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4
Bacteriophages as Surrogates for the Fate and
Transport of Pathogens in Source Water and
in Drinking Water Treatment Processes
Maria M.F. Mesquita and Monica B. Emelko
Department of Civil and Environmental Engineering, University of Waterloo,
Canada
1. Introduction
Less than 1% of the world's fresh water accessible for direct human uses is found in lakes,
rivers, reservoirs and those underground sources that are shallow enough to be tapped at an
affordable cost. Only this amount is regularly renewed by rain and snowfall, and is
therefore available on a sustainable basis (Berger, 2003).
More than a billion people have limited access to safe drinking water; over 2 million die
each year from water-related diarrhea, which is one of the leading causes of mortality and
morbidity in less economically developed countries (UNICEF and WHO, 2009). In more
economically developed countries, increasing demands on water resources raise concerns
about sustainable provision of safe drinking water. In 2008, supply and protection of water
resources was identified as the top strategic priority of North American water professionals
(Runge and Mann, 2008). This is not surprising given the rapidly expanding competition for
existing water supplies from industrial, agricultural and municipal development, as well as
the vital needs to protect human health and ecosystem functions. The challenge of
sustaining supply is further exacerbated by changes in water quality and availability as a
direct or indirect result of population growth, urban sprawl, climate change, water
pollution, increasing occurrence of natural disasters, and terrestrial and aquatic ecosystem
disturbance.
Most of the world population depends on groundwater for their supplies. Due to the
proximity of groundwater to sources of microbial contamination, the increasing occurrence
of extreme climate events and the lack of adequate disinfection, groundwater is responsible
for a large percentage of the waterborne outbreaks of disease worldwide (WHO, 2004; 2011).
For example, between 1999 and 2000, 72% of drinking water outbreaks of disease were
associated with groundwater. Although the number of groundwater-associated disease
outbreaks associated in the United States decreased during 2001–02, the proportion of
outbreaks associated with groundwater increased to 92% from 87% (Tufenkji and Emelko,
2011). As a result of such outbreaks and the economic implications of waterborne illness,
stricter water quality regulations to protect public health have been implemented in many
countries. Significant examples of such regulations include the Surface Water Treatment
Rules (SWTR -1989a; 2002) and the Ground Water Rule (2006) by the U.S. Environmental
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58
Bacteriophages
Protection Agency (USEPA); the revised Bathing Water Directive (2006/7/EC) and the Water
Framework Directive (200/60/EC) by the European Union. The pressure generated by such
regulations has increased the need to quantitatively understand and describe microbial
pathogen transport and survival in various natural and engineered environments, including
treatment systems.
Monitoring the fate and transport of all of the various microorganisms that can cause
outbreaks of waterborne disease is cost prohibitive; accordingly, representative organisms
such as “indicators” of pathogenic contamination or “surrogates” for the transport and
survival of pathogens in various environments are sought. While indicators often originate
from the same source and act as signals of pathogen presence, surrogates may or may not be
derived from the same source as pathogens and are often introduced into natural and
engineered environments to pseudoquantitatively assess pathogen fate and transport.
Commonly used surrogates for such investigations include several bacteria, aerobic and
anaerobic bacterial endospores, numerous bacteriophages, microbe-sized microspheres,
chemically inactivated protozoa, and nonpathogenic, fluorescently labeled bacteria and
protozoa (Tufenkji and Emelko, 2011). Bacteriophages meet many of the requirements of
“ideal” surrogates because they have many characteristics that are similar to those of
mammalian viral pathogens (i.e., size, shape, morphology, surface chemistry, isoelectric
points, and physiochemistry), are unlikely to replicate in environments such as the
subsurface due to a lack of viable hosts and other limiting factors, pose little risk to the
health of humans, plants, and animals, and are easier and less expensive to isolate and
enumerate relative to enteric viruses (Tufenkji and Emelko, 2011). All of these factors
contribute to the utility of bacteriophages as surrogates for microbial pathogen transport
and fate in source waters and in drinking water treatment processes.
This chapter focuses on the utility of bacteriophages as surrogates for the fate and transport
of microbial pathogens of health concern in source and drinking waters, with particular
reference to: (1) indicating the presence of enteric viruses in natural waters, (2) contributing
to microbial source tracking, (3) evaluating the effectiveness of water treatment processes
such as disinfection and filtration, and (4) elucidating the mechanisms involved in the fate
and transport of enteric viruses in natural or engineered filtration media. Present knowledge
acquired through laboratory and field approaches is reviewed and further research needs
are identified to respond to current and future challenges in this field.
1.1 Major waterborne microbial pathogens of concern
Although water-transmitted microbial pathogens include bacteria, protozoa, helminthes and
viruses, the groups of major threat to human health in freshwater supplies are pathogenic
protozoa and enteric viruses (Schijven and Hassanizadeh, 2000) (Table 1). The protozoans
Cryptosporidium and Giardia are among the major causal agents of diarrhoeal disease in
humans and animals worldwide, and can even potentially shorten the life span of
immunocompromised hosts (WHO, 2004). Their resistant forms (cysts or oocysts) are shed
in large numbers by infected animals or humans and are ubiquitous in surface water. They
are resistant to harsh environmental conditions and to chemical disinfectants at concentrations
commonly used in water treatment plants to reduce bacterial contamination (LeChevallier et
al., 1991; Rose, 1997; Karanis et al. 2002; Aboytes et al., 2004). Their small size (Giardia cysts 813 µm and Cryptosporidium oocysts 4-6 µm) and infectious dose (as low as a single organism -
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Bacteriophages as Surrogates for the Fate and Transport
of Pathogens in Source Water and in Drinking Water Treatment Processes
59
Health Canada, 2004), also contribute to waterborne disease transmission. Several studies
have revealed little or no correlation between bacterial fecal indicator and protozoan
(oo)cyst densities in source surface waters (reviewed by Health Canada, 2004). These
observations highlight the need for: (1) routine monitoring of surface waters for protozoan
(oo)cysts or for reliable indicators of their presence and infectivity, and (2) implementation
of improved drinking water technologies to effectively protect public health.
