Chapter 31
Antimicrobial Phages
David R. Harper1, Malcolm McConville1, Frank J. Anderson2 and Mark C. Enright3
1
AmpliPhi Biosciences Corporation, Sharnbrook, UK, 2Defence Threat Reduction Agency, Ft. Belvoir, VA, USA, 3University of Bath, Bath, UK
DISCOVERY
The first observation to suggest a potential for the use of
bacteriophages for antibacterial applications was reported
by Ernest Hanbury Hankin, although the nature of the
agent was unclear and was to remain so for many years.
In 1896 under the title ‘L’action bactericide des eaux de
la Jumna et du Gange sur le vibrion du cholera’ [1],
Hankin suggested that an antibacterial substance in the
River Ganges in India helped to control the spread of
cholera. Viruses were a new discovery at the time, and
were known only as infectious agents able to pass the fine
porcelain filters, developed to filter out bacteria, known
as Chamberland candles. The Russian scientist Dmitri
Ivanovsky had used these to demonstrate the sub-bacterial
nature of tobacco mosaic virus in 1892, but studies of
viruses were very much in their infancy and the nature
of the infectious agent was entirely unknown.
The next steps were taken independently, approxi-
mately 20 years later, by two workers. First, Frederick
Twort at the University of London reported the isolation
of an agent from Staphylococcus cultures which could
produce clear areas within bacterial colonies and which
could then be passed on to new bacterial cultures. Once
again, the ability of this agent to pass Chamberland filters
was key to its identification [2]. His studies were limited,
both due to funding issues and to the First World War,
during which he served in the Royal Army Medical
Corps. As a result, although Twort made and reported the
first direct observation of such agents, he did not follow
up on his original findings until these had been duplicated
elsewhere [3]. It is often stated that Twort’s finding
remained unknown, though this is difficult to reconcile
with the publication of his results in The Lancet in 1915.
The French Canadian Felix d’Herelle, working inde-
pendently of Twort at the Institut Pasteur in Paris in 1917,
isolated an agent from patients suffering from dysentery.
As with Twort’s work, this destroyed dysentery bacteria
Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00031-7
© 2015 Elsevier Ltd. All rights reserved.
and when passed through those same porcelain filters the
resultant filtrate was capable of killing bacteria [4]. Unlike
Twort, d’Herelle maintained and developed his interest in
his discovery. It was d’Herelle who invented the name bac-
teriophage to describe the new bacteria-destroying agent.
In 1919, d’Herelle extended his work to the investigation
of an epidemic of fowl typhoid, which is caused by
Salmonella gallinarum. His work demonstrated that bacter-
iophages had a role in natural control of the infection, and
also that they could be used to treat the disease. Moving on
from this, d’Herelle treated several patients with dysentery.
Safety was assessed by giving high doses to medical staff
before dosing the patient a somewhat more basic approach
than what is required in drug development today. The results
of these treatments were apparently successful, which led to
great interest in the potential of these agents. It should be
remembered that at this time the best available antibiotics
were compounds such as Salvarsan, an organic arsenic com-
pound. The appearance of a less toxic alternative was thus
well received. Reflecting this, the 1925 novel ‘Arrowsmith’,
written by Sinclair Lewis, was awarded the 1926 Pulitzer
Prize (though Lewis declined the accolade). The story was
based around the use of bacteriophages as antibacterial
agents at the fictional McGurk Institute in New York and on
the similarly fictional Caribbean island of St. Hubert.
Although d’Herelle performed a great deal of the early
work with bacteriophages, he was not the first to publish
on their use as antibacterial agents. That was achieved by
Bruynoghe and Maisin, who in 1921 published a report on
the treatment of six patients with staphylococcal skin dis-
eases [5]. It was shortly after this that d’Herelle published
his book, ‘Le Bactériophage son rôle dans l’immunité’,
which reported his earlier work in detail, and which also
defined much of the basis of modern virology [6].
Following on from these initial successes, matters
became rather more complex, with commercial exploitation
going alongside, and often ahead of, the development of sci-
entific knowledge.
567
568 PART | 5 Antibacterial Agents
FIGURE 31.1 Use of bacteriophages to prevent cholera deaths in Assam, India, 1927 1931. Adapted from reference [7].
EARLY USES
Following their discovery, and given the limited alternatives
available for treating bacterial diseases, bacteriophages
were used to treat a wide variety of infections including
typhoid, cholera and bubonic plague. In one of the largest
studies, Lieutenant-Colonel Morison directed large-scale
work aimed at controlling cholera in two regions of Assam,
India, which is often stated to have treated over a million
patients. However, since the population of the treated area
was around half that number at the time this is difficult to
understand [7]. Whatever the numbers, this was a massive
study and while the controls were not strictly segregated the
effect on cholera in the region where bacteriophages were
made widely available is genuinely striking (Fig. 31.1).
From 1927 onwards, commercial products were offered
for sale by multiple companies including Laboratoire de
Bactériophage/Robert et Carriere in France, Medico-
Biological Laboratories in the United Kingdom, Antipiol
in Germany, Saphal in Switzerland, and Lilly, Parke-
Davis, Squibb and Swan-Myers in the United States.
Unfortunately, all of this work was undertaken with
almost no understanding of what bacteriophages actually
were, let alone of how they worked.
Looking back, d’Herelle’s insistence based on the evi-
dence then available that this new agent was a virus that
infected and killed its bacterial targets seems entirely rea-
sonable. It must be remembered, though, that at that time
there was very limited understanding of what a virus
really was, or even of how genetic information was trans-
mitted. As a result, D’Herelle’s theory remained contro-
versial for many years. Other workers, notably Bordet and
Ciuca in Belgium and Kabeshima in Japan, proposed an
alternative hypothesis, in which ‘the bacteriophage’ was
actually a cascade of lytic enzymes produced by sequen-
tial lysis of its bacterial target, releasing more enzymes
with every cycle. Such theories found some support in
Twort’s work, which left open the possibility that the
activity was enzymic in nature.
There was also considerable controversy over who had
actually discovered what came for some time to be known
as the ‘Twort-d’Herelle Phenomenon’ [3]. Additionally,
d’Herelle’s interpretation of the role of bacteriophages
in bacterial disease was seen as denying the role of the
immune system in resolving disease, thus placing him in
opposition to established (and still current) medical views.
It has been suggested that d’Herelle’s didactic and very
personal insistence on this may have played a significant
role in distancing the use of bacteriophage preparations as
therapeutic antibacterial agents (commonly abbreviated to
‘phage therapy’) from mainstream medical practice [8].
It is often stated that the controversy over the nature of
bacteriophages was finally resolved when, in 1940, Ernst
Ruska used his newly developed electron microscope to
obtain the first direct images of bacteriophages [9], and that
these definitively showed them to be viruses. However,
the year after Ruska obtained these images, a major
and influential report for the US Council on Pharmacy and
Chemistry on ‘The Bacteriophage: Its Nature And Its
Therapeutic Use’ stated that while ‘d’Herelle built his
entire theory of bacteriophage around the concept that it
is a living ultramicrobe, parasitic on bacteria’, the preced-
ing 8 years of work had shown with ‘some finality’ that
‘Phage is a protein of high molecular weight’ [10].

