Phage Ecology: Harald Brüssow and Elizabeth Kutter

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6 Phage Ecology
1 2
Harald Brüssow and Elizabeth Kutter
1
Nestlé Research Center, Lausanne, Switzerland
2
Lab of Phage Biology, The Evergreen State College,
Olympia, WA
CONTENTS
1. Introduction.....................................................................................................130
2. General Principles.......................................................................................... 131
2.1. Phage Numbers in the Natural World..................................................131
2.2. Dynamic Phage-Host Relationships.....................................................132
2.3. Co-Evolution of Phages and Their Host Bacteria ...............................135
2.4. Effects of Host Physiology and Nutritional Status..............................136
2.5. Biofilms.................................................................................................137
3. Marine Phage Ecology ...................................................................................139
3.1. Marine Phage Impacts on the Food Web.............................................139
3.2. Marine Phage Characterization ............................................................141
3.2.1. Cyanophages............................................................................141
3.2.2. Genomic Analysis of Marine Phages......................................142
3.3. Marine Phage Host Specificity and Horizontal
Gene Transfer .......................................................................................142
4. Soil and Plant-Associated Phages ..................................................................144
5. Animal-Associated Phages .............................................................................145
5.1. Introduction...........................................................................................145
5.2. Enteric Phages ......................................................................................146
5.3. Other Phage Sites of Interest: Oral Cavity and Vagina.......................149
6. Industrial Phage Ecology................................................................................150
6.1. Dairy Industry.......................................................................................150
6.2. Non-Dairy Food Fermentation:
Sauerkraut and Sausage........................................................................155
7. Outlook ...........................................................................................................155
Acknowledgements................................................................................................156
References..............................................................................................................156
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130 Bacteriophages: Biology and Applications
1. INTRODUCTION
Felix d’Herelle, co-discoverer of phage, had a strikingly modern approach to biology.
Nearly 100 years ago, he used living organisms to control pests (diarrhea-causing
bacteria to halt locust epidemics) and disease (phage therapy of diarrheal diseases).
His approaches reflected ecological insights before this branch of biology became an
established scientific discipline. In fact, one might have predicted that phage research
would become a springboard for studies of microbial ecology. Instead, studies of phage
ecology were largely ignored and phage research became the cradle of molecular
biology. This turn in the history of biological research is not explained by any critical
technical breakthrough, but rather by a number of biographical reasons in the lives of
a handful of scientists. The second generation of Western phage researchers concen-
trated on a few phages from E. coli, the workhorse of bacterial genetics, in order to
better understand the basic nature of phages and of the phage infection process and
use this knowledge to explore fundamental aspects of biology at the molecular level.
Phage ecology was not within their conceptual framework. The diversity of phages
was better appreciated by medical microbiologists, who used phages for the typing of
clinical isolates of bacterial pathogens. However, in that field phages were exclusively
used as tools without intrinsic interest in their ecology or molecular characteristics.
Consequently, the first monograph on the distribution and behavior of bacterial viruses
in the environment appeared only in 1987 (Goyal).
Since the appearance of Goyal’s book, the study of phage ecology has funda-
mentally changed. One drastic reminder of the importance of phage in the ecosystem
was the surprising discovery of very large numbers of phage-like particles in the
ocean (Bergh et al., 1989). Phage ecology quickly became an intensively investigated
branch of marine microbiology, as documented by a recent review listing hundreds
of publications, mostly from the last decade (Wommack and Colwell, 2000).
Recently, general reviews have appeared on various major aspects of phage ecology
(Abedon, 2005; Ashelford et al., 2003; Azam and Worden, 2004; Breitbart et al.,
2002; 2003; Chibani-Chennoufi et al. 2004; Paul and Kellogg, 2000; Suttle, 2000b).
Scientists in two fields have developed a particularly keen interest in phage
ecology. One is the food industry, where fermentation techniques are used to trans-
form milk, vegetables, or meat into processed foods like cheese, sauerkraut, or salami.
This food production relies either on spontaneous fermentation or, in the case of
milk, on fermentation initiated by the addition of industrial bacterial starters. Phages
that infect these starters are the major cause of fermentation failures in the dairy
industry (Chapter 10). The high economic losses associated with phage infection
there motivated intense research into phages from lactic acid bacteria, which are the
major dairy starter organisms. As the dairy factory is a man-made environment, it
did not so much attract the interest of ecologists, but rather that of more technolog-
ically oriented microbiologists, focusing on the design of efficient starter rotation
systems and the construction of phage-resistant starter cells. In doing that, dairy
microbiologists had to investigate the factory ecology of phage infections, leading
to large systematic collections of dairy phages. However, these phages were char-
actarized more by sequence analysis and molecular biology than by classical eco-
logical approaches. The other emerging field is linked to the rekindling of interest
Phage Ecology 131
in phage therapy, as discussed in Chapters 13 and 14. The successful application of

this approach to the growing problem of antibiotic resistance depends on the avail-
ability of large collections of phages and a detailed knowledge of phage-host inter-
actions in different physiological compartments that necessitates sound ecological
knowledge of the interactions between the phage, bacteria, and plant or animal host.
It is also increasingly extending to interest in potential applications against plant
pathogens, bacteria in biofilms, and other complex real-world situations.
2. GENERAL PRINCIPLES
2.1. PHAGE NUMBERS IN THE NATURAL WORLD
A short 1989 Nature paper by a Norwegian group reported 107 phage-like particles
per ml of coastal and open ocean water, and even 20-fold higher concentrations in
a pre-alpine lake (Bergh et al., 1989). Two independent Nature papers in the fol-
lowing year confirmed the findings and provided experimental evidence that these
viruses limit the primary productivity of cyanobacteria, the major oceanic photo-
synthetic bacteria (Suttle, 1990; Proctor, 1990). Goyal’s 1987 Phage Ecology mono-
graph still started the marine chapter with a quote suggesting that phages isolated
from the oceans are not indigenous to the marine environment but are transported
to the sea by rivers or sewage. Now we know that phage are universally observed
in the open and coastal ocean all over the world; in surface water and in great depth
(Cochlan, 1993); in ocean ice (Maranger, 1994) and in ocean sediment. Even higher
phage concentrations have been detected in marine sediments than in the water
columns above them. Counts of up to 109 phage particles per ml of sediment were
reported by Danovaro and Serresi (2000). Meaningful studies of phage distribution
are much more difficult in terrestrial ecosystems, but such techniques as electron
microscopy suggest concentrations of the order of 107 viruses/gram in soil (Ashelford
et al., 2003) and in the feces of ruminants (Furuse, 1987). There seems now to be
broad agreement that phage are the most abundant life form on earth; the total
number is generally estimated at 1030 to 1032. Most of our detailed quantitative data
still comes from marine environments, where extensive ongoing mixing makes
meaningful sampling possible, but many of the principles and insights also seem
applicable to other environments.
In all but the most extreme environments, large numbers of different bacteria
and phages are found; there is growing suspicion that phages may represent the
largest unexplored reservoir of sequence information in the biosphere. For example,
in the case of Chesapeake Bay, Wommack et al. (1999) have estimated that there
are about 100–300 phage strains (with many variants of each), infecting 10–50
different bacterial species. Random sequencing of viral DNA from two uncultured
100-liter marine water samples suggested that they contained between 400 and 7000
different phage types (Breitbart et al., 2002); a similar analysis shows that the human
gut also contains hundreds of different phage genotypes (Breitbart et al., 2003).
Marine phage biologists have developed precise data about in situ burst sizes
(Borsheim, 1993), since this figure is essential for calculations of virus production
in the given environment and the level of virus-mediated mortality of bacterioplankton;
such determinations have not generally been possible in other natural habitats. The
burst size in the environment is generally smaller than that determined in the labo-
ratory by one-step growth experiments, reflecting the smaller size of bacteria in most
natural settings (Robertson and Button, 1989; Weinbauer and Peduzzi, 1994). The
nutrient level and temperature of the water sample and the morphology of the host
were the most important determinants of the burst size. In situ burst-size determi-
nation is mostly done by microscopic observation of virus particles within bacterial
cells (Hennes, 1995; Weinbauer et al., 1993). Alternatively, the in situ burst size is
calculated by balancing viral production with viral decay (Suttle and Chan, 1994),
assuming that if the phage concentrations are relatively stable over a given (short)
time period, then the rates of phage production and phage decay must be equal. This
lets one look at specific viable phage infecting a particular host. Quantitative data
on virus decay are important for other fields of research as they provide a measure
of the tenacity of a virus in the environment. Such information is crucial for such
diverse questions as public health evaluations of viral contamination of water sam-
ples, source tracking, the persistence of phages in industrial environments, and the
half-life of therapeutic phages.
2.2. DYNAMIC PHAGE-HOST RELATIONSHIPS