Table 1. Water-transmitted microbial pathogens of major concern in drinking water
(adapted from: Azadpour-Keeley et al., 2003; CDC, 2003).
The collective designation “enteric viruses” includes more than 140 serological types that
multiply in the gastrointestinal tract of both humans and animals (AWWA, 2006). Enteric
viruses associated with human waterborne illness include noroviruses, hepatitis A virus
(HAV), hepatitis E virus (HEV), rotaviruses and enteroviruses (polioviruses,
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Bacteriophages
coxsackieviruses A and B, echoviruses and four ungrouped viruses numbered 68 to 71)
(AWWA, 2006). Enteric viruses are widespread in sewage and some have been detected in
wastewater, surface water and drinking water (Gerba and Rose 1990; Payment and Franco,
1993; AWWA, 2006). Although they cannot multiply in the environment, they can survive for
several months in fresh water and for shorter periods in marine water (Health Canada, 2004).
Enteric viruses are the most likely human pathogens to contaminate groundwater because
they are shed in enormous quantities in feces of infected individuals (109 to 1010/g) (Melnick
and Gerba, 1980) and their extremely small size (20 to 100 nm) allows them to infiltrate soils,
eventually reaching aquifers (Borchardt et al., 2003) (Fig. 1). Depending on physicochemical
and virus-specific factors (e.g. size and isoelectric point), viruses can move considerable
distances in the subsurface environment (Vaughn et al., 1983; Bales et al. 1993) and persist
for several months in soils and groundwater (Keswick et al., 1982; Gerba and Bitton, 1984;
Yates et al., 1985; Sobsey et al., 1986; Gerba and Rose, 1990; John and Rose, 2005).
Enteroviruses also have been shown to be more resistant to disinfection than indicator
bacteria (Melnick and Gerba, 1980; Stetler, 1984; IAWPRC, 1991).
Fig. 1. Migration and survival of viruses and protozoa in the subsurface (adapted from
Keswick and Gerba 1980 with permission).
1.2 Source water protection and treatment
In general the multiple-barrier approach to water treatment including watershed or
wellhead protection, optimized treatment including disinfection, a well-maintained
distribution system, monitoring the effectiveness of treatment, and safe water storage, is the
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Bacteriophages as Surrogates for the Fate and Transport
of Pathogens in Source Water and in Drinking Water Treatment Processes
61
best approach for reducing the risk of infection to acceptable or non-detectable levels
(Health Canada, 2004). Surface and groundwater protection from microbial contamination
largely depend on adequate land use policies related to: (1) waste and wastewater
management practices, (2) the interaction of contaminated surface water with
groundwater supplies (including artificial recharge with treated wastewater) and (3) the
effective placement and protection of drinking water wells. Pathogenic protozoa and
enteric viruses are considered priority microbial contaminants in drinking water
legislation because of the significant role they play in waterborne disease outbreaks and
the associated risks to public health, their extended survival in the environment, their
considerable resistance to conventional water disinfection processes compared to bacteria,
and the often poor or lacking correlation with traditional bacterial water quality indicator
numbers.
Commonly used free chlorine concentrations and contact times applied in drinking water
treatment are effective in inactivating enteric viruses (Thurston-Enriquez et al. 2003; Health
Canada 2004). Ozone is generally considered more efficient against both protozoa and
enteric viruses than chlorine or chlorine dioxide (Erickson and Ortega 2006). UV light
disinfection, although highly effective for inactivation of protozoa, is not as efficient at
inactivating viruses as more traditional chlorine-based disinfection processes (Health
Canada, 2004). More recently, the combined performance of UV light and chlorine has been
suggested as more effective for reclaimed water disinfection than the use of each process
separately (Montemayor et al., 2008).
Effective “green” ways to remove existing and emerging pathogens and produce safe
drinking water at lower cost have received much attention in recent years. These include the
passage of surface water and/or groundwater through porous media in the subsurface
during processes such as riverbank filtration, dune recharge, aquifer storage and recovery,
and deep well injection. The need to develop regulations to protect public health coupled
with the infeasibility of concentration-based criteria for all known waterborne pathogens
has resulted in the evolution of regulatory approaches for water quality and treatment that
rely on performance indicators and surrogates and assume specific levels of pathogen
reduction through well-operated treatment systems (Tufenkji and Emelko, 2011).
1.3 Global quest for an effective pathogen indicator
Because routine monitoring for pathogens is usually costly and often unrealistic, the use
of surrogate parameters (i.e. microbial indicators) to predict the presence of pathogens in
water and model their behavior has long been pursued. For decades fecal bacterial
indicators (e.g. fecal coliforms and E.coli) have been useful to identify fecal contamination
to indicate the probable presence of microbial pathogens in water (Payment and Locas,
2011). However, their concentrations rarely correlate well with those of pathogens. Thus,
bacterial indicators may signal the probable presence of pathogens in water, but they
cannot predict precisely their level of occurrence (Payment and Locas, 2011). They are also
not reliable pathogen surrogates because when compared with both virus and protozoa,
bacterial indicators are less persistent in the aquatic environment and less resistant to
disinfection and removal by other water treatment processes (IAWPRC 1991; Payment
and Franco, 1993).
