in reclaimed water distribution systems
(Chapter 2).
• Develop a framework to improve management of reclaimed water distribution systems to
protect against bacterial regrowth issues and OPs (Chapter 2).
• Investigate how factors such as pipe material, nitrification, and disinfection treatment
influence the growth of N. fowleri in a simulated drinking water system (Chapter 3).
• For the first time, survey N. fowleri in private well water following a major flooding
event (Chapter 4).
• Understand how varying physicochemical factors, homeowner actions, and geospatial
circumstances may contribute to the colonization of N. fowleri in private wells and
premise plumbing (Chapter 4).
The overall thesis should improve understanding of how various reclaimed water treatment
strategies can influence the presence of OPs and which conditions may be conducive to the
growth and survival of N. fowleri in both municipal and private well drinking water.
9
1.4 Attributions
Chapter 2: Effect of biofiltration and disinfectant type on occurrence of opportunistic pathogens
in simulated reclaimed water distribution systems
The simulated reclaimed water distribution systems investigated in this study were constructed
by Ni (Joyce) Zhu and Dr. Sheldon Masters. Ni Zhu was responsible for overseeing
maintenance and operation of the systems, as well as collecting and analyzing samples for water
quality parameters including chlorine, pH, dissolved oxygen (DO), nitrogen species, total
organic carbon (TOC), flow cytometry, assimilable organic carbon (AOC), and biological
activity reaction tests (BARTs). Kandace Donaldson contributed to quantitative polymerase
chain reaction (qPCR) assays for Acanthamoeba spp.
Chapter 3: Effects of pipe material on the growth and survival of Naegleria fowleri in drinking
water
All experiments and data analyses were performed by the author.
Chapter 4: Naegleria fowleri in private well water after major flooding
Molecular qPCR assays for 16S rRNA, Legionella spp., and Legionella pneumophila were
performed by Dr. Dongjuan Dai. Survey data was organized and compiled by Dr. Adrienne
Katner and Dr. Kelsey Pieper.
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1.5 References
Addiss, D. G., Davis, J. P., LaVenture, M., Wand, P. J., Hutchinson, M. A., & McKinney, R. M.
(1989). Community-acquired Legionnaires’ disease associated with a cooling tower:
evidence for longer-distance transport of Legionella pneumophila. American Journal of
Epidemiology, 130(3), 557–568.
Allevi, R. P., Krometis, L.-A. H., Hagedorn, C., Benham, B., Lawrence, A. H., Ling, E. J., &
Ziegler, P. E. (2013). Quantitative analysis of microbial contamination in private drinking
water supply systems. Journal of Water and Health, 11(2), 244.
http://doi.org/10.2166/wh.2013.152
Bartrand, T. A., Causey, J. J., & Clancy, J. L. (2014). Naegleria Fowleri: An Emerging Drinking
Water Pathogen. Journal - American Water Works Association, 106(10), E418–E432.
http://doi.org/10.5942/jawwa.2014.106.0140
Blair, B., Sarkar, P., Bright, K. R., Marciano-Cabral, F., & Gerba, C. P. (2008). Naegleria
Fowleri in Well Water. Emerging Infectious Diseases, 14(9), 1499–1500.
http://doi.org/10.3201/eid1409.080143
Bright, K. R., Marciano-Cabral, F., & Gerba, C. P. (2009). Occurrence of Naegleria fowleri in
Arizona drinking water supply wells. Source Journal (American Water Works Association),
101(11), 43–50. Retrieved from http://www.jstor.org/stable/41313663
Brown, T. J., Cursons, R. T. M., Keys, E. A., Marks, M., & Miles, M. (1983). The occurence and
distribution of pathogenic free-living amoebae in thermal areas of the North Island of New
Zealand. New Zealand Journal of Marine and Freshwater Research, 17, 59–69.
http://doi.org/10.1080/00288330.1983.9515987
Capewell, L. G., Harris, A. M., Yoder, J. S., Cope, J. R., Eddy, B. A., Roy, S. L., … Beach, M. J.
11
(2015). Diagnosis, Clinical Course, and Treatment of Primary Amoebic
Meningoencephalitis in the United States, 1937-2013. Journal of the Pediatric Infectious
Diseases Society, 4(4), e68–e75. http://doi.org/10.1093/jpids/piu103
CDC. (2016). Notice to Readers: Final 2015 Reports of Nationally Notifiable Infectious Diseases
and Conditions. MMWR Morb Mortal Weekly Report, 65(46), 1306–1321.
CDC. (2017). Parasites — Naegleria fowleri — Primary Amebic Meningoencephalitis (PAM) —
Amebic Encephalitis. Retrieved August 12, 2017, from
https://www.cdc.gov/parasites/naegleria/illness.html#four
De Jonckheere, J., & Van De Voorde, H. (1976). Differences in Destruction of Cysts of
Pathogenic and Nonpathogenic Naegleria and Acanthamoeba by Chlorine. Applied and
Environmental Microbiology, 31(2), 294–297.
DeFelice, N. B., Johnston, J. E., & Gibson, J. M. (2016). Reducing Emergency Department
Visits for Acute Gastrointestinal Illnesses in North Carolina (USA) by Extending
Community Water Service. Environmental Health Perspectives, 124(10), 1583–1591.
Retrieved from https://ehp.niehs.nih.gov/wp-content/uploads/124/10/EHP160.alt.pdf
Esterman, A., Dorsch, M., Cameron I, S., Roder, D., Robinson, B., Lake, J., & Christy, P.
(1984). The Association Of Naegleria Fowleri With The Chemical, Microbiological And
Physical Characteristics Of South Australian Water Supplies. Water Research, 18(5), 549–
553. Retrieved from http://ac.els-cdn.com.ezproxy.lib.vt.edu/0043135484902021/1-s2.0-
0043135484902021-main.pdf?_tid=85b14000-8132-11e7-8160-
00000aacb35e&acdnat=1502744145_337f21001b3888b10e640e3abebf1761
Falkinham, J. (2015). Common Features of Opportunistic Premise Plumbing Pathogens.
International Journal of Environmental Research and Public Health, 12(5), 4533–4545.
12
http://doi.org/10.3390/ijerph120504533
Falkinham, J. O., Norton, C. D., & Mark, W. (2001). Factors Influencing Numbers of
Mycobacterium avium , Mycobacterium intracellulare , and Other Mycobacteria in
Drinking Water Distribution Systems. Applied and Environmental Microbiology, 67(3),
1225–1231. http://doi.org/10.1128/AEM.67.3.1225
García, M. T., Jones, S., Pelaz, C., Millar, R. D., & Abu Kwaik, Y. (2007). Acanthamoeba
polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection.
Environmental Microbiology, 9(5), 1267–77. http://doi.org/10.1111/j.1462-
2920.2007.01245.x
Garner, E., Zhu, N., Strom, L., Edwards, M., & Pruden, A. (2016). A human exposome
framework for guiding risk management and holistic assessment of recycled water quality.
Environ. Sci.: Water Res. Technol., 2, 580–598. http://doi.org/10.1039/C6EW00031B
Gensberger, E. T., Polt, M., Konrad-Köszler, M., Kinner, P., Sessitsch, A., & Kostić, T. (2014).
Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of
microbial water quality. Water Research, 67, 367–376.
http://doi.org/10.1016/j.watres.2014.09.022
Jjemba, P. K., Weinrich, L. A., Cheng, W., Giraldo, E., & LeChevallier, M. W. (2010a).
Guidance document on the microbiological quality and biostability of reclaimed water and
distribution systems (WRF‐05‐002). Alexandria, VA: WateReuse Research Foundation .
Jjemba, P. K., Weinrich, L. A., Cheng, W., Giraldo, E., & LeChevallier, M. W. (2010b).
Regrowth of potential opportunistic pathogens and algae in reclaimed-water distribution
systems. Applied and Environmental Microbiology, 76(13), 4169–4178.
http://doi.org/10.1128/AEM.03147-09
13
John, D. T. (1982). Primary Amebic Meningoencephalitis and the biology of Naegleria fowleri.
Ann. Rev. Microbiol., 36, 101–23. Retrieved from
http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.36.100182.000533
Kemble, S. K., Lynfield, R., Devries, A. S., Drehner, D. M., Pomputius Iii, W. F., Beach, M. J.,
… Danila, R. (2012). Fatal Naegleria fowleri Infection Acquired in Minnesota: Possible
Expanded Range of a Deadly Thermophilic Organism. Clinical Infectious Diseases, 54,
805–809. http://doi.org/10.1093/cid/cir961
Lee, J. V., Lai, S., Exner, M., Lenz, J., Gaia, V., Casati, S., … Surman-Lee, S. (2011). An
international trial of quantitative PCR for monitoring Legionella in artificial water systems.
Journal of Applied Microbiology, 110(4), 1032–1044. http://doi.org/10.1111/j.1365-
2672.2011.04957.x
Leoni, E., De Luca, G., Legnani, P. P., Sacchetti, R., Stampi, S., & Zanetti, F. (2005). Legionella
waterline colonization: detection of Legionella species in domestic, hotel and hospital hot
water systems. Journal of Applied Microbiology, 98(2), 373–379.
http://doi.org/10.1111/j.1365-2672.2004.02458.x
Louisiana Department of Health. (2013a). CDC Testing Confirms Presence of Rare Ameba in
One DeSoto Parish Water System | Department of Health | State of Louisiana. Retrieved
July 12, 2017, from http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/2889
Louisiana Department of Health. (2013b). DHH Confirms Death of a Child Associated with Rare
Amoeba Found in St. Bernard Parish Home | Department of Health | State of Louisiana.
Retrieved July 12, 2017, from
http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/2862
Louisiana Department of Health. (2017). LDH Confirms Naegleria Fowleri Ameba in North
14
Monroe, Schriever Water Systems | Department of Health | State of Louisiana. Retrieved
July 12, 2017, from http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/4284
Madarová, L., Trnková, K., Feiková, S. A., Klement, C., & Obernauerová, M. (2010). A real-
time PCR diagnostic method for detection of Naegleria fowleri. Experimental Parasitology,
126, 37–41. http://doi.org/10.1016/j.exppara.2009.11.001
Marciano-Cabral, F., Jamerson, M., & Kaneshiro, E. S. (2010). Free-living amoebae, Legionella
and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA.
Journal of Water and Health, 8(1), 71–82. Retrieved from
http://apps.webofknowledge.com.ezproxy.lib.vt.edu/full_record.do?product=WOS&search_
mode=GeneralSearch&qid=1&SID=1E4GaJT6WUkFmAaClKe&page=1&doc=9
Marciano-Cabral, F., MacLean, R., Mensah, A., & LaPat-Polasko, L. (2003). Identification of
Naegleria fowleri in Domestic Water Sources by Nested PCR. Applied and Environmental
Microbiology, 69(10), 5864–5869. http://doi.org/10.1128/AEM.69.10.5864-5869.2003
Miller, H. C., Morgan, M. J., Wylie, J. T., Kaksonen, A. H., Sutton, D., Braun, K., & Puzon, G.
J. (2016). Elimination of Naegleria fowleri from bulk water and biofilm in an operational
drinking water distribution system. Water Research, 110, 15–26.
http://doi.org/10.1016/j.watres.2016.11.061
Miller, H. C., Wylie, J., Dejean, G., Kaksonen, A. H., Sutton, D., Braun, K., & Puzon, G. J.
(2015). Reduced Efficiency of Chlorine Disinfection of Naegleria fowleri in a Drinking
Water Distribution Biofilm. Environmental Science & Technology, 49(18), 11125–11131.
http://doi.org/10.1021/acs.est.5b02947
Mull, B. J., Narayanan, J., & Hill, V. R. (2013). Improved Method for the Detection and
Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic
15
Separation and Real-Time PCR. Journal of Parasitology Research, 2013, 8.
http://doi.org/10.1155/2013/608367
Prevots, D. R., Shaw, P. A., Strickland, D., Jackson, L. A., Raebel, M. A., Blosky, M. A., …
Olivier, K. N. (2010). Nontuberculous Mycobacterial Lung Disease Prevalence at Four
Integrated Health Care Delivery Systems. American Journal of Respiratory and Critical
Care Medicine, 182(7), 970–976. http://doi.org/10.1164/rccm.201002-0310OC
Pruden, A., Edwards, M. A., & Falkinham III, J. O. (2013). State of the Science and Research
Needs for Opportunistic Pathogens in Premise Plumbing. Water Research Foundation.
Denver, CO. Retrieved from http://www.waterrf.org/PublicReportLibrary/4379.pdf
Puzon, G. J., Lancaster, J. a, Wylie, J. T., & Plumb, J. J. (2009). Rapid Detection of Naegleria
Fowleri in Water Distribution Pipeline Biofilms and Drinking Water Samples.
Environmental Science & Technology, 43(17), 6691–6696.
http://doi.org/10.1021/es900432m
Richards, C. L., Broadaway, S. C., Eggers, M. J., Doyle, J., Pyle, B. H., Camper, A. K., & Ford,
T. E. (2015). Detection of Pathogenic and Non-pathogenic Bacteria in Drinking Water and
Associated Biofilms on the Crow Reservation, Montana, USA. Microbial Ecology.
http://doi.org/10.1007/s00248-015-0595-6
Sarkar, P., & Gerba, C. P. (2016). Control of Naegleria fowleri by chlorine and ultraviolet light.
Journal - American Water Works Association, 104(3), E173–E180.
Sykora, J. L., Keleti, G., & Martinez, A. J. (1983). Occurrence and Pathogenicity of Naegleria
fowleri in Artificially Heated Waters. Applied and Environmental Microbiology, 45(3),
974–979. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC242399/pdf/aem00172-0248.pdf
16
Thayanukul, P., Kurisu, F., Kasuga, I., & Furumai, H. (2013). Evaluation of microbial regrowth
potential by assimilable organic carbon in various reclaimed water and distribution systems.
Water Research, 47, 225–232. http://doi.org/10.1016/j.watres.2012.09.051
Trolio, R., Bath, A., Gordon, C., Walker, R., & Wyber, A. (2008). Operational management of
Naegleria spp. in drinking water supplies in Western Australia. Water Science and
Technology: Water Supply, 8(2), 207–215. http://doi.org/10.2166/ws.2008.063
Tyndall, R. L., Ironside, K. S., Metler, P. L., Tan, E. L., Hazen, T. C., & Fliermans2, A. C. B.
(1989). Effect of Thermal Additions on the Density and Distribution of Thermophilic
Amoebae and Pathogenic Naegleria fowleri in a Newly Created Cooling Lake. Applied and
Environmental Microbiology, 55(3), 722–732. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC184187/pdf/aem00096-0188.pdf
US EPA. (2012). EPA Guidelines for Water Reuse. Washington, D.C. Retrieved from
http://nepis.epa.gov/Adobe/PDF/P100FS7K.pdf
US EPA. (2017). Private Drinking Water Wells. Retrieved July 29, 2017, from
https://www.epa.gov/privatewells
Weinrich, L. A., Jjemba, P. K., Giraldo, E., & Lechevallier, M. W. (2010). Implications of
organic carbon in the deterioration of water quality in reclaimed water distribution systems.
Water Research, 44, 5367–5375. http://doi.org/10.1016/j.watres.2010.06.035
Won, G., Gill, A., & LeJeune, J. T. (2013). Microbial quality and bacteria pathogens in private
wells used for drinking water in northeastern Ohio. Journal of Water and Health, 11(3),
555–562. http://doi.org/10.2166/wh.2013.247
Yoder, J. S., Hlavsa, M. C., Craun, G. F., Hill, V., Roberts, V., Yu, P. A., … Beach, M. J.
(2008). Surveillance for Waterborne Disease and Outbreaks Associated with Recreational
17
Water Use and Other Aquatic Facility-Associated Health Events — United States, 2005–
2006. Surveillance Summaries Centers for Disease Control and Prevention, 57, 2–38.
Retrieved from www.cdc.gov/mmwr
18
Chapter 2. Effect of biofiltration and
disinfectant type on occurrence of opportunistic
pathogens in simulated reclaimed water
distribution systems1
2.1 Introduction
Water reuse is expanding in practice, especially in drought-stricken and other water-strapped
regions where fresh water resources are limited. However, given that reclaimed water is sourced
directly from wastewater treatment plant effluent, there may be special concerns with respect to
risks posed by microbial contaminants. Especially important to consider is that microbes
occurring in reclaimed water can be subject to regrowth in distribution systems, which are
typically required to convey the water to the point of use. The most common use of reclaimed
water is for agricultural or recreational purposes, but it has also been applied for direct and
indirect potable reuse. While direct potable reuse water is treated to high drinking water quality
standards, given that reclaimed water is not intended for drinking, a looser spectrum of
treatments is typically applied to match capital and maintenance costs of treatment to the
intended reuse purpose (US EPA, 2012). However, while reuse purposes include groundwater
and wetland recharge, reclaimed water is also commonly used for a variety of other purposes
including cooling towers (Addiss et al., 1989), spray irrigation (Brissaud, Blin, Hemous, &
Garrelly, 2008; Rosenberg Goldstein et al., 2014), fire protection (Borboudaki, Paranychianakis,
& Tsagarakis, 2005), and toilet flushing (Gerba, Wallis, & Melnick, 1975), among others
(Garner, Zhu, Strom, Edwards, & Pruden, 2016; US EPA, 2012). Each of these uses have the
1 This work is intended for submission to the peer-reviewed journal, Environmental Science and
Technology. See page 8 for attributions.
19
potential to enhance production of aerosols, inhalation of which is the main human exposure
route for opportunistic pathogens (OPs). Other non-potable exposures to organic and chemical
contaminants, as well as emerging concerns, such as antibiotic resistant bacteria (ARB) and their
resistance genes (ARGs), have also been cited (Garner et al., 2016; Pruden et al., 2013) .
Therefore, it is essential to consider potential routes of exposure beyond traditional ingestion and
determine how current treatment standards can influence the regrowth of pathogens in reclaimed
water distribution systems from the point of treatment to the point of use.