Major fluctuations in phage and host numbers are observed over various periods. For
example, there is substantial seasonality in marine phage titers (Bratbak, 1990; Hennes,
1995; Mathias, 1995; Cochran and Paul, 1998). The levels are often 10-fold lower
in the winter than in the summer months, even though the bacterial densities do not
appear to fluctuate by a factor of more than 2–3, with no seasonal trend; the reasons
behind this apparent uncoupling of bacterial and phage populations are not yet clear,
but one explanation could be increased induction of prophages in the summer months
due to higher sunlight exposure. Another possibility is that phage production itself is
much more temperature sensitive, in general, than is bacterial growth. At the same
time, the UV in sunlight causes up to 5% loss in viable phage per hour for surface
water due to production of thymine dimers (Wommack, et al., 1996); this causes far
more loss in summer than in winter at more polar latitudes. A strong shift in balance
was seen over 6 months between the two major phage types infecting the rhizosphere
of sugar beets: from Siphoviridae with long latent period and big burst sizes to
Podoviridae with short latent periods and small burst sizes, apparently reflecting
changes with season in the availability and physiological state of the host bacteria and
plants (Ashelford et al., 1999). Variability was also shown over shorter time periods,
in one marine study even over half-hour time intervals, demonstrating a highly dynamic
relationship between phages and their hosts (Bratbak, 1996).
As discussed in Chapters 3 and 7 and by Abedon (2005), the phage lytic life
cycle has at least 4 key steps that are very relevant to phage ecology:
1. An extracellular search that is limited by diffusion rates and thus depen-

dent on host concentration.
2. A phage adsorption step that combines reversible phage binding, irrevers-
ible attachment, and genome transfer into the host, which typically occurs
Phage Ecology 133
rapidly following productive collision between a phage particle and a

phage-susceptible bacterium.
3. An infection step, during which host physiology is appropriately restruc-
tured, the phage genome is replicated, and phage particles are assembled.
4. For temperate phages, an indefinite period of lysogeny may be inserted,
during which the phage is inserted in the host genome and replicates with
it or replicates synchronously as a plasmid and the genes responsible for
lytic growth are repressed, followed by a step in which phage progeny
are released from the infected bacterium; except for filamentous phages,
this process involves cell lysis, which usually is carefully timed.
The attachment and lysis steps are generally rapid. Thus, most of the time the
phage is either:
1. Free during the extracellular search,

2. Trapped in some compartment where no bacteria are readily available,
such as bound to relatively inert particulate matter,
3. Actively infecting a bacterium, leading eventually to cell lysis or phage
secretion, or
4. Existing as a prophage
For the first two, the phage may become inactivated through capsid or genome
damage, but it also may remain infectious for many years, depending on environ-
mental conditions. The lytic infection phase seldom lasts more than a few minutes
to a day, though there are conditions like the one we call “hibernation” in which the
phage genome remains benignly inside until nutrients become available and the host
resumes more active growth. For virulent phages like T4, this differs markedly from
lysogeny, though, in that the phage genome then takes over and the only possible
outcome is host-cell death, accompanied by eventual phage production if nutrients
become available, as discussed in 2.4; no colonies are formed.
Our understanding of the phage infection process comes primarily from exper-
iments in which the laboratory researcher mixes a single phage strain with a single
bacterial strain at about 108 cells/ml. In contrast, in near coastal water, for example,
bacterioplankton concentrations are typically 106 cells/ml and the population nor-
mally consists of 100 different bacterial host species, yielding a mere 104 cells/ml
for the average host species (Murray and Jackson, 1992). Is this enough to maintain
an infection cycle? The answer is apparently yes, since no marine water samples
devoid of phage were ever reported, and enrichment procedures generally permit
the isolation of phage against any given host from the particular ecosystem. Yet
phage replication is clearly sensitive to effective cell concentration. Laboratory
experiments with T4 and Bacillus and Staphylococcus phages showed no phage
production until the host cell concentration reached 104 cells/ml (Wiggins and
Alexander, 1985). However, studies using Pseudomonas phages (Kokjohn et al., 1991)
showed evidence of lytic infection at cell concentrations as low as 102 cells/ml. In
some studies in natural marine environments, no intracellular phage were observed
when the number of rod-shaped bacteria fell below 105 cells/ ml (Steward et al., 1992;
Weinbauer and Peduzzi, 1994). However, some marine viruses replicated efficiently
down to 103 specific host cells/ml (Suttle and Chan, 1994; 1993). The ups and downs
of host cell concentration over a time series allowed the approximation that cyanoph-
age replication still occurred when the host cell concentration fell to 102 cells/ml
(Waterbury, 1993).
Such theoretical concerns related to the reproduction of virulent phages at low
host densities led to the hypothesis that temperate phages should outnumber virulent
phages in the ocean, since the production of temperate phages is independent of
host cell density. It has been proposed that lysogeny becomes the preferred strategy
when the cell density falls below the lower limit necessary for maintenance of the
phage density by repeated cycles of lytic infections (Stewart and Levin, 1984).
Lysogens might out-compete the non-lysogenic congeners by the selective advan-
tage conferred by lysogenic conversion genes contributed by many temperate
phages. Some of these are relatively universal, such as immunity functions and
superinfection-exclusion genes. Other prophages contribute genes that make the
lysogen competitive under special ecological situations, such as the serum resistance
conferred to the lysogen by the phage lambda bor gene under blood growth of
E. coli. This phenomenon is very marked in lysogenic bacterial pathogens, where
many virulence factors are encoded by prophages. However, even laboratory phages
like P1, P2, lambda, and Mu led to higher metabolic activity and faster and longer
growth than seen in non-lysogens (Edlin et al., 1975; Lin et al., 1977). With this
selective advantage even under laboratory conditions and the intrinsic difficulties
with the lytic life style, the prediction is a high concentration of lysogens in the
oceans. Indeed, two marine surveys revealed 40% mitomycin-C-inducible cells;
similar proportions of lysogens were identified in Pseudomonas colonies from lakes
(Ogunseitan et al., 1992). The surveys showed the trend for lysogeny to be more
prevalent in oligotrophic environments (Jiang, 1994; 1997). This observation fits
with theory since this setting is dominated by low densities of slow-growing
bacteria. However, other data contradict this explanation. Surveys in estuarine
waters showed a seasonal development of lysogeny with highs in the summer
months when eutrophic conditions were prevalent and lows in the winter months
when cells were at their minimum (Cochran and Paul, 1998). There are further
contradictions of expectations. First, spontaneous induction of prophages is generally
low (10−2 to 10−5 phage per bacterium per generation) (Stewart and Levin, 1984).
This release can only account for <1% of the phage concentrations in the ocean
(Jiang, 1997). Second, large phage surveys in the North Sea revealed that only 10%
of the phage isolates are temperate (Moebus, 1983).
Paul and Kellogg (2000) extensively explored the available research related to
the frequency of lysogeny in natural environments, the factors that can induce such
lysogens, and the roles of phages in bacterial genetic exchange in various ecosystems.
A variety of approaches have indicated that lysogeny and even polylysogeny are
common; the various microbial genome projects to date have confirmed this, with
at least half of the sequenced bacteria carrying prophages, and prophages or defective
prophages are responsible for many differences between isolates of the same species.
The extent of lysogeny varies between different kinds of bacteria; for example, nearly
100% of naturally occurring Pseudomonas are lysogenic. Prophages could be
Phage Ecology 135
induced from most bacteria in eutrophic lakes and estuaries, while induction was
far less common in offshore and northern-lake environments. The degree to which
this reflects prophage presence vs. metabolic state is not clear. Inducibility of lysis
and phage release by means of mitomycin C or UV are the most common criteria
for the presence of lysogeny; however, the two may not induce the same prophages,
and many prophages are not induced by either of them. Other inducing agents that
have been explored on natural isolates include sunlight, temperature, pressure, poly-
nuclear aromatic hydrocarbons (PAHs), fuel oil, and trichloroethylene. PAHs, a PCB
mixture, and Arochlor 1248 were the most efficient agents, giving effective induction
of prophages in 75% of the tested samples, vs. 50% for 254-nm UV radiation and
for mitomycin C. Raising the temperature to as little as 30 degrees for 30 min could
induce lysis; Paul and Kellogg suggest that this may help explain the 10-fold summer
increase in free phage. Induction also generally works less well when the host cells
are at a lower metabolic state, since most inducing agents act on replicating DNA.
In summary, it appears that a key aspect of the high prevalence of virulent
phages in the oceans is that the numbers of any given phage-host pair are constantly
fluctuating in any natural setting, probably coupled with the continual mixing and
astronomical total numbers involved. Phage replicate most rapidly on the most
abundant, fastest-growing host population in a given setting at a given time, where
new hosts are found most rapidly, thus, for example, terminating microbial blooms
(cf. Hennes and Simon, 1995). Various approaches in different aquatic environments
suggest that about 15% of the bacterioplankton are lysed by phages daily, leading
to the release of nutrients important to marine ecosystems. The fact that most phage
will not replicate to a significant degree at host concentrations below 103–105 per ml
assures the maintenance of microbial diversity despite the presence of phages that
can infect each potential host. A high fraction of the bacteria in the oceans, as in
many other habitats, also harbour one or more prophages, but free temperate phages
do not generally contribute substantially to the high concentrations of phages observed
in both marine and terrestrial environments.
2.3. CO-EVOLUTION OF PHAGES AND THEIR HOST BACTERIA