FIGURE 31.2 Advertisement for a bacteriophage therapeutic from the
1920s. Reprinted with the kind permission of Dr James Soothill.
With so little understanding of just what bacterio-
phages were and a similar lack of knowledge of how they
worked, many attempts to use bacteriophage-based medi-
cines were based more on guesswork than on sound
knowledge. A good example of this is seen in an adver-
tisement for ‘Enterofagos polyvalent bacteriophages’, as
supplied by Medico-Biological Laboratories of London
(Fig. 31.2). This lists a wide range of conditions that can
be treated with the product, including herpes (caused by a
virus), urticaria (an immune-mediated condition), and
gallstones (which are crystalline accretions of bile compo-
nents). It is unlikely to say the least that any specifically
antibacterial agent, however polyvalent, could have such
broad activity. This simply reflects an earlier era of medi-
cal advertising, when claims for the benefits of a particu-
lar medicine owed more to what might help to sell the
product than to any genuine medical evidence. It was this
kind of claim that led to the start of today’s tightly regu-
lated systems, notably with the passing of the Food, Drug
and Cosmetic Act in the United States in 1938.
Alongside inflated claims, early uses of bacterio-
phages as therapeutic agents were compromised by poor
understanding of the agents and their use in inappropriate
ways. As well as uses where no bacterial infection was
present, these included a widespread belief that ‘the bac-
teriophage’ was a single activity, capable of destroying
any bacterial target. In fact, bacteriophages are exquisitely
specific, with their activity very often restricted to spe-
cific strains within a single bacterial species. While some
bacteriophages may infect across multiple species, this
often reflects the presence of common, plasmid-borne
receptors [11], or simply the limitations of bacterial tax-
onomy, where definitions of a species can be highly vari-
able. Without knowledge of their highly specific nature, it
would be close to impossible to use them effectively. In
addition, many early preparations were manufactured
Chapter | 31 Antimicrobial Phages 569
with very poor quality control or the use of inappropriate
formulations which could inactivate their contents. There
are anecdotal reports that d’Herelle tested 20 bacterio-
phage preparations and did not find a single active bacte-
riophage in any of them.
One major problem with interpreting the results of
early studies was that most of the preparations used
were relatively impure, with high levels of bacterial mate-
rial remaining. Such material can produce significant
immune-mediated effects, complicating therapeutic out-
comes, and this was cited as a flaw with many early stud-
ies [12]. In this context it is interesting to note that
Delmont Laboratories Staphage Lysate (SPL), often cited
as a bacteriophage therapeutic which was available until
the 1990s, was actually classified by the manufacturer as
an immunotherapeutic, acting by virtue of its content of
bacterial lysate. In their 2000 submission to the US Food
and Drug Administration in respect of this product, the
manufacturer stated ‘The in-house research conducted by
Delmont indicates that SPL has the capacity to stimulate
the production of immunocompetent cells, thereby trig-
gering immune responses that might be useful in treating
certain diseases’.
CONCLUSIONS FROM EARLY WORK
All of the above factors, combined with a then unavoid-
able ignorance of what bacteriophages were and how they
worked, meant that much early work was of dubious
value. An extensive analysis of this work was carried out
by Monroe Eaton and Stanhope Bayne-Jones in 1934 in a
series of papers published in the Journal of the American
Medical Association [12,13]. Their analysis concluded
that these studies provided little convincing evidence for
efficacy, and that the majority of the published work had
a variety of flaws. In particular they noted a lack of sup-
porting data indicating efficacy in animal models, a lack
of adequate controls or patient numbers in human treat-
ments, and the potential for immunological effects caused
by bacterial components present in the crude preparations
in use at the time. As they stated in their final report,
‘The material called bacteriophage is usually a filtrate of
dissolved organisms, containing, in addition to the lytic
principle, antigenic bacterial substances, products of bac-
terial growth and constituents of the culture medium. The
effects of all these components must be taken into consid-
eration whenever therapeutic action is tested’ and that
‘The favourable results reported may have been due to
the specific immunizing action of bacterial proteins in the
material used and to non-specific effects of the broth fil-
trates’. They expressed concerns over whether activity
against bacteria in vitro actually occurred in vivo, and
concluded ‘Only in the treatment of local staphylococcic
infections and perhaps cystitis (due to colon bacilli and
570 PART | 5 Antibacterial Agents
staphylococci) has evidence at all convincing been pre-
sented.’ Overall, their conclusion was that the case had
not been made for the therapeutic use of bacteriophage by
the studies conducted to that time.
Even with the appearance of antibiotics, use of bacter-
iophages continued. The appearance of Prontosil (a sul-
phonamide precursor) in 1932 was followed by a range of
sulphonamide drugs, including sulphanilamide in 1936.
By the time of the Second World War American soldiers
were trained to treat any open wounds with topically
administered ‘sulfa powder’. In contrast, both the German
and the Russian armies used bacteriophage preparations,
in particular to treat dysentery (which was treated with
‘sulfa pills’ by the US military). However, despite its rela-
tively widespread use, the German therapeutic in particu-
lar was often criticized as being ineffective.
With penicillin arriving in the clinic in 1942, followed
by a steady stream of chemical antibiotics, the continuing
controversy over everything from what bacteriophages
were to whether they had any therapeutic value made
their use increasingly questionable. Flaws with much of
the early work with bacteriophages were thrown into
sharp contrast when compared with the development of
antibiotics such as streptomycin, the development of
which effectively defined the modern clinical trial in
1946 [14].
The arrival of safe, effective and properly trialled
chemical antibiotics effectively ended the first bacterio-
phage era. Despite some very careful and promising work
during and after the Second World War, in particular with
typhoid in North America [15,16], the use of bacterio-
phages as therapeutic agents was side-lined in Western
Europe and the United States. Antibiotics came to domi-
nate antibacterial therapy in the West, although some
small-scale applications of phage therapy continued. In
contrast, in the Soviet bloc this remained very much
mainstream medicine.
WORK IN THE EAST
Georgiy Eliava of the Georgian Institute of Microbiology,
had noticed that the water of the Mt’k’vari (in the local
Georgian language, ) River in Tbilisi had a bacte-
ricidal action, mirroring the earlier findings of Hankin
regarding the Ganges in India. Since d’Herelle’s work
had now suggested a method by which such action
occurred, Eliava travelled to Paris where he worked with
d’Herelle on this emerging technology.
d’Herelle had travelled widely, but was encouraged by
Eliava to collaborate with him to establish a centre for
bacteriophage research in the Georgian capital, Tbilisi.
Eliava was successful in persuading the Soviet govern-
ment to provide a large facility on the river Mt’k’vari to
which d’Herelle sent materials and equipment. D’Herelle
himself spent a year working in Tbilisi, and his book ‘The
Bacteriophage and the Phenomenon of Recovery’ was
translated into Russian and Georgian by Eliava. As was
normal with books published in the Soviet Union at that
time it was dedicated to ‘Comrade Stalin’. Despite this
and his leading position in this work, being an intellectual
and travelling to the West brought suspicion in its wake.
In 1937, at the height of the Stalinist purges, Georgiy
Eliava was executed as an ‘Enemy of the People’.
Unsurprisingly, d’Herelle never returned to Georgia, but
left as his legacy and that of Eliava a thriving industry
developing and manufacturing bacteriophage therapeutics
for use across the USSR.
Underlying this was the fact that while antibiotics
were becoming dominant in the West, their availability
and use in the Soviet bloc was much more limited. As a
result, bacteriophages were used, and used widely. From
Tbilisi and other manufacturing centres, bacteriophages
were exported across the Soviet bloc countries, and stud-
ies were carried out in Russia, Poland and elsewhere. The
studies conducted were of course in line with the local
requirements at the time. These were not the controlled
clinical trials that were now becoming common in the
West. Added to this, the reporting of the work was limi-
ted, and was rarely made widely available. At the time it
was also rare for these reports to be translated into lan-
guages suitable to aid their availability overseas.
It cannot be denied that by the standards applied at the
time of this work many patients were treated with suc-
cess. Despite this, evidence of safety and efficacy from
these studies is not in a form that is acceptable to Western
medical regulators. This contrasts sharply with attitudes
in the former Soviet bloc, and with the belief in those
regions where they have been widely used that bacterio-
phage therapeutics are both safe and effective.
BACK IN THE WEST
A decade or two after World War Two, the triumph of
chemical antibiotics in the West was effectively complete.
Despite this, concern was being expressed about the
phenomenon of antibiotic resistance as it had been from
the earliest days of their use. Alexander Fleming, the
discoverer of penicillin, stated in his 1945 Nobel Prize
lecture [15] that ‘there is the danger that the ignorant
man may easily underdose himself and by exposing his
microbes to non-lethal quantities of the drug make
them resistant’. This concern was echoed elsewhere, but
the outlook was still one of optimism that bacterial disease
was defeated. In a good indication of the spirit at the time,
it is widely reported that in 1969 the then US Surgeon
General, William Stewart, said, ‘It is time to close the
book on infectious diseases. The war against pestilence is
over.’ Despite many efforts it has proved impossible to
 Chapter | 31 Antimicrobial Phages 571

identify the source of this quote, and Stewart himself

never admitted saying it [16], but nonetheless it remains a
good indication of the optimism of the time.
It is undeniable that a combination of better sanitation,
hygiene, vaccination and effective drugs has removed
infectious diseases from their former position as the lead-
ing scourge of mankind. But that particular war is by no
means over.