OPs subject to regrowth in water distribution systems and premise plumbing include Legionella
pneumophila, Pseudomonas aeruginosa, Mycobacterium avium, Acanthamoeba spp., and
Naegleria fowleri. These organisms are relatively well-described in drinking water infrastructure,
characterized by their abilities to survive under low nutrient conditions, form biofilms, and resist
low levels of chlorine disinfectant (Falkinham 2015). Given that aerosolization is a primary
exposure route of concern for OPs, especially for L. pneumophila, M. avium, and P. aeruginosa,
benchmarking of reclaimed water standards to drinking water standards may not necessarily
represent the implied safety factor of this approach. This is particularly concerning as reclaimed
water is routinely aerosolized during spray irrigation (Falkinham 2015; Wang et al. 2012) and
other applications, such as fire-fighting, snowmaking, and toilet flushing. Additionally, several
free living amoebae (FLA) such as Acanthamoeba, Naegleria, and Vermamoeba (nee
Hartmanella) vermiformis, have been identified as important hosts for pathogenic amoeba-
resisting microorganisms (ARM) (Corsaro, Pages, Catalan, Loret, & Greub, 2010; J. M. Thomas
& Ashbolt, 2011; V. Thomas, Blanc, Bille, & Greub, 2005; Vincent Thomas, Loret, Jousset, &
Greub, 2008). When phagocytized, ARM have the capacity to replicate within the digestive
20
vacuoles of FLA, where they are protected from chlorine or chloramine disinfectant residuals
and other surrounding environmental pressures (De Jonckheere & Van De Voorde, 1976;
Kilvington & Price, 1990; Loret et al., 2008; Storey, Winiecka-krusnell, Ashbolt, & Stenström,
2004). Additionally, relative to drinking water, reclaimed water typically contains much higher
levels of nutrients, which can facilitate the regrowth of bacteria in the distribution system (P K
Jjemba, Weinrich, Cheng, Giraldo, & LeChevallier, 2010a). Although the U.S. EPA, along with
several states, have outlined guidelines and regulations for reclaimed water treatment, this study
aims to understand how variations in treatment, seasonality, and physicochemical water
characteristics, such as organic carbon levels, temperature, disinfectant strategy, and water age,
influence the growth of OPs in reclaimed water distribution systems (US EPA 2012; Gerba &
Rose 2003).
One nutrient source that is essential for regrowth of heterotrophic microbes is assimilable
organic carbon (AOC). AOC, as measured according to a few laboratory assay variations, is
intended to represent the fraction of organic carbon that is most readily bioavailable to
microorganisms and can vary depending on influent total organic carbon concentration and
upstream water treatment (Liu et al., 2015; Van der Kooij, 1992). In order to limit regrowth of
heterotrophic organisms, a maximum concentration of 10 µg/liter AOC has been recommended
for drinking water distribution systems without disinfectant and 100 µg/liter for those with
disinfectant. However, it is critical to recognize that reclaimed water systems tend to have
significantly higher levels of AOC compared to drinking water, ranging from 505-918 µg/liter
with moderate treatment (Garner et al., 2016; Karim & LeChevallier, 2005; Thayanukul, Kurisu,
Kasuga, & Furumai, 2013; Van der Kooij, 1992; Volk & LeChevallier, 2000; Weinrich, Jjemba,
21
Giraldo, & Lechevallier, 2010). The organic matter characterizing reclaimed water is also
distinct in nature, being derived largely from cellular debris and soluble microbial products
associated with biological treatment of wastewater, whereas drinking water AOC largely stems
from decay of natural organic matter. Disinfectants, such as chlorine, are typically strong
oxidants and have been found to increase AOC levels. Water age also influences AOC levels,
correlating with an increase bacterial regrowth but decreases in AOC over time (Liu et al., 2015;
Ryu, Alum, & Abbaszadegan, 2005; Thayanukul et al., 2013; Weinrich et al., 2010). However,
the effect of AOC on OPs is still unclear and effects vary among reported studies in the
literature. While one study indicated a strong correlation between mycobacteria and AOC in
drinking water (AOC range: 17-234 µg/liter) (J. O. Falkinham, Norton, & Mark, 2001), another
suggested that low levels of AOC (5-10 µg/liter) do not affect mycobacterial growth.(van der
Wielen & van der Kooij, 2013) For example, L. pneumophila has been shown to survive well
under conditions with high AOC in reclaimed water, but are also capable of surviving with low
AOC conditions in drinking water, leading to questions about optimal conditions for colonization
of Legionella (P K Jjemba, Weinrich, Cheng, Giraldo, & LeChevallier, 2010b; van der Wielen &
van der Kooij, 2013).
As is the case in drinking water, the primary disinfection as typically employed by most existing
reclaimed water systems consists of chlorination using free chlorine or chloramination using
chloramine disinfectant (US EPA, 2012). Chlorine is a stronger oxidant than chloramine, but
produces harmful disinfection byproducts and is less stable than chloramine in distribution
systems (Neden et al., 1992; Norton & Lechevallier, 1997; Zhang & Edwards, 2009). However,
as chloramine disinfectant decays, it provides a source of ammonia for nitrifying bacteria, which
22
grow best in warm water conditions (>25 °C), with the nitrite produced during nitrification
accelerating chloramine decay (Valentine, 1984; Zhang et al., 2010). Warmer conditions are
also associated with more rapid decay of chlorine and chloramine disinfectant residual and the
subsequent growth of microorganisms (Vieira, Coelho, & Loureiro, 2004). Seasonal temperature
shifts have also been known to alter disinfection efficacy and therefore the microbial
communities in drinking water and reclaimed water distribution systems. OPs in particular, have
also been known to thrive under warm temperature conditions, punctuated by the apparent
seasonality of most OP infections (CDC, 2015; Patrick K Jjemba, Johnson, Bukhari, &
LeChevallier, 2015; Tyndall et al., 1989; Wang et al., 2017).
Water age is another key factor of distribution systems that complicates treatment strategies.
Long distribution networks can introduce a variety of factors including varying pipe materials,
stagnant conditions, and temperature differentials (Masters, Wang, Pruden, & Edwards, 2015;
Wang, Masters, Edwards, Falkinham, & Pruden, 2014). These factors contribute to microbial
regrowth, creating redox zones, corrosion, and biofilm formation, each of which consume
disinfectant residual and degrade water quality (Masters et al., 2015). As reclaimed water has
much more variable water chemistry and microbiology than drinking water, treatment methods
must account for water age and provide adequate treatment to maintain target water quality
throughout the entirety of a distribution system.
The primary objective of this study was to assess how key physicochemical conditions influence
the growth and survival of OPs in reclaimed water distribution systems using controlled
laboratory-scale pipe rigs. Specifically, six parallel simulated reclaimed water distribution
23
systems (SRWDSs) were constructed to investigate the impact of temperature (14-30 °C), AOC
(high and low), disinfectant (free chlorine, chloramine, and no disinfectant), and water age (0-5
days) on the microbial composition of the bulk water and biofilm. This will provide (a) a better
understanding of how OPs behave in reclaimed water distribution systems and (b) a framework
to improve management of reclaimed water distribution systems in the field.
2.2 Materials and Methods
2.2.1 Simulated Reclaimed Water Distribution Systems (SRWDSs)
Six parallel SRWDSs were constructed to represent the extremes of organic carbon
concentrations, disinfection strategies, and operating temperatures exhibited in reclaimed water
distributions systems (Figure 2.1). Two targeted AOC levels (“Low AOC” and “High AOC”),
three disinfection strategies (no disinfectant control, 4 mg/liter free chlorine, 4 mg/liter
chloramine), and three temperatures (30 °C, 22 °C, and 14 °C) were tested in approximately
three-month intervals (Table 2.1). Each system consisted of three sections of 4” diameter
plumbing grade polyvinyl chloride (PVC) pipe (32.5”, 46.5”, 74.5”) connected by coils of 3/8”
small diameter high-density polyethylene (HDPE) tubing. Water flowed through the 31.6 L
systems at 0.25 L/hour, creating an overall water age of approximately 5.2 days (127 hours).
Bulk water sampling ports were installed on the large pipe sections to enable sampling at 0, 1,
2.5, and 5-day water ages while the small tubing that connected the large pipe sections provided
sacrificial biofilm samples. Small tubing was used to create a flow velocity more comparable to
real distribution systems (0.031 m/hour in large pipe vs 3.55 m/hour in small tubing).
2.2.2 Influent Water Treatment and Water Changes
Raw influent water was collected from the Town of Christiansburg Wastewater Treatment
Facility, which is a conventional activated sludge wastewater treatment plant that utilizes the
24
MLE denitrification process (recycles highly nitrified water to an anoxic zone for complete
denitrification). Water was collected monthly from the final effluent of the plant in 5 gallon
Figure 2.1. (a) Schematic of simulated reclaimed water distribution system (SRWDS) (b) Photo
of actual SRWDS set up with all six SRWDSs in temperature-controlled room. Water age is
indicated in red text and disinfectant condition in yellow text.
containers and was stored a 4 °C until use in the SRWDSs. The “High” AOC condition was
simulated using raw wastewater effluent and will be referred to as the Raw Water condition. The
Cl2
(b)
(a)
Reservoir
4°C
Day 0 Day 1
Day 2.5Day 5
18" PVC
Tubing
4" PVC Pipes
Sampling
Port
Effluent Collection
PeristalticPump
“Low” AOC or
GAC biofiltered
“High” AOC or
Raw Water
Day 0 Cl2
NH2Cl None
Day 1
Day 2.5 Day 5
Day 1
Day 5 Day 2.5
Day 0
None NH2Cl
Cl2
25
“Low AOC” condition was achieved by recirculating raw wastewater effluent through two
biologically-active granular activated carbon filters in series for 48 hours at room temperature
and will be referred to as GAC biofiltered water. Free chlorine was dosed directly to high and
low AOC influents from 5000 mg/liter stock solution of sodium hypochlorite to breakpoint
chlorinate all waters prior to adding additional disinfectant.
Table 2.1. Logistics of experiment. (a) Conditions of each SRWDS. (b) Timeline of experiment
including sampling dates. Note that the first 14 °C and 22 °C samplings did not include triplicate
sampling events. The data presented in this study was collected from the 30 °C temperature only.
Low AOC High AOC
Disinfection
Treatment
No Disinfectant No Disinfectant
Chlorine (4 mg/L) Chlorine (4 mg/L)
Chloramine (4 mg/L) Chloramine (4 mg/L)
*All conditions breakpoint chlorinated using sodium hypochlorite
For the associated conditions, chlorine was then dosed to 4 mg/liter and chloramines were dosed
to 4 mg/liter from a 1400 mg/liter stock solution to make 8-L influent reservoirs for each
SRWDS. Chlorine stock solutions were made from a dilution of Clorox™ bleach. Preformed
1000 mg/liter chloramine stock solution was made using the 5000 mg/liter chlorine stock, a
1,400 mg/liter stock solution of ammonium hydroxide, and DI water. All stock solutions were
Temperature Condition Time Period Sampling Dates
1st 2nd 3rd
14 °C* June – September 2015 9/14/15 - 9/18/15
22 °C* October 2015 – February 2016 2/3/16 - 2/4/16
30 °C February – July 2016 5/11/16-
5/12/16
5/17/16-
5/18/16
5/23/16-
5/24/16
22 °C July – November 2016 10/24/16-
10/25/16
11/7/16-
11/8/16
11/11/16-
11/12/16
14 °C November 2016 – March 2017 2/28/17-
3/1/17
3/14/17-
3/15/17
3/27/17-
3/28/17
(a)
(b)
26
stored at 4 °C between uses. The 8-L influent water reservoir feeding each system was stored at 4
°C to limit biological activity before entering the SRWDSs, and was replenished every 30 hours.
2.2.3 Sample Collection
Samples were collected in triplicate beginning after approximately three months of acclimation
(Table 2.1b). Biological triplicates were collected weekly or biweekly following initial
sampling. 500 mL bulk water samples were collected in sterile containers and remaining
disinfectant residuals were quenched with 100-300 µg of 0.01 mg/liter sodium thiosulfate.
Water samples were processed by filtering 250 mL, in duplicate, onto a sterile 0.22 µm-pore-size
mixed cellulose ester filter (Millipore, Billerica, MA). Filters were uniformly torn using sterile
tweezers and transferred to Lysing Matrix tube A from the FastDNA® SPIN Kit (Qbiogene, Inc.,
CA). Duplicate biofilm samples were collected from the small diameter PVC tubing by cutting
two 3.175 cm (1.25 inch) segments and further processed by disinfecting the outside of the tubes
with 70% (v/v) ethanol and cleaning with DNA Away (MBP, Inc., San Diego, CA). The tube
sections placed directly into DNA extraction tubes (Lysing Matrix tube A) containing sand, with
the ceramic bead removed to allow for sand penetration to the center of the tube and harvest as
much biofilm as possible.
2.3.4 DNA extraction & qPCR
DNA was extracted using the FastDNA® SPIN Kit and the FastPrep® Instrument (MP
Biomedical, Inc., Solon, OH) according to manufacturer protocols and stored at -20 °C.
Quantitative polymerase chain reaction (qPCR) was used to assess levels of various genes
including 16S rRNA, Legionella spp., Mycobacteria spp., Acanthamoeba spp., Vermamoeba
vermiformis, Pseudomonas aeruginosa, and Naegleria fowleri (Appendix Table 2.5). All qPCR
assays were carried out using a CFX96™ real-time system (Bio-Rad, Hercules, CA). Inhibition
27
was minimized by diluting samples 1:20 with molecular-grade water. In each qPCR run a
calibration curve was included with seven standard points included. The qPCR quantification
limit was dependent on the gene being assessed; 100 gene copies/reaction for 16S, 50-100 gene
copies/reaction for all other genes.
2.2.5 Water Chemistry Parameters
Several water chemistry parameters were monitored throughout the duration of this study.
Monitoring for chlorine/chloramine residuals, dissolved oxygen, AOC, and nitrogen species
were performed at regular intervals with these intervals, the associated method for each analysis
provided in Table 2.2.
Table 2.2. Water Chemistry parameters assessed with references to APHA Standard Method or
published method.
Analysis Instrument Method Reference
Free/Total Chlorine HACH DR/2400 Spectrophotometer
(HACH Co., Loveland, CO)
4500-Cl (APHA, AWWA,
1998)
Dissolved Oxygen Orion Star A326 pH/RDO/DO Meter
(Thermo Fisher Scientific Inc., Waltham, MA)
4500 O G (APHA,
AWWA, 1998)
pH Oakton pH 110 series meter
(Oakton Research, Vernon Hills, IL)
4500-H+ (APHA, AWWA,
1998)
Ammonia (NH4+-N) HACH DR/2400 Spectrophotometer
4500-NH3 (APHA,
AWWA, 1998)
Nitrite (NO2--N),
Nitrate (NO3--N)
Dionex® DX-120 Ion Chromatograph
(Dionex Co., Sunnyvale, CA)
4110 (APHA, AWWA,
1998)
Metals
Thermo Electron X-Series Inductively coupled plasma
mass spectrophotometer (ICP-MS)
(Thermo Fisher Scientific Inc., Waltham, MA)
3215-B (APHA, AWWA,
1998)
Assimilable Organic
Carbon (AOC)
BD Accuri C6 Flow Cytometer
(Accuri Cytometers, Inc., Ann Arbor, MI)
(F. Hammes, 2015; F. A.
Hammes & Egli, 2005)
Total Organic Carbon
(TOC)
Sievers 5310C Laboratory TOC Analyzer
(GE Analytical Instruments, Boulder, CO)
5310-A (APHA, AWWA
1998)
28
2.2.6 Statistical analysis
All statistical analyses were performed using JMP (SAS, Cary, NC) and RStudio (RStudio, Inc,
Boston, MA). Shapiro-Wilk tests were used to confirm normality for all data sets. For normally
and log-normally distributed data Student t-tests were used to assess variations in means. For
non-normally distributed data, non-parametric tests were used including, Wilcox signed-rank
tests, Kruskal-Wallis tests, and Spearman correlations.
2.3 Results & Discussion
Here we focus on the effect of GAC biofiltration and disinfectant conditions from the 30 °C
temperature setting, which had the highest potential for microbial re-growth and where most
consistent comparisons in data collected are available.
2.3.1 Water Chemistry data
2.3.1.1 Dissolved Oxygen
Dissolved oxygen was tracked throughout the experiment as an indicator of microbial activity
along the length of each SRWDS. The systems were operated in a manner to minimize the
potential for oxygen to externally enter the SRWDSs so, as anticipated, dissolved oxygen (DO)
decreased over time under all conditions due to oxygen demand by aerobic organisms (Figure
2.2). It was noted that the High AOC waters in the reservoirs and entering the SRWDSs
contained higher dissolved oxygen than the Low AOC conditions, indicating that prior GAC
biofiltration or the higher storage temperature allowed for biological activity, removing some
initial DO.
2.3.1.2 Chlorine residuals
At 30 °C, chlorine residuals for the chlorinated and chloraminated SWRDSs in both High and
Low AOC decreased rapidly with increasing water age, as anticipated due to the high chlorine
29
demand of reclaimed waters (Jjemba et al. 2010). The most rapid decrease occurred between the
reservoir (stored at 4 °C) and day 0 of the rigs, with chloramine (measured as total chlorine)
decaying faster than free chlorine (Figure 2.2). The faster decay of chloramine may have been
triggered by nitrification in chloramine fed pipe systems (Wang et al., 2014). All disinfectant
residuals were depleted by Day 2.5 in the rigs. The distance from the reservoir storage to the
entrance of the rigs was the greatest sink of chlorine or chloramine residual, indicating that
perhaps biological activity in the tubing biofilm, which was exposed to the ambient temperature
setting (30 °C), was consuming disinfectant residual.