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Bacteriophages
Some enteroviruses have been evaluated for monitoring environmental waters and tracking
sources of water pollution (Metcalf, 1978; Goyal, 1983; Payment et al., 1985). However, the
limitations associated with their use soon became apparent: (1) they are not constant
inhabitants of the intestinal tract and are excreted only by infected individuals and small
children, (2) laboratory methods for their detection and quantification are time-consuming,
expensive, require high expertise and are restricted to some enteroviruses subgroups, and
(3) virion size, surface characteristics and resistance to external agents such as disinfectants
vary among subgroups. Some studies have suggested using adenoviruses as an index of
human pollution because they have been shown to be more persistent and present in greater
numbers than enteroviruses in sewage and fecal contaminated aquatic environments (Pina
et al. 1998, Thurston-Enriquez et al. 2003).
When sewage is the source of enteric viruses and protozoa, spores of the anaerobic
bacterium Clostridium perfringens have been suggested as suitable indicators of the presence
and behavior of these pathogens in aquatic environments (Payment and Franco, 1993). Both
Bacillus spp. aerobic endospores and Clostridium perfringens spores have been used as
models for the removal of protozoa (oo)cysts and enteric viruses by drinking water
treatment processes (Payment and Franco 1993, Rice et al. 1996).
Increasing awareness of the shortcomings of fecal bacteria as indicators of the presence of
pathogenic viruses and protozoa in the environment has attracted attention to the potential
value of bacteriophages that infect enteric bacteria as indicators and surrogates for
evaluating the presence and behavior of human pathogenic viruses in aquatic environments
and during water treatment (Noonan and McNabb, 1979; Stetler, 1984; Gerba, 1987;
Havelaar, 1987; Havelaar et al., 1993). However, while phage meet many of the
requirements as surrogates for enteric viruses and are useful in certain situations, they are
not universal indicators, models or surrogates for enteric viruses in water environments
because several disadvantages can be associated with their use (further discussed in section
3). For example, enteric viruses have been detected in treated drinking water supplies that
yielded negative results for phages, even in presence–absence tests on 500 mL water
samples (Ashbolt et al., 2001).
Many years of research gradually elucidated that variations in pathogen input, dilution,
retention, and die-off in water environments result in conditions in which
relationships/correlations between any pathogen and any indicator may be random, sitespecific, and/or time-specific (Grabow, 1996; Payment and Locas, 2011). As a consequence,
the present general scientific consensus is that there is no universal indicator of microbial
water quality. Each specific situation, set of conditions, and objectives of study require a
great deal of judgment to select the best group(s) of pathogen indicator(s) and/or
surrogate(s) to be used most effectively (Table 2). Improved molecular detection techniques
(e.g. PCR amplification or hybridization) based on host specificity of targeted viral and
protozoan pathogens and surrogates in environmental samples may soon enable more
reliable source tracking and improved public health surveillance (Scott et al. 2002; Fong and
Lipp, 2005). Similarly, in-line microbial and chemical analytical systems installed at critical
treatment points may replace microbial indicators and may provide continuous monitoring
and reliable data, facilitating decision making. To further assist in process evaluation, efforts
also have been made to eliminate ambiguities in the term “microbial indicator”. Several
subgroups based on function have been recognized and are now commonly used in the
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Bacteriophages as Surrogates for the Fate and Transport
of Pathogens in Source Water and in Drinking Water Treatment Processes
63
literature, such as: process indicators or surrogates (useful for demonstrating the efficiency
of a process), fecal indicators (that indicate the presence of fecal contamination and imply
that pathogens may be present), and index or virus models (indicative of pathogen presence
and behavior respectively) (Ashbolt et al. 2001).
Table 2. Most commonly used pathogen surrogates and their uses (Sources: Havelaar et al.
1993, Health Canada 2004, Payment and Locas 2011)
2. Multifunctionality of bacteriophages
Estimated to be the most widely distributed and diverse entities in the biosphere (McGrath
and van Sinderen, 2007), bacterial virus, bacteriophages or phage can be found in all
environments populated by bacterial hosts, such as soil, water and animal guts. Their
unique characteristics bring several advantages to their use as pathogen surrogates (Table
3). Phages have been successfully used in a variety of environmental applications as follows:
As fecal indicators - the environmental occurrence and persistence of some groups
relate to health risks associated with fecal pollution and the potential occurrence of
enteric pathogens in aquatic environments (Havelaar, 1987; IAWPCR, 1991; Leclerc et
al., 2000; Morinigo et al., 1992; Lucena et al., 2006; Lucena and Jofre, 2010). As a result
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64
Bacteriophages
Advantages
i.
ii.
iii.
iv.
Have no known impact on the environment
Are non-toxic and non-pathogenic for humans animals or plants
Have a specific affinity to their bacterial host
Are reasonably similar to mammalian viral pathogens in size, shape, morphology,
surface properties, mode of replication and persistence in natural environments
v. Are colloidal in nature which makes them more adequate virus models then dissolved
tracers
vi. Are stable over periods of several months under laboratory conditions,
vii. Can be detected and enumerated by rapid and inexpensive methods with low
detection limits (1 to 2 phage per mL)
viii. Can be prepared in large quantities at high concentrations
ix. Specific phage groups are similar to specific pathogenic viral groups allowing the use
of phage cocktails to simultaneously target several groups of concern.
