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A Century of Bacteriophage Research and Applications: Impact on

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Received: 27 June 2018 Revised: 28 August 2018 Accepted article published: 3 September 2018 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.5810

A century of bacteriophage research and

applications: impacts on biotechnology,
health, ecology and the economy!
Erick J Vandammea* and Kristien Mortelmansb

Abstract
About a century ago, bacteriophages or phages – viruses that infect bacteria – were discovered and reported in the scientific
literature. This review aims at a comprehensive survey of bacteriophage discovery, research and applications since the 1920s,
and its impact on molecular biology, biotechnology, health, ecology and the economy. Phage therapy has been proven a
valuable asset to deal with pathogenic bacterial infections since the early 1920s, and has been practiced ever since, especially
in the former Soviet Union and in Eastern Europe. The Western world remained sceptical and resorted to the widespread use
of antibiotics since the 1940s. Now that antibiotic resistance among pathogenic bacteria has spread alarmingly and few really
novel antibiotic compounds are in the pipeline, renewed attention is being directed to the use of phages as antibacterial agents
in medicine. Because of this renewed interest in phage therapy in the Western world, novel applications with phages are being
pursued in the human health, environmental and the agri-food sectors. This review will focus on (1) the history of early phage
use and its successes and problems, (2) the study of phages as important tools in the development of molecular biology and
biotechnology, (3) current developments in phage research including the use of phage endolysins for use in antibacterial
treatment, (4) phage production systems including undesirable phage contamination of industrial fermentation processes
based on bacteria, (5) recent applications in phage therapy and in phage-based control, and (6) the roles of phages in nature
and in the human gut.
© 2018 Society of Chemical Industry

Keywords: bacteriophages; history of phage research; phage therapy; phage-based control; antibiotic resistance; phage preparations
for medical and agri-food use; endolysins; phage contamination in industrial fermentation processes; gut–skin–oral vaginal–urethral
phage viromes; phage ecology

BRIEF HISTORY OF BACTERIOPHAGE
DISCOVERY AND OF PHAGE THERAPY
PIONEERS
About 100 years ago, the existence of viruses that specifi-
cally infect and destroy bacteria was discovered and reported
in the scientific literature. These viruses are now known as bac-
teriophages, a term coined by Felix d’Herelle (1873–1949).
Bacteriophages, also known as phages or prokaryotic viruses,
infect Eubacteria as well as Archaea.1 The discovery was first
reported in 1915 by the British physician and biomedical scientist
Frederick W. Twort (1877–1950)2 (Fig. 1) and two years later, inde-
pendently, in 1917 by the French-Canadian self-taught biomedical
researcher Felix d’Herelle3 (Fig. 2). Since the 1880s, several papers
in the bacteriological literature had appeared that in hindsight
might have suggested the presence or activity of bacteriophages,
but bacterial autolysis, activity of bacteriolysins or bacteriocins
were believed to have acted as bacterial destroyers.4–6 It was in
1915 that Twort, Professor-Superintendent of the Brown Animal
Sanatory Institution in London, UK, reported an unusual glassy
form of spontaneous lysis in colonies of micrococci (Staphylo-
coccus albus), contaminating his agar plates, while studying the
pox vaccinia virus. He could with a bacterium-free filtrate of these
cultures transmit the lytic effect to fresh cultures of micrococci.
He considered the possibilities that the isolated bacteria might
carry an ultra-filterable virus or that an enzyme was involved with
‘the power of growth’. At that time the term virus was generally
used to designate any infectious disease agent, although the term
ultra-filterable virus gradually came into use for certain agents
that (i) could not be observed with the best available microscopes
at the time and (ii) were able to pass through unglazed porcelain
filters (e.g, the agents of tobacco mosaic disease, foot and mouth
disease, yellow fever and rabies).7 Another common property of
such agents was that they behaved as parasites because they
∗ Correspondence to: EJ Vandamme, Department of Biotechnology – Centre
for Industrial Biotechnology and Synthetic Biology, Faculty of Bioscience
Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
E-mail: erick.vandamme@ugent.be
Both authors are native Belgians, with lifetime careers spent in industrial micro-
biology/biotechnology (EJV) and medical microbiology/biotechnology (KM),
respectively, at both sides of the world. They have tried to include unbiased, for-
gotten, early and very recent Belgian scientific contributions to phage research
and applications.
a Department of Biotechnology-Centre for Industrial Biotechnology and Syn-
thetic Biology, Faculty of Bioscience Engineering, Ghent University, Ghent,
Belgium
b Biosciences Division, SRI International (Formerly Stanford Research Institute),
Menlo Park, California, USA

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 www.soci.org
Figure 1. Photo of Frederick Twort taken around 1900. From Wikimedia
Commons, the free media repository. Source: Obituary Notices of Fellows
of the Royal Society, Vol. 7, No. 20, November 1951, pp. 504–517. The file is
lacking author information. Because the work was published abroad before
1978 it is in the Public Domain in copyright terms in the United States.
could only propagate in the presence of a living host organism.
Whether they were organic molecules, such as enzymes, peptides
or toxins, or minuscule microbes was a matter of speculation.
At about the same time, Felix d’Herelle – then working as an
unpaid research assistant at the Institut Pasteur in Paris, France,
and unaware of Twort’s 1915 publication – observed a similar
phenomenon with cultures of the dysentery bacterium Shigella
dysenteriae. He published his findings in 1917 in a French language
article3 , where he used the term ‘bacteriophage’ for the first time
and described a microbe that was (i) antagonistic to bacteria, (ii)
lysed bacteria in liquid cultures and (ii) killed bacteria in discrete
patches when grown as a film layer on the surface of agar plates.
He termed these clearing zones ‘plaques’. In a following article,
he suggested ‘that it might involve a microorganism, belonging to
a new genus for which I propose the term Bacteriophagum, which
reflects its main property, with the germ found in the dysenteric
intestine, being Bacteriophagum intestinale’.8
d’Herelle’s work became widely known, in particular after he
published a monograph in French language in 1921, entitled
Le Bactériophage9 , whereas Twort’s paper remained initially
unnoticed. d’Herelle’s claim that the bacteriophage was a living
microorganism soon met with opposition, initially from a Japanese
colleague, Tamezo Kabeshima, also at the Institut Pasteur in Paris.
More opposition came from two scientists at the Institut Pasteur in
wileyonlinelibrary.com/jctb © 2018 Society of Chemical Industry
EJ Vandamme, K Mortelmans
Figure 2. Photo of Felix d’Herelle taken around 1910. Credit goes to the
Canadian Medical Hall of Fame and Irma Council (the artist). Approval
received 15 January 2018.
Brussels, Belgium, namely Jules Bordet (Fig. 3), Director and 1919
Nobel Prize laureate for Physiology or Medicine that involved
his work in immunology and work on Bordetella pertussis, the
causative agent of whooping cough (named after Bordet), and his
associate microbiologist André Gratia. Bordet proposed that the
causal agent was a normal secretion of bacteria, an endogenous
bacteriolytic enzyme, whose production was under some type of
auto-regulatory control. He also disagreed with d’Herelle‘s claim
that bacteriophages play a crucial role in the recovery of patients
with dysentery. For him, as an immunologist, antibodies and
complement were the foremost important defence factors. These
scientific disagreements escalated into personal friction, such
that in 1921 Bordet and Gratia10–12 rescued Twort’s observation
from oblivion and confronted d’Herelle to defend his position as
the discoverer of the bacteriophage. Another Belgian research
team at the Institut de Bactériologie of the Catholic University of
Leuven nearby, directed by Richard Bruynoghe (Fig. 4), defended
d’Herelle’s view against Bordet’s, challenging his Nobel-authority.
Bruynoghe and his group supported d’Herelle’s notion of an
ultramicroscopic microorganism,13 whereas Bordet found that the
proposal of a complex organism was unnecessary and saw the
bacteriophage as a simple endogenous bacterial enzyme, able
to induce its own secretion and ability to cause lysis of bacteria.
This Bordet–Bruynoghe controversy lingered on until the early
J Chem Technol Biotechnol (2018)
A century of bacteriophage research and applications
Figure 3. Photo of Jules Bordet. Source: Wellcome Library, London. Public
Domain, CC BY 4.0, via Wikimedia Commons. Date and photographer
unknown.