2.3.1.3 Assimilable Organic Carbon (AOC) and Total Organic Carbon (TOC)
The recommendation for drinking water maximum AOC to prevent bacterial regrowth is 100
µg/L in disinfected conditions (Volk & LeChevallier, 2000). However, in the reclaimed water
systems assessed in this study, the mean influent “High” and “Low” AOC concentrations at 30
ReservoirReservoir
Chlorine ResidualDissolved Oxygen(a) (b)
Figure 2.2 (a) Dissolved Oxygen (mg/liter) over time in all SRWDSs (b) Chlorine residuals (mg/liter) in chlorine-fed (free chlorine residual) and chloramine-fed (total chlorine residual) SRWDSs over time.
30
°C, after disinfectant was applied, were 510 µg/liter (±364 µg/liter) and 266 µg/liter (±264
µg/liter), respectively. Thus, while AOC was effectively reduced, it does not seem realistic to
achieve the ultra-low levels in reclaimed water that are recommended for microbial control in
drinking water. To obtain a Low AOC condition, as described above, raw wastewater effluent
was subject to GAC biofiltration for 48 hours, at room temperature. The expectation was that
GAC biofiltration would markedly decrease AOC in the Low AOC waters; however, there was
no significant difference between in the AOC measured in the Low and High AOC waters post
GAC biofiltration (p>0.05). There were, however, significant effects of disinfectants on AOC
levels noted, with elevated AOC measured following chlorine and chloramine disinfection when
compared to no disinfection (Kruskal-Wallis, Low AOC: p=0.015, High AOC: p=0.023; Figure
2.3). This is thought to be a result of the oxidation of complex organic matter by the disinfectants
into more bioavailable forms and is in agreement with previous studies, where higher AOC
levels were observed in conditions treated with chlorine or chloramine disinfectant than those
without (Polanska, Huysman, & Van Keer, 2005; Weinrich et al., 2010). But, in comparing Low
versus High AOC waters for the same disinfectant condition (i.e. Low AOC chlorine compared
to High AOC chlorine), there were no significant differences observed as a result of GAC
biofiltration (Wilcox test, p>0.05).
However, in contrast to AOC levels being insignificant between the raw and GAC biofiltered
waters, total organic carbon (TOC) was significant between these with the mean TOC across all
Raw Water conditions at 5.23 ppm (±0.61 ppm) and the mean TOC in GAC biofiltered
conditions at 2.20 ppm (±0.18 ppm). This result has multiple implications. It suggests that GAC
biofiltration is effective at removing nutrients from raw waters but that AOC was not
31
proportionately affected by this treatment. It also calls into question whether AOC methods
adapted for this study captured the full extent of AOC in the reclaimed waters. Further testing
should be done to analyze biodegradable dissolved organic carbon (BDOC) alongside these AOC
methods.
2.3.2 Relationships between Targeted Gene Markers for OPs
In order to assess relationships between OPs, FLA and total bacterial gene copy numbers (i.e.,
total 16S rRNA gene copies), gene copy numbers corresponding to various bacterial OPs
(Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa), amoebic OPs (Acanthamoeba
spp., N. fowleri), and non-pathogenic FLA (V. vermiformis) were enumerated via qPCR and
statistically compared with respect to various SRWDS conditions of interest. Legionella spp.,
Mycobacterium spp., and Acanthamoeba spp. were the most frequently detected (67-100% of
samples confirmed positive) and several moderate and strong Spearman rank correlations among
(µg/L
)
Figure 2.3. Assimilable organic carbon (AOC) over time for each
SRWDS.
32
the gene copy numbers of the targets were observed, particularly in high AOC bulk water
conditions (Table 2.3).
It was hypothesized that the FLA gene copies would be correlated with amoeba-resisting
bacterial OPs, such as Legionella and Mycobacterium (J. M. Thomas & Ashbolt, 2011). Among
the FLA assessed, Acanthamoeba was detected most frequently, being found in 67% of samples
assessed. In the bulk water, Acanthamoeba spp. were found to correlate with Mycobacterium
spp. in 60% of the conditions, with both genes tending to decrease with increasing water age
(Figure 2.4). Mycobacteria have been found to live successfully in Acanthamoeba in water
networks in previous studies (V. Thomas et al., 2005), suggesting that reclaimed water may be
another water environment that is beneficial for ARM to thrive.
V. vermiformis, a non-pathogenic FLA frequently associated with ARM colonization (J.
Falkinham, 2015), was detected in 11% of all samples, most commonly in chloramine-treated or
non-disinfected waters (Table 2.4). P. aeruginosa, a pathogen well known for its potential to
form biofilms and survive in stagnant conditions, was not detected in any collected samples at 30
°C (J. Falkinham, 2015; Palmer, Brown, & Whiteley, 2007; Rogers, Dennis, Lee, & Keevil,
1994).
On average, biofilm samples had fewer positive detections for the genes assessed, which may
have been due to inherent limitations of biofilm sample collection. However, significant
variations in gene copies/cm2 of tubing were detected among disinfectant conditions for
Mycobacterium spp. and Acanthamoeba spp. indicating that biofilm composition was impacted
by disinfectant (p<0.05).
33
Table 2.3. Spearman rank correlation (⍴) values for each of the highest quantified genes
assessed and associated p values. Dark red indicates a strong positive correlation and blue
indicates a strong negative correlation. Bold indicates 0.01<p<0.05, Bold and italicized
indicates p<0.01.
(a) High AOC Log Legionella spp. Log Mycobacterium
spp.
Log Acanthamoeba
spp. Spearman's ⍴ Spearman's ⍴ Spearman's ⍴
No
Dis
infe
ctan
t
Water
16S rRNA 0.7203 0.6643 0.7322
Log Legionella spp. 0.6573 0.6698
Log Mycobacterium spp. 0.8404
Biofilm
16S rRNA 0.3203 -0.028 -0.3229
Log Legionella spp. -0.1637 -0.4158
Log Mycobacterium spp. 0.4435
Ch
lori
ne Water
16S rRNA 0.8792 0.7531 -0.1037
Log Legionella spp. 0.7684 -0.0799
Log Mycobacterium spp. 0.0799
Biofilm
16S rRNA 0.7228 0.7738 0.2184
Log Legionella spp. 0.3778 0.4083
Log Mycobacterium spp. 0.0445
Ch
lora
min
e Water
16S rRNA 0.6713 0.9301 0.7631
Log Legionella spp. 0.7692 0.4243
Log Mycobacterium spp. 0.7809
Biofilm
16S rRNA 0.0709 -0.5035 -0.5491
Log Legionella spp. -0.3901 -0.2857
Log Mycobacterium spp. 0.2888
(b) Low AOC Log Legionella spp. Log Mycobacterium
spp.
Log Acanthamoeba
spp. Spearman's ⍴ Spearman's ⍴ Spearman's ⍴
No
Dis
infe
ctan
t
Water
16S rRNA 0.2308 0.5385 0.2382
Log Legionella spp. 0.0769 0.1086
Log Mycobacterium spp. 0.4168
Biofilm
16S rRNA 0.838 -0.3846 0.0426
Log Legionella spp. -0.5828 -0.0657
Log Mycobacterium spp. 0.4894
Ch
lori
ne Water
16S rRNA 0.461 0.9352 0.546
Log Legionella spp. 0.5258 0.0152
Log Mycobacterium spp. 0.6594
Biofilm
16S rRNA - 0.8441 0.4704
Log Legionella spp. - -
Log Mycobacterium spp. 0.4771
Ch
lora
min
e Water
16S rRNA 0.8741 0.0559 -0.0142
Log Legionella spp. 0.1818 0.1135
Log Mycobacterium spp. 0.8582
Biofilm
16S rRNA 0.5141 0.2308 -0.5945
Log Legionella spp. 0.4162 -0.4215
Log Mycobacterium spp. -0.2107
Color
Scale
1
0.67
0.33
0
-0.33
-0.67
-1
34
Table 2.4. Percentage of samples positive for a gene from each SRWDS conditions. Darker red
indicates higher presence. (a) Bulk water samples. (b) Biofilm samples. (n=12 for all
conditions).
(a) Water
No disinfectant Chlorine Chloramine
Low AOC High AOC Low AOC High AOC Low AOC High AOC
Legionella spp. 1.00 1.00 0.75 0.83 0.92 1.00
Mycobacteria spp. 1.00 1.00 0.83 0.83 1.00 1.00
Acanthamoeba spp. 0.92 1.00 0.83 0.33 0.75 0.92
Vermamoeba vermiformis 0.42 0.08 0.00 0.00 0.33 0.17
Naegleria fowleri 0.25 0.25 0.08 0.08 0.50 0.33
(b) Biofilm
No disinfectant Chlorine Chloramine
Low AOC High AOC Low AOC High AOC Low AOC High AOC
Legionella spp. 0.67 1.00 0.00 0.58 0.42 0.75
Mycobacteria spp. 1.00 1.00 0.75 0.67 1.00 1.00
Acanthamoeba spp. 0.75 0.58 0.42 0.08 0.75 0.67
Vermamoeba vermiformis 0.25 0.08 0.00 0.00 0.00 0.00
Naegleria fowleri 0.17 0.00 0.25 0.00 0.33 0.17
2.3.3 Effect of AOC on Targeted Gene Markers for OPs
While, the AOC levels measured in the all conditions were significantly greater than those
typical of drinking water, the actual difference in AOC concentration between the two
conditions, raw water and GAC biofiltered water, as applied in this study was not significant, as
discussed above. However, the differences in water quality introduced by GAC biofiltration of
the influent to produce “Low” AOC water still had a marked effect on the microbial community
associated with the bulk water and biofilm. A significant correlation was observed between 16S
rRNA gene copies and total cell counts, measured via flow cytometry (⍴=0.647, p=0.0008),
indicating that both means of measuring microbial numbers were in agreement. Still, there was
no significant difference in total cell counts in High AOC versus Low AOC water samples
(p>0.05) while there was a significant difference in total 16S rRNA gene copies between High
Fraction
Positive
1.00
0.75
0.50
0.25
0.00
35
and Low AOC conditions (p<0.0001). This could have been due to interference of particulates of
similar size as target cells in the analyzed reclaimed water that would not be picked up by 16S
rRNA qPCR.
Among the target OP marker genes quantified, one of the more striking differences observed
between the Low and High AOC conditions was in terms of Legionella spp. gene copies, with
differences both in bulk water and biofilm (p<0.0001). Conversely, no significant difference was
found between Mycobacterium spp. or Acanthamoeba spp. in the Low versus High AOC
conditions (p>0.05). These observations suggest that GAC biofiltration may select, remove, or
have no effect on certain OPs.
Spearman’s rank correlation analyses were carried out to compare target gene abundances of 16S
rRNA, Legionella spp., Mycobacterium spp., and Acanthamoeba spp. in the biofilm and water
phases of each of the three disinfectant x Low/High AOC conditions. It was found that 16 of 36
comparisons made were significant in the High AOC conditions while only 7 of 36 were
significant for the Low AOC conditions (Table 2.3). Only four correlations were conserved in
both High and Low AOC conditions: chlorine bulk water 16S rRNA genes vs. Mycobacterium,
chlorine biofilm 16S vs. Mycobacterium, chloramine bulk water 16S vs. Legionella, and
chloramine bulk water Mycobacterium vs. Acanthamoeba. These limited similarities between
High and Low AOC further indicate that GAC biofiltration greatly alters the biological
community of the influent water and provide insight into whether these organisms interact with
one another, and whether their growth patterns are similar to that of other bacteria in the systems.
Notably, in non-disinfected High AOC bulk water conditions, positive correlations are present
36
Figure 2.4. Four most abundant gene targets quantified by qPCR in this study, plotted as a
function of water age in the. SRWDS bulk water and biofilm are plotted in the top and bottom,
respectively, and Low and High AOC on the left and right, respectively, of each tile. (a) Total
Bacteria (16S rRNA) (b) Legionella spp. (c) Mycobacterium spp. (d) Acanthamoeba spp.
(a) (b)
(c)
(b)
(d)
37
across all genes, but no correlations exist for the corresponding Low AOC system. Similarly,
when comparing Low and High AOC conditions for chloramine disinfectant, all genes are
correlated in the High AOC water and only two sets are correlated in Low AOC waters.
2.3.4 Effect of Disinfectant on Targeted OP Gene Markers
The effect of disinfectant type on the occurrence and abundance of targeted OP gene copy
numbers was statistically compared across water and biofilm samples from both High and Low
AOC conditions. While the total bacterial 16S rRNA gene copy numbers in all chlorinated
systems (both High and Low AOC conditions) were significantly lower at day 0 than the
corresponding chloraminated and non-disinfected systems (Kruskal-Wallis, p<0.05), all
conditions reached a similar threshold level of bacteria over time. Trends over time show that
16S rRNA gene copy numbers in all three disinfectant conditions converged to approximately
the same total abundance by day 5 (Figure 2.4a).
Total bacteria results for biofilm indicate that, overall, disinfectant applications did not play a
significant role for High AOC raw water conditions but did have an effect in the Low AOC
waters where chlorine was significantly lower than chloramine and control conditions (Kruskal
Wallis, High AOC p>0.05, Low AOC p=0.02). Thus, it appears that GAC biofiltration may have
played a role in the efficacy of chlorine disinfectant in penetrating biofilm, but more needs to be
done to understand how particular organisms may be influenced by this penetration.
Overall, chlorine was the most effective at maintaining low copy numbers of the target genes
monitored in this study. While there was nearly a 100% detection rate of Legionella spp. in the
no disinfectant and chloramine bulk water samples, Legionella spp. detection rates were about
38
83% and 75% in the High and Low AOC conditions, respectively. Surprisingly, even though it
was detected in the bulk water, there was zero detection of Legionella spp. in biofilm of the
disinfected Low AOC conditions, either with chlorine or chloramines (Figure 2.4b). This was
particularly shocking due to the fact that previous studies have shown that Legionella is far more
susceptible as free-floating organisms in water than it is in biofilm (Jjemba et al. 2015; Cargill et
al. 1992). Assuming the Legionella spp./16S rRNA gene ratio measured in the bulk water for
these conditions remained constant and the levels of 16S rRNA genes detected in the biofilm,
Legionella spp. should have been detectable in the biofilm of the control and chloraminated Low
AOC conditions one to two orders of magnitude higher than detection limits. Therefore, lack of
detection of Legionella spp. in the biofilm was not because of methodological limitation. The
biofilms in the chlorinated condition, on the other hand, ranged from being greater than the
Legionella spp./16S rRNA ratio in water at lower water ages to 2-10 times lower than expected.
This suggests that the disinfected Low AOC conditions were actually prohibitive to Legionella
proliferation in the biofilm. Still, with an average of 58% less Legionella spp. gene markers in
the biofilm of the Low AOC relative to the High AOC condition, the clear trend was that the
chlorinated, Low AOC condition was optimal for Legionella control relative to the other
reclaimed water distribution system conditions investigated here.
Acanthamoeba spp., which have been shown generally to enhance the proliferation of both
Legionella spp. and Mycobacterium spp. in biofilms and carry resistance to chlorine disinfectant
(J. Falkinham, 2015; Lau & Ashbolt, 2009; J. M. Thomas & Ashbolt, 2011; V. Thomas et al.,
2005), were more consistently present in Low AOC conditions with chlorine than High AOC
with chlorine, contrary to the pattern observed for Legionella spp. No detections of V.
39
vermiformis were found in chlorinated waters suggesting that chlorination may have provided
adequate control for this amoeba. Unlike Legionella spp., Acanthamoeba spp. were also more
frequently detected in the Low AOC conditions. While viability assessments were not
performed for FLA in this experiment, correlations between FLA and ARM, were of interest.
The pathogenic FLA, N. fowleri, was detected in all bulk water and most biofilm conditions with
the highest rate of occurrence in chloraminated conditions, suggesting that microbial or chemical
changes introduced by chloramine were more conducive to the growth of N. fowleri (Table 2.4).
2.3.5 Effect of Water Age on Targeted Gene Markers for OPs
A range of water ages from 0 to 5 days was achieved along the length of the pipes to simulate the
time traveled in a real-world distribution system by employing wide diameter PVC pipes. In
general, water age is known to be associated with diminishing water quality in drinking water
distribution systems, conditions that would be expected to be exacerbated in reclaimed water
distribution systems. Here, a range of responses to increasing water ages along the lengths of the
pipes was observed across the different SRWDS conditions. Overall, total bacterial 16S rRNA
gene copies, particularly in the bulk water, did not vary with water age in the absence of
disinfectant. In the chlorinated SRWDS, total bacterial gene copy numbers were lowest at day 0,
as expected, likely of function of the fact that all chlorine residuals were consumed by day 2.5
(Figure 2.4). Beyond day zero in the chlorinated condition, all bulk water conditions achieved
similar levels of bacterial gene markers and remained constant through the 5-day water age. This
same general pattern was observed in both the High and Low AOC conditions, despite the high
AOC condition consistently sustaining higher levels of bacteria (Figure 2.4a).
40
Total bacterial 16S rRNA gene copy numbers in biofilm samples followed similar trends as bulk
water, with no significance found when comparing the disinfectant conditions (Kruskal-Wallis,
p>0.05).
While total bacterial DNA increased or generally remained constant with water age, OP and FLA
target gene copy numbers displayed different patterns. Both Mycobacterium spp. and
Acanthamoeba spp. gene copies decreased over time in bulk water samples with Acanthamoeba
spp. levels inversely correlating with water age in low AOC chlorine and chloramine conditions
(⍴=-0.610, p=0.035; ⍴=-0.610, p=0.01, respectively).
2.4 Conclusions
The following general conclusions are derived from this work and raise several questions
regarding adequate standards for treating reclaimed water in order to avoid growth of OPs.
• Disinfectant application did not control overall bacterial re-growth when distributing
reclaimed water.
• Chlorine disinfectant was far more effective at reducing OPs and FLA overall than
chloramine disinfectant. Chloramine disinfectant, in most cases, did not offer additional
disinfecting benefit relative to no disinfectant.
• While measured AOC levels were not significantly reduced by GAC biofiltration, overall
bacterial growth was still diminished downstream.
• GAC biofiltration appeared promising for the control of Legionella in reclaimed
distribution systems, particularly in the development of biofilms.