Disadvantages
i.
Are excreted by a certain humans and animals all the time while pathogenic viruses
are excreted by infected individuals for a short period of time (depending on the
epidemiology of viruses, outbreaks of infection, and vaccination). Consequently there
is no direct correlation between numbers of phages and viruses excreted by humans
ii. A wide range of different phage can be detected by methods for somatic coliphages
iii. At least some somatic coliphages may replicate in water environments
iv. Enteric viruses have been detected in water environments in the absence of coliphages
v. Pathogenic human enteric viruses are excreted almost exclusively by humans, while
bacteriophage used in water quality assessment are excreted by humans and animals.
vi. The microbiota of the gut, diet and physiological state of animals seems to affect the
numbers of coliphages in their feces
vii. The composition and numbers of phages excreted by humans is variable (e.g. patients
under antibiotic treatment excrete lower numbers than healthy or non- medicated
individuals)
viii. As water flows through porous media in the subsurface or engineered filtration
processes phage can attach, detach, and re-attach by physico-chemical filtration
mechanisms.
Table 3. Advantages and disadvantages of the use of bacteriophages as viral pathogen
surrogates and tracers in aquatic environments (Sources: Havelaar et al., 1993; Ashbolt et al.,
2001; Bateman et al., 2006).
phage infecting enteric bacteria are now accepted as useful indicators in water quality
control and included in some regulations as required parameters. For example, coliphages
are used in the US Water Ground Rule (USEPA, 2006), the drinking water quality
regulation for the Canadian Province of Quebec (Anonymous, 2001) and a few USA states
regulations regarding required quality for reclaimed water for certain uses (USEPA, 2003).
In microbial source tracking (MST) or identification of fecal contamination sources by
genotypic, phenotypic, and chemical methods, phage have proven useful based on their
host specificity (Hsu et al. 1995; Hsu et al., 1996; Simpson et al., 2003; Jofre et al., 2011).
By identifying problem sources (animal and human) and determining the effect of
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Bacteriophages as Surrogates for the Fate and Transport
of Pathogens in Source Water and in Drinking Water Treatment Processes
65
implemented remedial solutions MST is of special interest in waters used for recreation
(primary and secondary contact), public water supplies, aquifer protection, and
protection and propagation of fish, shellfish and wildlife (Simpson et al., 2003).
As process indicators phage groups are often successfully employed as enterovirus
surrogates in evaluating the effectiveness of water treatment processes and final
product quality. This is the case with filtration and disinfection (Stetler et al., 1984;
Payment et al., 1985; Havelaar et al., 1993; Durán et al., 2003; Davies-Colley et al., 2005;
Persson et al., 2005; Abbaszadegan et al., 2008).
As comprehensive pathogenic virus indices, phages are not very useful. This is because
their numbers seldom seem to correlate to pathogenic viruses numbers in water
samples when conventional statistics are applied (Lucena and Jofre, 2010). However, in
the future the application of advanced mathematical models to new databases may
reduce uncertainty and provide better information about relationships between phage
and pathogenic virus numbers (Lucena and Jofre, 2010).
As viral models and tracers, bacteriophages are often used at both field and laboratory
scales as biocolloids to estimate the fate and transport of pathogenic viruses in surface and
subsurface aquatic environments and through natural and manmade saturated and
unsaturated porous media. This use of phage as surrogates for pathogen transport applies
to protection of surface and groundwater supplies from microbial contamination,
assessment of potential health risk from pathogens in groundwater and design of more
efficient treatment systems in removing pathogens from drinking water supplies (Sen,
2011).
3. Main bacteriophage groups used in environmental studies
Three bacteriophage groups, somatic coliphages, male-specific F-RNA phages and Bacteroides
fragilis phages, have been proposed and are frequently used as surrogates for pathogenic
viruses in environmental studies (IAWPRC, 1991; WHO, 2004; Lucena and Jofre, 2010).
However, because each group has its pros and cons as a representative of enteric virus
presence and behavior in aquatic environments and water treatment processes, no agreement
has been reached on which of the three groups best fulfills the index/indicator function.
3.1 Somatic coliphages
Somatic coliphages are the most numerous and most easily detectable phage group in the
environment. It is a heterogeneous group whose members infect host cells (E.coli and other
Enterobactereacea) by attaching to receptors located in the bacterial cell wall. Their numbers
are low in human feces (often <10 g-1), but abundant in untreated domestic sewage (104 to
105 particles g-1) and in animal feces (Havelaar et al., 1986).
Somatic coliphages are not usually considered good fecal indicators because some of their
hosts are unlikely to be of fecal origin (Hsu et al. 1996), and some of these phage are able to
multiply in waters not subjected to fecal pollution (Gerba, 2006). However, some authors
argue that the number of somatic phage that replicate in environmental waters is negligible
(Jofre, 2009). Moreover, they are not predictive indicators of virus presence or absence in
groundwater (Payment and Locas, 2011), though some somatic phage such as T-4, T-7,
ΦX174, and PRD-1 have proven useful as viral surrogates of fate and transport in laboratory
investigations, pilot trials, and validation testing (WHO, 2004; Lucena and Jofre, 2010).