BACTERIOPHAGES AS A SCIENTIFIC TOOL FIGURE 31.3 Morphology of the tailed bacteriophages (Caudovirales).
From the 1940s onwards, bacteriophages become one of
the main tools used in developing the emerging science
of molecular biology, notably in the work of Max
Delbrück’s Phage Group [17]. The relative simplicity of
their bacterial hosts and the ease with which they could
be grown allowed studies using bacteriophages to identify
and explain many of the basic processes of life. As with
d’Herelle laying the basics of virology in the 1920s, this
work provided the tools on which much of our current
understanding of biology is based. Inevitably, as they
were used to clarify the processes of their replication, the
biology of the bacteriophages themselves also became
much more clearly understood.
Examples of findings from this work [18] include:
G The Hershey Chase experiment, that showed that
DNA is the genetic material
G Spelling out of the genetic code
G Demonstration of messenger RNA function
G Significant findings on the mechanisms of the control
of gene expression
G The discovery of genetic recombination
G Use of conditional lethal genes for genetic mapping
G Identification of bacterial restriction and modification
systems
G Identification of virus-induced metabolic pathways
G Understanding of the processes of virus assembly
G The discovery of lysogeny the latency of bacterio- FIGURE 31.4 Functional regions of a tailed bacteriophage
phages within the host genome. (Myoviridae).
As a result of this, by the time interest in bacterio-
phages as therapeutics returned in the 1980s, this was those identified to date fall into the broad category of
based on a level of understanding of their nature and the ‘tailed bacteriophages’, grouped as the virus order
function which those working 50 or 60 years earlier sim- Caudovirales. These all have double-stranded DNA gen-
ply could not have imagined. omes ranging in size from a few tens of thousands to sev-
The fundamental nature of this work with and on bac- eral hundred thousand base pairs. The order Caudovirales
teriophages was recognized in the award of the 1969 contains the families Myoviridae (with long, rigid, con-
Nobel Prize in Physiology or Medicine to Max Delbrück, tractile tails), Podoviridae (with short, non-contractile
Alfred D. Hershey and Salvador E. Luria ‘for their dis- tails) and Siphoviridae (with long, flexible, non-contractile
coveries concerning the replication mechanism and the tails) (Fig. 31.3). The basic functions of the components
genetic structure of viruses’. of a tailed bacteriophage are shown in Figure 31.4. The
It is now clear that there are many different types head contains the genome, the tail fibres mediate binding
of bacteriophage, with both DNA and RNA genomes of to receptor on the surface of the bacterial target, and the
varying size and structure. However, more than 90% of tail is involved in entry of the genome into the host cell
572 PART | 5 Antibacterial Agents
(although this involves different mechanisms for the three
families in the order).
SENSITIVITY OF THE BACTERIAL HOST
One aspect of bacteriophage biology, which is of parti-
cular relevance to phage therapy, is their high level of
specificity. This is still not fully understood although it is
not simply a property of the bacteriophages themselves.
A lot of attention is focused on the presence of the right
receptors at the bacterial surface, and this is of course
essential to allow a bacteriophage to bind to its target, but
this is by no means the whole story. Even once infection
of the host cell has occurred, bacteria have a number of
systems to combat bacteriophage infection. These range
from the restriction/modification systems which were dis-
covered in the 1950s and which underlie much of modern
genetic engineering technology [19], to the much more
recently discovered CRISPR/Cas adaptive response sys-
tems [20]. Both result in degradation of the invading
genome. Such systems have evolved in bacteria at least in
part to deter viral predation. Consequently, evolution of
the infecting viruses has occurred to sidestep these
defences [21], for example by the use of unusual base
sequences to prevent cleavage by restriction enzymes [22].
LYTIC OR LYSOGENIC
An essential part of understanding how bacteriophages rep-
licate was first reported in 1936. While some bacterio-
phages function much as was imagined by the early
workers in the field, infecting and then killing their hosts,
others do not have to destroy their target. Rather, these
bacteriophages can enter one of two basic lifecycles
lytic or lysogenic [23] (Fig. 31.5). In the lytic cycle, infect-
ing bacteriophages destroy (lyse) their bacterial target and
produce hundreds of new, infectious bacteriophages. This
can take as little as 20 minutes. Bacteriophages that can
only lyse their targets are referred to as obligately lytic, or
virulent. However, many bacteriophages can persist in the
FIGURE 31.5 The lytic and
lysogenic life cycles.
host cell, usually integrated into the host bacterial genome,
and persist there for a variable number of bacterial genera-
tions. This is known as a lysogenic infection. When acti-
vated by an appropriate triggering event, the lysogenic
bacteriophage can switch to a lytic infection and kill the
host cell. Bacteriophages that can be either lytic or lyso-
genic are known as temperate bacteriophages. A bacterio-
phage that was obligately lysogenic would of course cease
to be an independent bacteriophage and become a perma-
nent part of the bacterial genome many such remnants
are known to exist.
One significant issue in regard to phage therapy is that
lysogenic bacteriophages can pick up and transfer bacte-
rial DNA from around their insertion site by a process
known as specialized transduction. Uncertainty about
whether a temperate bacteriophage will actually kill its
bacterial target, along with the increased risk of bacterial
gene transfer means that lysogenic bacteriophages are
generally considered to be far less suitable for therapeutic
use. Knowledge of this is an important element in the
selection of a candidate therapeutic bacteriophage.
ANTIBIOTIC RESISTANCE AND THE
BACTERIOPHAGE RENAISSANCE
Resistance to antibiotics had been known about almost
since their first therapeutic use and even before that from
laboratory studies [15]. Despite this, awareness of the lim-
its this might place on the use of antibiotics as drugs was
much slower to emerge. However, by the 1990s, awareness
of the problem was widespread, and in the years that fol-
lowed a problem was to grow into a crisis [24].
The seeds of the on-going revival of interest in bacter-
iophages were sown in the 1980s. William Smith and
Ernest Huggins had conducted a well-controlled series of
experiments on mice and calves, looking at the efficacy
of bacteriophages against experimental infections with the
bacterium Escherichia coli [25 27]. Unlike those that
had received such criticism in the early days of phage
therapy, their experiments were both carefully designed