Classical experiments with E. coli and its phages generally showed a rapid outgrowth
of phage-resistant bacterial strains. The coexistence of susceptible bacteria and their
corresponding phages in the environment was thus a somewhat surprising observa-
tion. Further classical experiments showed a co-evolution, with bacteria and their
phages involved in a continuous cycle of resistance and counter-resistance mutations.
Theoretical ecologists pointed to an asymmetry in this relationship, since receptor
mutations arise more easily in bacteria than anti-receptor mutations in phages (Lenski
1984; 1985). Since there are far more phages than bacteria in many ecological
settings, phages may balance these differences out. The development of phage-
resistant cyanobacteria was demonstrated in the field (Waterbury, 1993). Other
researchers argued that virulent phages might survive due to the maintenance of
sensitive parental strains in the population if the parental strains have a slight growth
advantage. Most discussions of these issues use the terms resistant and sensitive as
if they are absolutes. In nature, one actually sees many cases in which a particular
host can be infected by a given phage, but with low efficiency; this can also lead to
co-survival of phage and host. For example, in spot testing nearly 100 phages against
a battery of hosts, 10 were identified as able to infect E. coli O157; however, the
EOP turned out to be only about 10−4 for most of them (Kutter lab, unpublished).
Only RB69 plated as efficiently on O157 as on B or K12. Parameters affecting the
efficiency of plating are discussed in the Appendix, section 4.1.2.
2.4. EFFECTS OF HOST PHYSIOLOGY AND NUTRITIONAL STATUS

The normal state of a marine bacterium was predicted to correspond to the nutritional
state of a laboratory bacterium under stationary-phase conditions (Kolter et al., 1993).
While gram-negative bacteria do not sporulate, as do some gram-positive bacteria,
they do undergo a variety of metabolic and structural changes in stationary-phase
conditions that contribute to long-term survival in hostile environments. These are
mediated by a new sigma factor, s s, which controls at least 30 genes that are
expressed during starvation and at the transition into stationary phase. RpoS mutants
survive much less well under laboratory conditions of carbon or nitrogen starvation
and fail to develop starvation-mediated cross protection to oxidative, osmotic, and
heat stresses (McCann et al., 1991). From laboratory studies, it has been widely
accepted that most phages cannot productively infect stationary-phase bacteria,
leading to great surprise at the observed phage concentration levels in the ocean and
illustrating gaps in our knowledge when we try to transfer our experiences from
laboratory phage-host interactions to the ecological situation.
Clearly part of the problem was the limited number of phage-host systems on
which the accepted model had been based. Woods (1976) had actually found that
Pseudomonas phages could infect starved host cells maintained for 40 days in natural
riverine conditions. Under these conditions, the latent period was lengthened and
the burst size greatly reduced when compared to logarithmic-phase infection.
Schrader et al. (1997) showed that coliphage T7 and the three Pseudomonas phages
he tested could replicate well in cells that were starved or had entered stationary
phase; the variety of patterns seen emphasizes the importance of avoiding general-
izations. Phage ACQ could even infect P. aeruginosa maintained in starvation con-
ditions for 5 years; the burst size was reduced whether the starvation was short or
long, and the latent period was extended severalfold. For T7, the latent period was
also lengthened severalfold, but, interestingly, the burst size increased from about
50 to 450 when the host had been starved for 24 hours. T4 cannot produce a burst
in stationary-phase cells. However, T4 can enter and persist in long-term stationary-
phase cells, but as long as the stationary-phase sigma factor is present, the infection
process is suspended at an early stage. Whereas T4 normally blocks all host-gene
transcription and translation within minutes, it enters what we call “hibernation
mode” and produces a stable infective center in these starved cells (Kutter lab,
unpublished results; Fig. 6.1); when nutrients become available, the usual host
outgrowth proteins are produced, but then the phage program takes over, all further
host protein synthesis is blocked, and progeny phage rather than colonies are pro-
duced after resumption of cell growth. This phenomenon could help explain the
persistence of many virulent phages within populations of non-growing cells.
Phage Ecology 137
FIGURE 6.1 Ability of bacteriophage T4 to survive in stationary-phase E. coli for extended

periods and still respond to form an infective-center plaque when transferred to nutrient-rich
plates. After growth in TSB for varying times (expressed in hours), 10 7 phage/ml were added
to samples of the culture. This ability to form stable infective centers in starved cells depended
on the presence of the stationary-phase sigma factor, rpoS; in otherwise-isogenic rpoS cells,
few infective centers were seen even at 4 hours (Kutter, 2001).
Complementary observations have been made in sporulating bacteria, illustrat-

ing some of the complexities of phage-host interactions under growth-limited con-
ditions in the wild. For example, phages j e and j29 are lytic when infecting growing
B. subtilis. However, each phage shows a narrow window during sporulation when
it can become entrapped in the spore and persist in a quiescent state which allows
later germination of the spore followed by formation of an infective center rather
than a bacterial colony (Sonenshein and Roscoe, 1969). Also, the complex life cycle
of the gram-negative bacterium Myxococcus xanthus includes the formation of
fruiting bodies containing relatively stable myxospores. Bacteriophage MX-1 is
virulent on exponential-phase cells but will not bind to myxospores (Burchard and
Dworkin, 1966). For an hour after the transition has been induced, MX-1 still binds
and injects its DNA but the phage genome becomes trapped in a persistent state;
again in this case, germination begins normally upon nutrient addition, but within
an hour the phage program takes over (Burchard and Voelz, 1972).
2.5. BIOFILMS
Most studies of the phage infection process have been carried out with bacteria
suspended free in liquid culture. However, at interfaces between solid surfaces and
aqueous environments, bacteria frequently aggregate to form complex attached com-
munities called biofilms (Fig. 6.2). Such biofilms are widespread and pervasive—
on river rocks, in pipes, industrial equipment and medical implants, lining the colon,
as dental plaque, in the lungs of cystic fibrosis patients (cf. Costerton, 1999).
Microorganisms undergo profound phenotypic developmental changes during the
FIG 6.2 Complex structures and water channels proposed for biofilm architecture. Drawn
by Kalai Mathee.
transition from free-floating planktonic to biofilm growth (O’Toole et al., 2000).

Due to these inherent physiological changes, their slow growth rates and the fact
that the cells are embedded in an extensive exopolysaccharide matrix with hetero-
geneous spatial distribution, cells living in biofilms tend to be at least 10-fold more
resistant than planktonic cells to most antibiotics and other antimicrobial treatments
(Anwar et al., 1992; Bagge et al., 2004).
Interest is growing in the natural roles of bacteriophages in modulating biofilms
and, particularly, in the potential for their use in controlling biofilms in a variety of
settings. Adams and Park (1956) first explored the properties of a phage polysac-
charide depolymerase, while Lindberg (1977) reported EM observation of these
enzymes as spikes attached to the phage baseplate. Here, the capsular material acts
as a secondary receptor to which the phage can bind, degrading the polymer until
it can reach its outer-membrane receptor and infect the cell (see Chapter 7, Fig. 7.4).
In exploring the role of cell death in Pseudomonas biofilm development, Webb
(1996) found evidence that an induced prophage actually played a significant role
in the process. They found that after a few days up to 50% of the microcolony
structures within biofilms formed by various bacteria showed areas of killing and
lysis in their centers, sculpting internal structures and releasing free-swimming
bacteria; this was paralleled by the release into the medium of a temperate phage
that seemed to be responsible for this process.
Studies have been carried out of phage interactions with biofilms involving such
bacteria as Pseudomonas aeruginosa, E. coli, Listeria monocytogenes, Enterobacter
agglomerans, and Staphylococcus aureus. Doolittle et al. (1995; 1996) demonstrated
lytic infection of E. coli biofilms by bacteriophage T4 and used fluorescent probes
to track the interactions of the phage with the biofilms. Hughes (1998), exploring
biofilm bacteria from a food processing factory, showed that E. agglomerans phage
SF153b has a polysaccharide depolymerase that can disrupt biofilms through
exopolysaccharide (EPS) degradation even when a phage mutation blocks cell infec-
tion and lysis. They partially purified the enzyme, an endoglycanohydrolase, and
confirmed that it could work in isolation and was specific for the Enterobacter EPS;
it could not attack similar biofilms formed by Serratia. As demonstrated by Hanlon
et al. (2001), appropriate phages could diffuse through alginate gels and produce a
2-log reduction in bacterial numbers in 20-day-old P. aeruginosa biofilms, reducing
Phage Ecology 139
the viscosity by as much as 40% despite the presence of EPS. Corbin et al. (2001)
used a chemostat coupled to a modified Robbins chamber and scanning confocal
microscopy and saw clear T4 effects on glucose-limited biofilms, at least at very
high multiplicities of infection. Here and in several of the previous cases, there was
no evidence for involvement of a degradative enzyme and it is not clear how the
phages got through the EPS layer to reach their receptors. McLean et al. (2001)
have explored useful techniques for studying the parameters affecting biofilm growth
and phage-biofilm interaction.
3. MARINE PHAGE ECOLOGY