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Bacteriophages
Phage
Family name Type
T2, T4, T6
Myoviridae
Somatic
Linear
ds-DNA
T5, λ
Siphoviridae
Somatic
Linear
ds-DNA
T3, T7
Podoviridae
Somatic
Linear
ds-DNA
PM2
Corticoviridae Somatic
Linear
ds-DNA
PRD-1 *
Tectiviridae
Somatic
Circular
ds-DNA
PR772**
Tectiviridae
MS2, Qβ
Leviviridae
φX174
Microviridae
F-specific
Linear
ds-DNA
F-specific
Linear
ss-RNA
Somatic
Circular
ss-DNA
SJ2, fd,
M13
Inoviridae
Bacteroides Siphoviridae
fragillis
phages
F-specific
Circular
ss-RNA
Linear
ds-DNA
Lipid pHzpc Hosts
Phage Size/Shape
(%)
0
—
E. coli and other Cubic capsid
Enterobateriaceae (icosahedral or
elongated), long
contractile tail, 95 x
65 nm (EM)
0
—
E. coli and other Cubic capsid
Enterobateriaceae (icosahedral), long
non-contractile tail
(150 nm), 54-60 nm
(EM)
0
—
E. coli and other Cubic capsid
Enterobateriaceae (icosahedral), short
non-contractile tail,
54-61 nm (EM)
13
7.3
Pseudomonas sp., Cubic capsid
Pseudoalteromona (icosahedral), with
s sp.
spikes in vertices, no
tail, 60 nm (EM)
S. typhimurium Cubic capsid
16
4.5
and other
(icosahedral), no tail,
Enterobactereaceae 63 nm (EM)
82 ± 6 nm (DLS)
—
3.8E. coli and other Cubic capsid
Enterobateriaceae (icosahedral), no tail,
4.2
63 nm (EM)
3.9; 5.2
0
E. coli and
Cubic capsid
Salmonella sp.
(icosahedral), no tail,
20-30 nm (EM)
0
6.6
Pseudomonas sp., Cubic capsid
Pseudoalteromona (icosahedral), with
s sp.
spikes in vertices, no
tail, 27 nm(EM)
0
—
E. coli and
Filamentous or rodSalmonella sp.
shaped, 810 x 6 nm
(EM)
Bacteroides
0
—
Icosahedral head (60
fragillis HSP40
nm), flexible noncontractile tail, 150 x
8 nm (EM)
EM - electron microscopy analysis (measures physical diameter of dry particles)
DLS - dynamic light scattering analysis (measures hydrodynamic size of particles in a fluid)
pHzpc - zeta potential charge
Table 4. Characteristics of bacteriophages commonly used as pathogenic virus surrogates in
environmental studies (adapted from Mesquita et al., 2010).
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Bacteriophages as Surrogates for the Fate and Transport
of Pathogens in Source Water and in Drinking Water Treatment Processes
67
Bacteriophage PRD-1 (Table 4) in particular has emerged as an important viral model for
studying microbial transport through a variety of subsurface environments. Its popularity is
due to its similarity to human adenoviruses in size (~62nm) and morphology (icosahedric),
its relative stability over a range of temperatures and low degree of attachment in aquifer
sediments (Harvey and Ryan, 2004; Ferguson et al., 2007).
3.2 F-RNA bacteriophages
F or male specific RNA bacteriophages are a homogeneous group of phage that attach to
fertility fimbriae (F-pili or sex-pili) produced by male bacterial cells (possessing an Fplasmid) in certain stages of their growth cycle. Since the F-plasmid is transferable to a wide
range of Gram-negative bacteria, F-specific bacteriophages may have several hosts besides
E.coli (Havelaar 1987). This group ranks second in abundance in water environments
although its persistence in surface waters, mainly in warm climates is low (Chung and
Sobsey, 1993; Mocé-Llivina et al., 2005).
F-RNA bacteriophages have been most extensively studied due to their similarity (in size,
shape, morphology and physiochemistry) to many pathogenic human enteric viruses,
namely enteroviruses, caliciviruses, astroviruses and Hepatitis A and E virus (Jofre et al.,
2011) (Table 4). These phages are infrequently detected in human and animal feces (103 g-1)
or in aquatic environments despite their frequent detection in wastewater (103 to 104 mL-1)
(Havelaar et al., 1986; Gerba, 2006). Further research is needed to clarify if their consistently
higher concentrations in sewage relative to feces are the result of direct environmental input
or multiplication. If the latter is true, F-RNA bacteriophages may not be acceptable fecal
pollution indicators (Havelaar et al., 1990). Jofre et al. (2011) suggested that the
environmental multiplication of these phages is unlikely, however, because F-pili
production only occurs at temperatures above 25oC and replication does not occur in
nutrient-poor environments and requires a minimum host density of 104 colony forming
units (cfu) per mL.
The presence of F-RNA phage in high numbers in wastewater and their resistance to
chlorination contribute to their usefulness as process indicators, indices of sewage pollution,
and conservative models of human viruses in water and shellfish (Havelaar et al., 1993;
Havelaar, 1993; Love and Sobsey, 2007). They are also promising in microbial source
tracking since they can be subdivided in four antigenically distinct serogroups. Because
those predominating in humans (groups II and III) differ from those predominating in
animals (groups I and IV), it is possible to distinguish between human (higher public health
risk) and animal wastes by serotyping or genotyping F-RNA coliphage isolates (Hsu et al.,
1995; Hsu et al., 1996; Scott et al., 2002).
F-RNA bacteriophages MS2 and f2 (Table 4) are morphologically similar to enteroviruses
and are frequently used to study viral resistance to environmental stressors, disinfection and
other treatment processes (Havelaar, 1986, Havelaar et al., 1993; WHO, 2004). These phage
have been shown to attach poorly to soil particles and survive relatively well in
groundwater (Goyal and Gerba, 1979; Yates et al., 1985; Powelson et al., 1990). As a result,
Havelaar (1993) described F-RNA phage as a “worst case” virus model for virus transport in
soil. Bacteriophage transport in the subsurface is reviewed in section 5 of this chapter.