1930s, with Bruynoghe gradually winning the debate. A detailed,
but forgotten, history of the contributions and arguments, pro
and contra (i.e. microbe particle versus enzyme), of these Belgian
pioneers of early phage research has been published recently.14
d’Herelle performed his initial phage therapy experiments in
early 1919, isolating phages from chicken faeces and successfully
treating an outbreak of chicken typhus. This success led him to
switch to trials on humans, including himself. A first patient was
healed of dysentery using phage therapy in August 1919 and oth-
ers followed.17 In the period 1920–1926, d’Herelle had travelled
on and off to South-East Asia, Egypt and India, to study and coun-
teract cholera and plague epidemics, He isolated phages from
plague-infected rats to successfully treat many human plague vic-
tims. For instance, in India he isolated phages from cholera victims
in the slums and dropped phage solutions in the village wells, an
action that drastically lowered the death toll. In 1924 he received
an honorary doctorate of the University of Leiden, The Nether-
lands, as well as the Antonie van Leeuwenhoek medal.
In 1934, while at the Institut Pasteur in Paris, d’Herelle met
with a visitor, the bacteriologist George Eliava (1892–1937). They
became friends and established a joint interest and cooperation
in phage research and therapy. Eliava was the founder in 1923
of the now Eliava Institute of Bacteriophages, Microbiology and
Virology (EIBMV), at the Georgian Academy of Sciences in Tbilisi,
in the former Soviet Republic of Georgia, where phage therapy has
been, and still is being, further developed until today.15,16 d’Herelle
J Chem Technol Biotechnol (2018) © 2018 Society of Chemical Industry
www.soci.org
Figure 4. Photo of Richard Bruynoghe taken around 1945. Source: www
.md.ucl.ac.be. Because the photo was taken before 1978 it is in the Public
Domain in copyright terms in the United States.
visited G. Eliava in Tbilisi for the first time in 1934 and collaborated
intensively with the Eliava Institute. However, G. Eliava came in
conflict with the Soviet regime and was executed in 1937.
Although phage therapy boomed in prewar times (1920–1940)
driven by the military on both sides, it happened that, in an effort
to keep the troops safe, at least from infections, the introduction of
penicillin killed phage therapy in the Western world. During WWII,
d’Herelle was kept under house arrest and wrote his memoirs. He
died rather forgotten in 1949.
PHAGES AS IMPORTANT TOOLS FOR
MOLECULAR BIOLOGY AND BIOTECHNOLOGY
DEVELOPMENT
In the 1930s, novel ultracentrifugation and filtration techniques
were developed that made easier the concentration and purifi-
cation of bacteriophages, and soon their main composition
of protein and nucleic acid was established.18 Two decades had
elapsed between bacteriophage discovery and their visualization
via electron microscopy in the early 1940s, which confirmed their
organization as supramolecular particles rather than an enzyme,
endorsing the viral hypothesis.19–21 Studies on the biology of
phage multiplication started in the late 1930s and 1940s at the
California Institute of Technology in Pasadena, CA, USA, first by
the physical chemist Emory L. Ellis who was soon joined by the
German émigré physicist Max Delbrück (Fig. 5), both being pro-
ponents of bacteriophages as biological entities.22,23 In 1940, this
American Phage Group also attracted the Italian physician Sal-
vador Luria (also shown in Fig. 5), who was trained in radiology. He
initially worked on phages at the University of Rome and moved
in 1938 to the Radium Institute in Paris. In 1940 he moved to the
USA, recruited to work initially at Columbia University in New York
with several radiation biologists. There he soon made contact with
Delbrück and his group. He also collaborated with Alfred Hershey,
a phage physiologist at Washington University in St Louis, MO,
who demonstrated that only the DNA of phages entered the
host bacteria. From 1943 to 1950, Luria was at Indiana University,
Bloomington, and moved in 1950 to the University of Illinois at
Urbana-Champaign. In 1959 he continued his research at the
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 www.soci.org
Figure 5. Photo of both Max Delbrück and Salvador Luria taken in 1969 at the Nobel Prize ceremony. Copyright © The Nobel Foundation 1969. The
complete illustration was provided by Pearson Education, Inc. with Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings.
Massachusetts Institute of Technology (MIT), Cambridge, MA.24
Guided by the organizational talent of Delbrück, this team decided
to focus on the type T1–T7 phages and in 1945 established an
annual phage course and meetings at the Cold Spring Harbor
Laboratories, Long Island, NY. These courses were attended by
students and senior scholars from all over the world and led to the
‘Delbrück School of phage research’. In 1969, Delbrück, Luria and
Hershey (shown in Fig. 6 that also shows Martha Chase, his labo-
ratory associate) received the Nobel Prize for their phage-based
research. The Luria–Delbrück fluctuation experiment was crucial
in this context. All of these events contributed to the foundation
of molecular biology as a novel discipline. In the 1950s virulent
phages were discerned from temperate phages at the Institut Pas-
teur in Paris, and the term prophage was introduced.25 Temperate
refers to a phage being dormantly present within bacteria that are
then in a state of lysogeny without direct lysis or killing effect. This
can transform harmless bacteria into pathogens; the diphtheria
phage is a notorious example of this. The temperate bacterio-
phage lambda, isolated from a particular Escherichia coli strain in
the Lederberg laboratory at the University of Wisconsin, Madison,
WI, became the tool to improve understanding of the physiology
of gene expression and the mechanisms of the lysogeny state
of bacteria (See Figs 7 and 8 for Esther and Joshua Lederberg,
respectively).26 The study of lysogeny with phage lambda led also
to the concept of gene regulation.27 By the 1960s, there was a
growing realization that phages were only one example of several
types of extrachromosomal genetic elements found in bacte-
ria, next to F (fertility) and R (resistance) factors and plasmids.28
Mutants of bacteria and phages could easily be detected by use
of the simple replica plate test.29
Subsequent detailed genetic analysis of phage genes led to
the concept of the triplet nature of the genetic code.30 During
studies on phage host-range mutations in the 1950s, host-induced
modification was observed, too. The host bacteria appeared to
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EJ Vandamme, K Mortelmans
modify the phage so that phages with the incorrect modification
were restricted in their replication in certain strains.31,32 It was only
20 years later that genetic and biochemical studies revealed that
this is caused by certain sequence-specific host endonucleases,
now universally in use as restriction enzymes. The existence and
role of messenger RNA was proven with phage-infected cells.33,34
The discovery of phages with RNA genomes in 196135 and the
experimental proof that these RNAs function as expected gave
strong support to Francis Crick’s statement that became ‘the
central dogma of molecular biology’, namely that information
goes from DNA to RNA to Protein (and never in the opposite
direction, a vision that was challenged in those days by, amongst
other things, the epigenetic phenomenon).
In 1976, Walter Fiers (Fig. 9), founder of the Laboratory of Molec-
ular Biology at Ghent University, Belgium, and his team were the
first to sequence a complete viral genome, namely that of the RNA
bacteriophage MS2.36,37 The complete genome sequencing of the
first DNA phage phi X174 followed soon in 1977 by Nobel laureate
Fred Sanger and his group in the UK.38
Further details about virulent phages versus temperate phages
and the induction of the lytic versus lysogenic cycle in their
host bacteria became elucidated in the following decades. This
research provided a paradigm of the genetic switch and devel-
opmental regulation in bacteria–phage interactions39–44 Phages
are now well-studied mobile and integrative genetic elements
(MIGEs), such as plasmids and transposons, that play a role in the
emergence of pathogenic bacteria. This is initiated by a process
named ‘phage conversion’ whereby phage-encoded virulence
factors convert their bacterial nonvirulent host into a virulent
strain. As a result, these virulent strains can produce potent toxins
such as diphtheria and botulinum toxin, pore-forming lysins and
effector proteins.41
Many phages form stable relationships with their bacterial hosts
and are referred to as lysogenic strains. In such strains lysis
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A century of bacteriophage research and applications www.soci.org

Figure 6. Photo of Alfred Hershey taken in 1969 Nobel Price award ceremony. Copyright © The Nobel Foundation 1969. The complete illustration was
provided by Pearson Education, Inc. with Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings.

Figure 7. Photo of Esther Lederberg in her Stanford Laboratory taken

January 1977. Permission to use: http://www.estherlederberg.com/
ColleaguesIndex.html.
does not occur, allowing for vertical and horizontal transmis-
sion within a bacterial population. Figure 10 illustrates schemat-
ically the pathways that lead to the lytic and lysogenic cycle.
The principles and mechanisms of temperate phage mediated
conversion of nonpathogenic bacteria into pathogenic strains
via expression of phage encoded virulent genes have recently
been extensively reviewed.41,45,46 Also reviewed are specific cases
involving Shiga-toxin-producing E. coli and toxin-producing Vibrio
cholerae.41,47
Especially from the 1980s onwards, basic research on DNA repli-
cation and its regulation (especially with phage lambda) has con-
tributed a lot to the understanding of the basic principles of DNA
replication control not only in phages, but also in bacteria and even
in eukaryotes. Several seminal and review papers48–53 have dealt
with these fundamental phage-related aspects and these will not
Figure 8. Photo of Joshua Lederberg in his laboratory at the University of
Wisconsin taken in October 1958. Source: http://profiles.nml.nih.gov/BB/B/
A/A/W/_bbaaw_.jpg. The work is in the public domain in the United States
under the terms of Title 17, Chapter 1, Section 105 of the US Code.
be detailed here. Also, information on bacteriophage classification
has been described recently19,54 and will deliberately not be cov-
ered here.