41
While the genes assessed in this study provided a baseline comparison to drinking water, further
analysis is required to understand which other microbes may play a key role in enhancing or
inhibiting the growth of pathogens in reclaimed water.
42
2.5 Acknowledgements
This study was funded by the U.S. National Science Foundation (Award # 1437118), the Alfred
P. Sloan Foundation—Microbiology of the Built Environment Program, and the Virginia Tech
Institute for Critical Technology and Applied Science (ICTAS). The findings do not represent
the views of the sponsors.
43
2.6 References
Addiss, D. G., Davis, J. P., LaVenture, M., Wand, P. J., Hutchinson, M. A., & McKinney, R. M.
(1989). Community-acquired Legionnaires’ disease associated with a cooling tower:
evidence for longer-distance transport of Legionella pneumophila. American Journal of
Epidemiology, 130(3), 557–568.
APHA, AWWA, and W. (1998). Standard Methods for Examination of Water and Wastewater
(20th ed.). Washington, D.C.: APHA.
Borboudaki, K. E., Paranychianakis, N. V., & Tsagarakis, K. P. (2005). Integrated wastewater
management reporting at tourist areas for recycling purposes, including the case study of
Hersonissos, Greece. Environmental Management, 36(4), 610–623.
http://doi.org/10.1007/s00267-004-0115-9
Brissaud, F., Blin, E., Hemous, S., & Garrelly, L. (2008). Water reuse for urban landscape
irrigation: aspersion and health related regulations. Water Science and Technology : A
Journal of the International Association on Water Pollution Research, 57(5), 781–7.
http://doi.org/10.2166/wst.2008.162
CDC. (2015). Naegleria fowleri — Primary Amebic Meningoencephalitis (PAM).
Corsaro, D., Pages, G. S., Catalan, V., Loret, J.-F. O., & Greub, G. (2010). Biodiversity of
amoebae and amoeba-associated bacteria in water treatment plants. International Journal of
Hygiene and Environmental Health, 213, 158–166.
http://doi.org/10.1016/j.ijheh.2010.03.002
De Jonckheere, J., & Van De Voorde, H. (1976). Differences in Destruction of Cysts of
Pathogenic and Nonpathogenic Naegleria and Acanthamoeba by Chlorine. Applied and
Environmental Microbiology, 31(2), 294–297.
44
Falkinham, J. (2015). Common Features of Opportunistic Premise Plumbing Pathogens.
International Journal of Environmental Research and Public Health, 12(5), 4533–4545.
http://doi.org/10.3390/ijerph120504533
Falkinham, J. O., Norton, C. D., & Mark, W. (2001). Factors Influencing Numbers of
Mycobacterium avium , Mycobacterium intracellulare , and Other Mycobacteria in
Drinking Water Distribution Systems. Applied and Environmental Microbiology, 67(3),
1225–1231. http://doi.org/10.1128/AEM.67.3.1225
Garner, E., Zhu, N., Strom, L., Edwards, M., & Pruden, A. (2016). A human exposome
framework for guiding risk management and holistic assessment of recycled water quality.
Environ. Sci.: Water Res. Technol., 2, 580–598. http://doi.org/10.1039/C6EW00031B
Gerba, C. P., & Rose, J. B. (2003). International guidelines for water recycling: microbiological
considerations. Water Science & Technology: Water Supply, 3(4), 311–316. Retrieved from
http://www.reclaimedwater.net/data/files/114.pdf
Gerba, C. P., Wallis, C., & Melnick, J. L. (1975). Microbiological hazards of household toilets:
droplet production and the fate of residual organisms. Applied Microbiology, 30(2), 229–
237.
Hammes, F. (2015). Assimilable Organic Carbon (AOC): Filtration Method (No. 2.1).
Hammes, F. A., & Egli, T. (2005). New Method for Assimilable Organic Carbon Determination
Using Flow-Cytometric Enumeration and a Natural Microbial Consortium as Inoculum.
Environmental Science and Technology, 39(9), 3289–3294.
http://doi.org/10.1021/ES048277C
Jjemba, P. K., Johnson, W., Bukhari, Z., & LeChevallier, M. W. (2015). Occurrence and Control
of Legionella in Recycled Water Systems. Pathogens (Basel, Switzerland), 4(3), 470–502.
45
http://doi.org/10.3390/pathogens4030470
Jjemba, P. K., Weinrich, L. A., Cheng, W., Giraldo, E., & LeChevallier, M. W. (2010a).
Guidance document on the microbiological quality and biostability of reclaimed water and
distribution systems (WRF‐05‐002). Alexandria, VA: WateReuse Research Foundation .
Jjemba, P. K., Weinrich, L. A., Cheng, W., Giraldo, E., & LeChevallier, M. W. (2010b).
Regrowth of potential opportunistic pathogens and algae in reclaimed-water distribution
systems. Applied and Environmental Microbiology, 76(13), 4169–4178.
http://doi.org/10.1128/AEM.03147-09
Karim, M., & LeChevallier, M. W. (2005). Microbiological quality of reuse water; Internal
Report. Voorhees, NJ.
Kilvington, S., & Price, J. (1990). Survival of Legionella pneumophila within cysts of
Acanthamoeba polyphaga following chlorine exposure. Journal of Applied Bacteriology,
68(5), 519–525. http://doi.org/10.1111/j.1365-2672.1990.tb02904.x
Lau, H. Y., & Ashbolt, N. J. (2009). The role of biofilms and protozoa in legionella
pathogenesis: Implications for drinking water. Journal of Applied Microbiology, 107(2),
368–378. http://doi.org/10.1111/j.1365-2672.2009.04208.x
Liu, X., Wang, J., Liu, T., Kong, W., He, X., Jin, Y., & Zhang, B. (2015). Effects of Assimilable
Organic Carbon and Free Chlorine on Bacterial Growth in Drinking Water. PLOS ONE,
10(6), e0128825. http://doi.org/10.1371/journal.pone.0128825
Loret, J. F., Jousset, M., Robert, S., Saucedo, G., Ribas, F., Thomas, V., & Greub, G. (2008).
Amoebae-resisting bacteria in drinking water: risk assessment and management. Water
Science & Technology, 58(3), 571. http://doi.org/10.2166/wst.2008.423
Masters, S., Wang, H., Pruden, A., & Edwards, M. A. (2015). Redox gradients in distribution
46
systems influence water quality, corrosion, and microbial ecology. Water Research, 68,
140–149. http://doi.org/10.1016/j.watres.2014.09.048
Neden, D. G., Jones, R. J., Smith, J. R., Kirmeyer, G. J., Foust, G. W., for Bacter Douglas G Ned
Kirmeyer, C. C., & Capilano Coquitlam, S. (1992). Comparing Chlorination and
Chloramination for Controlling Bacterial Regrowth Physical and chemical characteristics of
untreated waters. Journal of the American Water Works Association, 84(7), 80–88.
Retrieved from http://www.jstor.org/stable/41293789
Norton, C. D., & Lechevallier, M. W. (1997). Chloramination: its effect on distribution system
water quality. Journal of the American Water Works Association, 89(7), 66–77. Retrieved
from http://www.jstor.org/stable/41295964
Palmer, K. L., Brown, S. A., & Whiteley, M. (2007). Membrane-bound nitrate reductase is
required for anaerobic growth in cystic fibrosis sputum. Journal of Bacteriology, 189(12),
4449–4455. http://doi.org/10.1128/JB.00162-07
Polanska, M., Huysman, K., & Van Keer, C. (2005). Investigation of assimilable organic carbon
(AOC) in flemish drinking water. Water Research, 39, 2259–2266.
http://doi.org/10.1016/j.watres.2005.04.015
Pruden, A., Larsson, D. G. J., Amézquita, A., Collignon, P., Brandt, K. K., Graham, D. W., …
Zhu, Y.-G. (2013). Management options for reducing the release of antibiotics and
antibiotic resistance genes to the environment. Environmental Health Perspectives, 121(8),
878–85. http://doi.org/10.1289/ehp.1206446
Rogers, J., Dennis, P. J., Lee, J. V, & Keevil, C. W. (1994). selection on biofilm formation and
growth of Legionella pneumophila in a model potable water system containing complex
microbial Influence of Temperature and Plumbing Material Selection on Biofilm Formation
47
and Growth of Legionella pneumophila in a Model . Applied and Environmental
Microbiology, 60(5), 1585–1592.
Rosenberg Goldstein, R. E., Micallef, S. a, Gibbs, S. G., He, X., George, A., Sapkota, A. A. R.
A. A. R., … Sapkota, A. A. R. A. A. R. (2014). Occupational exposure to Staphylococcus
aureus and Enterococcus spp. among spray irrigation workers using reclaimed water.
International Journal of Environmental Research and Public Health, 11(4), 4340–55.
http://doi.org/10.3390/ijerph110404340
Ryu, H., Alum, A., & Abbaszadegan, M. (2005). Microbial Characterization and Population
Changes in Nonpotable Reclaimed Water Distribution Systems. Environ Sci Technol, 39,
8600–8605. http://doi.org/10.1021/es050607l
Storey, M. V., Winiecka-krusnell, J., Ashbolt, N. J., & Stenström, T. (2004). The Efficacy of
Heat and Chlorine Treatment against Thermotolerant Acanthamoebae and Legionellae.
Scandinavian Journal of Infectious Diseases, 36(9), 656–662.
http://doi.org/10.1080/00365540410020785
Thayanukul, P., Kurisu, F., Kasuga, I., & Furumai, H. (2013). Evaluation of microbial regrowth
potential by assimilable organic carbon in various reclaimed water and distribution systems.
Water Research, 47, 225–232. http://doi.org/10.1016/j.watres.2012.09.051
Thomas, J. M., & Ashbolt, N. J. (2011). Do Free-Living Amoebae in Treated Drinking Water
Systems Present an Emerging Health Risk? Environmental Science & Technology, 45(3),
860–869. http://doi.org/10.1021/es102876y
Thomas, V., Blanc, D., Bille, J., & Greub, G. (2005). Biodiversity of Amoebae Resistant Micro-
Organisms in an Hospital Water Network. Abstracts of the Interscience Conference on
Antimicrobial Agents and Chemotherapy, 45(4), 318.
48
http://doi.org/10.1128/AEM.72.4.2428
Thomas, V., Loret, J.-F., Jousset, M., & Greub, G. (2008). Biodiversity of amoebae and
amoebae-resisting bacteria in a drinking water treatment plant. Environmental
Microbiology, 10(10), 2728–2745. http://doi.org/10.1111/j.1462-2920.2008.01693.x
Tyndall, R. L., Ironside, K. S., Metler, P. L., Tan, E. L., Hazen, T. C., & Fliermans2, A. C. B.
(1989). Effect of Thermal Additions on the Density and Distribution of Thermophilic
Amoebae and Pathogenic Naegleria fowleri in a Newly Created Cooling Lake. Applied and
Environmental Microbiology, 55(3), 722–732. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC184187/pdf/aem00096-0188.pdf
US EPA. (2012). EPA Guidelines for Water Reuse. Washington, D.C. Retrieved from
http://nepis.epa.gov/Adobe/PDF/P100FS7K.pdf
Valentine, R. L. (1984). Disappearance of Monochloramine in the Presence of Nitrite. In Water
Chlorination, Volume V: Chemistry, Environmental Impact and Health Effects. Chelsea,
Michigan: Lewis Publisher Inc.
Van der Kooij, D. (1992). Assimilable Organic Carbon as an Indicator of Bacterial Regrowth.
American Water Works Association, 84(2), 57–65. http://doi.org/10.2307/41293634
van der Wielen, P. W. J. J., & van der Kooij, D. (2013). Nontuberculous mycobacteria, fungi,
and opportunistic pathogens in unchlorinated drinking water in the Netherlands. Applied
and Environmental Microbiology, 79(3), 825–834. http://doi.org/10.1128/AEM.02748-12
Vieira, P., Coelho, S., & Loureiro, D. (2004). Accounting for the influence of initial chlorine
concentration, TOC, iron and temperature when modelling chlorine decay in water supply.
Journal of Water Supply Research and Technology - Aqua, 53(7), 453–467. Retrieved from
http://apps.webofknowledge.com.ezproxy.lib.vt.edu/full_record.do?product=WOS&search_
49
mode=GeneralSearch&qid=31&SID=2EtnL8ojMZgOxmFAFbm&page=1&doc=3
Volk, C. J., & LeChevallier, M. W. (2000). Assessing Biodegradable Organic Matter. Journal -
American Water Works Association, 92(5), 64–76. Retrieved from
https://www.awwa.org/publications/journal-awwa/abstract/articleid/14205.aspx
Wang, H., Edard, E. B., Ele Pr Evost, M., Camper, A. K., Hill, V. R., & Pruden, A. (2017).
Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A
review. Water Research, 117, 68–86. http://doi.org/10.1016/j.watres.2017.03.046
Wang, H., Edwards, M., Falkinham, J. O., & Pruden, A. (2012). Molecular survey of the
occurrence of legionella spp., mycobacterium spp., pseudomonas aeruginosa, and amoeba
hosts in two chloraminated drinking water distribution systems. Applied and Environmental
Microbiology, 78(17), 6285–6294. http://doi.org/10.1128/AEM.01492-12
Wang, H., Masters, S., Edwards, M. A., Falkinham, J. O., & Pruden, A. (2014). Effect of
Disinfectant, Water Age, and Pipe Materials on Bacterial and Eukaryotic Community
Structure in Drinking Water Biofilm. Environmental Science & Technology, 48(3), 1426–
1435. http://doi.org/10.1021/es402636u
Weinrich, L. A., Jjemba, P. K., Giraldo, E., & Lechevallier, M. W. (2010). Implications of
organic carbon in the deterioration of water quality in reclaimed water distribution systems.
Water Research, 44, 5367–5375. http://doi.org/10.1016/j.watres.2010.06.035
Zhang, Y., & Edwards, M. (2009). Accelerated chloramine decay and microbial growth by
nitrification in premise plumbing. Journal AWWA, 101(11), 51–62.
Zhang, Y., Edwards, M., Pinto, A., Love, N., Camper, A., Rahman, M., & Baribeau, H. (2010).
Effect of Nitrification on Corrosion in the Distribution System. (W. R. Foundation,
Ed.)Water Research Foundation.
50
Appendix A
Table 2.5. qPCR primers, probes, and assay conditions used in this study.
Targeted
organisms
Targeted
genes Sequences (5'-3')
Program
Amplicon
(bp) Reference Initial
denaturation
and enzyme
activation
Denaturing
/
Annealing/
Extension
Legionella
spp.
23S
rRNA
Leg23SF:
CCCATGAAGCCCGTT
GAA
Leg23SR:ACAATCAGC
CAATTAGTACGAGTT
AGC
Probe: HEX-
TCCACACCTCGCCTAT
CAACGTCGTAGT
95 °C for 2
min
40 cycles
of 95 °C
for 5 s and
58.5 °C for
10 s
92 Nazarian
et al.
2008
Mycobacteri
um spp.
16S
rRNA
110F:
CCTGGGAAACTGGGT
CTAAT
I571R:
CGCACGCTCACAGTT
A
H19R:
FAM-
TTTCACGAACAACGC
GACAAACT
95 °C for 2
min
45 cycles
of 95 °C
for 5 s, 55
°C for 15 s,
and 72 °C
for 10 s
462 Radomski
et al. 2010
V.
vermiformis
18S
rRNA
Hv1227F:
TTACGAGGTCAGGAC
ACTGT
Hv1728R:
GACCATCCGGAGTTC
TCG
98 °C for 2
min
40 cycles
of 98 °C
for 5 s and
72 °C for
18 s
502 Kuiper et
al. 2006
Acanthamoe
ba spp.
18 rRNA TaqAcF1:
CGACCAGCGATTAGG
AGACG
TaqAcR1:
CCGACGCCAAGGACG
AC
Probe: FAM-
TGAATACAAAACACC
ACCATCGGCGC
95 °C for 2
min
40 cycles
of 95 °C
for 5 s and
60 °C for
10 s
66 Rivière et
al. 2006
P.
aeruginosa
ecfX &
gyrB
ecfX-F:
CGCATGCCTATCAGG
CGTT
ecfX-R:
GAACTGCCCAGGTGC
TTGC
Probe:
HEX-
ATGGCGAGTTGCTGC
GCTTCCT
gyrB-F:
95 °C for 2
min
50 cycles
of 95 °C
for 5 s and
60 °C for
10 s
63 (ecfX)
220
(gyrB)
Anuj et al.
2009
51
Appendix References:
Anuj, S. N., Whiley, D. M., Kidd, T. J., Bell, S. C., Wainwright, C. E., Nissen, M. D., & Sloots,
T. P. (2009). Identification of Pseudomonas aeruginosa by a duplex real-time polymerase
chain reaction assay targeting the ecfX and the gyrB genes. Diagnostic Microbiology and
Infectious Disease, 63(2), 127–131. http://doi.org/10.1016/j.diagmicrobio.2008.09.018
Kuiper, M. W., Valster, R. M., Wullings, B. A., Boonstra, H., Smidt, H., & Van Der Kooij, D.
(2006). Quantitative Detection of the Free-Living Amoeba Hartmannella vermiformis in
Surface Water by Using Real-Time PCR. Applied and Environmental Microbiology, 72(9),
5750–5756. http://doi.org/10.1128/AEM.00085-06
Mull, B. J., Narayanan, J., & Hill, V. R. (2013). Improved Method for the Detection and
Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic
Separation and Real-Time PCR. Journal of Parasitology Research, 2013, 8.