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Bacteriophages
Together, somatic and F-specific bacteriophages counts in water samples are usually
designated as “total coliphage count”. Some bacterial strains can be used to enumerate both
simultaneously (Guzmán et al., 2008). Their enumeration may be a good alternative for
determination of viral contamination in poorly contaminated waters such as groundwater
and drinking water or in double disinfection water treatments (Lucena and Jofre, 2010).
3.3 Bacteriophages of Bacteroides fragilis
Bacteriophages of Bacteroides fragilis and other Bacteroides species rank third in abundance in
natural waters. They have been suggested as potential indicators of human viruses in the
environment by Tartera and Jofre (1987). Their host Bacteroides fragilis is a strict anaerobic
bacterium abundant in human feces. These bacteriophages attach to the host bacteria cell
wall and have narrow host range. They occur only in human feces (108 g-1) and in
environmental samples contaminated with human fecal pollution (Havelaar et al., 1986).
Consequently they are useful in microbial source tracking, helping to differentiate human
from animal contamination (Ebdon et al. 2007; Lucena and Jofre, 2010). In contrast with
other phage they are absent from natural habitats and unable to multiply in the
environment (Tartera et al., 1989). They also decay in the environment at a rate similar to
that of enteric viruses. The main drawbacks associated with their use as routine fecal
indicators, are that: (1) their host is a strict anaerobe requiring complex and tedious
cultivation methodology, (2) their numbers in water may be low requiring concentration
from large volumes, and (3) different hosts are needed for different geographic areas. Within
this group, the most commonly used bacteriophages in environmental and treatment
resistance studies are B40-8 and B56-3 (Lucena and Jofre, 2010).
4. Available methodology for bacteriophage detection, enumeration and
propagation
Relatively simple and reliable methods for detection, isolation, enumeration and
characterization of bacteriophages from natural sources are available in the literature. These
include classic culture-based techniques using liquid or solid bacteriological media, as well
as more recent physico-chemical, immunological, immunofluorescence, electron
microscopy, and molecular methods. However, a lack of methodology standardization and
quality control has for decades limited the use of phage data for comparison studies. This
situation has improved since the publication of standardized plaque assays and
presence/absence methods in the USA and Europe. For somatic coliphages (APHA, EWWA,
and WEF, 2005; EPA, 2001a; 2001b), F-specific RNA phages (ISO, 1995; ISO, 2000; EPA,
2001a; 2001b) and bacteriophages infecting Bacteroides fragilis (ISO, 2001).
Sobsey et al. (1990) developed a simple, inexpensive and practical procedure for the
detection and recovery of F-RNA bacteriophages from low turbidity water using mixed
cellulose and acetate filters with 47 mm diameter and 0.45 um pore size. A slightly modified
version of this method has shown excellent performance for recovery of somatic and Fspecific phages, and bacteriophages of Bacteroids fragilis in up to 1L water samples (Mendez
et al., 2004). Rapid bacteriophage detection methods involving enrichment steps followed by
latex agglutination or bioluminescence (Love and Sobsey, 2007) and molecular approaches
have also been developed and recently reviewed by Jofre et al. (2011).
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Specific methods for the production of the large-volume, high-titer purified bacteriophage
suspensions that are necessary for many types of environmental fate and transport studies
were, until very recently, difficult to find in the refereed literature. Given that system
chemistry and other surface-related characteristics of phage particles, may substantially
contribute to observations of their environmental fate and transport behavior in many types
of porous media filtration systems used for water treatment (Pieper et al., 1997; Harvey and
Ryan, 2004; Cheng et al., 2007), it is critical to consider the impacts of the
propagation/purification protocol on those factors. In response to this need, a selected
sequence of rapid, reliable, and cost-effective procedures to propagate and purify high-titer
bacteriophage suspensions has recently been proposed (Mesquita et al., 2010). This
methodology emphasizes the most important factors required to ensure maximum
bacteriophage yields, minimum change on phage particles surface characteristics, and low
dissolved organic carbon (DOC) concentration in the final suspensions.
Many of the methods routinely used to quantify microscopic discrete particles such as
bacteriophages are known to yield highly variable results arising from sampling error and
variations in analytical recovery (i.e., losses during sample processing and errors in
counting); thereby leading to considerable uncertainty in particle concentration or log10reduction estimates (Emelko et al., 2008; 2010; Schmidt et al., 2010). For example, sampling
error is substantially greater than analytical error when organisms are present in relatively
low concentrations; in these cases, improved sampling (i.e., resulting in counts of
approximately 10 or more organisms in a sample or, in some cases, several replicates)
substantially contributes to reducing uncertainty. In contrast, when organisms are present in
higher and homogeneous concentrations, uncertainty in concentration estimates can be
reduced by decreasing analytical errors (Emelko et al., 2008; 2010). Emelko et al. (2010)
demonstrated that uncertainty in concentration and removal estimates derived from
microbial enumeration data can be addressed when these errors are properly considered
and quantified. The development and use of such quantitative approaches is an essential
component of strategies (e.g., the monitoring of surrogate parameters/pathogens,
experimental design, and data analysis) for better evaluating microorganism transport and
fate in source and treated drinking waters.