All of these achievements point to the important impact of
basic phage research on the development of molecular biol-
ogy and modern biotechnology in general. Now the favour is
returned with novel molecular biotechniques such as clustered
regularly interspaced short palindromic repeats (CRISPR)-Cas9
being used to modify industrial biotechnologically important bac-
terial strains, such as Bacillus subtilis and E. coli, to increase their
phage resistance during large-scale fermentation processes.55–61
Also, viral metagenomics, complete phage genome sequenc-
ing, phage-mediated efficient gene transfer and molecular

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 www.soci.org
Figure 9. Photo of Walter Fiers (center) with his wife Arlette and Erick
Vandamme taken in 1990 in the townhall of Ypres (Ieper), Belgium, (his
birthplace). He was being recognized as a famous Belgian/Ghent University
biotechnologist and bio-engineer by the Royal Flemish Society of Engi-
neers (KVIV), of which he became then an Honorary Member. Erick Van-
damme was his former student and then young professor to give the
laudatio-speech at the event. Photo provided by Erick Vandamme.
characterization of phage lytic enzymes (endolysins) are now rou-
tinely studied in view of improved applications of phages to com-
bat pathogens and spoilage bacteria in the medical and agri-food
sector.62–66
ECOLOGICAL ROLE OF PHAGES AND
RESISTANCE OF BACTERIA TO PHAGES
Bacterial resistance to phages has been observed ever since they
were put to use, and it has been and is often used as a reason
not to pursue phage therapy or phage-based control any further.
This resistance phenomenon was observed during early antibiotic
therapy and even Alexander Fleming, during his Nobel Prize
lecture, warned against the emergence of antibiotic-resistant
strains/mutants, thus instructing that there should be a ‘prudent’
use of the antibiotics.67–69 Nobody would argue with stopping
searching for new antibiotic compounds and not to administer
antibiotics any further in medical settings, with the ever-increasing
spread of bacterial multidrug/antibiotic resistance (MDR). On the
contrary, this search should continue, although big pharma is
no longer much involved in infectious diseases as a therapeutic
focus.70–75
Evidence is accumulating for the coevolution of antibiotic resis-
tance and antibiotic biosynthesis genes in soil bacteria such as
Streptomyces species and similarly for a coevolution of bacteria and
phage populations in nature as well as in experimental batch cul-
tures and in chemostat microcosms.56,62,76–78 In this context it is
essential to understand resistance build-up in function of the bac-
terial composition or sequence and selection of the ‘best practice’
use of single phages or of phage cocktails.17,79,80
Next to developing and applying novel antibiotics and antimi-
crobials as well as antibodies and probiotics, phages have been
shown to really combat pathogenic bacteria and the diseases or
spoilage they cause.81–83 However, bacterial strains can also resist
phage infections and turn into phage-resistant bacteria at any
stage of the phage infection process by different mechanisms
described below.
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EJ Vandamme, K Mortelmans
• Phage adsorption inhibition: adsorption/contact with the phage
particle can be prevented by bacteria via blocking of phage
receptors, mediated by either the presence of inhibitors or syn-
thesis of a protective biofilm.84–86 Biofilm formation by bacte-
ria on human tissues, on medical devices, fomites, on indus-
trial equipment, in water treatment plants and in nature is
indeed a well-known survival strategy to fend off antiseptics,
sanitizers and antimicrobial compounds. However, several stud-
ies indicate that phages can overcome this hurdle by penetrat-
ing through the biofilm and/or by producing lytic enzymes that
degrade the bacterial exopolysaccharide matrix.84,87–93
• Injection blocking of the phage genome: the phage genome is
not able to inject its genetic material into the potential host
bacterium.94
• Restriction modification: bacterial endonucleases recognize and
can destroy the injected foreign phage nucleic acid, although
some phages can evade this mechanism.95
• Abortive infection: this encompasses a range of phage resistance
mechanisms causing phage-infected bacterial cells to die off
before completing the lytic cycle.94
• Action of CRISPRs: these are genetic loci, found in the genome
of most bacteria that function as a prokaryotic immune
system in conferring acquired immunity (CRISPR-Cas inter-
ference) towards exogenous genetic elements like plasmids
and phages.60,96
In phage therapy and phage-based control applications, the
screening for and isolation of new phage mutants/variants from
the environment or specialized niches that are active against resis-
tant bacterial strains that survive a phage attack, is already com-
mon practice. It is rather quick and easy to perform and is routinely
applied.97 Phages have indeed always been universally present in
all environments and ecosystems on Earth. They are now consid-
ered as our most abundant biological entities with an important
ecological role, consisting in fact in the modulation of ecologi-
cal equilibria and the prevention of bacterial strain overgrowth in
nature. These environments encompass soil, oceans and their sed-
iments, river and underground water, plant/decaying plant and
animal biomass, waste streams from agricultural and food indus-
tries, households, human waste, hospitals, schools, airports and
in municipal sewage. In this context, the presence of phages has
recently been reported in 14th century fossilized faecal samples
(coprolites) recovered from excavated latrines in the city of Namur,
Belgium. By using a combination of electron microscopy, high
throughput sequencing and PCR approaches, Christelle Desnues
et al.62 from Aix-Marseille University, Marseille, France, were the
first to analyse this ancient DNA phage metagenome. They iden-
tified various genes coding for toxins and virulence and observed
that the phage DNA sequences are related to those found in con-
temporary stools, indicating that the viral/phage community has
played a fundamental role within the human gastrointestinal tract
for many centuries.66,98
In general, the ecological importance of phages relates to: (i)
prokaryotic evolution by gene transfer, such as transduction and
lysogenic conversion; (ii) controlling and modulating bacterial lev-
els in nature and in the variety of microbiota throughout animals,
including in the gut, the oral cavity, the lungs, the skin and the
vagina; and (iii) their role in recycling nutrients via lysis of bacterial
cells in different ecological niches.62,66,98–103
Tailor-made genetically engineered phages can now be con-
structed, aiming at specific pathogenic or spoilage bacteria. In
addition, their lytic enzymes (endolysins) are being studied at the
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A century of bacteriophage research and applications
Figure 10. Illustration of the lysogenic and lytic cycle of bacteriophages. Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings.
molecular level and as to their potential application in medical set-
tings and in the food sector.63,65,104–107
THE PHAGE PRODUCTION PROCESS BY
SMALL-AND LARGE-SCALE FERMENTATION
Wider application of bacteriophages demands larger scale pro-
duction processes of phages, using strictly controlled fermenta-
tion bioreactors, rather than petri dishes and shake flask cultures.
This controlled production of bacteriophages in large quantities
became even more important due to their increasing use as trac-
ers and for phage typing, for production of peptides and anti-
bodies (referred to as phage display), and for their increasing use
in phage therapy and phage-based control. The importance of
phage display was highlighted by the 3 October 2018 announce-
ment of the Nobel Prize for Chemistry that was awarded to and
shared by the following two laureates: George P. Smith, University
of Missouri, Columbus, OH, USA, and Sir Gregory P. Winter, MRC
Laboratory of Molecular Biology, Cambridge, UK, in recognition of
their pioneering work on phage display of peptides and antibod-
ies. A third laureate also received the 2018 Nobel Prize for Chem-
istry, namely Frances H. Arnold, California Institute of Technol-
ogy, Pasadena, CA for her work on directed evolution of enzymes.
Generally, intact viruses and phages and virus-like particles (VLPs)
are recovered from microbial and cell communities and fermen-
tation broths via physical separation and purification techniques.
These VLP particles consist mainly of the empty viral capsids that
recently have attracted attention as to their potential use as tem-
plates for nanomaterials and as vehicles or carriers for vaccines,
bio-imaging labels or for drug delivery.108–110
Generally, information about key parameters and process vari-
ables in large-scale fermentations, such as temperature and pH
profile, dissolved oxygen levels, carbon dioxide evolution rate,
nutrient medium composition, inoculum size, foaming agent and
vessel pressure, are difficult to find in the research literature and
often represent proprietary knowledge.111 This is all the more
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true in the case of phage production on a larger fermentation
scale.112,113 It is important to mention here that most successes
with medicinal phage therapy were obtained with an individual-
ized phage production procedure, where the causal pathogenic
bacterial strain was first isolated from the patient and then used to
screen for its lytic phage(s). This bacterial strain was then cultured
on a small scale with subsequent infection with the phage(s),
followed by the process of producing a suitable phage prepara-
tion. This successful individual approach does not lend itself to the
need for a large-scale phage production facility. The situation is
quite different when large bacterial infection outbreaks are to be
dealt with.17 In this context, at the Eliava Institute in Tbilisi, Geor-
gia, fermenter vessels up to 500 L volume are in use.114 In order
to produce phages on a larger scale, the pathogenic host bacte-
rial cells must first be cultivated in fermenter vessels under strictly
contained conditions.