CCTGACCATCCGTCGC
CACAAC
gyrB-R:
CGCAGCAGGATGCCG
ACGCC
Probe:
FAM-
CCGTGGTGGTAGACC
TGTTCCCAGACC
N. fowleri ITS JBVF: AGG TAC TTA
CGT TAG AGT GCT
AGT
JBVR:
ATGGGACAATCCGGT
TTTCTCA
Probe: FAM-
ACGCCCTAGCTGGTT
ATGCCGGATT-BHQ1
95 °C for 15
min
45 cycles
of 95 °C
for 15 s and
63 °C for
33 s
123 Mull et al.
2013
Total
Bacteria
16S
rRNA
BACT1369F:
CGGTGAATACGTTCY
CGG
PROK:
GGWTACCTTGTTACG
ACTT
98 °C for 2
min
40 cycles
of 98 °C
for 5 s and
55 °C for 5
s
124 Suzuki et
al. 2000
52
http://doi.org/10.1155/2013/608367
Nazarian, E. J., Bopp, D. J., Saylors, A., Limberger, R. J., & Musser, K. A. (2008). Design and
implementation of a protocol for the detection of Legionella in clinical and environmental
samples. Diagnostic Microbiology and Infectious Disease, 62(2), 125–32.
http://doi.org/10.1016/j.diagmicrobio.2008.05.004
Radomski, N., Lucas, F. S., Moilleron, R., Cambau, E., Haenn, S., & Moulin, L. (2010).
Development of a real-time qPCR method for detection and enumeration of Mycobacterium
spp. in surface water. Applied and Environmental Microbiology, 76(21), 7348–51.
http://doi.org/10.1128/AEM.00942-10
Rivière, D., Szczebara, F. M., Berjeaud, J.-M., Frère, J., & Héchard, Y. (2006). Development of
a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts. Journal
of Microbiological Methods, 64(1), 78–83. http://doi.org/10.1016/j.mimet.2005.04.008
Suzuki, M. T., Taylor, L. T., & DeLong, E. F. (2000). Quantitative analysis of small-subunit
rRNA genes in mixed microbial populations via 5’-nuclease assays. Applied and
Environmental Microbiology, 66(11), 4605–14. http://doi.org/10.1128/AEM.66.11.4605-
4614.2000
53
Chapter 3: Effects of pipe material on the
growth and survival of Naegleria fowleri in
drinking water
3.1 Introduction
N. fowleri is an ameboflagellate and opportunistic pathogen (OP) that can cause the fatal disease,
primary amoebic meningoencephalitis (PAM), when water containing the amoeba gets “up the
nose” and is nasally aspirated. Discovery of this free-living “brain-eating” amoeba in municipal
drinking water and associated deaths have raised concerns about how to safely manage this
pathogen as a drinking water contaminant. As such, N. fowleri is now included on the U.S.
Environmental Protection Agency Candidate Contaminant List (CLL 3; US EPA 2017). Risk
associated with N. fowleri proliferation in drinking water distribution systems is expected to
continue to increase into the future, given that it is a warm water microorganism and there is an
association with its increasing prevalence and trends towards warming regional temperatures.
Such trends are believed to be contributing to the expansion of the spread of this pathogen into
higher latitudes and longer through extended summer seasons in southern latitudes.
Not until 2003 did investigations of PAM cases in Arizona begin to link N. fowleri to drinking
water (CDC, 2015; Marciano-Cabral, MacLean, Mensah, & LaPat-Polasko, 2003). Since then,
warm drinking water distribution systems have frequently been subject to N. fowleri
contamination, particularly in areas such as Australia and the Middle East, but the first PAM
cases that were confidently verified to be associated with contaminated municipal drinking water
were two cases in Louisiana in 2011, sourced from two different municipalities, St. Bernard
Parish and DeSoto Parish (Yoder et al., 2012). In 2013, another death due to PAM occurred in
54
St. Bernard Parish after a child’s exposure to municipal drinking water on an outdoor play slide
(Louisiana Department of Health, 2013b). DeSoto Parish was also found to be positive for N.
fowleri in their distribution system in 2013 and very recently, in June 2017, two more water
systems in Louisiana tested positive for the presence of the amoeba (Louisiana Department of
Health, 2013a, 2017). In all cases where N. fowleri was detected in Louisiana drinking water
systems, stringent chlorination protocols were implemented in an attempt to eliminate the
amoeba from the distribution system. However, due to high chlorine demand in the water,
particularly in areas of the distribution system near the N. fowleri cases, have made it hard to
maintain measureable residual. The unlined cast iron mains typical of this part of Louisiana
likely exacerbates this problem.
Although N. fowleri poses a serious health risk, there exist significant knowledge gaps in
understanding key influences of water chemistry and microbiology on N. fowleri growth and
control. Adequate free chlorine disinfectant residuals (>0.5 mg/liter) have been shown to
effectively inactivate N. fowleri trophozoites in drinking water (Cursons, Brown, & Keys, 1980;
De Jonckheere & Van De Voorde, 1976b). Still, residuals can be challenging to maintain, and
other general factors contributing to N. fowleri prevalence in municipal drinking water are not
well understood. As N. fowleri is a warm water organism (25-45°C), increasing regional and
global temperatures have been suggested as a possible cause in the increase of positive detection
of the amoebae (CDC 2017). However, it is important to consider the role of other
physicochemical factors as well. In addition to disinfectant type and residual concentrations
maintained, the presence of nitrifying bacteria and aging drinking water distribution systems are
also important factors that could contribute to an environment amenable to N. fowleri. While N.
55
fowleri can be adequately disinfected using standard chlorination, monochloramine has been
growing in popularity in recent years and is now used by about 50% of U.S. drinking water
utilities. Thus, it is important to determine if monochloramine is an effective control for N.
fowleri (Bartrand, Causey, & Clancy, 2014), particularly under real-world conditions that could
trigger its decay. Specifically, nitrification is a significant impediment to successful application
of chloramine. Nitrification is stimulated by the residual free ammonia, substantial quantities of
which can remain after it is added at the treatment plant and be oxidized by nitrifying bacteria to
nitrite, which in turn reacts with chloramine and contributes to its decay water conditions (Zhang
& Edwards, 2009; Zhang, Love, & Edwards, 2009). Because nitrifying bacteria are slow-
growing autotrophs, nitrification tends to be particularly problematic during warm seasons.
Additionally, aging iron drinking water mains and distribution lines impart a chlorine demand,
making it extremely difficult to maintain an effective chlorine residual (Zheng, Chunguang He,
& Qiang He, 2015).
While N. fowleri have been shown to survive a wide pH range (pH 3.3-9.6), they tend to grow
best under slightly acidic conditions (Brown, Cursons, Keys, Marks, & Miles, 1983; John, 1982;
Sheehan, Fagg, Ferris, & Henson, 2003). This is particularly noteworthy as both iron corrosion
and nitrification can cause slight acidification in water, with enhanced pH reduction in the
presence of chloramine disinfectant as opposed to chlorine (Masters, Wang, Pruden, & Edwards,
2015).
While attention to the brain-eating amoeba has increased in recent years, it is clear that the
factors that influence the prevalence of N. fowleri in drinking water have not been adequately
56
investigated. Therefore, this study seeks to investigate how factors such as pipe material,
nitrification, and disinfection treatment influence the growth of N. fowleri in a simulated
drinking water system.
3.2 Materials & Methods
3.2.1 Simulated warm water batch reactors (SWWBR)
The SWWBR were constructed to test how varying pipe material, disinfectant strategies, and
levels of nitrification may influence the growth and survival of N. fowleri in drinking water. The
reactors consisted of 48, 125-mL wide-mouth straight-sided glass jars with phenolic PTFE lids
equipped with rubber liners. All reactors had 1 inch sections of 1 inch diameter PVC pipe
coupons fixed with epoxy to the bottom of the glass jars. Half (n=24) of the reactors also had
two 1.5-gram iron coupons fixed to opposite sides of the PVC coupons (Figure 3.1).
Figure 3.1. 125 mL simulated warm water batch reactors containing PVC only (left) and PVC
with iron coupons (right).
3.2.2. Influent water and water changes
Initially, nitrifying organisms were established in annular reactors by inoculating each reactor
with 125 mL of granular activated carbon filtered Blacksburg tap water known to have nitrifying
57
organisms (influent water from Rhoads et al., 2015) and incubating at 30 °C for 7 days.
Ammonia was dosed to influent water using ammonium sulfate at 5 mg/liter to initiate
nitrification. Nitrification was confirmed by measuring complete oxidation of ammonia. After
four weeks, the water source was switched to water from St. Bernard Parish, Louisiana and
nitrification continued to occur. Water was shipped to Virginia Tech from St. Bernard Parish
approximately every 5 weeks. The St. Bernard Parish distribution system is composed of
different pipe materials and uses primarily chloramine disinfection to achieve a disinfectant
residual within the distribution system. St. Bernard Parish water was collected from a site near
the location where N. fowleri was detected in 2015 in order to best simulate the water conditions
that facilitated the growth of N. fowleri. It is important to note, however, that water chemistry
might have been altered after several iron pipe fixtures were removed and replaced with PVC
materials after the PAM cases were identified. Total chlorine residuals were measured upon
collection in St. Bernard Parish and water was shipped overnight in coolers to Blacksburg, VA.
Upon arrival, the total chlorine residual was measured again as well as total ammonia, nitrite,
and pH. Waters were then dosed with sodium thiosulfate to neutralize chloramine residuals and
stored at 4 °C prior to water preparation.
The SWWBR underwent 50% water changes with minimal mixing three times per week in order
to simulate stagnant warm water conditions. With each water change, they were replenished with
approximately 55 mL of St. Bernard Parish water. To optimize N. fowleri growth conditions,
reactors were incubated at 35°C and agitated at 100 rpm to simulate turbulent flow characteristic
of water distribution systems and the warm water conditions similar to that of St. Bernard Parish
in summer months.
58
N. fowleri strains (ATCC 30894 and the CDC Davis strain) were grown on Nelson’s agar (saline
agar, CDC) with lawns of E. coli (K5808) at 42 °C for at least 48 h. Phase contrast microscopy
was used to detect advancing fronts, fronts were flooded with WB Saline (CDC), and the
amoebae were scraped and transferred into a sterile 15 mL conical tube. Using a Thermo
hemocytometer, cells were counted to determine concentration and all SWWBR were inoculated
with approximately 1500 cells/reactor. Water changes continued three times per week for
approximately six months. Immediately prior to water change, all St. Bernard Parish water was
filtered through a biologically-active granulated activated carbon to biofilter the water, heated to
approximately 35 °C, and dosed with CO2 gas to reduce influent to pH 7 (±0.25). Reducing the
pH with CO2 took place in order to lower reactor’s pH to the optimal conditions for N. fowleri.
The water was heated in order to avoid thermal shock. Prior to implementing the different
experimental conditions that were the subject of this study, reactors were with the same pipe
materials were cross-inoculated, where 50% of the waters were pooled and redistributed among
reactors along with 50% fresh water in order to establish similar baseline conditions upon
commencement of the experiments.
3.2.3 Water Chemistry Analyses
Throughout the experiment, various water chemistry variables were measured. Analyses,
summarized in Table 3.1, provided insight into the conditions most suitable for the growth of N.
fowleri. All samples collected for chemical analysis, following inoculation of N. fowleri to the
reactors, were filter-sterilized through 0.45 µm luer-lok syringe filters fitted to 30 mL syringes
prior to analysis.
59
Table 3.1. Water Chemistry parameters assessed with references to APHA Standard Method or
other published standard method.
Analysis Instrument Method Reference
Free/Total Chlorine HACH DR/2400 Spectrophotometer
(HACH Co., Loveland, CO) 4500-Cl
Dissolved Oxygen
Orion Star A326 pH/RDO/DO Meter with Optical DO
Probe
(Thermo Fisher Scientific Inc., Waltham, MA)
4500 O G
pH (water) Oakton pH 110 series meter
(Oakton Research, Vernon Hills, IL) 4500-H+
pH (surface) Orion Star A326 pH/RDO/DO Meter w/ Orion
PerpHect Ross Combination pH Micro Electrode 4500-H+
Ammonia (NH4+-N),
Nitrite (NO2--N)
HACH DR/2400 Spectrophotometer 4500-NH3
Nitrite Hach Method 8507
Nitrite (NO2--N),
Nitrate (NO3--N)
Dionex® DX-120 Ion Chromatograph
(Dionex Co., Sunnyvale, CA) 4110
Total Dissolved Iron HACH DR/2400 Spectrophotometer Hach Method 8008
3.2.4 Microbiological Analyses
3.2.4.1 Sample Concentration
To sample for later analysis of DNA, approximately 55 mL of water from each SWWBR was
collected and filtered onto sterile 0.22 μm-pore size mixed cellulose ester filters (Millipore,
Billerica, MA). Filters were uniformly torn under aseptic conditions using sterile tweezers and
transferred to DNA extraction tubes (Lysing Matrix tube A). DNA was extracted using the
FastDNA® SPIN Kit and the FastPrep® Instrument (Qbiogene, Inc., CA) according to
manufacturer protocols and stored at -20°C.
3.2.4.2 DNA Extraction & qPCR
Quantitative polymerase chain reaction (qPCR) was used to quantify a 123-bp section of the ITS
gene, specific to N. fowleri. This assay was chosen due to its capacity to quantitative detect shifts
in the presence of N. fowleri under varying water conditions. The primers included JBVF, 5-
AGG TAC TTA CGT TAG AGT GCT AGT-3, JBVR, 5-ATG GGA CAA TCC GGT TTT CTC
60
A-3 and the (FAM-) labeled probe JBVP, 5-FAM-AC GCC CTA GCT GGT TAT GCC GGA
TT-BHQ1-3. All qPCR assays were performed using the CFX96™ real-time system (Bio-Rad,
Hercules, CA). Cycle parameters were 95 °C for 15 minutes followed by 45 cycles of 95 °C for
15 seconds and 63 °C for 33 seconds (Mull, Narayanan, & Hill, 2013). A standard curve
including seven serial diluted standards and negative control were included in every assay.
3.2.4.3 Culture
Culturing for viable N. fowleri was conducted using Nelson’s agar with established lawns of E.
coli (K5808). Plates were incubated at 42°C and were observed daily for up to 5 days under a
phase-contrast microscope at 100x magnification to observe advancing fronts. For qPCR
confirmation of N. fowleri, advancing fronts were collected using a sterile loop and resuspended
in 250 µL of WB saline followed by DNA extraction.
3.2.5 Statistical Analysis
All statistical analyses were performed using RStudio (RStudio, Inc, Boston, MA). All data was
assessed for normality using the Shapiro-Wilks test. Student’s t-tests were used to compare
means for normally distributed data and Wilcoxon tests were used for non-parametric
comparison of two data sets.
3.3 Results & Discussion
3.3.1 Nitrification
To ensure that nitrification would be consistent across all reactors, nitrifying bacteria were
allowed to establish for several months before N. fowleri was introduced. Initial influent
ammonia concentrations from the St. Bernard Parish water averaged 1.12 mg/liter NH3-N (±0.16
mg/liter NH3-N) and after one month of water changes, ammonia was fully oxidized (<0.01
mg/liter NH3-N) within 2 days.
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3.3.2. Effect of iron
Once nitrification was established, as determine by complete oxidation of added ammonia after 2
days, viable N. fowleri cells were inoculated to the reactors and conditions maintained constant
for six months, optimized for amoeba growth. Confirmation of amoeba viability occurred
monthly, along with qPCR verification and quantification. Initially, qPCR confirmed
quantifiable levels of N. fowleri in all reactors only for the PVC condition. This was likely due to
inhibition of PCR as a result of accumulation of iron corrosion products in the reactors. No level
of dilution of DNA extracts was able to eliminate the effects of qPCR inhibition, resulting in the
potential for false negatives, raising the detection limit, and limiting confidence in the
quantitative value of the results for the iron condition. However, verification of positive
detection based on qPCR of culture-enriched N. fowleri was performed to confirm that viable N.
fowleri were only viable in reactors containing iron. After six months of water changes, samples
were again collected for qPCR and culture, and, while inhibition was interfering with
quantification, it was confirmed that viable N. fowleri was still present in all reactors with iron
(n=24), but was no longer present in reactors with PVC only (n=24) (Chi-squared, p<0.0001;
Table 3.2).
Several reasons for this shift in viability to only conditions containing iron were hypothesized.
First, the pH of waters in PVC only compared to PVC with iron were significantly different
(Wilcox test, p<0.0001) with the average pH 8.26 (±0.42) for PVC only and pH 7.56 (±0.55) for
PVC with iron, despite lowering influent waters to pH 7 prior to water changes.
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Table 3.2. Comparison of 24 replicate PVC only and PVC with iron reactors. Red-filled boxes
indicate positive confirmation via both qPCR and culture. Yellow-filled boxes represent negative
confirmation via qPCR.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
PV
C o
nly
rea
cto
r
PV
C w
ith
Iro
n r
eact
or
Dissolved oxygen (DO) levels were also significantly lower in iron compared to PVC reactors (t-
test, p=0.007). Average DO for PVC only reactors was 5.45 mg/liter (±0.18 mg/liter) and for
PVC with iron reactors 3.69 mg/liter (±0.99 mg/liter).
Iron levels were consistently high in all reactors, as corrosion was extreme enough to discolor
waters and create sediment and granules. Average dissolved iron was 114 mg/liter (±72
mg/liter), which is over 380 times greater than the EPA secondary MCL, but is not unusual in
particularly corrosive water conditions (Teng et al. 2008; Pieper et al. 2017).
3.3.3. Disinfectant conditions
The next phase of the experiment introduced a range of disinfectant and nitrification conditions
to the reactors for replicated comparison in parallel. However, continued issues with qPCR
inhibition resulted in a lack of quantifiable assessment of N. fowleri over time. The conditions
tested are summarized in Table 3.1 and were chosen to represent varying levels of nitrification
and disinfectant using chlorine and chloramine, but these will not be discussed in full as it was
Positive Detection via qPCR and culture Negative Detection via qPCR
63
deemed impossible to attain quantitative data from qPCR. Instead, it was considered that a
positive detection was a true positive, while negative detections were suspect to the potential of
false negatives due to PCR inhibition.
Table 3.3. Experimental conditions for second phase of experiment.