5. Bacteriophages contribution to predicting pathogen transport in filtration
porous media
In the last two centuries a large number of field studies have evaluated the transport of
bacteriophages in the subsurface (especially through the vadose zone) at different field sites
around the world (Rossi, 1994; Collins et al., 2006; Pieper et al., 1997; Bales et al., 1997; Dowd
et al., 1998; Rossi et al., 1998; Sinton et al., 1997; Ryan et al., 1999; Auckenthaler et al., 2002;
McKay et al., 2000; Schijven and Hassanizadeh, 2000; Schijven, 2001; Harvey and Harms,
2002; Ryan et al., 2002; Harvey and Ryan, 2004; Blanford et al., 2005; Harvey et al., 2007;
Ferguson et al., 2007). PRD-1, MS-2 and ΦX174 have also been extensively used at controlled
laboratory conditions to elucidate physicochemical effects on virus transport through a
variety of porous media (Bales et al., 1991; Bales et al., 1993; Schulze-Makuche et al., 2003;
Zhuang and Jin, 2003; Han et al., 2006; Sadeghi et al., 2011).
Based on existing data, major environmental factors affecting enteric viruses and phage
survival and transport through soil, porous media and in groundwater have been identified
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(Table 5). Due to the complexity of interactive factors controlling survival and transport
there is great variability among study outcomes, however. It is, at present, generally
accepted that the main processes for viral removal in water filtration through porous media
Factors
Findings
1.
Temperature - a major controlling factor for virus inactivation usually with greater
inactivation at temperatures above 20°C. This may be due to more rapid denaturation
of viral capsid proteins or potential degradation of extracellular enzymes with
increased temperature
2. Native Microbial activity - Inactivation rates have often been reported to be lower in
the absence of groundwater bacteria possibly because bacterial enzymes and protozoa
may destroy viral capsid protein. However, other studies have found the opposite to
be true.
3. Moisture content - Different viruses and phage (MS2 and PRD-1) have been reported
to have different inactivation rates in groundwater, saturated, unsaturated and dry
soils. Migration seems to increase under saturated flow conditions.
4. Nutrients - addition when native organisms are present seems to determine decreased
viral inactivation. Possibly because the nutrients offered protection from inactivation
by enzymatic attack or acted as alternate nutrient sources for the native bacteria
5. Aerobic and anaerobic condition - Anaerobic conditions have been shown to slow
down poliovirus and coxsackievirus inactivation. It has been suggested this is
potentially an interactive factor with the impact of native microorganisms since low
oxygen will minimize negative microbial activity.,
6. pH - most enteroviruses are stable over a pH range of 3 to 9, survival may be
prolonged at near neutral; low pH favors virus attachment and high pH detachment
from soil particles
7. Salt species and concentration - some viruses are protected from inactivation by
certain cations: the reverse is also true. Generally increasing the concentration of ionic
salts and cation valences enhances virus attachment.
8. Association with soil and other particles – in many cases viral survival is prolonged
by attachment to soil, although the opposite has also been observed. Usually virus
transport through the soil is slowed or prevented by association with particles.
However, attachment to solid surfaces appears to be virus-type-dependent
9. Soil properties – effects on survival are probably related to the degree of virus
attachment: greater virus migration is usually observed in coarse-textured soils, while
there is a high degree of virus retention by the clay fraction of soil.
10. Virus type – particle-structure may be a deciding factor in attachment/detachment
and inactivation by physical, chemical and biological factors.
11. Organic matter (OM) – may protect virus from inactivation or reversibly retard virus
infectivity. Soluble OM seems to compete with virus particles for attachment sites on
soil.
12. Hydraulic conditions – increasing hydraulic loads and flow rates usually increase
virus transport.
Table 5. Major factors determining viral survival and transport in the subsurface and in
groundwater (adapted from: Azadpour-Keeley et al., 2003; John and Rose, 2005).
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are physio-chemical attachment/detachment and inactivation (Keswick and Gerba, 1980;
Yates et al., 1987; Bales et al, 1991; 1997; Gitis et al., 2002; Tufenkji and Emelko, 2011). Virus
attachment and inactivation depend on the type virus, as well as on the physico-chemical
properties of the water and soil or filtration media grain (Schijven and Hassanizadeh, 2000;
Tufenkji and Emelko, 2011). Physical and physico-chemical processes such as advection,
dispersion, diffusion, and physico-chemical filtration all contribute to attenuation of virus
concentrations (Schijven and Hassanizadeh 2000; Tufenkji and Emelko, 2011). Various
physico-chemical forces may be involved in the attachment of viruses to soil or filtration
media particles including, hydrogen bonding, electrostatic attraction and repulsion, Van der
Waals forces and covalent ionic interaction (Murray and Parks; 1980). Straining (i.e. physical
blocking of movement) may come into play in some environments as well (Bradford et al.,
2006).
The unsaturated or vadose zone (i.e. the layer between the land surface and the
groundwater table) where much of the subsurface contamination originates, passes through,
or can be eliminated before it contaminates surface and subsurface water resources has
gained particular attention in recent years. In unsaturated conditions, additional and more
complex mechanisms are involved in pathogen transport such as: variability in ionic
strength , pH and water content, particle capture at the water-gas interface, particle capture
at the solid-water-gas interface, and preferential flow or retention in the immobilization
zone (Sen, 2011). Biological processes such as growth and decay, active attachment or
detachment, survival, random mobility and chemotaxis are also believed to strongly affect
virus transport in saturated and unsaturated porous media (Sen, 2011). Less information is
available regarding the fate of pathogenic protozoa in the vadose zone (Harvey et al., 1995;
Harvey et al., 2002; Hancock et al., 1998; Brush et al., 1999; Harter et al., 2000; Darnault et al.,
2004; Davies et al., 2005), however, the physico-chemical processes that affect virus fate and
transport also apply to protozoan cysts and oocysts during soil transport, albeit to a
different extent (Schijven and Hassanizadeh, 2000).