This is similar to the technology used for bacterial vaccine
production, but here deliberate contamination with the bacterial
viruses is initiated during the mid-log phase growth of the bacte-
ria. Parameters such as temperature, pH profile, dissolved oxygen
tension and inoculum size are important factors here. Production
of E. coli phages is well-documented and phage titres of >1013
plaque forming units per millilitre (PFU mL−1 ) were obtained.
Aeration rate and timing of addition of the phage inoculum to the
actively growing E. coli cells were found to be critical.112,113,115,116
After the phage replication stage, and resultant lysis of the bac-
terial cells, the phages need to be separated from the cell debris
and purified from the fermentation broth and then further formu-
lated, that includes ability to ‘survive’ during the storage phase.
If cultures are left too long after the phage lysis phase, the cul-
ture broths can become overgrown with resistant bacterial cells,
complicating the further phage recovery steps. This all requires
sophisticated bioprocess technology and downstream processing
facilities.
Phage production and preparation is so far mostly performed
as a rather simple technology and relatively cheap process, and
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the question remains whether drug companies or government
agencies are willing to invest in well-known, but more expensive,
advanced and sophisticated technologies. Indeed, in most cases,
crude phage lysates are used that are obtained after their sepa-
ration from their host bacterial residues by membrane filtration
of the fermentation broth. It would be far better, but much more
expensive, to use purified phage stock preparations to exclude
possible side effects such as immunological reactions caused by
lysed bacterial host or spent broth components, such as those
induced by endotoxins or peptidoglycan fragments. However, no
significant changes in eosinophil blood counts were observed in
patients during phage therapy, which suggests that it rather does
not induce allergic reactions.117 Before it can be approved for the
market, a successful phage therapy product must be safe, repro-
ducibly effective and stable. The overall regulatory approval of a
phage preparation remains a major hurdle in addition to patenting
it as well, because phages are considered neither a chemical nor
a living cell. As of now, no regulatory framework deals with these
aspects adequately.114,118–121
A first official application or (re)implementation step has just
been officially described and adopted by Belgium, whereas sev-
eral neighboring countries actually have the intention to copy
it.122 This pragmatic phage therapy framework centres on the
‘magistral preparation’ of tailor-made phage medicines. In Euro-
pean law, a magistral preparation, called compounded prescrip-
tion drug product in the USA, is defined as ‘any medical product
prepared in a pharmacy in accordance with a medical prescription
for an individual patient’. Magistral preparations are prepared
from their constituent ingredients by a pharmacist for a given
patient according to a prescription by a physician and follow-
ing the technical and scientific standards of the pharmaceutical
practice. The magistral formula is a practical way for a medical
doctor to personalize patient treatments to specific needs and
to make medications available that do not exist commercially.
This pragmatic mechanism is already in use or in demand for
other medicines, such as natural hormone combinations, aller-
gens and rare disease treatments that are not produced commer-
cially because they lack patent protection. As such phage-therapy
treatment is to be seen as a form of applying the principle of
personalized medicine.122 This allows also for ongoing discus-
sions with the authorities to work on a widely adapted phage
therapeutic framework and guidelines enabling better large-scale
study designs, and exchange of genetic resources and phage
strains based on the Nagoya Protocol (NP) and implementation in
practice.123–126
PHAGE THERAPY IN PRACTICE
From first use to successful practice in Eastern Europe
The few bacteriologists involved in early phage research quickly
realized that these bacterial viruses could be successfully applied
in human and animal medicine to combat pathogenic bacteria
and the infectious diseases they may cause. The initial ongo-
ing controversy on the biological nature of phages was soon
overshadowed by their therapeutic potential. Earliest research
on therapeutic use of phages was conducted in 1919 on animals
by d’Herelle himself, with field studies on a poultry epidemic,
namely a fowl/avian typhoid epidemic caused by Salmonella
var. gallinarum.9 He also conducted laboratory studies at the
Institut Pasteur in Paris on human bacillary dysentery caused by
S. dysenteriae. Subsequent animal studies and field trials in rural
areas of France and The Netherlands (on avian typhosis) and in
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Indochina (on bovine hemorrhagic septicemia caused by Pas-
teurella multicoda), indicated that phage therapy really is effective
and could be applied in practice. Studies on humans to test the
safety of phage preparations started also with d’Herelle together
with some of his family members and some of his colleagues. In
these studies Shigella phages were taken orally or via injection as
well, without any ill effects. Also deliberate administration of E. coli
phages or Staphylococcus phage preparations had generally no
major adverse effects on human volunteers. Recently intravenous
phage administration was shown to be effective against a bac-
teremic episode in a patient at the military hospital Queen Astrid
in Brussels, Belgium127 and other similar cases have been recently
described.128 A very first and successful phage therapy treatment,
orally administered as a Shigella-bacteriophage culture, was per-
formed by d’Herelle at the Hôpital des Enfants-Malades in Paris
in August 1919 on five children suffering dysentery. These events
are described in his unpublished memoirs by Hausler in 2006.129
The first recorded therapeutic use was reported by Bruynoghe
and Maisin in 1921130 and was based on a staphylococcal phage
for topical skin treatment of cutaneous boils in a single patient. In
1925, d’Herelle treated patients with bubonic plague successfully
with anti-plague phage in Alexandria, Egypt. He was then invited
to visit India in 1926 to apply and spread his phage therapy to
treat cholera epidemics in the decade to follow. Only in the late
1960s did the World Health Organization (WHO) organize a phage
therapy trial for cholera in Dhaka, Bangladesh, that was based
on international standards. There was a substantial reduction of
excretion of V. cholerae in the stools of patients, but it did not lead
to an overall clinical improvement. The diversity of Vibrio serotypes
and the rapid transit of ingested phages may have limited the
desirable effects.102,131 Faruque et al.132 reported in 2005 on the
self-limitation of seasonal cholera epidemics in Bangladesh and
on the continuous phage/bacterial ‘arms-race’, which makes the
phage therapeutic approach exactly a ‘sustainable antibacterial
treatment approach’. In hindsight, phages have always been
ingested by all of us unintentionally, because they are present, for
example, in our oral cavity, our gut, faecal matter, drinking water,
air and foodstuffs. They are a most important component of the
human virome with an estimated 1015 phage particles populating
the human gut.62,66,98 In nature, their predator–prey relationship
with bacteria has played a key role in balancing bacterial life on
Earth for over 3.5 billion years.133 They are abundantly present on
Earth, at total numbers estimated at >1031 for aquatic environ-
ments, >107 per gram of soil and >108 –1010 phages per millilitre
of sewage.134–138
The pioneering work of d’Herelle at the Institut Pasteur in Paris
that continued at the now EIBMV in Tbilisi, Georgia, led to the
successful application of phage therapy. About 120 scientists
and 700 technicians worked at the Eliava Institute. Between
1930 and 1955 some 800 publications on the subject appeared,
most of them in Russian and Georgian language, making it dif-
ficult to access and interpret these data in the West.139 Until
now, research and applications of phage therapy as well as the
supply of phage preparations are still going on there, more
recently in collaboration with Belgian and Canadian phage
researchers, with their results being published in internationally
peer-reviewed and respected journals.16,114,140,141 Also several
research institutes in the former Soviet Union (Russia) were, and
still are, involved in phage research and applications of phage
therapy.
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A century of bacteriophage research and applications
Georgian and Russian experience with phage therapy
In contrast to the West, this Georgian medicinal phage therapy
has been implemented widely since the 1930s and is still in prac-
tice in Eastern Europe today. Physicians in the Russian and German
armies used phages in WWII to treat their war-wounded soldiers.