Treatment Condition PVC with Iron
Reactor
Control 1 2 3
Control 1 2 3
BPC*, no ammonia 1 2 3
BPC*, 1 mg/L NH3 1 2 3
0.5 mg/L Chlorine** 1 2 3
0.1 mg/L Chloramine** 1 2 3
0.5 mg/L Chloramine** 1 2 3
1 mg/L NH3 + chlorite** 1 2 3
*BPC is breakpoint chlorinated water which is then heated to remove residual
and the pH is lowered back to ~pH 7 using CO2
**Conditions take place following BPC
Previous studies have shown that N. fowleri, in its trophozoite form, is susceptible to chlorine
disinfectant (Cursons et al., 1980; De Jonckheere & Van De Voorde, 1976a). Therefore, it was
anticipated that N. fowleri would most likely begin to disappear from conditions dosed with
chlorine first. While only 2 of the three replicates were positive for N. fowleri using qPCR after
46 days, it appears that the greatest decreases in positive detections occurred in the control
condition and 0.1 mg/L chloramine conditions (Table 3.3).
This could be due to a variety of reasons, including that the samples may not have been detected
due to inhibition of qPCR in the collected samples. A significant takeaway from this analysis is
that inhibition can vary in samples independent of iron levels. This will be further discussed
below.
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Table 3.4. Summary of positive detections via qPCR in the iron reactors following shifting them
to the indicated conditions for the time period indicated. Green yellow indicate that all replicates
were positive for N. fowleri using qPCR while orange/red indicate that some samples were
negative.
Day 0 (Before
Treatment) Day 11 Day 31 Day 46
Control* 6 6 6 3
BPC, no ammonia 3 2 3 3
BPC, 1 mg/L NH3 3 3 3 3
0.5 mg/L Chlorine 3 3 3 2
0.1 mg/L Chloramine 3 3 2 1
0.5 mg/L Chloramine 3 3 3 2
1 mg/L NH3 + chlorite 3 3 3 3
*n=6 for Control, n=3 for all others
3.3.4. Inhibition Troubleshooting
The iron coupons utilized in half of the SWWBR corroded significantly in the reactors over time,
producing iron sediment and granules and turning the glass reactors visibly orange. Therefore,
filter concentration of reactor water samples concentrated high levels of undissolved iron
species. The speciation of iron was not determined. Iron has been shown to inhibit PCR
reactions, but the standard recommendation for reducing inhibition is dilution, which can reduce
sensitivity and raise detection limits (Tsai & Olson, 1992; Wilson & Wilson, 1997). A recent
study published a simplified method of using the chelating agent, EDTA, along with excess
MgCl2 to improve efficiencies of PCR reactions by sequestering available iron remaining in the
DNA extract (Teng et al. 2008). There was no significant increase in qPCR results with EDTA
added and those without. While this method was shown to be effective in PCR reactions, the
small and limited volumes utilized in qPCR made the direct conversion of this method
ineffective.
65
In another trial, attempts were made to remove iron from the samples upon filtration onto the
0.22 µm filters. Samples were dosed with hydroxylamine hydrochloride, which has been shown
to reduces ferric (III) iron to ferrous (II) iron allowing the soluble ferrous iron to pass through the
filter (Gopala & Somidevamma, 1959). This reduction in iron accumulation on the filter was
hypothesized to reduce iron concentrations below inhibitor concentrations for qPCR. Beginning
with 6% v/v and increasing to 16% v/v hydroxylamine hydrochloride, iron concentrations were
measured before and after filtration of samples and filters were collected and DNA extracted as
described above. However, iron particulates did not dissolve even at 16% v/v hydroxylamine,
qPCR results did not show any increase in gene copies as a result of this method.
3.4 Conclusions
Though this study was eventually limited by the methodology, an important finding was that
high iron levels were conducive to the growth and viability of N. fowleri and therefore that iron
could be a potential indicator for the prevalence of N. fowleri in drinking water. There is a four-
fold reason why high iron could potentially contribute to the proliferation of N. fowleri,
diminishing overall water quality and presenting a danger to those exposed.
• Iron is an essential nutrient for N. fowleri and enhances viability.
• Iron reduces disinfectant residuals and high temperatures can increase the rate of removal
of disinfectant.
• Iron lowers dissolved oxygen and N. fowleri can survive under lower oxygen conditions
than some other protozoa.
• Iron causes fluctuations in pH and corrosion and therefore lowers overall pH.
Another important consideration is the fact that systems that utilize chloramine disinfectant
introduce a potential for nitrification to occur in the distribution system, which can further
66
decrease dissolved oxygen and pH (Zhang et al., 2010), but this variable must be further
researched to verify any correlations with nitrification. Lastly, inhibition due to iron corrosion
played a key role in this experiment and is something that is not adequately addressed in
literature. Future work, particularly regarding drinking water samples from aged iron water
pipes, would merit from investigations of ways to reduce iron-induced inhibition in qPCR.
3.5 Acknowledgements
This study was supported by the Alfred P. Sloan Foundation – Microbiology of the Built
Environment program and the Virginia Tech Institute for Critical Technology and Applied
Science. The findings do not represent the views of the sponsors.
67
3.6 References
Bartrand, T. A., Causey, J. J., & Clancy, J. L. (2014). Naegleria Fowleri: An Emerging Drinking
Water Pathogen. Journal - American Water Works Association, 106(10), E418–E432.
http://doi.org/10.5942/jawwa.2014.106.0140
Brown, T. J., Cursons, R. T. M., Keys, E. A., Marks, M., & Miles, M. (1983). The occurence and
distribution of pathogenic free-living amoebae in thermal areas of the North Island of New
Zealand. New Zealand Journal of Marine and Freshwater Research, 17, 59–69.
http://doi.org/10.1080/00288330.1983.9515987
CDC. (2015). Naegleria fowleri — Primary Amebic Meningoencephalitis (PAM).
Cursons, R. T. M., Brown, T. J., & Keys, E. A. (1980). Effect of disinfectants on pathogenic
free-living amoebae: In axenic conditions. Applied and Environmental Microbiology, 40(1),
62–66.
De Jonckheere, J., & Van De Voorde, A. H. (1976a). Differences in Destruction of Cysts of
Pathogenic and Nonpathogenic Naegleria and Acanthamoeba by Chlorine. Applied and
Environmental Microbiology, 31(2), 294–297. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC169762/pdf/aem00002-0158.pdf
De Jonckheere, J., & Van De Voorde, H. (1976b). Differences in Destruction of Cysts of
Pathogenic and Nonpathogenic Naegleria and Acanthamoeba by Chlorine. Applied and
Environmental Microbiology, 31(2), 294–297.
Gopala, G., & Somidevamma, G. (1959). Volumetric Determination of Iron (III) with
Hydroxylaminc as a Reducing Agent. Fresenius’ Zeitschrift Für Analytische Chemie,
165(6), 432–436. Retrieved from
https://link.springer.com/content/pdf/10.1007%2FBF00462146.pdf
68
John, D. T. (1982). Primary Amebic Meningoencephalitis and the biology of Naegleria fowleri.
Ann. Rev. Microbiol., 36, 101–23. Retrieved from
http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.36.100182.000533
Louisiana Department of Health. (2013a). CDC Testing Confirms Presence of Rare Ameba in
One DeSoto Parish Water System | Department of Health | State of Louisiana. Retrieved
July 12, 2017, from http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/2889
Louisiana Department of Health. (2013b). DHH Confirms Death of a Child Associated with Rare
Amoeba Found in St. Bernard Parish Home | Department of Health | State of Louisiana.
Retrieved July 12, 2017, from
http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/2862
Louisiana Department of Health. (2017). LDH Confirms Naegleria Fowleri Ameba in North
Monroe, Schriever Water Systems | Department of Health | State of Louisiana. Retrieved
July 12, 2017, from http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/4284
Marciano-Cabral, F., MacLean, R., Mensah, A., & LaPat-Polasko, L. (2003). Identification of
Naegleria fowleri in Domestic Water Sources by Nested PCR. Applied and Environmental
Microbiology, 69(10), 5864–5869. http://doi.org/10.1128/AEM.69.10.5864-5869.2003
Masters, S., Wang, H., Pruden, A., & Edwards, M. A. (2015). Redox gradients in distribution
systems influence water quality, corrosion, and microbial ecology. Water Research, 68,
140–149. http://doi.org/10.1016/j.watres.2014.09.048
Mull, B. J., Narayanan, J., & Hill, V. R. (2013). Improved Method for the Detection and
Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic
Separation and Real-Time PCR. Journal of Parasitology Research, 2013, 8.
http://doi.org/10.1155/2013/608367
69
Pieper, K. J., Tang, M., & Edwards, M. A. (2017). Flint Water Crisis Caused By Interrupted
Corrosion Control: Investigating “Ground Zero” Home. Environmental Science &
Technology, 51, 2007–2014. http://doi.org/10.1021/acs.est.6b04034
Rhoads, W. J., Ji, P., Pruden, A., & Edwards, M. A. (2015). Water heater temperature set point
and water use patterns influence Legionella pneumophila and associated microorganisms at
the tap. Microbiome, 3(1), 67.
Sheehan, K. B., Fagg, J. A., Ferris, M. J., & Henson, J. M. (2003). PCR Detection and Analysis
of the Free-Living Amoeba Naegleria in Hot Springs in Yellowstone and Grand Teton
National Parks PCR Detection and Analysis of the Free-Living Amoeba Naegleria in Hot
Springs in Yellowstone and Grand Teton National Parks. Society, 69(10), 5914–5918.
http://doi.org/10.1128/AEM.69.10.5914
Teng, F., Guan, Y. T., & Zhu, W. P. (2008). Effect of biofilm on cast iron pipe corrosion in
drinking water distribution system: corrosion scales characterization and microbial
community structure investigation. Corrosion Science, 50(10), 2816–23.
http://doi.org/10.1016/j.corsci.2008.07.008
Teng, F., Guan, Y., & Zhu, W. (2008). A simple and effective method to overcome the inhibition
of Fe to PCR. Journal of Microbiological Methods, 75(2), 362–364.
http://doi.org/10.1016/j.mimet.2008.06.013
Tsai, Y.-L., & Olson, B. H. (1992). Detection of Low Numbers of Bacterial Cells in Soils and
Sediments by Polymerase Chain Reaction. Apllied and Environmental Microbiology, 58(2),
754–757.
US EPA. (2017). Contaminant Candidate List 3. Retrieved September 7, 2017, from
https://www.epa.gov/ccl/contaminant-candidate-list-3-ccl-3
70
Wilson, I. G., & Wilson, I. a N. G. (1997). Inhibition and Facilitation of Nucleic Acid
Amplification Inhibition and Facilitation of Nucleic Acid Amplification, 63(10), 3741–
3751.
Yoder, J. S., Straif-Bourgeois, S., Roy, S. L., Moore, T. A., Visvesvara, G. S., Ratard, R. C., …
Beach, M. J. (2012). Primary Amebic Meningoencephalitis Deaths Associated With Sinus
Irrigation Using Contaminated Tap Water. Clinical Infections Diseases, 55(9), e79-85.
http://doi.org/10.1093/cid/cis626
Zhang, Y., & Edwards, M. (2009). Accelerated chloramine decay and microbial growth by
nitrification in premise plumbing. Journal AWWA, 101(11), 51–62.
Zhang, Y., Edwards, M., Pinto, A., Love, N., Camper, A., Rahman, M., & Baribeau, H. (2010).
Effect of Nitrification on Corrosion in the Distribution System. (W. R. Foundation,
Ed.)Water Research Foundation.
Zhang, Y., Love, N., & Edwards, M. (2009). Nitrification in Drinking Water Systems. Critical
Reviews in Environmental Science and Technology, 39(3), 153–208.
http://doi.org/10.1080/10643380701631739
Zheng, M., Chunguang He, & Qiang He. (2015). Fate of free chlorine in drinking water during
distribution in premise plumbing. Ecotoxicology, 24, 2151–2155.
http://doi.org/10.1007/s10646-015-1544-3
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Chapter 4: Naegleria fowleri in private well
water after major flooding
4.1 Introduction
Private wells are the primary drinking water source for approximately 15% of Americans, but it
is not subject to Safe Drinking Water Act regulations, including control for microbial
contaminants (US EPA, 2002). Therefore, in the event that a private well is contaminated with
surface water, it is the responsibility of homeowners to maintain and decontaminate their own
drinking water source. However, many homeowners are unaware of the degree and type of
contamination, limiting their ability to take appropriate steps toward disinfection and leaving
themselves at risk for water-borne illnesses.
In August 2016, a 1000-year flooding event took place in rural southern Louisiana near Baton
Rouge, which led to the temporary relocation of over 30,000 people and the of damage more
than 90,000 homes (Bel Edwards & Nungesser, 2017; Di Liberto, 2016). Over 500,000 people
in the state of Louisiana rely on private well water as their drinking water source with almost
400,000 people served by private wells in the most heavily impacted parishes (those that
received individual FEMA assistance) (FEMA.gov, 2016; Louisiana Department of Health,
2017b; U.S. Geological Survey, 2016). Well water has the potential to be a major source of
harmful chemical elements and compounds such as arsenic and nitrates, but the biggest issue
facing private well owners is microbial contamination (Craun et al., 2010; DeFelice, Johnston, &
Gibson, 2016).
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Microbial contamination of well water can occur due to fecal contamination or surface runoff
and standard testing only indicates the presence of total coliforms and E. coli, which the EPA
requires for public water systems supplied by groundwater, but not for private wells (US EPA,
2006). In addition to fecal coliforms, recent studies suggest that opportunistic pathogens (OPs)
and free-living amoeba (FLA) in private well water could be a source of infection for those who
use well water as their primary drinking water source (Baquero et al., 2014; Blair, Sarkar, Bright,
Marciano-Cabral, & Gerba, 2008; Richards et al., 2015). OPs such as Legionella,
Mycobacterium, Pseudomonas aeruginosa, Acanthamoeba, and Naegleria fowleri are of great
concern in drinking water due to their abilities to grow in household plumbing conditions under
low nutrient conditions, resist low levels of disinfectant, form biofilms, and grow under stagnant
conditions. Bacterial OPs are also known to be capable of colonizing FLA, contributing to
protection of bacteria from disinfectant and providing a reservoir for growth (Falkinham, 2015).
Due to the lack of regulatory oversight for private well water quality and minimal understanding
of OPs in well water, a great deal must be done to determine how existing preventative measures
influence the growth of OPs and how these measures might be changed to decrease risk exposure
of those using private wells for their water supply.
Both the EPA and the State of Louisiana provide recommendations for disinfecting private wells
after flooding or the laboratory detection of bacteria; however, these recommendations are
untested and can vary depending on the agency. Recommendations involve running water
through a faucet or spigot until it appears clear followed by pouring 1 gallon of chlorine bleach
(EPA), or 4 gallons of chlorine bleach solution (1 gallon of bleach into 3 gallons of water; State
of Louisiana) down the well. Then, homeowners are instructed to run water through all faucets
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until they are able to smell chlorine. They are then instructed to allow 6-24 hours (EPA) or 24
hours (State of Louisiana) of contact time in their plumbing in order to disinfect the entire system
(Office of Public Health.; Jindal & Greenstein, 2014; Office of Water, 2005). This procedure has
several limitations, including inadequate long-term disinfectant residual, potentially inducing
corrosion in metal plumbing fixtures (Cantor, Park, & Vaiyavatjamai, 2000; Seiler, 2006), and
the inconvenience of inaccessibility to a homeowner’s water source for 6-24 hours. Additionally,
there are no known studies investigating the effectiveness of this “shock chlorination” technique
in disinfecting and preventing regrowth of bacteria in homes served by private wells. Particularly
in circumstances where warm surface waters are allowed to infiltrate well water columns and/or
aquifers, the study of pathogenic microbial constituents is essential to understanding how
homeowners may be impacted by fecal coliforms and OPs and the impact that regulations might
have on the disinfection of homeowner’s wells following natural disasters.
Given the warm climate of Louisiana and recent outbreaks in drinking water, the “brain-eating
amoeba,” Naegleria fowleri, was chosen as the focus of this study. This amoeba, causes the fatal
disease, primary amoebic meningoencephalitis (PAM) when contaminated water is forced up the
nasal cavity (CDC, 2015). N. fowleri is a thermophilic organism which, in recent years, has
become a major concern in the U.S. due to its emergence in northern locations as well as the
increasing awareness and detection of the amoeba in both recreational waters, the most common
source of infection, and municipal drinking water distribution systems (Bartrand, Causey, &
Clancy, 2014; Kemble et al., 2012; Martinez & Visvesvara, 1997; Puzon, Lancaster, Wylie, &
Plumb, 2009; Yoder et al., 2012).
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For decades, N. fowleri has been detected in warm (>25 °C) surface waters, presenting a risk to
those using the water for recreational purposes and causing numerous PAM infections (Ettinger
et al., 2002; John & Howard, 1995.; Kemble et al., 2012; Sykora, Keleti, & Martinez, 1983;
Wellings, Amuso, Chang, & Lewis, 1977). In the past 3 years, 6 utilities supplied by
combinations of groundwater and surface water in the state of Louisiana have reported positive
detection of N. fowleri in their drinking water with 4 deaths occurring due to PAM infections
contracted from drinking water (Louisiana Department of Health, 2013a, 2013b, 2017a).
Therefore, this raised concerns that flooding of private wells in Louisiana from warm surface
water, particularly in the month of August, could lead to contamination of N. fowleri into private
wells, putting homeowners at risk of infection. While some studies have investigated municipal
well water and high-volume private well water companies for N. fowleri, there are no known
studies investigating N. fowleri in single home private wells (Blair et al., 2008).
In order to assess the breadth of microbial contamination that occurred in areas damaged within
the August 2016 Louisiana flood zone, a sampling effort took place in October 2016, targeting
homes with flooded wells in Livingston Parish and Ascension Parish. Homeowner sampling for
metals, total coliforms/E. coli, and DNA extraction for OP-targeted qPCR took place. Sampling
kits included samples for three water types: first draw cold (premise plumbing), first draw hot
(premise plumbing), and cold flushed (well water). The collection of these three water samples
allowed for and in-depth understanding of well water column contamination compared to
colonization and contamination in the home plumbing systems. While a broad spectrum of
opportunistic pathogens were assessed from the sampling survey, this paper focuses on N.