3.1. MARINE PHAGE IMPACTS ON THE FOOD WEB
A number of parameters of the phage-bacterium interaction have been determined
in quantitative detail for marine phages, making them currently the best-characterized
phage ecology system. The production and distribution of marine viruses is, not
surprisingly, determined by the productivity and density of the host populations
(Boehme, 1993; Jiang, 1994; Weinbauer, 1995). The usual virus-to-bacterium ratio
falls between 3 and 10 and depends clearly on the nutrient level: bacterioplankton
produce more phages under environmental conditions favouring fast bacterial growth
and productivity (Hara, 1996; Maranger, 1994; Steward, 1996). The quantitative
relationships led to numerical calculations of the degree of bacterial mortality caused
by marine phages (Binder, 1999) and mathematical modeling of the energy flow in
marine food webs. Phage predation of marine bacteria now enters into models of
global biogeochemical cycling of carbon (Proctor, 1990). Bacterial and algal viruses
are established members of the microbial loop in the oceans with profound effects
on the cycling of carbon and nitrogen and on the marine food web (Fuhrman, 1999).
The change from discovery of their existence to such a prominent place in ecological
modelling could not be more dramatic.
When a bacterium is lysed by phage infection, probably 99% of the cell contents
enter the dissolved organic matter pool (Fuhrman, 1992). Phage are thus efficient
drivers in the biomass-to-dissolved-organic-matter conversion. They are important
sources of bacterial mortality in the sea, along with bacterial grazing by zooplank-
ton. The result of phage lysis of marine bacteria is, paradoxically, a stimulation of
bacterial growth. The rationale is the following: The presence of phage stimulates
the growth of bacteria in comparison to a situation where phages are lacking.
Predation of bacteria by protists results in the transfer of bacterial biomass into the
next layer of the food web with no feedback (in the literal sense) to bacteria. Phage
lysis, in contrast, releases nutrients from the lysed cells that become available to
the bacterial community (Middelboe et al., 1996). In one model, viral lysis causes
a net loss of 25% in nanozooplankton production (Fuhrman and Suttle, 1993). To
complicate the matter further, some protists like dinoflagellates also prey on viral
particles.
Substantial theoretical and experimental research efforts were undertaken to
determine the quantitative degree of virus-mediated bacterial mortality and to assess
the ratio of phage lysis versus grazing by higher organisms. Not surprisingly, the
greatest impact of phage lysis was in oligotrophic environments. Furthermore, the

ratio of lysis versus grazing changed with water depth both in the ocean and in
lakes (Steward, 1996; Weinbauer and Hoefle, 1998). To summarize a large body of
literature, different approaches in different environments yielded a remarkably
stable rate of virus-mediated bacterioplankton mortality of about 15% per day
(Suttle, 1994). The rate seems to be higher for heterotrophic bacteria than for the
autotrophic cyanobacteria (Suttle, 1994). Even if these figures seem to suggest only
a modest effect of phages on bacteria, a 15% bacterial mortality can still have a
profound effect on the relative proportions of different species or strains in a
community. Models of virioplankton that control host community diversity have
been developed. For a eutrophic estuarine environment, the basic numbers were
approximated as follows: bacteria at a density of 106/ml with about 50 different
species and viruses with a concentration of 107/ml and 200 different strains
(Wommack et al., 1999). One concept is that of “killing the winner populations,”
i.e. phage expand on the fastest-growing host population in the given ecological
setting (Thingstad and Lignell, 1997). The epidemic ceases when the diminished
host population no longer supports efficient phage replication. Blooms have been
observed where up to 80% of the total bacterial population is represented by a
single bacterioplankton strain, although lower peak levels are more frequent. There
is strong indirect evidence that some bloom collapse is mediated by viral lysis. The
most convincing data are from the Lake of Constance, where transient increases in
bacterial abundance were closely followed by peaks in the frequency of infected
bacteria and free phage (Hennes, 1995).
Phage infection in the ocean leads to better retention of nutrients in the euphotic
zone because more organic material remains in non-sinkable bacterial form (Murray
and Eldridge, 1994; Thingstad et al., 1993). In contrast, lesser phage infection allows
a transfer of organic material upwards in the food chain, into organisms that even-
tually either sink themselves or are compacted in the fecal pellets of organisms with
guts (Fuhrman, 1992). These processes transport organic masses from the euphotic
zone to the deep sea. They have substantial impact on global climate models via
CO2 fixation from the atmosphere into marine biomass and eventual transfer to the
ocean sediment, with the net result of a reduction of this important greenhouse gas
(Wilhelm and Suttle, 1999); phage infection helps counteract this. Marine viruses
may have an additional effect on the shaping of the global climate by inducing the
release of dimethyl sulfide (DMS) from lysed phytoplankton (Malin et al., 1998).
DMS is a gas that nucleates cloud formation and thus affects the radiative properties
of the atmosphere.
As discussed in section 2.1, much quantitation has been carried out in the
marine environment, but extrapolation of data from the marine field to others is
often problematic. Ocean water often contains substances that protect viruses from
inactivation, such as adsorption to clay particles (LaBelle and Gerba, 1982; Smith
et al., 1978), as well as heat-labile uncharacterized virucidal substances that show
geographical variation (Suttle and Chen, 1992). In principle, two processes must
be differentiated: the destruction of phage particles and the loss of infectivity.
There is a consensus that unattenuated sunlight is the dominant factor controlling
the decay of viral infectivity in surface waters (Garza and Suttle, 1998). Inactivation
Phage Ecology 141
by sunlight was significant down to a depth of 200 m. UV-A (320 to 400 nm)
has the greatest impact (Murray and Jackson, 1993). UV-mediated dimer forma-
tion of adjacent pyrimidines was the principal photodamage (Wilhelm et al., 1998).
However, host- and phage-encoded photorepair systems could still recover some
of the lost infectivity (Bernstein, 1981). Phages with smaller capsid size turned
out to be more sensitive than phages with capsids >60 nm (Heldal and Bratbak,
1991; Mathias, 1995). One to five percent infectivity loss per hour was the average,
and no marked differences were observed between marine phages and reference
laboratory phages (Wommack et al., 1996); under surface light conditions, a one-
log loss of infectivity was observed over a day of natural sunlight exposure.
Differences were observed between distinct phage isolates infecting the same
host, demonstrating that environmental persistence is a trait particular to a given
phage strain. Non-native phages experienced a greater sunlight inactivation than
native phages, suggesting adaptation of phages to local conditions (Noble and
Fuhrman, 1997).
3.2. MARINE PHAGE CHARACTERIZATION

3.2.1. CYANOPHAGES
Cyanobacteria are among the most important primary producers and nitrogen fixers
on earth, responsible for much of the primary production in oceans, lakes, and other
aqueous environments. They are ubiquitous, found in extreme environments from
hot springs to polar lakes as well as in ponds for waste stabilization and for raising
fish. They can be differentiated into marine and freshwater forms, into those using
phycoerythrin vs. those using phycocyanin as their primary photosynthetic pigment,
and into those that are unicellular vs. those that grow as filaments. While they are
clearly bacteria, their ecological roles are more closely tied to those of eukaryotic
algae than to those of heterotrophic bacteria (Suttle, 2000a; Suttle, 2000b).
Cyanophages infecting filamentous freshwater cyanobacteria were first isolated
by Safferman and Morris (1963), and stimulated the hope of using such viruses to
control cyanobacterial blooms. Phage infecting filamentous cyanobacteria generally
cause rapid invagination and destruction of the host’s photosynthetic membranes,
whereas such destruction is only seen very late in the infection cycle with those
infecting unicellular cyanobacteria, for which successful infection seems to depend
on ongoing photosynthesis.
Demuth et al. (1993) reported that nearly all of the phages they saw in Lake
Plubsee had tails, the majority being Siphoviridae; in those studies, the phage levels
were so high that they simply floated the TEM grids on the samples and let the phages
adsorb. Half of the phages seen in Chesapeake Bay by Wommack et al. (1992) also
had tails. Myoviridae are the most common characterized from marine waters and
are also often isolated from fresh-water species. Five Synechoccus cyanophages
isolated by Wilson et al. (1993) included two myoviruses and one siphovirus. Head
proteins of one group of marine phages even have clear sequence relationships to
T4-like coliphages, as discussed below. The similarities are particularly interesting
since cyanobacteria diverged from other bacteria billions of years ago.
3.2.2. Genomic Analysis of Marine Phages
Various genomics approaches have given new insights into the field of phage ecology.
A large-scale random sequencing effort of two uncultured marine water samples
demonstrated that 65% of the sequences lacked matches to the database (Breitbart
et al., 2002). The database hits were mostly with viruses, covering all major families
of tailed phages and some algal viruses. A careful statistical analysis of the sample
revealed between 400 and 7000 different viral types in the two 100-litre samples,
with the most abundant type representing 3% of the total viral population. Over 200
Vibrio parahaemolyticus phages were isolated from various locations and seasonal
periods in Florida and Hawaii (Paul and Kellogg, 2000). All observed isolates were
Myoviridae and shared some genetic determinants, giving 83%–100% identity for
one sequenced 500 bp region, but on the basis of restriction patterns they could be
divided into at least 7 groups for the Florida isolates, one of which was consistently
dominant (71%), plus a pair of Hawaii isolates.
In striking contrast to the careful ecological work performed with marine viruses,
only a handful of marine phages have actually been sequenced. However, these few
examples delivered surprises. A Pseudoalteromonas phage with a 10 kb genome
became the type phage of a new family, called Corticoviridae, of lipid-containing
phages. Cyanophage P60 and phage SIO1, infecting the marine heterotroph Roseo-
bacter, resembled coliphage T7 closely in their genome organization. Three Syn-
echoccus cyanophages were found to share distant head-gene sequence relationships
with coliphage T4 (Fuller et al., 1998). Zhong et al. (2002) then designed primers
to amplify capsid assembly protein gp20 from both isolated marine cyanophages
and natural virus communities and looked at 114 different gp20 homologues. He
found these cyanophages to be a highly diverse family, with 65%–96% sequence
similarity among the cyanophage gp20’s (and 50%–55% similarity with T4). They
fall into 9 different phylogenetic groups (Fig. 6.3), with up to 6 clusters and 29
genotypes found in a single sample.
3.3. MARINE PHAGE HOST SPECIFICITY