They were mainly used in the hospitals for treatment of primary
as well as nosocomial infections and were administered as liquids,
sprays and tablets, and were also in use as vials in medical kits, be
it often in combination with the then known antibacterial com-
pounds. After WWII, the wide availability of antibiotics and their
successful use led to a halt in phage therapy trials in Western
Europe and in the USA, but not in the Soviet Union and Eastern
European countries. Indeed, the development of phage therapy
since the 1930s was left largely practiced in the former Soviet
Union, where a number of phage therapies are at present still
exploited in a clinical setting. Over the years, multidrug-resistant
pathogenic bacterial strains such as Staphylococcus, Proteus, Pseu-
domonas, Acinetobacter and Clostridium, were sent to Tbilisi, where
phages were isolated from different environments such as soil,
hospital sewage and wastewater as well as from patients, in order
to combat these pathogenic strains. Such phage preparations for
therapy or prophylaxis are still distributed in the former Soviet
Union. The Georgian EIBMV is still a renowned phage research
centre and hosts a large collection of about 830 phages for ther-
apy. The Phage Therapy Centre in Tbilisi, a spin-off of the Eliava
Institute, produces three main types of therapeutic phage prepara-
tions, targeted at: (i) acute and chronic infections (e.g. bladder and
ear infections, infected burns and intestinal infections); (ii) infec-
tions where blood circulation is poor (e.g. diabetic foot and bed
sores); and (iii) infections caused by antibiotic-resistant bacteria.16
Phage therapy research and applications in Central and
Western Europe
During the pioneering early years, the medicinal phages and their
preparations were not well characterized. In addition, most exper-
iments were poorly set up, and the successful practice in the East
was not known to the West. However, in the 1950s phage epi-
demiological studies had started at the Ludwik Hirszfeld Institute
of Immunology and Experimental Therapy (HIIET) at the Polish
Academy of Sciences, in Wroclaw, Poland. In 1957 HIIET published
a paper in Polish on phage use and reported in 1967 on phage
use/therapy in prophylaxis of Shigella sonnei.142,143 In fact, phage
therapy had been applied earlier in Poland since 1923 and phages
were applied in Warsaw hospitals during WWII.117,144 Research on,
and application of, phage therapy has continued until today in a
systematically and scientifically well-documented way. HIIET has
provided sound scientific- and application-based understanding
for phage therapy and remains at present an excellent and emi-
nent centre of phage therapy. Over a period of about 50 years,
this institute has published many high-standard phage therapy
research papers and reviews. Their research work as well as clinical
results are published on a regular basis in peer-reviewed journals
and books.117,142,145–148 They also did important research on the
immunological aspects of phage therapy as well as on observa-
tions on potential anti-oncological effects of some phages.149,150
Activities include production of phages and application of phage
therapy, based on a large collection of selected phages and
pathogenic bacterial strains that are tested for their susceptibil-
ity to their phages.151 In addition, HIIET has treated over 2000
patients with bacterial infections resistant to antibiotic treatment
with an overall success rate from 30% to 90%, depending on the
pathogen involved and clinical setting. This is still one of the best
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sets of solidly documented phage therapy research and clinical
data available today directly related to the spread of antibiotic
resistance.117,142,145–148 After accession of Poland to the European
Union in 2004, the rules for conducting phage therapy had to be
adapted to new regulations. HIIET performs phage therapy under
the rules of a therapeutic experiment on the basis of the respective
Polish regulations as well as – more generally – the Declaration of
Helsinki. In this Declaration it is stated that a therapeutic experi-
ment is defined to occur when a physician introduces new or only
partially tested diagnostic, therapeutic or prophylactic methods
for the direct benefit of the person being treated. Such therapeutic
experiments may be implemented only when available treatment
has failed. They presented data indicating that phage therapy is no
longer a treatment of ‘last resort’ but also allows for significant sav-
ings in the overall costs of infection treatments.146 Some of their
phage preparations have been produced by Biomed SA, Krakow,
Poland, under good manufacturing practice (GMP) conditions.117
From the 1980s on, researchers at the Houghton Poultry
Research Station, Institute of Animal Research in the UK, treated
infected animals such as mice, lambs, piglets and calves, under rig-
orous conditions, with an E. coli strain and a specific E. coli phage,
with very positive results.152–155 They focused on a well-known
E. coli strain that was pathogenic to calves, and exploited phage
receptor biology by selecting phages that require the E. coli K1
antigen for infection. Their encouraging results inspired other
groups to apply phage therapy with success to treat infections in
mice, chickens and fish.156–159 However, other early reports dealing
with an attempt to reduce bacterial contamination of beef with
a phage pool160 , and one to prevent rabbit diarrhoea induced by
an enteropathogenic E. coli strain were not successful.161 This may
have been due to phage-resistant bacteria. In 2009, researchers
at Biocontrol Ltd, London, reported, as a first, on a double-blind
placebo-controlled clinical trial in 12 human patients with a bacte-
riophage mixture, targeting chronic (otitis) ear infections caused
by an antibiotic-resistant Pseudomonas aeruginosa. Phage admin-
istration proofed to be safe and resulted in a significant reduction
in clinical symptoms as compared to the placebo-treated group.162
Also the group of Harald Brussöw at Nestlé Research Centre,
Nutrition and Health Department in Lausanne, Switzerland, per-
formed safety tests on phage administration to human volun-
teers with no adverse effects recorded.163 Other rather sporadic
studies on phage therapy has originated from laboratories in
Romania.164–169
WANING INTEREST IN PHAGE THERAPY AND
REGAINING BASIC PHAGE RESEARCH IN THE
WESTERN WORLD
The widespread interest in phage therapy displayed mainly in East-
ern Europe, Poland, UK and Switzerland contrasts with the overall
low interest in the rest of the West. In the Western world, initial
excitement about phage therapy was counteracted by scepticism
and doubts of effectiveness. Furthermore, the phage therapy prin-
ciple was soon marginalized, especially in the West, in the 1940s
by the use of penicillin, earlier discovered by Alexander Fleming
in 1928 in London at Queen Mary’s Hospital. The newly discov-
ered antibiotic was developed into a miracle molecule in the early
1940s by heroic efforts of British scientists at Oxford University,
and by a research and development team at Northern Regional
Research Laboratories (NRRL), United States Department of Agri-
culture (USDA) in Peoria, IL.170 The resulting widespread use of
penicillin during WWII to successfully treat bacterial infections did
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rapidly lead to a lack of nearly all interest in phage therapy, at least
in the USA and the West.133
The subsequent use of other early antibiotics such as strepto-
mycin and tetracycline, introduced in the medical profession since
the 1945s, significantly further reduced Western interest in phage
therapy.
In the 1930s several major American pharmaceutical companies,
such as Eli Lilly and Company, E.R. Squibb, Abbott Laboratories, and
Park-Davis and Company, produced medicines based on phages
that met with varying success. The still poor understanding of
the biological nature of phages was a major concern expressed
in a 1934 report of the USA Council on Pharmacy and Chemistry,
together with the lack of standards for the purity and potency of
phage preparations.171 These USA company efforts on phage ther-
apy were soon phased out following the introduction of penicillin,
streptomycin and other antibiotics.
However, as indicated above, although the scientific and medical
attention for phage therapy diminished in those years, phages and
viruses became from then on favourite objects of basic study for
biochemists, microbiologists, geneticists and molecular biologists.
Early molecular biology research benefited greatly from the tools
that arose from the work of phage biologists.172
THE RATIONALE TO REVISIT PHAGE THERAPY
AND PHAGE-BASED CONTROL
Over the last four to five decades, many pathogenic bacteria that
threaten the health of individuals in developed as well as in under-
developed countries all over the World have become increasingly
resistant to the well-known and widely used broad spectrum
antibiotics. Resistance also has developed to recently introduced
antibiotic compounds, such as imipenem, ceftazidime, linezolid,
daptomycin and synercid.75,173 Also, the occurrence of hospital
acquired nosocomial infections (HAIs) is growing alarmingly high.
This spreading and accelerating emergence of antibiotic resis-
tance was actually observed as soon as antibiotics were introduced
into medical practice over 75 years ago.67 For example, over 30%
of Streptococcus pneumoniae strains have become resistant not
only to penicillin, but also to broad spectrum antibiotics such as
azithromycin, clarithromycin, cefprozil and cefuroxime. The spread
of vancomycin-resistant Enterococcus sp. (VRE) also has evolved
rapidly, even though vancomycin was precisely introduced as
one of the last resort antibiotics. The situation in less developed
countries is even more alarming, with multiple antibiotic-resistant
pathogens and HAIs spreading within these areas and from
there towards more developed countries. The recent emergence
in the West of carbapenemase producing Enterobacteriaceae
(CPE) strains, such as E. coli and Klebsiella pneumoniae, and
multidrug-resistant Mycobacterium tuberculosis, originating from
countries such as India and Pakistan, are but a few examples of
many antibiotic-resistant pathogenic bacteria.174 Other threats
are multiresistant Gram-positive cocci such as methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus
(VRSA) and Clostridium difficile strains, as well as extended spec-
trum beta lactamase (ESBL) producing Gram-negative bacteria
such as E. coli, Acinetobacter baumannii and P. aeruginosa. The use
of a combination of beta lactam antibiotics such as penicillins
and cefalosporins, such as ceftozolane, and monobactams with
beta lactamase inhibitors such as tazobactam, clavulanic acid and
avibactam may currently help to stop these threats, but eventually
resistance will pop up.175 In this context, the launch in 2013 of
the Journal of Global Antimicrobial Resistance (JGAR), now already
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EJ Vandamme, K Mortelmans
in its tenth volume, will enable timely coverage of all aspects of
the latest developments related to the struggle against the global
spread of antibiotic-resistant microbes.