and meticulously conducted. Their work led to some very
positive conclusions; the title of one of their papers sets
this out very clearly: ‘Successful treatment of experimen-
tal Escherichia coli infections in mice using phage: its
general superiority over antibiotics’ [25]. Unfortunately,
at that time the antibiotic crisis was still forming. In addi-
tion, the veal calves, which formed the basis of their
expanded work, are an economically driven market with
different concerns to those caused by antibiotic-resistant
infections in human patients. As a result of these factors
this work, while recognized as important, did not lead
directly to any commercial development.
By the 1990s, as recognition of need grew and with
Smith and Huggins’ data in hand, there was a revived
interested in the apparent success of bacteriophage thera-
peutics in the Soviet bloc countries. Taking a commercial
lead in this was the US company, Phage Therapeutics,
which tried to access this experience by setting up colla-
borations with Georgian phage researchers. Other compa-
nies worked with groups as far afield as Poland, Moscow
and Bashkortostan. Unfortunately, very little came of this
surge of enthusiasm. Certainly the Eastern European
groups had bacteriophages and a great deal of experience
in their use. However, the requirements of moving
from the procedures, materials and facilities that were
available in Eastern Europe at the time to what would be
needed to satisfy Western regulators such as the US
Food and Drug Administration (FDA) and the European
Medicines Agency (EMA, formerly EMEA) were simply
too challenging to be practical. There were also significant
clashes of attitude between the newly arrived Western
companies and the Eastern researchers. As a result of
these problems, none of these companies was successful
in progressing this technology into clinical trials in the
West at that time. However, matters have now progressed
and in 2009 the food multinational company Nestlé began
a locally regulated trial in Bangladesh comparing Western
and Russian bacteriophage preparations for the treatment
of diarrhoea caused by E. coli, following on from earlier
work carried out in Switzerland [28].
An alternative approach was taken by another group
of companies. These elected to be informed and encour-
aged by the Eastern European work, but to proceed with
these approaches based entirely upon Western technolo-
gies, on occasion with the recruitment of Eastern
European staff. It was this group of companies that was
prominent in carrying out (though not always reporting)
the first Western clinical trials.
REACHING THE CLINIC
The use of bacteriophage therapeutics continues in the
former Soviet bloc countries, and work at a clinic in
Poland is continuing as a locally regulated trial [29].
Chapter | 31 Antimicrobial Phages 573
In addition to this, in recent years a number of bacterio-
phage preparations have been entered into clinical trials in
Western Europe and the USA. Most of these are phase 1
safety trials, and to date only one phase 2 trial of efficacy
has been completed.
While it is of course necessary to treat patients with
the target bacterial infection to evaluate the efficacy of a
drug, for bacteriophage therapeutics this is also true in
most cases for evaluation of safety. With dosing in the
range of nanograms to micrograms in the trials reported
to date, bacteriophage replication is required to generate
the therapeutic dose so that the actual level, which will be
attained in clinical use, is not reached unless such replica-
tion takes place. This is similar to the situation with live
vaccines, where a failure of the small administered dose
to replicate generally prevents any detectable effect. This
means that safety trials in patients lacking the bacterial
host will usually deliver a significantly lower dose than
what would be seen in infected patients. Thus, a key stage
to developing the field as a whole was the completion of
a fully regulated phase 2 clinical trial. The first such trial
was reported in 2009 [30].
Details of the clinical trials undertaken in Western
Europe and the USA are given below.
EBI
The US Dutch company Exponential Biotherapies
Incorporated laid claim to carrying out the first trial con-
forming to current Western standards. The details of this
trial have not been reported, but personal communications
indicate that this was a phase 1 (safety) trial involving the
intravenous administration of 5 3 106 bacteriophages tar-
geting Enterococcus to 12 healthy volunteers. The trial
was carried out in the United Kingdom in 1999. This
work was based on previous studies adapting bacterio-
phages to survive in the blood [31]. However, all of the
administered bacteriophage was cleared by 9 days after
treatment (though it should be noted that this was in the
absence of its bacterial target, so that it could not repli-
cate). This was apparently not a promising enough result
for the work to proceed.
Nestlé
The first published report of a modern clinical trial came
from the food multinational Nestlé. In 2005 they reported
a small phase 1 safety study among Nestlé workers in
Switzerland [28]. Fifteen healthy volunteers received a
single dose containing 1.5 3 105 or 1.5 3 107 bacterio-
phages orally in mineral water. A single type of bacterio-
phage T4, targeting E. coli was used. No adverse events
were attributed to the study product. Bacteriophage was
detected in the stools, indicating that it had successfully
574 PART | 5 Antibacterial Agents
passed through the acidified stomach environment.
Bacteriophage titres recovered from stool provided evi-
dence of bacteriophage replication in vivo, presumably in
E. coli in the normal gut flora.
This is now being followed by a series of trials in
Bangladesh which are still underway at the time of writ-
ing, and which are planned to involve a total of 450
patients treated with rehydration alone, or with rehydra-
tion plus a Russian-sourced bacteriophage mixture, or
rehydration plus a novel proprietary bacteriophage mix-
ture, both mixtures targeting E. coli [32]. These report-
edly incorporate both local phase 1 safety trials and a
larger phase 2 safety and efficacy trial in the target group
of infants with diarrhoea.
Southwest Regional Wound Care Center
Another safety (phase 1) trial was reported by Rhoads
et al. in 2009 from a study carried out in Texas using bac-
teriophages supplied by the US company Intralytix [33].
Forty-two patients with chronic venous leg ulcers were
treated, of whom 39 completed the trial. Patients were
treated with 4 3 109 infectious units of each of eight
bacteriophages once a week for up to 12 weeks, the
mixture containing individual bacteriophages targeting
Pseudomonas aeruginosa, Staphylococcus aureus and E.
coli. Treatment was co-administered with other ulcer ther-
apies and no adverse events were attributed to the study
product. Although this was a phase 1 trial, some monitor-
ing of efficacy was undertaken, but since only a small
minority of patients had the relevant bacterial flora
(Wolcott, personal communication) no evidence of effi-
cacy was obtained.
Belgian Army
A small phase 1 trial carried out by the Belgian military
used a single dose containing 3 3 109 units of each of
three bacteriophages (two targeting P. aeruginosa and
one targeting S. aureus) applied to burn wounds. The trial
has never been formally reported, although a short section
in a paper on the production of the bacteriophage mixture
used refers briefly to the trial, noting that eight patients
had been treated, with no adverse effects reported. The
bacteriophages used were sourced from Georgia, but were
produced locally.
Biocontrol
The first trial of the efficacy of a bacteriophage therapeu-
tic was reported in 2009 [30] targeting chronic, antibiotic-
refractory ear infections in a combined phase 1/2 clinical
trial. This was a double-blind randomized trial, involving
24 patients. Twelve patients were treated with a single
dose containing 1 3 105 infectious units of each of six
bacteriophages targeting P. aeruginosa, and 12 with pla-
cebo. The therapeutic appeared to be safe and well toler-
ated, with no adverse events attributed to the study
product. Improvement was apparent in the majority of test
group patients with apparent resolution of disease in 25%
within 6 weeks, even though patients in this group had
proved refractory to conventional antibiotics and had
been ill for up to 40 years. Bacterial numbers were also
reduced.
Phico
A phase 1 safety trial conducted by the UK company
Phico was not actually a trial of a bacteriophage therapeu-
tic, but rather of a non-replicating, bacteriophage-based
gene vector that carried a gene capable of shutting down
bacterial DNA metabolism. The outcomes of the trial
have not been reported to date.
CLINICAL TRIALS AND THE REGULATORY
AUTHORITIES
It is clear that work in Eastern Europe maintained interest
in the therapeutic use of bacteriophages during the period
since the discovery and exclusive use of antibiotics to
treat infections in the West.
However, despite intense efforts by both Western and
Eastern researchers to bring this technology to clinical
trials in Western Europe and the USA, it became clear
that the materials, techniques and clinical procedures used
in the historical work in these locations differed from cur-
rent Western requirements. It was also clear that it would
require a great deal of expensive and technically demand-
ing work to modify the on-going work to meet current
EU (EMA) or US (FDA) regulatory requirements, and
that even the inclusion of historical data will not be
viewed favourably in submissions to these regulators.
In this context, it is essential to define what will be
required for a bacteriophage therapeutic to progress
through clinical trials that will be acceptable to the EMA
or the FDA. The answer to this is quite simple: the same
as is required for any new medicine. Thus production will
require careful product characterization and compliance
with good manufacturing practice (GMP). Studies must
move from high-quality preclinical studies into well-con-
trolled, adequately powered clinical trials using appropri-
ately defined end points. These must progress from the
small-scale phase 1 or phase 1/2 trials into the larger
phase 3 trials that will provide sound statistical evidence
for safety and efficacy and support applications for mar-
keting approvals. In all cases this will require on-going
interactions with the relevant regulatory bodies and com-
pliance with established processes of drug development.
 Chapter | 31 Antimicrobial Phages 575