AND HORIZONTAL GENE TRANSFER
In the large majority of the phages investigated in the laboratory, host species
specificity is the rule (Ackermann, 1987); phages generally also display strain
specificity within a host species due to using a variety of different receptors. Phages
with broad host ranges have been described, but they are the exceptions. There was
some expectation that marine phages might show broader host range to facilitate
their multiplication at the frequently low host densities seen in ocean environments,
as discussed above. However, the results to date indicate that most of them are host-
species specific; many also demonstrate strain-specificity (Baross et al., 1978a;
Bigby and Kropinski, 1989; Koga et al., 1982; Moebus, 1992). Striking host range
differences were seen between phages recovered east or west from the Azores islands
in the Atlantic Ocean (Moebus and Nattkemper, 1981). Broad host range was more
prevalent in cyanophages, but Hennes (1995) demonstrated that fluorescence-
labelled cyanophages attached specifically only to their known host and not to other
Phage Ecology 143
FIGURE 6.3 Neighbor-joining tree showing the phylogenetic affiliation of cyanophage iso-
lates and representative clones from all six of the natural virus communities studied. The tree
was constructed on the basis of a 176-amino-acid sequence alignment with T4 as the outgroup.
Each value in parentheses is the number of different nucleotide sequences in the same cluster
and same community as the representative clone. Clusters A through F and I through III were
assigned on the basis of phylogenetic relatedness. Bootstrap values of less than 50 were not
shown. The scale bar indicates 0.1 substitution per site.
bacteria of the natural consortium. However, some observations suggested that

bacteriophages isolated from very low-nutrient marine habitats showed a trend
toward increased breadth of host range. If confirmed, this could represent an adap-
tation to the low host cell concentrations.
The still-unsettled subject of possible selection for broader host specificity in
nutrient-poor marine environments is of substantial scientific relevance. If phages
with unusually broad target ranges are indeed more widely distributed in the marine
environment than we expect from laboratory infections, transfer of genetic material
between marine bacterial species via transduction might occur at even higher fre-
quency than currently anticipated on the basis of the high concentrations of phages
and bacterial cells and the tremendous volume of water in the oceans. Transduction,
the accidental transfer of host DNA via a phage particle, occurs at varying rates for
different phages, but is about once in every 108 phage infections in several well-
studied systems. A mathematical treatment of experimental data from the estuary of
the size of the Tampa Bay led to an estimation of 1014 transduction events occurring
annually (Jiang and Paul, 1998). If even a minute fraction of this DNA is travelling
between different bacterial species, it is clear that marine phages open up enormous
possibilities for horizontal DNA transfer. The probability of transformation between
unrelated species may well also be increased by the liberation of free bacterial DNA
during lysis of infected cells. Current bacterial genomic analyses underline the
important impact of lateral gene transfer, but despite the theoretical importance of
transduction, few transduction studies have been conducted in the marine environ-
ment. Experiments with Pseudomonas revealed that higher transduction rates were
obtained when both the donor and the recipient were lysogenic (Morrison et al.,
1978). The reason is that a lysogenic strain can accept foreign DNA from a trans-
ducing phage, but is protected from lysis due to the immunity functions of the
resident prophage if the two phages are related. In freshwater samples, suspended
particles increased the transduction frequency since they allow adsorption of the
donor and recipient cells on a solid phase.
4. SOIL AND PLANT-ASSOCIATED PHAGES

The constant mixing found in aqueous contexts plays a major role in our ability to
make generalizations about marine phages. Phages are also present at high levels in
a variety of soils, but are much harder to study in detail and such work is in its
infancy. Conditions in terrestrial environments vary far more drastically than they
do in marine environments. One is really talking about enormous numbers of microen-
vironments, affected by position within soil particles, patterns of rain, drought, and
temperature, and diurnal and seasonal variations, often with low and variable levels
of exchange between them. Bacteria have many physiological adaptations for dealing
with these changes, and these in turn affect host-phage interactions in ways that we
are scarcely beginning to understand. In addition, phage have the challenge of finding
a new bacterial host under conditions where the soil may often be only partially
hydrated and the phage may be trapped in biofilms, bound to clay, or inactivated by
acidity or other properties of the soil.
High phage concentrations comparable to those in marine environments have
also been reported in terrestrial environments. For example, rhizosphere soil from
a sugar beet field revealed 107 phage per gram by using transmission electron
microscopy (Ashelford, 2003) and reconstitution experiments suggested that this
figure underestimates the true number by nearly a factor of 10 for technical reasons.
Phage from non-pathogenic bacteria like thermophilic Bacillus species are easily
isolated from soil, compost, silage, and rotting straw, suggesting a tight association
Phage Ecology 145
of phage with plants (Sharp et al., 1986). A wide variety of phages were observed,
most of them strain-specific within a given Bacillus species. Hybridization experi-
ments with Serratia and Pseudomonas colonies from the soil showed that at least
5% of the bacteria are actually phage-infected. There are also reports of plants and
plant extracts that can induce bacterial lysogens (Erskine, 1973; Gvozdyak, 1993;
Sato, 1983). Soil phages, like their aquatic counterparts, are thus likely to be impor-
tant in controlling bacterial populations and mediating gene transfer.
Various reports indicate that phage-host interactions can be quite complicated
in the soil. For example, phages had only a minimal impact on net growth of
Streptomycetes in the soil (Burroughs et al., 2000). In a combination of experimental
observations and mathematical modeling, spatial heterogeneity in phage-host inter-
action, and temporal changes in susceptibility to phages were explored as determi-
nants in bacterial escape from phage lysis in the soil. It turned out that germinating
spores were more susceptible to phage infection than hyphae of developed mycelia.
Mature resistant mycelia adsorb most of the Streptomyces-specific soil bacteria and
thus protect younger susceptible hyphae from infection.
A number of labs have explored phages for biocontrol of plant pathogenic
bacteria, as discussed in Chapter 13. One popular candidate is Erwinia amylovora,
the cause of fire blight disease of apple and pear trees. Fire blight has generally been
fought with limited success by antibiotics. Biological control by apathogenic
Pseudomonas or by Erwinia phage Ea1 has been explored. Erwinia-specific phages
like Ea1 were prevalent in orchards affected by fire blight, demonstrating a wide
distribution of this phage; other genetically distinct phages, some with very broad
host ranges on the fire blight pathogen, were also detected (Schnabel and Jones,
2001). The logistics of studying phage treatment of tree pathogens has been very
challenging, but work is also going on in Guelph, Tbilisi, and elsewhere in applying
phage against Erwinia species that infect important but more experimentally tractable
model systems such as potatoes, carrots and ornamental flowers. In all cases, it has
been possible to isolate a range of relevant phages from the wild, once again
emphasizing their ubiquity in the environment. Phages against Leuconostoc and
Lactobacillus have been isolated from numerous spontaneous fermentation pro-
cesses, including coffee, pickled cucumbers, sauerkraut, cereals, and wine. These
presumably represent phage that are normally associated with the respective plants
being used for the fermentation processes.
5. ANIMAL-ASSOCIATED PHAGES
5.1. INTRODUCTION
When the draft sequence of the human genome arrived at the finishing line, it
provided only a small part of the genetic material that makes up a human being. In
fact, we harbour in our gut more bacterial cells than we have human cells. Not
surprisingly, these gut bacteria are associated with their specific phage communities
(Breitbart et al., 2003). This situation is not peculiar to humans; phage concentrations
up to 109 per gram of feces were detected in cattle and sheep (Furuse, 1987). Other
vertebrates (birds, fish) also contained appreciable phage concentrations in the gut
content. Phages have also been isolated from fecal pellets of many invertebrates
belonging to diverse taxonomical groups (earthworms, bees, flies, mussels), as
discussed by Ackermann and Dubow (1987). Oysters are filtering water and retain
material suspended in the water, so it is no surprise that up to 106 pfu of vibriophages
were found per g of oyster tissue (Baross et al., 1978b).
A number of studies have reported large phage populations in the rumens of
sheep and cattle, affecting the complex balance among the bacteria that convert grass
into nutrients to support ruminant growth. This is one area where research can be
carried out at a level of sophistication comparable to that seen in the marine and the
dairy environments. Animals with permanent rumen cannulae have facilitated non-
invasive sampling and allowed studies over time that give new insight into rumen
ecology—an important field in science-based livestock husbandry, which aims to
optimize the efficiency of converting feed into meat and milk. Klieve and Bauchop
(1988) partially purified phages from fluid samples collected through a nylon stock-
ing from the rumens of cattle and sheep and studied them by electron microscopy.
They found mainly tailed phages, with a high range of diversity as determined by
head shape and size and tail morphologies: at least 14 different kinds of isometric-
headed myoviridae, 4 podoviruses, and 4 isometric and 2 prolate-headed siphovi-
ruses, including an astonishing giant phage with a head measuring 85 × 238 nm.
The tails ranged from 25 to 1050 nm long. The ruminal phage DNA varied in size
from 10 to 850 kbp. Klieve and Swain (1993) saw discrete bands of a wide variety
of sizes, each essentially homogeneous, that differed in distribution from sample to
sample, against a broad background of DNAs between 30 and 200 kbp; they showed
that the latter represented a large, mixed population of intact DNAs, not degraded
DNA from larger bands. The total phage population was determined to be about
1010 per ml—significantly higher than most earlier estimates. Klieve et al. (1989)
also explored the incidence of mitomycin C-inducible temperate phage in the rumen.
Of the 38 different ruminal bacteria that they analyzed, only 9 produced phage-like
particles, all but one of them siphoviridae.
Phage can be isolated from the feces of most animals. In fact, an extended
scientific discussion deals with the question of whether phages can be used as
surrogate measures of fecal contamination levels in the environment. Since F+-
specific E. coli phages were mainly isolated from animal feces and Bacteroides
fragilis phages only from human feces, specific phage detection methods can poten-
tially differentiate the origin of environmental fecal contamination and phages were
used as tracers to follow the intrusion of polluted surface waters into groundwater.
The recent excitement about phage therapy of human and animal diseases has
renewed the interest of microbiologists in the ecology of phage-bacterium interaction
in the context of their hosts. Here, we will discuss three ecological niches: the gut,
the oral cavity, and the skin.
5.2. ENTERIC PHAGES