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fowleri due to increase awareness of its presence and high concern over its potential for survival
in well water.
The objectives of this study were (a) to be the first survey of N. fowleri in private well water
following a major flooding event and (b) to understand how varying physicochemical factors,
homeowner actions, and geospatial circumstances may have contributed to the colonization of N.
fowleri in private wells and premise plumbing.
4.2 Methods
4.2.1 Study Design
In October 2016, a surveying effort took place to assess opportunistic pathogens and water
chemistry characteristics of private wells in rural southern Louisiana. A total of 150 well users
reporting being impacted by the recent flood participated in the study with 113 completing the
sampling and survey. All residents were given water sampling kits and written sampling
instructions as well as verbal instructions to collect water from their kitchen sink tap.
Specifically, each resident was instructed to collected three samples from their kitchen sink cold-
water tap: (1) a 250-mL first draw sample from the kitchen tap after 6+ hours of stagnation
(targets premise plumbing corrosion), (2) an additional 250-mL sample after 5 minutes of
flushing (targets corrosion within the well and characterizes physiochemical parameters), and (3)
a 100-mL microbial sample (targets TC and E. coli). An additional subset of 40 residents
volunteered to collect additional samples for in-depth microbial analysis for OPs. These
residents received three additional 1-L sterile sampling bottles, which were collected from the
cold-water tap following the first draw sample, first draw of the hot-water tap, and the cold-water
flushed tap following the microbial sample. Upon return of samples the morning that they were
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collected, all microbial samples were placed on ice and pH measurements were recorded from
the 250-mL well water samples. All microbial samples were transported overnight to Virginia
Tech. Each participant received a water quality analysis packet within one month. Residents
with OPs were contacted by a member of the research team to inform them of the potential risks
posed by associated OPs and provided information on best practices to disinfect their well and
prevent regrowth.
4.2.2 Sample Analysis
4.2.2.1 Water Analysis
All pH measurements were collected per Standard Method 4500-H+ with an Oakton pH 110
series meter (Oakton Research, Vernon Hills, IL; (APHA, AWWA, 1998). Samples were
analyzed for a spectra of metals concentrations using a Thermo Electron X-Series inductively
coupled plasma mass spectrometer (ICP-MS) per Standard Method 3125-B (APHA, AWWA,
1998). Samples and calibration standards were prepared in a matrix of 2% nitric acid by volume.
Total coliforms and E. coli were quantified using the IDEXX Colilert 2000 method (IDEXX
Laboratories Inc., Westbrook, MN, USA). For data quality assurance, quality control blanks and
spikes of known concentrations were measured every 10-15 samples for all analyses, except in
the case of Colilert, where a positive and negative control were included for each resident’s
sample set. All 1-L OP samples were processed by filter concentrating through sterile 0.22 um-
pore-size mixed cellulose ester filters (Millipore, Billerica, MA). Filters were uniformly torn
using sterile tweezers and were transferred to Lysing Matrix tube A from the FastDNA® SPIN
Kit (Qbiogene, Inc., CA).
77
4.2.3 DNA extraction & qPCR
DNA was extracted using the FastDNA® SPIN Kit and the FastPrep® Instrument (MP
Biomedicals, Inc., Solon, OH) according to manufacturer protocols and stored at -20 °C.
Quantitative polymerase chain reaction (qPCR) was used to assess 16S rRNA, Legionella spp.,
L. pneumophila, and Naegleria fowleri gene copies (Table 4.3). All qPCR assays were carried
out using a CFX96™ real-time system (Bio-Rad, Hercules, CA). Inhibition was minimized by
diluting 1:10 with molecular-grade water. In each qPCR run a calibration curve was included
with seven standard points included. The qPCR quantification limit was dependent on the gene
being assessed; 100 gene copies/reaction for 16S, 50-100 gene copies/reaction for all other
genes.
4.2.4 Statistical Analysis
Statistical analyses were performed in R studio and JMP. A Shapiro-Wilk test was used to
determine normality of each data set. Non-parametric Wilcoxon tests (p<0.05) were used to
compare two groups and Spearman rank correlation tests (p<0.05) were used to assess non-
parametric correlations.
4.3 Results & Discussion
4.3.1. Correlations among bacteria
4.3.1.1 Total Coliforms & E. coli
Fecal coliforms, including E. coli can cause serious illness when consumed, and are indicators
for surface water or septic tank contamination in well water (Allevi et al., 2013; EPA, 2013;
Macler & Merkle, 2000). In Louisiana, all 113 of 150 (75% return rate) distributed sampling kits
were returned and assessed for total coliforms and E. coli. Of the 113 homes sampled, 25% were
found to be positive for total coliforms. However, only 4% of homes were positive for E. coli
78
(Table 4.1). Overall, when compared to other studies of total coliforms and E. coli in private
wells this is not an unusually high rate of detection. A subset of studies report a range of 35.7%-
67% positive samples for total coliform and 1.37%-54.5% positive samples for E.coli (n=11-
16138) (Allevi et al., 2013; DeFelice et al., 2016; Maran et al., 2016; Sousa, Taveira, & Silva,
2015; Won, Gill, & LeJeune, 2013). This detection rate was unexpected as surface water from
the flooding was anticipated to introduce coliforms and fecal contaminants. However, these
samples were collected in late October 2016, two months following the flooding, indicating that
perhaps coliforms had dissipated through the flushing of water systems over time. The lack of
high occurrence of total coliforms and E. coli suggests that minimal or no colonization of well
systems occurred due to surface water inundation during the flooding or that post-flood flushing
reduced colonization.
This study was also limited by not having pre-flood sampling results to compare and future
studies would benefit through pre- and post-flood sampling to perform a robust comparison of
the impacts of the flood.
Table 4.1. Positive detection of bacteria and OP DNA in three sample types. *Positive detection
in a home = positive detection in at least one water sample
1: n=113; 2: n=38, two samples were lost; 3: n=37; 4: n=37; 5: n=40
Positive detection Well water Stagnant cold
water
Stagnant hot
water
Homes*
Total coliform 25%1 / / 25%1
E. coli 4%1 / / 4%1
Legionella spp. 45%2 43%3 54%4 75%5
L. pneumophila 5%2 3%3 11%4 15%5
N. fowleri 5%2 11%3 14%4 23%5
79
4.3.1.2 Naegleria fowleri
While total coliform tests present a standard method for understanding potential surface water
contamination and well integrity issues, OPs, which are not fecally transmitted, are known to
colonize home plumbing and are uncommonly assessed in well water. In order to understand the
occurrence of OPs in homes served by private wells, a random subset of 40, out of 113 total
homes, were sampled. The samples for bacterial DNA detection included stagnant cold water,
stagnant hot water, and flushed cold well water. Though several OPs, including Legionella spp.
and Legionella pneumophila were assessed, this study focuses primarily on the brain-eating
amoeba, Naegleria fowleri, due to the recent increased occurrence of detection in Louisiana
municipal drinking water (Bartrand et al., 2014; Louisiana Department of Health, 2013a, 2013b,
2017a; Yoder et al., 2012). Of the 112 samples collected from 40 homes (3 samples per home
with 8 samples not collected or removed), 11 (9.8%) were found to be positive for N. fowleri via
qPCR. These positive samples were collected from eight of 40 homes (20%). While not all
samples were within quantifiable limits of qPCR (>5 gene copies/mL), detection was confirmed
in each of these samples via multiple qPCR assays. The quantifiable range of N. fowleri ranged
11-610 gc/mL, with an average of 285 gc/mL (±176 gc/mL). Two of the homes had multiple
samples positive for N. fowleri with all three samples in one home, averaging 256 gene
copies/mL (±12.4 gc/mL). While viability culturing was not performed on these samples, the
detection of N. fowleri in all of the samples collected from a home suggests that the
homeowner’s well was contaminated and N. fowleri successfully colonized the home’s premise
plumbing.
No correlation was observed between the N. fowleri gene copies and total bacterial DNA (16S
rRNA; Spearman’s ρ= 0.29, p>0.05) or between N. fowleri and Legionella spp. (ρ=-0.20,
80
p>0.05). However, there was a positive correlation between Legionella spp. and 16S rRNA gene
copies/mL (ρ=0.7818, p=0.0045). Total bacterial DNA levels in samples positive for N. fowleri
ranged 1000-1.14x107 gc/mL (±3.58x106 gc/mL; Figure 4.1). The wide range of total bacterial
DNA in N. fowleri positive samples suggest that a threshold level of bacteria may exist to sustain
N. fowleri in well water but a viability assessment would be required to confirm this.
Figure 4.1. Comparison of log10(16S rRNA) and log10(N. fowleri+1) gene copies. No
correlation was found between total bacterial DNA and N. fowleri (Spearman’s ρ=0.29,
p>0.05). Samples at ‘0’ gene copies per mL were below quantification limits but verified to be
present. Sample types are indicated by color and shape. Red circles indicate samples that were
collected from the same home.
4.3.2 Naegleria fowleri & iron
Iron is essential for the growth of most all organisms, and has been shown to promote the growth
of some OPs, such as Legionella (Wang et al., 2012; Wang et al. 2014). Very few studies have
suggested that N. fowleri growth and viability could be increased by iron (John, 1982; A. L.
Newsome & Wilhelm, 1981; Anthony L Newsome & Wilhelm, 1983), but recent work has
indicated that this association could be a key trigger for N. fowleri proliferation. First drawn and
81
flushed well water samples were analyzed for metal concentrations. Among samples collected,
no positive correlation was shown between first draw cold water iron concentrations and flushed
well water iron concentrations and Legionella spp. (ρ =-0.111, p>0.05; ρ =-0.352, p>0.05,
respectively), but a correlation was seen between first draw iron levels and N. fowleri (ρ=0.7083,
p=0.015; Figure 4.2). There was no statistically significant difference in iron or pH in homes
when comparing positive and negative N. fowleri homes (p=0.05).
Figure 4.2. Comparison of log10(N. fowleri+1) gene copies versus first draw (cold stagnant)
total iron concentrations. Samples at ‘0’ gene copies per mL were below quantification limits
but verified to be present. A strong correlation exists between these two variables (Spearman’s
ρ=0.7083, p=0.015).
It is unknown whether the homes with high iron levels had iron plumbing fixtures in their home,
and while there was no significant difference in iron levels in flushed versus first draw iron
samples (paired Wilcox-test, p>0.05), four of the nine homes had first draw iron levels 1.4-2.9
82
times higher than the flushed iron values. This suggests the possibility of iron plumbing fixtures
in the premise plumbing of these homes, but further investigation in needed to verify the impact
of premise plumbing on the water quality and presence of N. fowleri.
4.3.3 Survey data correlations
In addition to the samples collected from 113 homes, homeowners were provided a survey to
indicate descriptions of their home, well, observable water quality, actions before and after the
flood, as well as several questions targeted at understanding how they acquire information in the
event of a natural disaster like the flooding. Answers to survey questions were assessed
alongside analytical results to understand what conditions may have been conducive to the
growth of coliforms or OPs.
4.3.3.1 Geospatial data
The 40 homes sampled for OPs represented homes from 13 zip codes in the flooded region, four
of which contained homes with N. fowleri positive samples. The most frequent sampled zip code
comprised 30% of the sampled homes (n=40) with 33% of homes from that zip code positive for
N. fowleri (n=12). From the three most common zip codes sampled, 28% of samples were N.
fowleri positive (n=25).
4.3.3.2 Treatment and Maintenance
To assess the effect on colonization from treatment and maintenance, homeowners were
provided questions relating to continuous treatments (e.g. whole house filters, tap filters) or one-
time treatments or maintenance (e.g. shock chlorination, filter replacement, pump replacement).
An assessment of maintenance and specific actions taken before and after flooding suggests that
the flooding did not influence the behavior of homeowners in terms of how to treat their water to
ensure that it was safe to drink (Table 2 & Table 3). There is a difference of only one
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homeowner that reported maintenance after the flood compared to before, and over 70% of
homeowners report either no maintenance before or after or gave no answer (n=113). Similarly,
there was no change in action after the flood for those who reported no maintenance before, and
there was only a 1% increase in maintenance action in those who reported maintenance before
the flood (Table 1). Only one additional homeowner reported chlorination of their system after
the flooding when they hadn’t done so before and only 2 homeowners reported having had any
testing done on their water following the flooding. Note that discrepancies among percentages in
Tables 1 and 2 are due to inconsistency in homeowner responses to survey questions. Further
investigations of this survey data have been presented and are pending publication (Brown,
Katner, Pieper, O’Rear, & Edwards, 2017; Katner et al., 2017; Pieper et al., 2017; Pieper &
Katner, 2017).
Of the nine homes positive for N. fowleri, three reported using continuous water treatment on
their private well water; two homes reported using a water softening unit (one with a salt system
included) and one reported a sediment/activated carbon filter. Interestingly, while previous
studies have indicated that N. fowleri can be removed from water treated with activated carbon
filtration, it has also been shown to colonize carbon filters (Thomas, Loret, Jousset, & Greub,
2008). Following the flooding, five of the nine homeowners positive for N. fowleri reported
taking any action. These actions included replacing pumps in two homes, adding salt to the
home with a salting system, changing the filter in the home with a sediment and activated carbon
filter, installing new PVC plumbing, and shock chlorination. The only N. fowleri positive home
that reported taking action to bleach their system was the home with detectable N. fowleri in all
three samples collected from the home.
84
Table 4.2. Homeowner reported maintenance before and after flooding
MAINTENANCE
BEFORE (%)
MAINTENANCE
AFTER (%)
YES 25.7 26.5
NO 66.4 64.6
NA 8.0 8.8
(n=113)
Table 4.3. Maintenance, treatment, or action reported before and after flooding. Mechanical
maintenance includes replacing or servicing well pumps or well tanks and bleach/chlorination
includes reports of shock chlorination using liquid bleach or chlorine tablets.
BEFORE
FLOOD (%)
AFTER
FLOOD (%)
MECHANICAL MAINTENANCE 3.5 7.1
BLEACH/CHLORINATION 15.9 16.8
REPLACE/SERVICE/INSTALL FILTER 3.5 5.3
WATER TESTING 0.0 1.8
OTHER (FILTER & RO, FLUSHING, ADD SALT) 2.7 2.7
NA 75.2 72.6
(n=113)
4.4 Conclusions
Private well water is an essential resource for millions of Americans, but proper standards must
be implemented to reduce risk of water-borne disease in those exposed, especially when harmful
OPs could colonize a system. This study began to investigate ties between well water chemistry,
home owner actions, and the presence of N. fowleri, after a natural disaster left wells vulnerable
to contamination. The following conclusions can be made regarding this work:
• Warm surface water inundation via flooding may increase risk of N. fowleri
contaminating a well column and/or premise plumbing (although non-flooded wells were
not investigated in this study).
85
• Iron in stagnant water (premise plumbing) correlates with quantification of N. fowleri
suggesting that iron increases viability of N. fowleri in non-disinfected drinking water.
• Shock chlorination, performed by private well owners is inadequate at disinfecting a well
and/or premise plumbing long-term and other solutions must be considered, particularly
if there is risk for N. fowleri colonization.
• Education for private well owners in Louisiana following natural disasters is inadequate
at informing residents and encouraging them to take action to disinfect their wells.
Performing thorough investigations of private wells is a complex task and presents unique
challenges to scientists. In many cases it may not be feasible, but it is essential to develop a
more scientifically founded basis of the risks that residents are exposed to in order to adequate
inform and develop infrastructure to assist in safe maintenance and operation of private wells.
4.5 Acknowledgements
This study was supported by the National Institute of Food and Agriculture, U.S. Department of
Agriculture (#2016-67012-24687) and a National Science Foundation RAPID grant (#1556258).
The findings do not represent the views of the sponsors.
86
4.6 References
Allevi, R. P., Krometis, L.-A. H., Hagedorn, C., Benham, B., Lawrence, A. H., Ling, E. J., &
Ziegler, P. E. (2013). Quantitative analysis of microbial contamination in private drinking
water supply systems. Journal of Water and Health, 11(2), 244.
http://doi.org/10.2166/wh.2013.152
APHA, AWWA, and W. (1998). Standard Methods for Examination of Water and Wastewater
(20th ed.). Washington, D.C.: APHA.
Baquero, R. A., Reyes-Batlle, M., Nicola, G. G., Martín-Navarro, C. M., López-Arencibia, A.,
Guillermo Esteban, J., … Lorenzo-Morales, J. (2014). Presence of potentially pathogenic
free-living amoebae strains from well water samples in Guinea-Bissau. Pathogens and
Global Health, 108(4), 206–211. http://doi.org/10.1179/2047773214Y.0000000143
Bartrand, T. A., Causey, J. J., & Clancy, J. L. (2014). Naegleria Fowleri: An Emerging Drinking
Water Pathogen. Journal - American Water Works Association, 106(10), E418–E432.
http://doi.org/10.5942/jawwa.2014.106.0140
Bel Edwards, J., & Nungesser, B. (2017). State of Louisiana Proposed Action Plan Amendment
No. 1 for the Utilization of Community Development Block Grant Funds in Response to the
Great Floods of 2016. Retrieved from http://www.doa.la.gov/OCDDRU/Action Plan
Amendments/Great_Floods_2016/APA 1 Floods Master Action Plan
Amendment_FINAL_English_31Jan2017.pdf
Blair, B., Sarkar, P., Bright, K. R., Marciano-Cabral, F., & Gerba, C. P. (2008). Naegleria
Fowleri in Well Water. Emerging Infectious Diseases, 14(9), 1499–1500.
http://doi.org/10.3201/eid1409.080143
Brown, K., Katner, A., Pieper, K. J., O’Rear, L., & Edwards, M. A. (2017). The Great Louisiana
87
Flood of 2016: Private well owner knowledge, response and needs. In American College of
Epidemiologists (ACE) Annual Meeting: Methods of Translating and Disseminating
Epidemiology into Public Health. New Orleans, LA.