‘The world may soon be faced with previously treatable diseases
that have become untreatable just as in the pre-antibiotic era’.
Incredibly, this warning of a growing menace to all people was
released 15 years ago by a US interagency task force, namely the
Food and Drug Administration (FDA), Centers for Disease Control
and Prevention (CDC) and the National Institutes of Health (NIH).
Sadly, the situation has grown worse ever since. Ironically it was
exactly this spread of antibiotic resistance, especially among
nosocomial bacterial infections, that triggered the revival of
the older principle of phage therapy. Numerous scientifically
sound publications by authoritative scientists and reviews have
appeared over the last 15 years, focusing on different aspects of
this menacing antibiotic resistance and the potential of phage
therapy as an alternative specific and selective antibacterial
weapon.1,40,73,81,114,117,139,176–181 Also, the World Health Organi-
zation recently published a global action plan to cope with
antimicrobial resistance.182,183 It warned that ‘too few antimicro-
bials are in the pipeline to tackle the global crisis of drug resistance,
responsible for the rise of almost untreatable infections around the
world’ and ‘antimicrobial resistance is a global health emergency
that seriously will jeopardize progress in modern medicine’.
This still-ongoing, alarming, spread of antibiotic-resistant bac-
teria, and the scarcity of really novel antibiotic compounds in
the pipeline emphasizes the need for an urgent revisiting of the
potential of phage therapy and phage-based control of undesir-
able microbes by use of their own viruses.75,125,184–186 It is only
recently, with the frightening rise of antibiotic-resistant bacteria,
that researchers in the West, based on and in combination with
the experience gained over decades in the East, have started to
reconsider phage therapy or other nonmedical phage-based con-
trol applications. This applies not only for human, but also for
animal medical treatment, as well as for phage-based control in
agricultural practices, such as crop protection, and in the food and
sanitation sectors. This effort should indeed not be restricted to
the medical field, but it is equally necessary in veterinary practice,
in agriculture and aquaculture practice in general, as well as in
the related food, health and environmental sectors. This is espe-
cially important in crop protection and the agricultural field where
phage-based control applications could reduce and maybe even-
tually replace the use of chemical pesticides. Phage-based prac-
tices also may eliminate the use of feed antibiotic compounds,
some of which have been linked to adverse health effects, spread-
ing of antibiotic resistance to humans and to negative environ-
mental effects.182,183 However, this area urgently demands further
basic and applied phage research so as to avoid similar problems
as is experienced now with the overuse of antibiotics.
REBORN WORLDWIDE AND WESTERN
INTERESTS AND DOUBTS ABOUT PHAGE
THERAPY: ADVANTAGES VERSUS
DISADVANTAGES
Notwithstanding the apparent usefulness and recorded successes
of phage therapy over so many years, its efficacy as an effective
therapy is still questioned, and much more so in the West than
in the East. Due to the calamitous spread of antibiotic-resistant
pathogenic bacterial strains in medical settings and in nature,
the ever-increasing ineffectiveness of antibiotic treatment
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A century of bacteriophage research and applications
to combat once common and easily cured infections also has
intensified research in the field of phage therapy, revealing
benefits to be exploited and dangers still to be tackled.
Phage therapy advantages over antibiotic treatment
• Generally, medicinal phages are far more specific than antibi-
otics in that they have a very narrow host range and kill only
the intended group of bacterial pathogens. Unlike some broad
spectrum antibiotics, phage therapy does not affect bacteria
such as the beneficial commensal and gut bacteria that make up
the human microbiome.187–190 The same is true for ecologically
important bacterial species.
• In this context, because of their very narrow bacterial host cell
specificity, bacteriophages have for a long time also been used
to identify and classify bacteria (so called phage typing) and
as indicators to demonstrate the presence of bacteria that are
difficult to isolate, for instance in sewage, soil and sediments.
This also is the principle that is used to test the sensitivity of
clinical isolates towards a range of phages to be potentially used
in phage therapy. Well-known exceptions to the principle of
phage specificity include phage Felix 01 that is known for its
ability to infect and lyse almost all Salmonella serotypes and
phages including P1 and T4, that propagate on many strains
of E. coli and other Gram-negative hosts.191 To counteract the
lack of phage specificity, a phage cocktail can eventually be
prepared and administered.79,80
• Phages are a self-replicating medicine at the expense of their
bacterial hosts through the lytic cycle. A single initial phage
dose is theoretically sufficient but usually, in practice, it seems
that several applications are best to treat infections successfully.
Phage replication occurs when the host’s replicating machinery
is taken over after entry of the phage DNA or RNA with the
release at the end of the lytic cycle of about 100 phage particles
(burst size), each of which in turn is able to infect a new
host cell. Therefore, an initially small dose of phage – typically
between 106 and 109 PFU mL−1 – is sufficient to stop a bacterial
infection or contamination. Phages stop functioning after all
target bacteria are destroyed and do not harm any further
the desirable microbiota, nor the mammalian cells.172 They
will be excreted in the faeces and/or urine, but in the first
instance eliminated by the reticulo-endothelial system (RES) of
the spleen.
• They can be administered in various ways – topically, orally,
by nebulization/inhalation, directly into body tissues or intra-
venously. In addition, patients that are allergic to antibiotics can
be treated with phages, usually without adverse effects.192
• The production of phages as well as phage dose preparations
is a well- known biological technology that is based on fermen-
tation processes that are quite similar to the production of vac-
cines and a range of fine biochemicals.
• Thanks to the enormous ubiquity and variety of phages in
nature, there is far less danger for resistance development than
with the current antibiotic (mis)use in the medical and agri-
cultural sectors.193 If phage-resistant bacteria develop during
treatment, a screening round for new phages, active against the
resistant strain, can easily and quickly be set up.17
Disadvantages of phage therapy
• Because of the high host specificity of phages, the diagnosis
and identification of the pathogenic bacterial species involved
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in the infection/contamination must be known very pre-
cisely beforehand. Consequently, it takes some time before
one can intervene with the right phage, or phage cocktail
with its lytic spectrum known. However, the emergence of
rapid culture-independent bacterial detection/identification
techniques should circumvent this disadvantage.
• Phages also can act as antigens, especially in the case of sys-
temic infections, and thus may evoke undesired immunological
reactions such as phage inactivation.194 The use of a different
phage type can then in a second instance provide a solution.
• Moreover, phages can be inactivated for instance by leuko-
cytes and other nonspecific blood factors, as well as by gastric
juices and by antiseptics.195 Therefore, the use of alkalinizing
agents such as sodium bicarbonates is introduced as pre-phage
application.196
• Phages also can mutate, encode toxin genes and lysogenize
bacteria. Also, their behavior under strictly anaerobic conditions
(i.e. in the gut) has not been sufficiently studied.41,66,98
• Relatively little is known about the different proteins produced
by phages, including their toxins. At most 70% of the proteome
functions of E. coli phage T4 are known, yet it is one of the
best-studied phages.66
These negative or understudied aspects of phages need to be
urgently addressed in view of solving these lacunae in basic phage
research and leading to improved phage therapy applications.
Revival of phage therapy in the West
The revival of interest in phage therapy in the West has led to the
emergence of several small companies, established since the late
1990s, that specialize mainly in medicinal phage therapy, but also
on phage-based control.197,198
Below are listed some current players.
• Exponential Biotherapeutics (USA) develops mutant phages
that circulate much longer in the human body. They are
referred to as long-circulating phages. With phage therapy
in life-threatening systemic infections the wild-type phages
are normally eliminated by the RES. Such mutant phages
are not inactivated by the RES and still kill the causal bacte-
ria such as vancomycin-resistant (VRE) Enterococcus faecium,
S. pneumoniae and MRSA. Phages against Propionibacterium
acnes, the causative agent of acne, are being studied in this
context as well.
• In collaboration with the Center for Medical Polymers and
Biomaterials of the Georgian Technical University, Phage
International Inc. (USA) has developed an ‘artificial skin’, named
PhagoBioDerm. It is currently under the intellectual prop-
erty and marketing rights of PolymerPharm. It consists of a
biodegradable polymer, impregnated with a mix of phages
in addition to ciprofloxacin, benzocaine and 𝛼-chymotrypsin.
It is targeted towards killing a range of Gram-positive and
Gram-negative bacteria for the healing of skin ulcers and burn
wounds.
• Bohemia Pharmaceuticals Ind. (Czechia) produces ‘Stafal’ (dis-
tributed in Slovakia); it is a registered drug containing anti-staph
phage.