There have been a number of attempts to lobby for
modification of the regulatory process for bacteriophages,
based on the accumulated evidence from nearly 100 years
of therapeutic use. However, there are many medicines
with long histories and with expectations of efficacy
based upon apparently successful historical uses, such as
immunotherapy or vaccination. But each new medicine
has had to prove itself through fully regulated clinical
trials before gaining widespread acceptance. The same
needs to be done for bacteriophages.
SUITABILITY OF PHAGE THERAPY FOR
SPECIFIC INFECTIONS
When considering whether an infection may be
suitable for treatment with a bacteriophage therapeutic,
there are a number of issues that need to be considered.
A summary of these is presented in Table 31.1.
If, after considering these, the use of a bacteriophage
therapeutic is found to be appropriate, it is necessary to
consider how to develop this. The three basic approaches
taken are detailed in Table 31.2 and the applicability of
these is under active discussion [34]. Bacteriophage cock-
tails have been used in the majority of trials (including all
efficacy trials) undertaken to date, not least because of
experiences with the use of single antibiotics and the con-
sequent generation of resistance. Cocktails in this setting
are directly analogous to the use of combinations of anti-
biotics to prevent the development of drug-resistant bacte-
ria. With ‘phage banks’, this could allow for ‘personalized
medicine’ with a therapeutic tailored in individual infec-
tions. However, for such uses issues regarding the produc-
tion and approval of acceptable collections and the
definition of suitably predictive in vitro tests remain to be
addressed in detail.
Another relevant consideration is that where very low
concentrations of the bacterial target are present (as, for
example, on a recently cleaned wound surface) it is likely
to be necessary to use much higher levels of bacterio-
phage simply to ensure that bacteriophage and target

TABLE 31.1 Considerations in the Therapeutic Use of Bacteriophages

Issue Explanation Examples

There should be a clear clinical
need
In many instances conventional antibiotics
remain effective, making development of
entirely new agents difficult and often
commercially unviable
Multidrug resistant Gram-negative infections
have few remaining treatment options. Biofilm-
based infections are likely to show increased
resistance to antibiotics. Antibiotic resistance is
increasing worldwide, generating an
increasingly urgent need for new approaches

Pathology should be caused by
one species of bacterium or
possibly by a small number of
species
Bacteriophages are highly specific and usually
multiple bacteriophages are needed even to
target a single species (though exceptions do
exist). The number of bacteriophages needed
to target a polymicrobial infection would be
difficult to manage
Pseudomonas aeruginosa in mid-stage cystic
fibrosis lung and in late stage ear infections is
the dominant infection. By contrast early stage
ear infections are polymicrobial and thus less
suited to bacteriophage treatment

Bacteriophages must be The ease with which specific bacteriophages Isolating bacteriophages that are effective
available or able to be isolated may be isolated for individual bacterial targets against Streptococcus mutans or
is very variable Mycobacterium tuberculosis is difficult

Bacteriophages should be For some bacterial species most Bacteriophage targeting Acinetobacter
broadly active bacteriophages affect only a few strains within baumannii may have a narrow range of
the species or only target bacteria from specific activity
geographical locations

Bacteriophages should be lytic,
not lysogenic
Although lytic bacteriophages are common for
some targets, for others these may be difficult
to find. Some types of bacteriophage (such as
the filamentous Inoviridae) may not produce
lytic effects at all
Bacteriophages reported to date for
Clostridium difficile appear to be lysogenic
rather than lytic; for Staphylococcus aureus
both types are found

Bacteriophages should not be
associated with toxin transfer or
pathogenic effects
Some bacterial species have bacteriophage-
borne toxins, although these are often
associated with very specific types of
bacteriophage
Shiga toxin (dysentery) and cholera toxin
(cholera) are known to be transferred by
specific lysogenic bacteriophages.
Interestingly, these are two of the earliest
infections to be treated using bacteriophage
therapy, with apparent success
576 PART | 5 Antibacterial Agents

TABLE 31.2 Approaches to the Production of Bacteriophage Therapeutics

Approach Explanation Advantages Disadvantages

Superphage Uses a single bacteriophage with Only one type of bacteriophage Difficult to find, resistance can
very broad specificity needed give complete immunity to
therapy

Phage bank Bacteria from patient tested against Proof of efficacy in vitro before use Need for an approved phage bank
a large collection of in vivo of proven safety. Requirement for
bacteriophages, and those that are extrapolation from in vitro to
effective in vitro are used in vivo activity

Phage Uses a mixture of bacteriophages to Required coverage easier to develop, Multiple bacteriophages needed,
cocktail achieve desired specificity can target multiple species, resistance may require adaptation to novel
still likely to leave some bacterial strains
bacteriophages effective

actually encounter each other. One food hygiene product
using live bacteriophages has been sold in bottles each of
which contained twenty trillion bacteriophages. However,
in a clinical setting where an active infection is targeted
the concentration of the bacterial target is likely to be
much higher. This can allow even a few hundred bacterio-
phages (a picogram range dose) to establish a productive
infection and thus to produce therapeutic benefit [35].
This approach, using a low initial dose of bacterio-
phages and relying on amplification in vivo to generate
the therapeutic dose (similar to the use of a live, replicat-
ing vaccine where replication is required for efficacy) is
classed as ‘active’ phage therapy. As noted above, this
can allow dosing in the sub-nanogram range with subse-
quent amplification only where the bacteriophage encoun-
ters its bacterial host, thus greatly reducing concerns over
toxicity of the input dose. An alternative approach of
using swamping numbers of bacteriophages and not rely-
ing on amplification but rather on direct killing from the
input bacteriophage is known as ‘passive’ therapy. At
high enough doses this can be by ‘lysis from without’
where the process of infection itself is lethal to the bacte-
rial host [36]. This has seen much less use in clinical set-
tings, although it would be required if the bacteriophage
used was unable to replicate, as would be the case in
some experimental approaches using genetically engi-
neered bacteriophages.
ADVANTAGES AND DISADVANTAGES
Like all therapeutic approaches, the use of bacteriophages
as antibacterial agents has both advantages and disadvan-
tages. Some of these are summarized in Table 31.3.
One increasing trend in combating bacterial infection
is that of the informed use of highly specific antibiotic
drugs. This actually fits well with the high specificity of
bacteriophage therapeutics. This, along with the low doses
that can be used due to in vivo amplification aids their
safety profile, which from trials to date appears to com-
pare favourably with that of conventional antibiotics.
A potential advantage that bacteriophages have over
conventional antibiotics is in the countering of resistance.
Inevitably, bacteria will adapt to resist any antibacterial
agent, from penicillin to copper. With a chemical anti-
biotic, such resistance is then able to counter any use of
that antibiotic. With bacteriophages, a process of antago-
nistic co-evolution occurs [37]. By this means, as bacteria
evolve resistance, bacteriophages themselves develop
means to counter this by simple Darwinian principles [21].
Those bacteriophages that can grow in the newly resistant
bacteria are able to amplify themselves (and destroy their
bacterial host), while those that cannot perish.
Another potential advantage of bacteriophages is their
ability to destroy bacteria within biofilms. The presence
of bacteria within a biofilm can raise the concentration of
antibiotic required for a significant antibacterial effect to
a level well in excess of that which can be attained in
clinical use [38]. Since they have evolved to infect their
hosts in vivo, bacteriophages have the ability to target
bacteria in this commonly encountered form, which is
reportedly found in the majority of bacterial infections.
Bacteriophages appear to have a number of ways to kill
bacteria within biofilms which are not available to con-
ventional antibiotics. Penetration of antibiotics into a bio-
film is variable. Furthermore, if only a low dose
penetrates into the biofilm, that may even be counterpro-
ductive, failing to kill the bacteria while actively selecting
for resistance. Unlike conventional antibiotics, if bacterio-
phages infect the cells within a biofilm, hundreds of new,
infectious bacteriophages can be released from every
infected cell. Following sequential cycles of infection and
release this can lead to very high levels of localized
antibacterial activity. Bacteriophages also appear to be
able to induce the production of enzymes that can
destroy the biofilm matrix, either by coding for these
directly or by inducing their expression in infected