The human gut is a complex ecosystem, colonized by about 400–500 microbial
species, 30–40 of which account for 99% of the total population. A first understanding
of human colonic phages comes from recent metagenomic analyses of an uncultured
Phage Ecology 147
FIGURE 6.4 Genomic overview of uncultured viral community from human feces based on
TBLASTX sequence similarities (Breitbart, 2003).
phage community from human feces of a single subject (Fig. 6.4) (Breitbart
et al., 2003). DNA analysis using pulse field gel electrophoresis showed major
bands at 15 and 90 kbp with minor bands at 30, 40, and 60 kbp and an average size
of about 30 kbp—a significantly different distribution than that observed in seawater
or the rumen, as described above; the dominant band at only 15 kbp was especially
unusual. Extracted DNA was cloned and sequenced. No significant GenBank hits
were seen for 59% of the 532 sequences. Half of the positive hits were related to
bacterial genes, while a quarter were with phage genes, mainly to structural proteins
and terminases; one might expect that a large fraction of the sequences without
GenBank hits were from phages, since more information is available from bacterial
genome projects and sequenced phages often show a very large fraction of unique
sequences. There were few matches to T7-like podoviridae or to l-like siphoviridae,
which were the most abundant species observed in the marine environment. Many
were to phages infecting gram-positive bacteria, in agreement with the observation
that 62% of the cells detected in human feces with specific rRNA probes were
gram-positive bacteria. Since earlier studies exploring gut phages against E. coli,
Salmonella and Bacteroides have shown substantial differences among individuals
that did not correlate with age or sex, it will be very important to carry out more
studies of this nature and also look for correlations with such factors as dietary
patterns and prior antibiotic use.
E. coli and its phages belong for molecular biologists to the most carefully
investigated phage systems, yet surprisingly few reports have investigated the gut
ecology of coliphages or even the ability of coliphages to replicate under anaerobic

conditions. Kutter et. al. (1994) reported that phage T4 can successfully infect
anaerobically as long as the bacteria are already pre-adapted to anaerobic growth.
In this connection, it is important to distinguish anaerobic fermentation, in which
bacteria use an organic byproduct of metabolism as the terminal electron acceptor,
from anaerobic respiration, in which they use an electron acceptor such as nitrate
or fumarate. The former is found, for example, in the rumen of grass-eating mam-
mals, where much glucose is available, while the latter predominates in the colon,
where virtually no fermentable substrates remain. For T4-like phages, we find quite
different patterns of infection in comparing anaerobic respiration, anaerobic fermen-
tation and aerobic respiration, even when largely similar defined media are used;
burst sizes are generally lower anaerobically and lysis is often delayed for many
hours, even though cells lysed with chloroform at 50 minutes after infection show
good phage production.
In one large study, stool samples from 600 healthy patients and 140 patients
suffering from traveller’s diarrhea were investigated for the presence of coliphages
on 10 different E. coli indicator strains (Furuse, 1987). From healthy subjects, 34%
of the stool samples contained phages but only 1% showed high amounts. Most of
them were classified as temperate phages related to phages phi80, lambda, and phi28.
In comparison, 70% of the stools from diarrhea patients (half of whom might have
had an enterotoxigenic E. coli infection) contained phages, 18% in high concentra-
tions. About half of these isolates were composed of virulent phages related to T4
and T5. This change of phage composition between healthy subjects and diarrhea
patients seemed to reflect some disturbance of their intestinal bacterial flora. In
another study with stool samples from 160 pediatric diarrhea patients from a clinic
in Dhaka Bangladesh, a third of the patients were phage-positive when tested on
two E. coli indicator cells (Chibani-Chennoufi et al., in preparation). Notably, in this
clinic about 30% of cases are caused by E. coli (Albert et al., 1995). The phages
were nearly exclusively T4-like phages by genome size and morphology. However,
restriction analysis, diagnostic PCR and partial sequencing demonstrated substantial
genetic diversity between the phage isolates. The stool samples from the Bangladeshi
children in the convalescent phase yielded no higher phage counts than the stool
samples from the same patients during the acute diarrhea episode, but the phages
were not screened on the infecting E. coli serotypes. Strain-specific phages might
thus suffer from underreporting. In animal experiments (Chibani-Chennoufi et al.,
2004) and recent human adult volunteer trials at the Nestle Research Centre (Bruttin
et al., manuscript in preparation), T4 and T4-like phages were added to the drinking
water. The phages survived the gastric passage and were recovered from the feces
in titres comparable to the dose orally fed to the subjects.
This suggests that these phages transit through the entire gut relatively unscathed,
but no significant replication on the endogenous E. coli gut flora was observed despite
the fact that many fecal strains were susceptible to the phage infection in vitro; fecal
counts of the endogenous E. coli flora also remained relatively unchanged both in
mouse and in man. These experiments raise doubts about too-simple models of phage-
host interaction in the mammalian gut. The metabolically active E. coli dwelling as
microcolonies in the intestinal mucus layer covering the mucosa might be physically
Phage Ecology 149
protected against lumenal phage (Poulsen, et al., 1995; Krogfelt, et al. 1993). Mouse
experiments suggested that freshly added E. coli applied by mouth were susceptible
to luminal phage. Actually, we know relatively little about the ecology of E. coli in
the human gut. E. coli might even be a misnomer since the pathogenic strains cause
their diarrhea effects by interacting with the small intestine and not the colon. Mucosa-
associated E. coli in the small intestine might acquire sufficient oxygen from the
blood vessels to facilitate host and phage growth. Here again, research areas at the
borderline between microbiology, ecology and physiology are key. It is clearly very
important to develop a better general understanding of phage infection under such
conditions and of the roles of phage in gut and rumen ecology. These ecological
considerations are important for phage therapy approaches in the alimentary tract.
(The Hungate technique, an inexpensive and fairly simple system for carrying out
anaerobic phage infection studies in vitro, is described in the Appendix.)
E. coli and its phages can be easily isolated from the environment. For RNA
coliphages, the most common sources were sewage, both from domestic drainage
and sewage treatment plants, followed by feces of man, domesticated animals
(cows, pigs), and zoo animals. Much less rich sources were environmental water
samples (Furuse, 1987). This led to the proposal that coliphages could be a surrogate
measure for fecal contamination of recreational waters or other waters of public
health interest (el-Abagy et al., 1988). Recently it has become technically possible
to screen for human viruses in water samples. However, the majority of the med-
ically important human enteroviruses are RNA viruses, with the single exception
of adenovirus. The most sensitive PCR techniques thus cannot be applied. Testing
in coastal waters in California impacted by urban run-off water revealed that four
out of twelve sites contained adenovirus. However, coliform bacteria and coliphages
did not correlate with the adenovirus, calling for a reevaluation of both indicator
organisms for the monitoring of recreational waters. In contrast F-specific RNA
coliphages showed a good correlation with adenoviruses (Jiang et al., 2001). There
is some ecological knowledge on these RNA coliphages in the environment (Furuse,
1987). They are found with strikingly variable prevalence in domestic drainage
from different geographical areas. These phages were also found in the feces of
humans and domesticated animals. The fact that the feces from cows and pigs
contained large amounts of RNA coliphages suggested that these phages were
actually propagated in the intestines of the animals. Different groups of RNA
coliphages were found with distinct frequencies in humans and animals, suggesting
some specificity that probably reflected the distinct composition of the gastrointes-
tinal microbial flora. This fact supports the idea that the intestine of mammals may
constitute one of the natural habitats of coliphages despite the fact that E. coli
represents only a minor constituent of the normal bacterial flora in the human
alimentary tract
5.3. OTHER PHAGE SITES OF INTEREST: ORAL CAVITY AND VAGINA