• Pherecydes Pharma is a French company that develops ther-
apeutic lytic phages against E. coli and P. aeruginosa to treat
severely burned patients. A clinical trial, named PhagoBurn, is
coordinated by military hospitals in France and Belgium, and
eight civilian hospitals in France, Belgium and Switzerland.199
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Clean Cells, a French pharmaceutical company is producing the
phages and phage cocktails in line with GMP.
• Another phage therapy company, Intralytix Inc. (USA), together
with PhageBiotech (Israel), is now focusing on phages
that destroy bacteria that are pathogenic to livestock (e.g.
Salmonella spp., E. coli 0:157:H7, Bacillus anthracis, Pasteurella
spp.) and pathogens that contaminate food, feed and pet food
(e.g. Listeria monocytogenes, Salmonella spp., Campylobacter
spp., and E. coli). Their phage cocktail ListShield ™ is approved
to control L. monocytogenes on food contact surfaces.85
• Phi Therapeutics (USA) is specialized in topical probiotic per-
sonal care products. One of their developments relates to the
formulation of a phage, selected from facial comedones and
plated for effectiveness against different inflammatory P. acnes
strains. Using a nonionic gelling agent such as hydroxyethylcel-
lulose, the phage formulation tolerated typical pharmaceutical
preservatives such as potassium sorbate, methyl- and propyl-
paraben very well over several weeks.200
• Also, EBI Food Safety/Micreos BV (The Netherlands) commercial-
izes Listeria-killing Listex P100™ phage preparations. These were
FDA-approved in 2006 for food preservation. The spray-on prod-
uct is intended for use with meat, raw fish, seafood, cheese and
ready-to-eat (RTE) food. The above phage preparations, specific
for Listeria, E. coli and Salmonella have been approved as food
additives, decontaminating agents on food processing surfaces
and are awarded GRAS status by FDA and EPA.201
The following companies are now also active in research and
applications of phage therapy/phage-based control: Biophage
Pharma Inc. (Canada), Hexal Genentech (Germany), Technophage
(Portugal), Novolytix Ltd (UK), Special Phages Services (Australia),
GangaGen (India/USA), and Biomed S.A., Biophage Pharma S.A.
and Proteon Pharmaceuticals in Poland. Additional companies
that are based in the USA are: AmpliPhi Biosciences, EpiBiome
Inc., New Horizon Diagnostics, PhageLux, Contrafect and Phage
International Corporation. There also are many other companies
in Poland.
PHAGE-BASED CONTROL AND APPLICATIONS
IN THE AGRICULTURAL, FOOD AND
ENVIRONMENTAL SECTORS
• Agriculture/crop protection: Apart from medicinal/therapeutical
applications, phage-based control is now also being used in
the agro-food and environmental sectors, to protect food (veg-
etables, fruits, meat, fish), flowers and water from bacterial
spoilage or (phyto)pathogenic contamination.202,203 phages can
be exploited also in agriculture, horticulture and crop pro-
tection, to combat phytopathogenic bacteria such as Xan-
thomonas citri causing citrus canker, Agrobacterium sp. and
Erwinia amylovora causing fire blight of pome fruit, and Ralsto-
nia solanacearum, Xanthomonas sp. and Clavibacter sp. causing
disease in tomato plants.204,205 Phytopathogen-targeted phages
against bacterial rotting diseases in potato, leek, cauliflower and
other crops also are under development. An example is Erwinia
carotovora, a phytopathogen that represents bacterial soft rot
(BSR), that is usually associated with carrots.198,206,207
• Food safety: Phage sprays are under development as a prophy-
lactic treatment of eggs, poultry, meat, fish, cheese and general
food produce, and water supply systems in the food industry
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EJ Vandamme, K Mortelmans
to combat contamination caused by E. coli, Salmonella, She-
wanella, Campylobacter, Listeria and Legionella species.65,85,208
This FDA-approved preventive phage bioprocessing of food is
now gaining increased interest from food microbiologists, as
well as from food technologists.207,209,210
• Farm animal and bee protection: Phage-based control has now
also been introduced on farms and outdoor settings in a pre-
ventative way to restrict animal pathogens from spreading and
causing disease.177,198 Bacteriophages also may help to directly
or indirectly control the bee pathogenic bacteria Paenibacil-
lus larvae or Melissococcus plutonicus, responsible for causing
American or European foulbrood disease in honeybees, by
infecting and killing their larvae and leading to the collapse of
the entire hives. Indirect ‘bystander’ phage-biocontrol is based
on phage-induced bactericidal toxin formation in the beehive
using commensal Brevibacillus laterosporus, that then kills veg-
etative cells of the pathogen, but not the endospores.211–213
• Environmental applications: An example of an environmental
application where phages could potentially be used is to com-
bat the bacterial marine pathogen Thalassomonas loyana, the
causative agent of coral bleaching that is widespread in warm
ocean regions.212,214 Other settings for phage control are treat-
ment of cyanobacterial blooms, biofilm formation and biofoul-
ing of surfaces, and of medical and industrial devices.84,215
An important, challenging and novel application that should be
considered is the use of fungal viruses, referred to as mycoviruses.
They could be harnessed as a biopesticide to combat fungi/yeasts
that affect staple crops such as wheat, barley, sorghum, corn,
potatoes and rice. Mycoviruses, containing double-stranded and
single-stranded RNA, have a persistent intracellular nature. Most
are symptomless (cryptic), whereas some are beneficial in confer-
ring a hypovirulence phenotype, with reduced fungal virulence.
However, thus far relatively little is known about the potential
and practical uses of mycoviruses as biocontrol agents for attack-
ing (phyto)pathogenic fungi that cause crop diseases. Even less is
known about how to apply them as therapeutic agents to treat
animal and human fungal infections. This is another challenge for
microbial biotechnology.216–219
RECENT RESEARCH AND DEVELOPMENTS IN
PHAGE THERAPY AND PHAGE-BASED
CONTROL
Most bacteriophages use endolysins which are lytic enzymes
attacking the bacterial cells from within by hydrolysing the bacte-
rial peptidoglycan layer. In addition, they use a lysin helper-protein
named holin to first penetrate the plasma membrane. These
phage mechanisms lead to the lysis of the host bacteria that
results in their release. Five different groups of phage lysins have
been recognized according to their cleavage site specificity within
the peptidoglycan structure.106,107,220,221 These phage lysins can
be cloned and produced and might be used in a purified form
to kill Gram-positive bacteria in a specific way, with quick lethal
action, being nontoxic and developing no resistance in the aimed
bacteria.12,63,104,177
Combined use with an outer membrane permeabilizer such
as EDTA or linking hydrophobic amino acids makes them active
also towards Gram-negative bacteria, as pioneered by Rob
Lavigne’s group at the Catholic University Leuven, Belgium.51
New Horizon Diagnostics Corporation (USA) also studies
these phage-associated lytic enzymes and has coined them
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A century of bacteriophage research and applications
‘enzybiotics’.222 Construction of lysin-deficient phage mutants has
a potential use against certain bacteria containing intracellular
toxins (i.e. Clostridium difficile toxin A and toxin B), thereby killing
the host, but without liberating the toxin.
Other ongoing experimental developments are the construction
of recombinant therapeutic phages that are highly lytic and engi-
neered for different bacterial cell receptor specificities, or that have
extra genes coding for host lethal toxin proteins to enhance their
killing effect.177 Conversely, lysogenic phages could be genetically
modified so as to improve the degradation of dietary components
in the gut or to contribute to essential nutrients.66 Phages also
have been labelled with photosensitizers to improve their outdoor
bactericidal action.179 However, these approaches raise ethical and
social questions of whether such engineered phages can be intro-
duced in medical and agri-food practice, considered to be con-
ducted in open environments. Such questions feed into the often
vigorous debate about the pros and cons of genetic modification.
This debate is even more relevant to the deliberate introduction of
recombinant phages into contaminated water and food systems
as phage-based control or as biotracers.223
UNDESIRABLE PHAGE CONTAMINATION OF
INDUSTRIAL FERMENTATION PROCESSES
It goes without saying that the occurrence of, in this case,
unintentional phage contamination caused by phage infection
and prophage induction in the fermentation industry needs to be
avoided at all cost. Microbial fermentation processes are playing
a crucial and increasing role in the transition from a fossil-based
economy to a bio-based one.71,111 This phage contamination
phenomenon has been recognized in the fermentation industry
resulting in faulty or sluggish fermentations, especially since the
1960s, and it results in reduced product quality, lower production
capacity and financial loss. Up to 1012 to 1013 PFU mL−1 can be pro-
duced and then unintentionally spread through the fermentation
plant and its up- and downstream facilities.