TABLE 31.3 Advantages and Disadvantages in the Use of Bacteriophages as Antibiotics
Advantages
High specificity
Avoids killing of non-target or ‘friendly’ bacteria
Lack of reported side effects
Due to high specificity and low initial dose
Amplifies in vivo at site of infection
Able to produce therapeutic dose only at infected sites from a very
small input dose, allowing dose-independent administration
Able to counter the development of resistance
Bacteriophages undergo antagonistic co-evolution with their bacterial
targets, countering the development of resistance by a variety of means
Bacteriophages can be grown to high titres in vitro
Facilitates manufacture
Able to target and destroy bacteria in biofilms
By amplification within the biofilm, induction of biofilm dissolution by
infected bacteria, and ability to infect persister cells
Destroy their target bacteria by entirely different routes to existing
antibiotics
Bypassing existing antibiotic resistance mechanisms
Able to complement conventional antibiotics
By opening up biofilms to drugs that would otherwise be ineffective
bacteria. Finally, bacteriophages are able to target the
inactive ‘persister cells’ deep within the biofilms that,
being metabolically inactive, are resistant to antibiotics,
infecting them and lysing them once they activate [39].
One potential disadvantage that has to be considered
results from the simple size of bacteriophages, which are
nucleoprotein complexes, massing from one to hundreds
of megadaltons, and at least 50 megadaltons for the tailed
bacteriophages of the Caudovirales. As a consequence of
this they have very different diffusion properties to small-
molecule antibiotics, and this must be considered in eval-
uating how they should be delivered [40].
It is possible to administer bacteriophages directly
onto body surfaces or to the infected site [30,33,35]. Oral
administration for delivery to the gut has also been
evaluated [30], while the potential for aerosolized
delivery into the lung has also been demonstrated in
experimental models [41,42].
Systemic delivery via the blood is more complex and
challenging. Bacteriophage therapy in Eastern Europe
routinely utilized delivery to body surfaces and oral
administration, but reports of systemic use were much
Chapter | 31 Antimicrobial Phages 577
Disadvantages
High specificity
Need to know the nature of infection and cause of pathology
Delivery of viable agent required
Cold chain likely to be required
Regulatory issues
No bacteriophage therapeutic has yet progressed to market
in the West, so the pathway is as yet undefined at later stages
Potential for gene transfer
Need to avoid lysogenic bacteriophages and confirm lack of
transduction
Requirement to use multiple bacteriophages
Need to assemble multiple agents into a single therapeutic
more limited [43,44]. However, in animal models it has
been shown that bacteriophages can be delivered systemi-
cally and are then able to penetrate barriers within the
body, as exemplified by the successful use of intraperito-
neally administered bacteriophages to control systemic
infections with P. aeruginosa [45 48]. The evaluation of
such delivery methods for therapeutic use is continuing,
underpinned by studies of clinical efficacy where direct
delivery is possible.
A significant issue with the systemic use of bacterio-
phages is their interaction with the various effectors of the
immune system. Bacteriophages, as virus-sized masses of
protein, clearly have the ability to induce specific, adap-
tive immune responses given sufficient time and loading,
but what is not known is how ablative these would be on
any realistic therapeutic use. Delivery of nanogram to
microgram quantities of unadjuvanted protein has a lim-
ited probability in and of itself to generate major immune
response, but the potential for combination of amplified
bacteriophage with immunogenic bacterial components
remains to be clarified. It may be possible to use modifi-
cations such as pegylation [49], which is used to prolong
578 PART | 5 Antibacterial Agents
the life of antibodies in the circulatory system. It is also
possible that such effects will be moderated by the induc-
tion of immune tolerance by exposure to bacteriophages
in the gut content, where they are present at around
107 109 virus particles per millilitre [50].
FORMULATION OF BACTERIOPHAGE
THERAPEUTICS
A variety of formulations have been evaluated in experi-
mental studies. These include the simple solutions used in
most clinical trials to date [28,30,33], as well as attach-
ment to solid matrices and encapsulation for aerosolized
oral delivery [51]. Delivery has traditionally involved
neutralization of stomach acid, though other approaches
such as encapsulation or rectal delivery are possible.
However, it is clear that at least some bacteriophages are
able to survive transit through the un-neutralized acid
environment of the stomach [28] and pass into the gut
even at relatively low doses.
When considering formulation, the dose-independent
nature of a self-amplifying agent must be borne in mind.
The initiation of even a single productive infection can at
least theoretically result in the rapid delivery of a thera-
peutic dose in sites where the host bacterium is available.
Thus it is possible to take the position that the only dose
that is too low is no bacteriophage at all, even though
higher efficacy might result from more substantial deliv-
ery. Trials reported to date have used between 1.5 3 105
and 3.2 3 1010 infectious units in total, with from one to
eight bacteriophage strains administered. Animal studies
have used as few as 400 infectious units [35]. When opti-
mized bacteriophage cultures in vitro can readily produce
1012 PFU per litre, thus simply increasing the input dose
may make more sense than seeking a more efficient but
complex formulation.
THE REGULATORY POSITION
While there are multiple products approved for agricul-
tural or food safety applications [52], as yet there are no
bacteriophage therapeutics approved for human or veteri-
nary therapeutic use in the United States (by the FDA) or
in Western Europe (by the EMA). Until such approvals
are in place there will always be uncertainties regarding
the regulatory path. However, it is undeniable that many
of the accepted ‘facts’ regarding phages have turned out
to be incorrect. Before bacteriophage therapeutics
entered modern clinical trials there was a substantial
body of opinion that held that the regulators would be
unwilling to accept the complexities of biological agents
in such applications. Unsurprisingly, given the very real
successes of live viral vaccines (which are of course live
viruses and unlike bacteriophages, are capable of
growing within the cells of the vaccinated individual) in
controlling and even eliminating human disease, this was
not the case. It is also informative to note the success of
monoclonal antibodies. Like bacteriophages, there are
many billions of potential therapeutics (1031 is an often
quoted number for the global population of bacterio-
phages, but is of necessity a rather broad estimate), each
one of which is a complex biological agent, with applica-
tions deriving from a technology base that was originally
in the public domain. Despite this, the combined market
value for monoclonal antibody drugs is now many tens
of billions of dollars per annum, with high market
growth rates.
Another issue which turned out to be far less signifi-
cant than was anticipated by some was that of the use of
mixtures of bacteriophages known in this context as
‘cocktails’ that may be necessary to achieve useful cov-
erage of the varying strains of the bacterial target. It had
been suggested that the regulators would be reluctant to
accept such mixtures. Again, by analogy with live viral
vaccines such as the trivalent polio vaccine or the com-
bined measles-mumps-rubella-varicella vaccine (MMRV),
it is unsurprising that this proved not to be the case, with
the majority of trials to date using such cocktails in both
the EMA and the FDA jurisdictions.
It is the progression of bacteriophage therapeutics
through careful product characterization and well-
controlled clinical trials, and from there onto the market
that all questions can be answered. This process is now
well under way and should, once completed, produce a
much needed and powerful weapon against bacterial dis-
ease at a time when the need has rarely been greater.
PHAGE GENOMICS
Bacteriophage genomes were the first to be sequenced.
The sequence of the single-stranded 3569 base MS2 geno-
mic RNA was reported in 1976 [53] followed by that of
the single-stranded 5375 base ΦX174 genomic DNA [54].
Sequencing capability has moved on considerably since
that time. Ten years later, a 125 000 base pair double-
stranded DNA genome took several years to sequence.
With pyrosequencing and the even faster approaches now
available a genome of the same size can easily be
sequenced in a single day.
In determining the suitability of bacteriophages for
therapeutic use, a genome sequence has always been
regarded as the gold standard in determining lysogenic
potential (by the presence or absence of the relevant genes
and control sequences) and confirming the absence of
toxin genes and bacterial DNA. With costs for the
sequencing of a whole genome by commercial organiza-
tions now down to approximately $1000 (and likely to
drop further in the near future), there is little reason not to