The growing database of information concerning phage attachment, inactivation and
transport behavior in porous media has led to their use as viral surrogates in mathematical
models used to describe viral transport within physically or geochemically heterogeneous
granular media at environmentally-relevant field scales (Rehmann et al., 1999; Schijven and
Hassanizadeh, 2000; Schijven et al., 2000; Bhatacharjee et al., 2002; Schijven et al., 2010). As
they continue to improve, such models may become useful tools in decision making related
to in public health protection because they may ultimately be incorporated into quantitative
microbial risk assessment to: (1) access groundwater vulnerability, especially of highly
vulnerable geological settings (i.e. fractured rock aquifers, cross-connecting bore holes, or
leaking well cases in sandstone and shale aquifers) in combination with significant sources
of contamination (i.e. wastewater treatment plants, septic tanks and animal manure), (2)
simulate the transport of viruses from a contamination source at or near the surface to a
groundwater abstraction well, and (3) evaluate set back distances from abstraction wells
from potential contamination sources for source protection (Schijven et al. 2010).
6. Conclusions and recommendations for future research
Considerable progress has been made in understanding how suitable bacteriophages are as
surrogates for pathogenic enteric viruses. As a result, they have become invaluable tools in
environmental research and are often successfully used in a variety of applications, namely:
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The use of somatic and F-specific coliphages as indices of water contamination by
sewage and as process indicators in the evaluation of drinking water and the efficacy of
drinking water treatment processes.
The use of F-specific bacteriophages as indices and models of human enteric viruses in
contaminated water, shellfish and agricultural products and in microbial source
tracking.
The use of particular somatic and F-specific bacteriophages to improve the understanding
of the multiple physical, chemical and biological processes affecting biocolloid transport
in saturated and unsaturated subsurface environments.
The use of bacteriophages of B. fragilis as indicators of human fecal contamination and
in microbial source tracking.
Additional research efforts are needed in the following areas:
Use of more sensitive and reliable methodologies (i.e. standardized cultural procedures,
molecular and other techniques) to minimize the variance between reported and actual
numbers of bacteriophages in field and laboratory studies and allow the development
of more complete and reliable databases.
Use of more consistent experimental procedures to reduce variability among
researchers’ findings. Standardized protocols are required for the preparation
(propagation, concentration and purification) of bacteriophages to be used in laboratory
and field scale studies, as well the use of phage from well known sources such as the
American Type Culture Collection (ATCC) or the Canadian Felix d’Herelle Reference
Center for Bacterial Viruses to avoid differences in the viruses themselves.
Evaluation of the complex interactions of native groundwater organisms with
introduced enteric microbes (including enteric bacteriophage) and the environmental
factors that influence them.
Evaluation of the impact of viral structure and surface properties on attachment/
detachment and inactivation of virus particles in various environments.
Improved understanding of the transport and survival of both bacteriophages and
pathogenic enteric viruses in surface water and the subsurface is needed; not only at
laboratory scale to clarify the generic mechanisms involved, but also at field scale at
settings with specific environmental conditions (water matrixes, flow regimes,
hydrogeological and filtration media characteristics, etc.) in an attempt to clarify
conflicting evidence previously reported on the extent of inactivation and
immobilization of viruses by some physico-chemical and biological factors.
Development of sound databases reflecting the occurrence, persistence and transport of
viral particles in natural environments and water treatment systems that can be used to
improve mathematical models of microbial fate and transport.
Development of microbial fate and transport models taking into account the many
factors affecting virus fate and transport under various conditions applicable to:
improve viral contamination control in specific environments, ensure compliance with
current water quality regulations, help in the selection and control of treatment
processes and ultimately improve public health protection.
Further investigation of the usefulness of bacteriophages for source tracking purposes.
Taking advantage of the stringent host specificity of some phage groups and the speed,
high specificity and sensitivity of molecular detection methods in order to better
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characterize sources of contamination in aquatic environments so that appropriate and
cost-effective water quality remediation plans can be developed.
In the future, the progress of such applications will reveal the true potential of
bacteriophages as viral pathogen surrogates in water and water treatment.
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Bacteriophages
Edited by Dr. Ipek Kurtboke
ISBN 978-953-51-0272-4
Hard cover, 256 pages
Publisher InTech
Published online 14, March, 2012
Published in print edition March, 2012
Bacteriophages have received attention as biological control agents since their discovery and recently their
value as tools has been further emphasized in many different fields of microbiology. Particularly, in drug design
and development programs, phage and prophage genomics provide the field with new insights.
Bacteriophages reveals information on the organisms ranging from their biology to their applications in
agriculture and medicine. Contributors address a variety of topics capturing information on advancing
technologies in the field. The book starts with the biology and classification of bacteriophages with subsequent
chapters addressing phage infections in industrial processes and their use as therapeutic or biocontrol agents.
Microbiologists, biotechnologists, agricultural, biomedical and sanitary engineers will find Bacteriophages
invaluable as a solid resource and reference book.
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of Pathogens in Source Water and in Drinking Water Treatment Processes, Bacteriophages, Dr. Ipek Kurtboke
(Ed.), ISBN: 978-953-51-0272-4, InTech, Available from:
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