Bacteriophages have also been isolated from other parts of the alimentary tract, for
example the oral cavity. This anatomic site is richly colonized with many mainly
commensal bacteria, but pathogenic strains like Streptococcus pneumoniae and
S. pyogenes were also detected. The actual species composition varies from precise
anatomic site to site. Bacteriophages play an important role for these pathogens,
since a majority of the clinical isolates of S. pneumoniae are lysogenic (Severina
et al., 1999; Ramirez et al., 1999) and in the case of S. pyogenes the prophages
contribute a set of virulence factors to the cell that directly influence the epidemi-
ology of the clinical isolates (Beres et al., 2002). Phage lysins applied to the oral
cavity can potentially diminish the degree of colonization of the oral cavity with
these pathogenic bacteria and the seeding of these pathogens into the respiratory
tract (see Chapter 12). In about 3% of dental patients, dental plaque yielded both
Actinomyces and the corresponding phages (Tylenda et al., 1985). The phages could
be re-isolated from most of the patients up to a month later, suggesting that they
belonged to the local microbial community. In another study, about 10% of the oral
washings from dental patients allowed the isolation of virulent phages directed
against Veillonella strains, a resident constituent of the oral cavity. Enterococcus
faecalis phages were isolated from human saliva (Bachrach et al., 2003), but the
ecological role of all these oral phages is still unsettled.
Recent data suggest that phages may play an important role in the ecology of
the vagina as well, in a way that is attracting significant medical attention (Kilic
et al., 2001). Lactobacilli constitute the dominant vaginal bacterial flora and are
beneficial to women’s health, since they inhibit the growth of harmful microorgan-
isms by producing lactic acid, hydrogen peroxide and other antimicrobial substances
(Redondo-Lopez et al., 1990). Bacterial vaginosis, linked to various medical condi-
tions, is observed when anaerobic bacteria outnumber lactobacilli in the vagina.
About 30% of lactobacilli isolated from healthy women from the United States or
Turkey were lysogenic. This rate was 50% in women with bacterial vaginosis. Many
of these lysogens could be induced by mitomycin C, releasing infectious phage,
some at high titer, that could lytically infect lactobacilli belonging to multiple species
(L. crispatus, jensenii, gasseri, fermentum, and vaginalis). The authors note further
that smoking is a risk factor for bacterial vaginosis, and the mutagen benzopyrene,
which is created by smoking tobacco, could induce phages from lysogenic lactoba-
cilli at the concentrations found in vaginal secretions of smoking women (Pavlova
and Tao, 2000). They suggest that smoking may reduce vaginal lactobacilli by
promoting phage induction, leading to a replacement of lactobacilli by anaerobic
bacteria and precipitating bacterial vaginosis.
6. INDUSTRIAL PHAGE ECOLOGY

6.1. DAIRY INDUSTRY
Phage contamination is a constant problem for fermentation-dependent industries
such as the dairy industry. In fact, phages are the primary cause for fermentation
delays in yogurt and cheese production and can in extreme cases lead to the loss of
the product. These cost considerations were a powerful incentive for the dairy
industry to design elaborate phage control measures. An overview of this activity is
provided in the flow scheme of Fig. 6.5. The first steps in these control measures
are ecological surveys of phages in the factory environment. The industry needs to
Phage Ecology 151
FIGURE 6.5 Phage control in industrial food fermentation. The flow scheme illustrates the
approach in the company of one of the authors using Streptococcus thermophilus as an
example. The different steps depicted in this flow diagram have been the subject of a number
of publications. A recent review summarizing this work can be found in Brüssow (2001).
know the extent of the problem, the prevalence and titers of the phages, and their
distribution in space and time. The dairy industry has accumulated substantial knowl-
edge in the field of phage factory ecology, but these data are generally not published
as the ecology of phages in a man-made environment has not attracted many aca-
demic ecology-oriented microbiologists. However, industrial microbiologists soon
realized that it is not sufficient to explore the distribution of phages in the factory.
The space around the factory and the material delivered into the factory (milk and
starters) had to be studied as well. Classically, the major focus of this industrially-
oriented work is the selection of appropriate combinations of bacterial starters
showing non-overlapping phage susceptibility patterns to allow the design of an
efficient starter strain rotation system, which is still the most-used means of coping
with the industrial phage problem.
Over the last decade, substantial efforts have been conducted to design phage-
resistant starter strains (see also Chapter 10). In the case of cheese production using
Lactococcus lactis as a starter, this task was facilitated by the availability of many
natural phage resistance systems in this bacterium. This allowed the construction of
phage-resistant starters by the transfer of plasmids using methods that are not
considered genetic engineering. The situation is different for Streptococcus thermo-
philus, the bacterial starter used in yogurt production. Few strains contain plasmids
and even fewer possess plasmids with phage-resistance functions. Therefore, indus-
trial microbiologists did substantial sequencing of phage genomes to obtain genetic
elements that interfere with the phage replication cycle when the starter is super-
infected with a phage. These inhibitory elements included genes (phage repressor,
superinfection exclusion proteins), non-coding DNA (origin of phage replication)
and anti-messengers of phage genes. These systems suffer from the fact that they
are metabolically costly to the cell; they are most efficient when present on high
copy number plasmids, and frequently do not work when integrated into the bacterial
chromosome as a single copy. In addition, food-grade plasmid vectors have only
been developed for some bacterial starters (L. lactis).
In S. thermophilus, this problem could be circumvented by genetic engineering
approaches where a plasmid is forced into chromosomal integration, leading to the
disruption of bacterial genes. Phage-resistant mutants are then selected and charac-
terized. The most powerful resistance mechanism was the disruption of a membrane
protein that was apparently used by the phage for the injection of its DNA into the
cell (see Chapter 7). Serial passaging led to loss of the plasmid, while the IS element
of the plasmid remained in the bacterial DNA and disrupted the expression of the
targeted bacterial gene. As the plasmid IS element occurs naturally in dairy bacteria,
phage resistance can thus be achieved by self-cloning. However, none of these
approaches in S. thermophilus were introduced into industrial practice. European,
in contrast to US, legislation requires the labelling of starters as GMO (genetically
modified organisms) that were modified by self-cloning and thus contain only
species-specific DNA. The persistent scepticism of the European consumer towards
GMO in food production led to a substantial reduction of research activity in the
dairy sector, both in industry and in EU-funded public research. Nevertheless, the
search for practical solutions to the industrial phage problems made dairy phages
one of the best-investigated phage systems with respect to both phage genomics
analysis and phage ecology. Pertinent ecological features are analysed in the fol-
lowing paragraphs. As this research was conducted in the private sector, only part
of the data has been published.
Two basic kinds of ecological situations can be distinguished in the industrial
food-fermentation environment, as characterized by the yogurt factory and the
cheese factory. Even if the same bacterial starter is used (yogurt and mozzarella
cheese fermented by added S. thermophilus), the two dairy factories differ in several
basic respects. Milk for yogurt production undergoes treatment at 90°C, which kills
all phages (Quiberoni et al., 1999), while raw or pasteurised milk is used in cheese
fermentation. Furthermore, yogurt production is a relatively aseptic process where
the fermented product has minimal exposure to the factory environment. In contrast,
during cheese making the factory experiences a massive daily aerosol contamination
during cheese whey separation (Budde-Niekiel et al., 1985). In Europe, industrial
yogurt factories are generally smaller than cheese factories (about 50,000 vs.
500,000 liters of milk processed daily per factory). Phages are thus seldom seen
in yogurt production, though they may unknowingly be introduced into the factory
either by the starter cells or by interventions that compromise the physical barrier
separating the product from the environment. Phage problems are still sufficiently
frequent to make them the primary source of fermentation failure in yogurt
Phage Ecology 153
production—mostly in the form of fermentation delays or product alterations, but

occasionally also as complete product loss. Cheese factories, in contrast, are char-
acterized by the coexistence of phage in the milk and bacterial starter and thus this
is a constant problem.
Yogurt samples in a factory reporting occasional fermentation delays yielded
phage-positive samples with titres up to 800 pfu/ml. Aerosols containing phage
were the likely vehicle for phage transmission within the factory. In the literature,
two potential sources of phage were identified: raw milk (Bruttin et al., 1997) and
lysogenic starter strains (Heap et al., 1978). These strains spontaneously release
phage that can lead to phage amplification if susceptible starter cells are used in the
same factory. Fermentation becomes prolonged when the phage titres mount beyond
a critical threshold of 1,000 or 10,000 pfu/ml. When a rotation system is used, regular
fluctuations of the phage titres synchronized with the starter strain rotation can be
observed. This situation can be maintained over some time. When the titre of the
phage rises beyond 106 pfu/ml, a fermentation failure is the likely consequence and
cannot be buffered by a rotation system. The milk samples inoculated with the starter
will no longer coagulate and the product is altered in its technological properties or
entirely lost. The production line must then be carefully cleaned to eliminate the
phages. If available, starters insensitive to the phage of the fermentation failure are
sometimes used to prevent a recontamination of the production line by residual
phage in the factory.
Persistent phage infection is frequently observed in cheese factories. In mozza-
rella fermentation using a complex mixture of S. thermophilus starter strains, phage
titres in the cheese whey were normally 105 pfu/ml or higher (peak titres: 107 pfu/ ml)
(Bruttin et al., 1997). All cheese whey samples contained between four and eight
different phage strains. Some phage types were frequently observed and showed
high titres while others were only occasionally seen, at low titres. No apparent
regularities could be deduced from the temporal cycling of the specific phage titres
in the whey samples. In fermentation simulations, cell counts dropped when phage
were added at an moi (multiplicity of infection) of 0.01 and cells were lost and no
coagulation occurred when the cells were infected at a moi of 0.1. At low moi, two
successive waves of phage replication were observed. High yields of progeny phage
were only obtained with cells in active growth. Phage yields dropped by 5 orders
of magnitude when cells were infected in the late logarithmic or stationary phase.
However, when these cells were resuspended in fresh medium and growth resumed,
renewed phage replication was observed, leading to lysis and phage release. In
contrast, infected cells maintained in the stationary phase failed to produce sizable
amounts of progeny phage.
No phage-resistant cells are selected in the factory ecology, since the fermenta-
tion process is restarted each time with a frozen standard starter culture. A repetition
of the classical Delbrüeck-Luria phage challenge experiment (Luria and Delbrück,
1943) yielded only very few outgrowing cells that had lost the capacity to adsorb
the challenge phage. These mutant cells grew poorly, suggesting a metabolic cost
for mutation to phage receptor loss in S. thermophilus (unpublished results).
S. thermophilus is found in raw milk after enrichment techniques (heating
at 60°C) and has never been isolated from any sources not in contact with milk.
In contrast, its closest relative, S. salivarius, is an oral commensal. However, total