sequence the genome of any interesting bacteriophage at
an early stage in preclinical development.
While no specific measure is available to quantify the
increasing number of unpublished sequences, an NIH
Entrez search of bacteriophage genomes present in two
major databases (GenBank and RefSeq) showed 582 cur-
rently accessible sequences, though this number is likely
to be a significant underestimation, since Entrez is not
automatically updated for new submissions [55]. The
European ACLAME database lists 463 characterized
bacteriophage genomes [56]. The SEED, a project of
the PHANTOME.org and the Fellowship for the
Interpretation of Genomes, provides an alternative data-
base of 660 genomes, in various states of protein anno-
tation [57]. Overlap between the three databases is
significant, but many genomes present in one database are
not present in the others. Still, GenBank is the standard
reference for all published genomes, in that the accession
number format is ubiquitous and easily parsed by most
software. All other databases provide cross-references to
GenBank when available.
GENOMIC SIZE AND PROTEIN CODING
POTENTIAL
Among the SEED’s database of bacteriophages, the
genomic size varies between 2.5 kb (Pseudomonas phage
phi2954) and 316 kb (Pseudomonas phage 201phi2-1).
They encode, respectively, 4 and 461 proteins. The larg-
est currently known bacteriophage genome, that of
Bacillus phage G, is estimated at 498 kb, somewhat
smaller than early estimates of 670 kb. However, it
remains the second largest virus genome sequenced to
date. Comparisons of results from other sites indicate
these are the rough upper and lower limits for genome
size among bacteriophages.
The shortest genomes tend to be single-stranded RNA,
with just a handful of protein-coding genes. One particu-
larly small genome, that of Enterobacteria phage GA
(a member of the Leviviridae, the same family as MS2),
contains just three genes: a coat protein, a maturation
protein and a replicase protein. The largest genomes, like
that of 201phi2-1, contain dozens of tRNA genes, as well
as sophisticated regulatory networks, with time-regulated
transcriptional phases and advanced scaffolding genes to
increase the speed of procapsid assembly. Small genomes
correspond with very simple ssRNA folds and simple
morphology (as in the Leviviridae), while the largest gen-
omes are among members of the Myoviridae with com-
plex tailed virions with dsDNA genomes, along with
significant articulation of the tail, extensively variable tail
fibres for increased adhesion, and membrane-piercing
structures to raise the rate of cellular penetration.
Chapter | 31 Antimicrobial Phages 579
THE PROBLEM OF RE-ANNOTATION
A recognized problem within the genomics community is
the static nature of most GenBank submissions. Annotated
genomes tend to have their original annotations present in
the reference GenBank file, even after subsequent schol-
arly work has demonstrated more nuanced explanations,
more convincing characterizations, and more specific bio-
chemical interactions.
While a mechanism to resubmit genomes is available
through GenBank, it is infrequently utilized for bacterio-
phage genome submission. One causative factor is the
rapid progress of automated annotation software, such as
RAST [58]. While a specifically bacteriophage-oriented
version is still in development (phi-RAST), plasmid-based
methods use ever-improving Hidden Markov Models to
position suggested annotations. New comparative tools
such as the Artemis Comparison Tool [59] and
Phamerator [60] allow comparison of similar bacterio-
phages to help detect cryptic or degenerate genes which
are no longer coding, or non-protein-coding genes like
tRNA genes.
The pace of annotation is ever quickening, resulting in
vastly more accurate and sensitive predictions for newly
characterized phages, while some of the early sequences
languish in obscurity. With no clear solution on the hori-
zon for a more lifecycle-based system of annotation, the
current state can best be viewed as a snapshot of an
evolving process of annotation.
SELECTIVE SCREENING OF CANDIDATE
PHAGES
With respect to the practical considerations raised above,
phage genomics can still very much be considered the
gold standard of certainty where questions of lysogeny
and toxin-generating potential are concerned. Without
recourse to expensive clinical trials, the genome can be
screened for any significant similarity to not just known
toxins and lysogenic factors, but to the intragenic domains
that provide the conformation required for such activity.
The resolution allowed by this approach is superior to
observational studies in terms of economics and the safety
of subjects, although these do retain value alongside more
molecular approaches.
Detailed knowledge of the makeup of common bacte-
riophage regulatory systems continues to grow, with
widely available databases as mentioned previously.
Extremely accurate gene predictions have been made, and
the cross-calibration of similar genomes provides an
enhanced ability to detect not just known problems, but
the likelihood of identifying undesirable traits in unknown
and uncharacterized genomes.
580 PART | 5 Antibacterial Agents
BACK TO THE ROOTS
In an intriguing return to its origins, a series of papers by
Faruque and co-workers published in the Proceedings of
the National Academy of Sciences [61 63] has expanded
our understanding of the role that bacteriophages may
play in the control of cholera epidemics in Bangladesh
(due to political changes in the Indian subcontinent, the
region studied by Morison (see Fig. 31.1) is now split
between India and Bangladesh). The conclusions from
these studies [63] support and expand on those reached
over a hundred years previously by Hankin [1], stating
‘in vivo phage amplification in patients and subsequent
phage predation in the environment may explain the self-
limiting nature of seasonal cholera epidemics in
Bangladesh’. It would seem that what is now under devel-
opment for therapeutic use is already happening in the
wider world, and has been for many years.
REFERENCES
[1] Hankin EH. L’action bactericide des Eaux de la Jumna et du
Gange sur le vibrion du cholera. Annals de L’Institute Pasteur
1896;10:511.
[2] Twort FW. An investigation on the nature of ultramicroscopic
viruses. Lancet 1915:1241.
[3] Duckworth DH. Who discovered bacteriophage? Bacteriol Rev
1976;40:793 802.
[4] D’Herelle F. Sur un microbe invisible antagonistic des bacilles
dysenteriqes. Comptes Rendus Acad Sc Paris 1917;165:373 5.
[5] Bruynoghe R, Maisin J. Essais de therapeutique au moyen du bac-
teriophage du staphylocoque. Comptes Rendus des Séances et
Mémoires de la Société de Biologie 1921;85:1120 1.
[6] d’Herelle F. Le Bacteriophage Son Role dans l’Immunite.
Paris: Masson et Cie; 1922.
[7] Summers WC. Félix D’Herelle and the Origins of Molecular
Biology. New Haven, CT: Yale University Press; 1999.
[8] Fruciano DE, Bourne S. Phage as an antimicrobial agent:
d’Herelle’s heretical theories and their role in the decline of phage
prophylaxis in the West. Can J Infect Dis Med Microbiol
2007;18:19 26.
[9] Ruska E. (1986). Nobel Lecture The development of the elec-
tron microscope and of electron microscopy. Available at:
,http://www.nobelprize.org/nobel_prizes/physics/laureates/1986/
ruska-lecture.html. .
[10] Krueger AP, Scribner EJ. The bacteriophage: Its nature and its
therapeutic use. JAMA 1941;116:2160 7.
[11] Olsen RH, Thomas DD. Characteristics and purification of PRR1,
an RNA phage specific for the broad host range Pseudomonas
R1822 drug resistance plasmid. J Virol 1973;12:1560 7.
[12] Eaton MD, Bayne-Jones S. Bacteriophage therapy: Review of the
principles and results of the use of bacteriophage in the treatment
of infections. JAMA 1934;103:1769 76.
[13] Eaton MD, Bayne-Jones S. Bacteriophage therapy: Review of the
principles and results of the use of bacteriophage in the treatment
of infections. JAMA 1934;103:1847 53.
[14] Streptomycin in Tuberculosis Trials Committee. Streptomycin
treatment of pulmonary tuberculosis. A Medical Research Council
investigation. Br Med J 1948;2:769 82.
[15] Fleming A. In: Nobel Lectures: Physiology or Medicine
1942 1962, Singapore: World Scientific; 1999.
[16] Spellberg B. Dr. William H. Stewart: mistaken or maligned? Clin
Infect Dis 2008;47:294.
[17] Cairns J, Stent GS, Watson JD, editors. Phage and the Origins of
Molecular Biology. New York: CSHL Press; 2007.
[18] Matthews CK. Bacteriophage T4. The Encyclopedia of Life
Sciences (online). Chichester: John Wiley and Sons; 2006.
[19] Bertani G, Weigle JJ. Host controlled variation in bacterial
viruses. J Bacteriol 1953;65:113 21.
[20] Horvath P, Barrangou R. CRISPR/Cas, the immune system of
Bacteria and Archaea. Science 2010;327:167 70.
[21] Stern A, Sorek R. The phage-host arms race: shaping the evolu-
tion of microbes. Bioessays 2011;33:43 51.
[22] Warren RA. Modified bases in bacteriophage DNAs. Annu Rev
Microbiol 1980;34:137 58.
[23] Burnet FM, Lush D. Induced lysogenicity and mutation of bacteri-
ophage within lysogenic bacteria. Aust J Exp Biol Med Sci
1936;14:27 38.
[24] Chan M. Antimicrobial resistance in the European Union and the
World. Keynote address at the Conference on Combating
Antimicrobial Resistance: Time for Action, Copenhagen,
Denmark, 14 March 2012.
[25] Smith HW, Huggins MB. Successful treatment of experimental
Escherichia coli infections in mice using phage: its general superi-
ority over antibiotics. J Gen Microbiol 1982;128:307 18.
[26] Smith HW, Huggins MB. Effectiveness of phages in treating
experimental Escherichia coli diarrhoea in calves, piglets and
lambs. J Gen Microbiol 1983;129:2659 75.
[27] Smith HW, Huggins MB, Shaw KM. The control of experimental
Escherichia coli diarrhoea in calves by means of bacteriophages.
J Gen Microbiol 1987;133:1111 26.
[28] Bruttin A, Brussow H. Human volunteers receiving Escherichia
coli phage T4 orally: a safety test of phage therapy. Antimicrob
Agents Chemother 2005;49:2874 8.
[29] Clinicaltrials.gov. Experimental phage therapy of bacterial infec-
tions, Trial ID NCT00945087; 2013. Available at: ,http://www.
clinicaltrials.gov/ct2/show/NCT00945087. .
[30] Wright A, Hawkins CH, Anggard EE, Harper DR. A controlled
clinical trial of a therapeutic bacteriophage preparation in chronic
otitis due to antibiotic-resistant Pseudomonas aeruginosa; a pre-
liminary report of efficacy. Clin Otolaryngol 2009;34:349 57.
[31] Merril CR, Biswas B, Carlton R, Jensen NC, Creed GJ, Zullo S,
et al. Long-circulating bacteriophage as antibacterial agents. Proc
Natl Acad Sci USA 1996;93:3188 92.
[32] Clinicaltrials.gov. Antibacterial treatment against diarrhea in oral
rehydration solution, Trial ID NCT00937274; 2012. Available at:
,http://www.clinicaltrials.gov/ct2/show/NCT00937274. .
[33] Rhoads DD, Wolcott RD, Kuskowski MA, Wolcott BM, Ward
LS, Sulakvelidze A. Bacteriophage therapy of venous leg ulcers in
humans: results of a phase I safety trial. J Wound Care
2009;18:240 3.
[34] Pirnay JP, De Vos D, Verbeken G, Merabishvili M, Chanishvili N,
Vaneechoutte M, et al. The phage therapy paradigm: pret-a-porter
or sur-mesure? Pharm Res 2011;28:934 7.