Cantor, A. F., Park, J. K., & Vaiyavatjamai, P. (2000). The Effect of Chlorine on Corrosion in
Drinking Water Systems. Retrieved from
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.137.1011&rep=rep1&type=pdf
CDC. (2015). Naegleria - Illness. Retrieved October 19, 2015, from
http://www.cdc.gov/parasites/naegleria/infection-sources.html
Craun, G. F., Brunkard, J. M., Yoder, J. S., Roberts, V. A., Carpenter, J., Wade, T., … Roy, S. L.
(2010). Causes of outbreaks associated with drinking water in the United States from 1971
to 2006. Clinical Microbiology Reviews, 23(3), 507–528.
http://doi.org/10.1128/CMR.00077-09
DeFelice, N. B., Johnston, J. E., & Gibson, J. M. (2016). Reducing Emergency Department
Visits for Acute Gastrointestinal Illnesses in North Carolina (USA) by Extending
Community Water Service. Environmental Health Perspectives, 124(10), 1583–1591.
Retrieved from https://ehp.niehs.nih.gov/wp-content/uploads/124/10/EHP160.alt.pdf
Di Liberto, T. (2016, August 19). August 2016 extreme rain and floods along the Gulf Coast.
NOAA Climate.gov. Retrieved from https://www.climate.gov/news-features/event-
tracker/august-2016-extreme-rain-and-floods-along-gulf-coast
EPA. (2013). Federal Register 40 CFR Parts 141 and 142. Federal Register (Vol. 78). Retrieved
from https://www.gpo.gov/fdsys/pkg/FR-2013-02-13/pdf/2012-31205.pdf
Ettinger, M., Webb, S., Harris, S., McIninch, S., Garman, G., & Brown, B. (2002). Distribution
of free-living amoebae in James River, Virginia, USA. Parasitology Research, 89(1), 6–15.
88
http://doi.org/10.1007/s00436-002-0707-3
Falkinham, J. (2015). Common Features of Opportunistic Premise Plumbing Pathogens.
International Journal of Environmental Research and Public Health, 12(5), 4533–4545.
http://doi.org/10.3390/ijerph120504533
FEMA.gov. (2016). Louisiana Severe Storms and Flooding (DR-4277). Retrieved August 8,
2017, from https://www.fema.gov/disaster/4277
Health, O. of P. (n.d.). Private Water Well Testing in Louisiana - What you Need to Know to
Protect Your Water. Baton Rouge, LA. Retrieved from
http://new.dhh.louisiana.gov/assets/oph/Center-
EH/sanitarian/onsitewastewater/PrivateWaterWellTestinginLouisiana.pdf
Jindal, B., & Greenstein, B. D. (2014). What To Do If Your Private Well Was Flooded. Baton
Rouge, LA. Retrieved from http://dhh.louisiana.gov/assets/oph/Center-
EH/sanitarian/onsitewastewater/mkvPrivatewells08272012.pdf
John, D. T. (1982). Primary Amebic Meningoencephalitis and the biology of Naegleria fowleri.
Ann. Rev. Microbiol., 36, 101–23. Retrieved from
http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.36.100182.000533
John, D. T., & Howard, M. J. (1995). Seasonal distribution of pathogenic free-living amebae in
Oklahoma waters. Parasitology Research, 81(3), 193–201.
http://doi.org/10.1007/bf00937109
Katner, A., Pieper, K. J., Brown, K., Dai, D., O’Rear, L., & Edwards, M. (2017). Examining well
water quality and emergency resource needs after a flooding event. In Water and Health
Conference. Chapel Hill, NC: Water Institute.
Kemble, S. K., Lynfield, R., Devries, A. S., Drehner, D. M., Pomputius Iii, W. F., Beach, M. J.,
89
… Danila, R. (2012). Fatal Naegleria fowleri Infection Acquired in Minnesota: Possible
Expanded Range of a Deadly Thermophilic Organism. Clinical Infectious Diseases, 54,
805–809. http://doi.org/10.1093/cid/cir961
Louisiana Department of Health. (2013a). CDC Testing Confirms Presence of Rare Ameba in
One DeSoto Parish Water System | Department of Health | State of Louisiana. Retrieved
July 12, 2017, from http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/2889
Louisiana Department of Health. (2013b). DHH Confirms Death of a Child Associated with Rare
Amoeba Found in St. Bernard Parish Home | Department of Health | State of Louisiana.
Retrieved July 12, 2017, from
http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/2862
Louisiana Department of Health. (2017a). LDH Confirms Naegleria Fowleri Ameba in North
Monroe, Schriever Water Systems | Department of Health | State of Louisiana. Retrieved
July 12, 2017, from http://www.dhh.louisiana.gov/index.cfm/newsroom/detail/4284
Louisiana Department of Health. (2017b). Private Well Initiative. Retrieved July 29, 2017, from
http://new.dhh.louisiana.gov/index.cfm/page/1407
Macler, B. A., & Merkle, J. C. (2000). Current knowledge on groundwater microbial pathogens
and their control. Hydrogeology Journal, 8(1), 29–40. http://doi.org/10.1007/PL00010972
Maran, N., Crispim, B., Iahnn, S., Araújo, R., Grisolia, A., & Oliveira, K. (2016). Depth and
Well Type Related to Groundwater Microbiological Contamination. International Journal
of Environmental Research and Public Health, 13(10), 1036.
http://doi.org/10.3390/ijerph13101036
Martinez, A. J., & Visvesvara, G. S. (1997). Free-living, Amphizoic and Opportunistic Amebas.
Brain Pathology, 7(1), 583–598. http://doi.org/10.1111/j.1750-3639.1997.tb01076.x
90
Newsome, A. L., & Wilhelm, W. E. (1981). Effect of exogenous iron on the viability of
pathogenic Naegleria fowleri in serum. Experientia, 37. Retrieved from https://link-
springer-com.ezproxy.lib.vt.edu/content/pdf/10.1007%2FBF01989894.pdf
Newsome, A. L., & Wilhelm, W. E. (1983). Inhibition of Naegleria fowleri by Microbial Iron-
Chelating Agents: Ecological Implications. APPLIED AND ENVIRONMENTAL
MICROBIOLOGY, 45(2), 665–668. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC242341/pdf/aem00171-0323.pdf
Office of Water. (2005). What to Do After the Flood. Retrieved from
https://www.epa.gov/sites/production/files/2015-05/documents/epa816f05021.pdf
Pieper, K., Dai, D., Katner, A., Rhoads, W., Pruden, A., & Edwards, M. (2017). Characterizing
private well communities after a historic flooding event. In American Water Works
Association’s Water Quality Technology Conference and Exposition. Portland, OR.
Pieper, K., & Katner, A. (2017). Response and Recovery for the Great Louisiana Flood of 2016.
In The Private Well Conference: First Annual National Stakeholder Meeting. Champaign,
IL.
Puzon, G. J., Lancaster, J. a, Wylie, J. T., & Plumb, J. J. (2009). Rapid Detection of Naegleria
Fowleri in Water Distribution Pipeline Biofilms and Drinking Water Samples.
Environmental Science & Technology, 43(17), 6691–6696.
http://doi.org/10.1021/es900432m
Richards, C. L., Broadaway, S. C., Eggers, M. J., Doyle, J., Pyle, B. H., Camper, A. K., & Ford,
T. E. (2015). Detection of Pathogenic and Non-pathogenic Bacteria in Drinking Water and
Associated Biofilms on the Crow Reservation, Montana, USA. Microbial Ecology.
http://doi.org/10.1007/s00248-015-0595-6
91
Seiler, R. L. (2006). Mobilization of lead and other trace elements following shock chlorination
of wells. Science of the Total Environment, 367, 757–768.
http://doi.org/10.1016/j.scitotenv.2006.01.020
Sousa, A., Taveira, M., & Silva, L. (2015). Groundwater from Private Drinking Water Wells:
Imminent Public Health Issue. Water Resources, 42(4), 517–524.
http://doi.org/10.1134/S0097807815040144
Sykora, J. L., Keleti, G., & Martinez, A. J. (1983). Occurrence and Pathogenicity of Naegleria
fowleri in Artificially Heated Waters. Applied and Environmental Microbiology, 45(3),
974–979. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC242399/pdf/aem00172-0248.pdf
Thomas, V., Loret, J.-F., Jousset, M., & Greub, G. (2008). Biodiversity of amoebae and
amoebae-resisting bacteria in a drinking water treatment plant. Environmental
Microbiology, 10(10), 2728–2745. http://doi.org/10.1111/j.1462-2920.2008.01693.x
U.S. Geological Survey. (2016). USGS Water Use in the United States - Estimated Use of Water
in the United States County-Level Data for 2010. Retrieved August 8, 2017, from
https://water.usgs.gov/watuse/data/2010/index.html
US EPA. (2002). Drinking Water From Household Wells. Washington, DC. Retrieved from
https://nepis.epa.gov/Exe/ZyPDF.cgi/200024OD.PDF?Dockey=200024OD.PDF
US EPA. (2006). Ground Water Rule: A Quick Reference Guide. Office of Water. Retrieved from
https://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=P100156H.txt
Wang, H., Masters, S., Edwards, M. A., Falkinham, J. O., & Pruden, A. (2014). Effect of
Disinfectant, Water Age, and Pipe Materials on Bacterial and Eukaryotic Community
Structure in Drinking Water Biofilm. Environmental Science & Technology, 48(3), 1426–
92
1435. http://doi.org/10.1021/es402636u
Wang, H., Masters, S., Hong, Y., Stallings, J., Falkinham, J. O., Edwards, M. A., & Pruden, A.
(2012). Effect of Disinfectant, Water Age, and Pipe Material on Occurrence and Persistence
of Legionella , mycobacteria , Pseudomonas aeruginosa , and Two Amoebas.
Environmental Science & Technology, 46(21), 11566–11574.
http://doi.org/10.1021/es303212a
Wellings, F. M., Amuso, P. T., Chang, S. L., & Lewis, A. L. (1977). Isolation and Identification
of Pathogenic Naegleria from Florida Lakes. APPLIED AND ENVIRONMENTAL
MICROBIOLOGY, 34(6), 661–667. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC242727/pdf/aem00227-0059.pdf
Won, G., Gill, A., & LeJeune, J. T. (2013). Microbial quality and bacteria pathogens in private
wells used for drinking water in northeastern Ohio. Journal of Water and Health, 11(3),
555–562. http://doi.org/10.2166/wh.2013.247
Yoder, J. S., Straif-Bourgeois, S., Roy, S. L., Moore, T. A., Visvesvara, G. S., Ratard, R. C., …
Beach, M. J. (2012). Primary Amebic Meningoencephalitis Deaths Associated With Sinus
Irrigation Using Contaminated Tap Water. Clinical Infections Diseases, 55(9), e79-85.
http://doi.org/10.1093/cid/cis626
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Appendix B
Table 4.4. qPCR primers, probes, and assay conditions used in this study.
Appendix References:
Mull, B.J., Narayanan, J. & Hill, V.R., 2013. Improved Method for the Detection and
Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic
Separation and Real-Time PCR. Journal of Parasitology Research, 2013, p.8.
Nazarian, E.J. et al., 2008. Design and implementation of a protocol for the detection of
Legionella in clinical and environmental samples. Diagnostic microbiology and infectious
disease, 62(2), pp.125–32.
Targeted
organisms
Targeted
genes Sequences (5'-3')
Program
Amplicon
(bp) Reference Initial
denaturation
and enzyme
activation
Denaturing/
Annealing/
Extension
Legionella
spp.
23S rRNA Leg23SF:
CCCATGAAGCCCGTTG
AA
Leg23SR:ACAATCAGCC
AATTAGTACGAGTTAG
C
Probe: HEX-
TCCACACCTCGCCTAT
CAACGTCGTAGT
95 °C for 2
min
40 cycles of
95 °C for 5 s
and 58.5 °C
for 10 s
92 Nazarian
et al. 2008
N. fowleri ITS JBVF: AGG TAC TTA
CGT TAG AGT GCT
AGT
JBVR:
ATGGGACAATCCGGTT
TTCTCA
Probe: FAM-
ACGCCCTAGCTGGTTA
TGCCGGATT-BHQ1
95 °C for 15
min
45 cycles of
95 °C for 15
s and 63 °C
for 33 s
123 Mull et al.
2013
Total
Bacteria
16S rRNA BACT1369F:
CGGTGAATACGTTCYC
GG
PROK:
GGWTACCTTGTTACGA
CTT
98 °C for 2
min
40 cycles of
98 °C for 5 s
and 55 °C
for 5 s
124 Suzuki et
al. 2000
94
Suzuki, M.T., Taylor, L.T. & DeLong, E.F., 2000. Quantitative analysis of small-subunit rRNA
genes in mixed microbial populations via 5’-nuclease assays. Applied and environmental
microbiology, 66(11), pp.4605–14.
95
Chapter 5: Conclusions and Final Thoughts
5.1 Conclusions and Contributions
While the scope of this work is broad, each topic has broken ground on an understudied facet of
environmental and water research.
5.1.1 Reclaimed Water
Reclaimed water is an emerging water resource and will only expand in use around the world as
it promotes sustainability and can be used for many purposes. This lab-scale study is the first of
its kind and, when compared to field study data will contribute to the growing pool of knowledge
regarding proper treatment of reclaimed water. Limitations were abundant in this study due to
lack of replicability, but even so, the data collected has risen new questions regarding reclaimed
water and the lab-scale study design.
One of the key questions that has arisen is whether approaching reclaimed water from a drinking
water lens is appropriate. This project has brought light to the fact that reclaimed water is not
only vastly different from drinking water, it is also highly variable in and of itself. Therefore, it
can allow for the growth of different bacteria and, particularly, different pathogens that could
pose greater health risks than the standard drinking water OPs that were investigated. This is not
to say that drinking water OPs are unimportant; they pose a great risk to humans coming into
contact and could, in fact, pose a higher risk than those based on typical exposure to reclaimed
water, but next steps in research should attempt to target the OPs of greatest concern in
reclaimed water without defaulting to drinking water as a standard.
96
5.1.2 Naegleria fowleri
While the presence of N. fowleri in surface water has been well known for the past 50 years,
shockingly little is known about the factors that contribute to its growth in the environment.
Less is known about its presence in drinking water and recent studies do more to investigate its
presence or absence rather than the reasons why it resides in certain waters. This is a noteworthy
gap in our knowledge and must be addressed. If the key factors that trigger N. fowleri in drinking
water are identified, drinking water infrastructure and/or treatment methods could be
appropriately adapted in problematic areas.
5.1.2.1 Municipal Drinking Water
This study provided a firm a conclusion that the presence of iron can significantly increase
viability of N. fowleri in drinking water. This also occurred in the presence of nitrification,
suggesting that this may have played a part in the growth of amoeba. While the investigation of
disinfection and nitrification did not succeed, the findings from the first phase of the experiment
should spark further studies to note the importance of iron and study it further. Iron pipes
present a dynamic and complex environment that can cause drastic shifts in water chemistry, any
one of which can alter the microbial environment. The fact that iron contributes to not only N.
fowleri viability, but other OPs as well, further adds to the conclusion that aging infrastructure in
the United States is dangerous to public health.
5.1.2.2 Private Well Water
The lack of understanding of opportunistic pathogens in private well is perhaps the most
significant knowledge gap observed in this thesis. Untreated drinking water presents a vastly
different physicochemical and microbial environment that cannot be justified in studies of treated
groundwater. However, this presents a great challenge to scientists, as private well water is
highly variable and, to grasp the microbial environment, a great deal of sampling must occur
97
coupled with interactions with homeowners. While this is not enticing from a research
standpoint as it challenges replicability and standardization, this topic merits investigation from a
public health perspective.
5.2 Future Work
5.2.1 Reclaimed Water
This project involved the collection of a great deal of data over two years, which is only
beginning to be analyzed. Future OP analysis will include quantification of OP genes such as L.
pneumophila and M. avium, as well as quality analyses and comparison of samples collected at
22 °C and 14 °C to understand the influence of temperature on OP presence. Additionally,
Biological Activity Reaction Tests (BARTs) were performed at 30 °C to detect activity of
several organisms including sulfate-reducing bacteria (SRBs), iron-reducing bacteria (IRBs), and
nitrifying bacteria have yet to be analyzed. These tests will be analyzed alongside OP data to
further understand the overall microbiome of the SWRDSs. A concurrent analysis of OP data
with metagenomic data assessing antibiotic resistance genes (ARGs) will be used to understand
the reclaimed water conditions that may be suitable for both of these contaminants. Nanopore
sequencing will begin to reveal potential connections between OPs and ARGs as amplicon
genome sequencing is performed.
On a broader scale, future studies should seek to isolate factors that make the reclaimed water
microbiome unique, treating it as a separate water source than drinking water or wastewater.
This will allow for deeper understanding of the microbiological, chemical, and physical factors
that contribute to the complexity of reclaimed water and provide better conclusions regarding
health risks and exposure.
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5.2.2 N. fowleri in drinking water
As this study was unable to be completed in its entirety, investigations must continue to
understand what role nitrification and varying disinfectants play on N. fowleri proliferation in
drinking water. These could be essential variables that could change policy regarding drinking
water treatment in warm regions. Already iron has been shown to enhance viability but, if
nitrification is proven to provide similar benefits to N. fowleri, chloramine disinfectant may be
rethought in vulnerable systems. Further work to understand iron inhibition in qPCR reactions
could bring this study to fruition if sample quantification becomes possible.
5.2.2 N. fowleri in private well water
Viability assessments are the next key step to understand N. fowleri viability in private wells.
This study broke ground to study presence or absence of N. fowleri DNA, but viability is
required to form concrete conclusions of the conditions most suitable for its growth. Future
studies of private wells should seek to not only look for indicator organisms like E. coli, but to
sample for the full spectrum of drinking water OPs.