bacterial counts in uncontaminated raw milk are relatively low: about 1000 cfu/ml.
It is thus not surprising that S. thermophilus phages can only be isolated from raw
milk in low titers, ranging from undetectable (<10/ml) in most samples to a maxi-
mum of 130 pfu/ml (Bruttin et al., 1997). This poses a dilemma: How can phages
be maintained in the environment when they have only such a small pool of sus-
ceptible cells? The most logical explanations might be lysogeny and wide host range.
However, the analysis of hundreds of S. thermophilus phage isolates from cheese
factories showed very narrow host ranges. All but two phages were only able to
infect the strain on which they were isolated (Bruttin et al., 1997). This pattern of
strain-specificity was also observed in larger industrial strain collections (Le Marrec
et al., 1997). Furthermore, lysogenic strains are rare in industrial strain collections;
only about 1% of strains can be induced by mitomycin C, for example. Southern
hybridization with DNA of the two major classes of S. thermophilus phages con-
firmed a low lysogeny rate. Only a single survey reported a 10% rate of lysogenic
cells by hybridization (Fayard et al., 1993). In addition, less than one per cent of
S. thermophilus phages from major strain collections are temperate phages (Brüssow
et al. 1994; Le Marrec et al., 1997; Lucchini et al., 1999b). However, the genome
maps of the major virulent S. thermophilus phages betray their origin from temperate
parental phages (Lucchini et al., 1999a; Bruttin and Brüssow, 1996). The prepon-
derance of virulent phages in our collections might therefore represent an adaptation
to the abundance of host cells in the dairy environment. In fact, serial passage of a
temperate S. thermophilus phage quickly resulted even in the laboratory in its replace-
ment by a virulent derivative deletion mutant. The rare raw milk S. thermophilus
phages are nevertheless the source of phage contaminations in the cheese factory.
In a large intervention trial, one starter combination was replaced by a second that
was insensitive to the resident phages of the factory. The intervention resulted in a
nearly immediate disappearance of the resident phages: 70% of the milk samples
lacked any phages and 30% contained phages detectable only on the old starters
(apparently a washout from the previous high phage contamination level since the
titres were low). However, by 5–7 days after the intervention the first three phages
infecting the new starters were detected. Restriction analysis of the phage DNA
traced the origin of the new phages to the rare raw milk samples delivered to the
factory during the intervention period.
Cheese factories using Lactococcus lactis as a starter frequently yielded high
levels of phages; one study reported up to 109 pfu/ml whey without great fluctuation.
Phages were ubiquitous in the factory, and up to 105 pfu/m3 phages were detected
in the factory air (Neve et al., 1994). Like some streptococcal phages, lactococcal
phages were reported to survive pasteurisation. L. lactis strains were frequently
detected in the raw milk while lactococcal phages were only a rare observation.
Many lactococcal phages showed very restricted host ranges, limited to one or a
few starter strains within the L. lactis species. A clear difference from S. thermophilus
phages is the much greater morphological and genetic variability of lactococcal
phages; in some (unpublished) surveys, shifts in the predominant morphological
types of phages were observed in the whey samples that correlated with the change
of the starter culture.
Phage Ecology 155
6.2. NON-DAIRY FOOD FERMENTATION:

SAUERKRAUT AND SAUSAGE
Like most vegetable fermentations, sauerkraut fermentation is spontaneous and relies
on bacterial epiphytes present on cabbage. Food ecological studies demonstrated a
succession of two groups of lactic acid bacteria. In the initial heterofermentative
stage, Leuconostoc species dominate the fermentation. When the pH decreases, they
are followed by the more acid-tolerant Lactobacillus species; L. plantarum eventu-
ally becomes the most abundant species. An ecological survey also demonstrated a
succession of two phage populations corresponding to the replacement of Leuconostoc
by Lactobacilli (Yoon et al., 2002; Barrangou et al., 2002; Marchesini et al., 1992;
Lu et al., 2003b). The Leuconostoc phages represented a number of distinct Sipho-
and Myoviridae, while the Lactobacillus phages included in addition Podoviridae.
The Myoviridae showed genome sizes in the 40–50-kbp range. The Leuconostoc
phages showed very narrow host ranges, infecting only selected strains of L. fallax
species. In contrast one Lactobacillus phage could infect both of the ecologically-
related species L. brevis and L. plantarum. Leuconostoc and Lactobacillus phages
have been isolated from numerous other spontaneous fermentation processes: coffee,
pickled cucumbers, cereals, and wine (Lu et al., 2003a; Lu and Dahlquist, 1992).
Apparently, phage infections are not limited to food fermentation initiated with
bacterial starter cultures.
Lactobacillus plantarum is also used as an industrial starter in meat fermentation.
Phage infections were documented in salami production, but they were of no indus-
trial impact. After an initial rise, the phage titres dropped and the initial phage-
sensitive starter population was replaced by a phage-insensitive mutant derivative
strain. From meat fermentation, a L. plantarum myovirus with large genome size
was isolated (Chibani-Chennoufi et al., 2004). The 130-kbp genome showed close
sequence similarity with Bacillus phage SPO1 at the protein level in regions encoding
structural and DNA replication proteins. Both have been recently shown to be related
to phages infecting other gram-positive bacteria such as Staphylococcus and Listeria,
defining a broad new genus of Myoviridae, as discussed in Chapter 5. Phage infec-
tions were without consequence in salami fermentation using Staphylococcus car-
nosus starters that do not quickly develop phage resistance. Two reasons were quoted
for the limited impact of phages on meat fermentation: The staphylococcal starter
showed only a modest growth and the solid food matrix prevented the spread of
phage in the food product (Marchesini et al., 1992).
7. OUTLOOK
All in all, recent ecological surveys in a variety of environments have underlined
the notion that we live in a sea of bacteriophages, as they are common biological
parts of the soil we stand on, air we breathe, water we drink, and food we eat. There
is also an increased appreciation that phages play a key role in controlling bacterial
population levels everywhere in the environment, and in the genetic diversification
of bacterial strains and species. These observations clearly have profound basic- and
applied-science implications, leading to important theoretical insights and concepts
for ecology and medicine. With the addition of such new techniques as metagenomic
analyses, it is clear that this field will explode in the next few years, providing many
new insights.
ACKNOWLEDGEMENTS
We express special appreciation to Steve Abedon, Jason Gill, Sandra Chibani-
Chennoufi, Mya Breitbart, Gautam Dutta, Raul Raya, and Burton Guttman.
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fm Page 129 Saturday, October 30, 2004 10:14 AM

130 Bacteriophages: Biology and Applications

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in phage therapy, as discussed in Chapters 13 and 14. The successful application of

132 Bacteriophages: Biology and Applications

2.2. DYNAMIC PHAGE-HOST RELATIONSHIPS

1. An extracellular search that is limited by diffusion rates and thus depen-

Phage Ecology 133

rapidly following productive collision between a phage particle and a

1. Free during the extracellular search,

134 Bacteriophages: Biology and Applications

Phage Ecology 135

2.3. CO-EVOLUTION OF PHAGES AND THEIR HOST BACTERIA

136 Bacteriophages: Biology and Applications

2.4. EFFECTS OF HOST PHYSIOLOGY AND NUTRITIONAL STATUS

Phage Ecology 137

FIGURE 6.1 Ability of bacteriophage T4 to survive in stationary-phase E. coli for extended

Complementary observations have been made in sporulating bacteria, illustrat-

138 Bacteriophages: Biology and Applications

transition from free-floating planktonic to biofilm growth (O’Toole et al., 2000).

Phage Ecology 139

3. MARINE PHAGE ECOLOGY

140 Bacteriophages: Biology and Applications

greatest impact of phage lysis was in oligotrophic environments. Furthermore, the

Phage Ecology 141

3.2. MARINE PHAGE CHARACTERIZATION

142 Bacteriophages: Biology and Applications

3.2.2. Genomic Analysis of Marine Phages

3.3. MARINE PHAGE HOST SPECIFICITY

Phage Ecology 143

bacteria of the natural consortium. However, some observations suggested that

144 Bacteriophages: Biology and Applications

4. SOIL AND PLANT-ASSOCIATED PHAGES

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146 Bacteriophages: Biology and Applications

5.2. ENTERIC PHAGES

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ecology of coliphages or even the ability of coliphages to replicate under anaerobic

Phage Ecology 149

5.3. OTHER PHAGE SITES OF INTEREST: ORAL CAVITY AND VAGINA

150 Bacteriophages: Biology and Applications

6. INDUSTRIAL PHAGE ECOLOGY

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production—mostly in the form of fermentation delays or product alterations, but

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In contrast, its closest relative, S. salivarius, is an oral commensal. However, total

Phage Ecology 155

6.2. NON-DAIRY FOOD FERMENTATION:

156 Bacteriophages: Biology and Applications

Phage Ecology 157

158 Bacteriophages: Biology and Applications

Phage Ecology 159

160 Bacteriophages: Biology and Applications

Phage Ecology 161

162 Bacteriophages: Biology and Applications