[35] Marza JA, Soothill JS, Boydell P, Collyns TA. Multiplication of
therapeutically administered bacteriophages in Pseudomonas aeru-
ginosa infected patients. Burns 2006;32:644 6.
[36] Abedon ST. Lysis from without. Bacteriophage 2011;1:46 9.
[37] Buckling A, Rainey PB. Antagonistic coevolution between a
bacterium and a bacteriophage. Proc Biol Sci 2002;269:931 6.
[38] Moskowitz SM, Foster JM, Emerson J, Burns JL. Clinically
feasible biofilm susceptibility assay for isolates of Pseudomonas
aeruginosa from patients with cystic fibrosis. J Clin Microbiol
2004;42:1915 22.
[39] Pearl S, Gabay C, Kishony R, Oppenheim A, Balaban NQ.
Nongenetic individuality in the host phage interaction. PLoS
Biol 2008;6:e120.
[40] Harper DR, Anderson J, Enright MC. Phage therapy: delivering
on the promise. Ther Deliv 2011;2:935 47.
[41] Carmody LA, Gill JJ, Summer EJ, Sajjan US, Gonzalez CF,
Young RF, et al. Efficacy of bacteriophage therapy in a model of
Burkholderia cenocepacia pulmonary infection. J Infect Dis
2010;201:264 71.
[42] Golshahi L, Lynch KH, Dennis JJ, Finlay WH. In vitro lung delivery
of bacteriophages KS4-M and PhiKZ using dry powder inhalers for
treatment of Burkholderia cepacia complex and Pseudomonas aerugi-
nosa infections in cystic fibrosis. J Appl Microbiol 2011;110:106 17.
[43] Sulakvelidze A, Alavidze Z, Morris Jr JG. Bacteriophage therapy.
Antimicrob Agents Chemother 2001;45:649 59.
[44] Weber-Dabrowska B, Mulczyk M, Gorski A. Bacteriophage ther-
apy of bacterial infections: an update of our institute’s experience.
Arch Immunol Ther Exp (Warsz) 2000;48:547 51.
[45] Soothill JS. Treatment of experimental infections of mice with
bacteriophages. J Med Microbiol 1992;37:258 61.
[46] Wang J, Hu B, Xu M, Yan Q, Liu S, Zhu X, et al. Use of bacterio-
phage in the treatment of experimental animal bacteremia from
imipenem-resistant Pseudomonas aeruginosa. Int J Mol Med
2006;17:309 17.
[47] Watanabe R, Matsumoto T, Sano G, Ishii Y, Tateda K, Sumiyama Y,
et al. Efficacy of bacteriophage therapy against gut-derived sepsis
caused by Pseudomonas aeruginosa in mice. Antimicrob Agents
Chemother 2007;51:446 52.
[48] Vinodkumar CS, Kalsurmath S, Neelagund YF. Utility of lytic bacte-
riophage in the treatment of multidrug-resistant Pseudomonas aerugi-
nosa septicemia in mice. Indian J Pathol Microbiol 2008;51:360 6.
[49] Kim KP, Cha JD, Jang EH, Klumpp J, Hagens S, Hardt WD, et al.
PEGylation of bacteriophages increases blood circulation time and
reduces T-helper type 1 immune response. Microb Biotechnol
2008;1:247 57.
Chapter | 31 Antimicrobial Phages 581
[50] Letarov A, Kulikov E. The bacteriophages in human- and animal
body-associated microbial communities. J Appl Microbiol
2009;107:1 13.
[51] Puapermpoonsiri U, Spencer J, van der Walle CF. A freeze-dried
formulation of bacteriophage encapsulated in biodegradable
microspheres. Eur J Pharm Biopharm 2009;72:26 33.
[52] Harper DR. Biological control by microorganisms. Encyclopedia
of Life Sciences (on line). Chichester: John Wiley & Sons; 2006.
[53] Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D,
Merregaert J, et al. Complete nucleotide sequence of bacterio-
phage MS2 RNA: primary and secondary structure of the replicase
gene. Nature 1976;260:500 7.
[54] Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA,
et al. Nucleotide sequence of bacteriophage φX174 DNA. Nature
1977;265:687 95.
[55] Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL.
GenBank. Nucleic Acids Res 2008;36:D25 30.
[56] Leplae R, Hebrant A, Wodak SJ, Toussaint A. ACLAME:
CLAssification of Mobile genetic Elements. Nucleic Acids Res
2004;32:D45 9.
[57] Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY,
Cohoon M, et al. The subsystems approach to genome annotation
and its use in the project to annotate 1000 genomes. Nucleic
Acids Res 2005;33:5691 702.
[58] Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA,
et al. The RAST Server: rapid annotations using subsystems tech-
nology. BMC Genomics 2008;9:75.
[59] Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell
BG, Parkhill J. ACT: the artemis comparison tool. Bioinformatics
2005;21:3422 3.
[60] Hatfull GF, Cresawn SG, Hendrix RW. Comparative genomics of
the mycobacteriophages: insights into bacteriophage evolution.
Res Microbiol 2008;159:332 9.
[61] Jensen MA, Faruque SM, Mekalanos JJ, Levin BR. Modeling the
role of bacteriophage in the control of cholera outbreaks. Proc
Natl Acad Sci USA 2006;103:4652 7.
[62] Faruque SM, Islam MJ, Ahmad QS, Faruque AS, Sack DA, Nair GB,
et al. Self-limiting nature of seasonal cholera epidemics: Role of
host-mediated amplification of phage. Proc Natl Acad Sci USA
2005;102:6119 24.
[63] Faruque SM, Naser IB, Islam MJ, Faruque AS, Ghosh AN, Nair
GB, et al. Seasonal epidemics of cholera inversely correlate with
the prevalence of environmental cholera phages. Proc Natl Acad
Sci USA 2005;102:1702 7.