Undesirable phages would kill off the industrially useful bacterial
strains that are deliberately cultivated in fermentation processes
for the large-scale production of, for instance, antibiotics (Bacillus
spp., Streptomycetes spp.), proteins and peptide-hormones (rec
E. coli, Bacillus spp.), industrial enzymes (Bacillus spp.), organic
acids (lactic, acetic and propionic bacteria), vitamins224 , sol-
vents such as acetone-butanol (Clostridium spp.),201 amino acids
(Corynebacterium spp., Brevibacterium spp.) and many other
bio-based fine and bulk chemicals such as 1,3 -propanediol (E. coli,
K. pneumoniae).60,225,226
In addition, the production of starter cultures needed for var-
ious large-scale produced dairy fermented foods and probiotic
drinks must proceed phage-free, as must the resulting fermented
foods/drinks producing processes.227 A wide range of different
lactic acid bacterial strains such as Lactobacillus spp., Leuconos-
toc spp. and Bifidobacterium spp. are used in this economically
very important food sector to produce a range of yoghurts and
cheeses. Indeed, lytic phage infection and contamination of the
starter cultures or their ensuing fermentation processes can slow
down or arrest the desirable fermentation course, with large eco-
nomic losses as a result.228–230 Also vegetable fermentations like
sauerkraut from cabbage that are based on a correct sequence of
lactic acid bacteria such as Leuconostoc mesenteroides and Lacto-
bacillus plantarum, can suffer from phage infection.231 Vinegar fer-
mentation, based on Gluconobacter strains, is quite susceptible to
phage contamination. Phages at levels of 108 to109 PFU mL−1 have
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www.soci.org
been found in failed fermentations in different European vinegar
plants.232,233 In such industrial fermentation processes, the use of
phage-free and/or phage-resistant strains is essential.55,94,234,235
Originally a range of antiphage chemical agents have been
used to prevent or limit phage accumulation and loss of prod-
uct yield.236–238 Spontaneous mutation to multi-phage resistance
has been successfully used to protect the vancomycin fermen-
tation process with Amycolatopsis orientalis.239,240 Phage-resistant
lactic bacteria production strains241 and spoilage or pathogenic
bacterial strains, such as Listeria monocytogenes242 , important for
the food sector have been studied and developed via classical
strain improvement approaches based on chemical or UV-based
random mutagenesis and subsequent screening. Phage infections
in E. coli fermentations could be minimized by cloning a 4-cutter
restriction system from Haemophilus influenzae.243 Novel molec-
ular biotechniques such as CRISPR-Cas are now being applied to
modify industrially used bacterial strains, such as B. subtilis and
E. coli, to generate phage resistance during large-scale fermen-
tation processes.55–58,60,61,239,244 Strict sterility of equipment, cul-
ture media and phage-free heated air/oxygen/gas supply and ade-
quate filter systems also is of utmost importance.245–247
THE HUMAN GUT BACTERIOPHAGE VIROME,
OR PHAGEOME
Phages are now recognized as the most abundant biological enti-
ties on Earth, with an estimated 1031 phage particles, and they
are present wherever bacteria are found.248 They are represented
by at least 100 million species. The human gut represents one
of the most densely populated microbial communities and is
referred to as the human gut microbiome. The make-up of the
microbiome consists of bacteria, being the most abundant mem-
bers, archaea, protozoa and fungi amounting to microbes. With
the recent advances made in high-throughput deep sequencing
(metagenomics) the discovery was made that in addition to con-
taining human viruses, the gut microbiome also harbours viruses
that infect gut bacteria, now referred to as gut phages. Both bac-
teria and phages represent the most abundant members in the
gut with an estimated 1014 bacteria and 1015 phages. Considering
the high number of phage particles present in the gut, they repre-
sent an important component of the human gut virome that also
includes human viruses. Together they are referred to as the ‘other
microbiome’.
Close association between gut bacteria and phages
As a whole, the human bacterial microbiome and bacteriophage
virome are believed to play an important role in human health
and disease. Because of the versatility of phages they are believed
to have an effect on human health by contributing to, or chang-
ing, the make-up of the resident bacterial microbiome. In healthy
individuals there is a dynamic equilibrium between the micro-
bial and phage members. Gut phages tend to be an aggregate
of unique and shared phages in individuals and encompass tem-
perate phages that exist as prophages, their DNA being inte-
grated in the host chromosome, and lytic phages.249 In addition,
an absence of a balanced microbial ecology leads to a dysbiotic
state that has been correlated with multiple diseases and con-
ditions such as inflammatory bowel disease, obesity, allergic dis-
orders, type 1 diabetes and colorectal cancer in human and ani-
mal models. Evidence for the support for the close association
between phages and their host bacteria has been provided by the
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 www.soci.org
finding that phage DNA is incorporated in the bacterial host
chromosome as ‘clustered regularly interspaced short palindromic
repeats’ (CRISPR) that is used as a form of immunity against super-
infection by closely related phages.250
From marine phage to human gut phages
In 2013, an important finding was made by Dr Forest Rohwer, a
marine biologist at San Diego State University, CA, who was study-
ing phages associated with coral reefs in the Pacific Ocean. His
work led to the discovery that in a marine environment, phages
outnumber bacterial populations by a factor of 10. His laboratory
also discovered how phages interact with coral reefs – they attach
to the mucus layers that are made up of glycoproteins (mucins)
that cover the surface of coral reefs.251 Additional work using
epifluorescence microscopy revealed that the phage-to-bacteria
ratio (PBR) in mucus samples from diverse environments such
as sea anemones, fish and human gum was on average 4.4-fold
higher than respective adjacent environments. Speculation was
then made that phages in the human gut might similarly attach
to the mucus layers that cover the gut epithelial lining. They used
techniques similar to those used to study the interaction of marine
phages. As mentioned above it was the use of metagenomics of
the human microbiome that led to the discovery that, in addition
to containing human viruses, the gut microbiome also harbours
viruses that infect bacteria. The great advantage of using metage-
nomics was that it allowed for the unravelling of the composition
of the gut bacterial microbiota that no longer required the cultur-
ing of the gut bacteria.
Attachment of phages to mucin
A number of in vitro studies were performed to determine if it is
mucus or some other component associated with the surface of
gut epithelial cells that is responsible for phage attachment thus
leading to a more concentrated phage density.251 These studies
involved the use of mucus- and nonmucus-producing tissue cul-
ture cells that were exposed to a mucus-adherent phage T4. In
addition, phage T4 also was plated on agar plates coated with
mucin and other macromolecules such as DNA and protein. For
these agar plate assays, a T4 phage-sensitive E. coli strain was used
that allowed for plaque formation. The results of these studies
indicated that phage T4 adhered more to the mucus-producing
cells which was in contrast when similar experiments were per-
formed with nonmucus-producing tissue culture cells. When using
the plaque assay on agar plates with E. coli the presence of
mucin significantly increased phage adherence. Thus, the con-
clusion was made by the authors that phages adhere to mucin
glycoproteins.
Discovery of Ig-like phage surface proteins
Additional in vitro experiments were performed by the Rohwer
laboratory that revealed that the surface of the T4 phage head
contains some Ig-like proteins that are similar to antibodies and
T-cell receptors. These Ig-like phage surface proteins act as hooks
to mucin. The impetus for this work was a paper by Minot et al.252
that described the presence of phage DNA in stool samples from
12 healthy individuals. The DNA encoded for genes that were
predicted to belong to Ig-superfamily proteins. Homologues of
these Ig-like domains are found in many phages from many
environments, especially those adjacent to mucosal surfaces. Four
of these Ig-like domains are part of the highly antigenic outer
capsid protein (Hoc) of the T4 phage. Experiments performed with
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EJ Vandamme, K Mortelmans
hoc+ and hoc− T4 phages revealed that hoc+ phages adhered to
many different glycans in experiments with mucin coated agar
plates, but showed a preference for the O-linked glycan residues
that are typically found in mucin glycoproteins.
Bacteriophage adherence to mucus immunity model
In 2013, the Rohwer laboratory proposed the ‘bacteriophage
adherence to mucus’ (BAM) immunity model.251 This model is
based on the notion that phages provide a nonhost-derived
antimicrobial defense on the mucosal surfaces of different meta-
zoan hosts. As the mucus layer is considered to be part of the
innate immune system, it also attracts phages and is considered
to provide physical and chemical antimicrobial defenses. A viral
carpet is produced as phages attach to the mucus that prevents
commensal and pathogenic bacteria from crossing the epithelial
mucus layer. This is considered to be a dynamic process as there
is a continuous removal of mucus with adhering phages while
the epithelial cells continuously produce mucus to which phage
progeny and bacteria attach.
Models for phage–bacteria interaction
Several models have been proposed for how gut phages interact
with their bacterial hosts. These are described in detail and are
mentioned briefly below253 (see also98 ):
Model 1: ‘Kill the winner’ believed to operate in environments
with a high bacterial density. Overgrowth by one particular
species will be curtailed by a specific phage so as to return to a
more balanced make-up of the bacterial microbiome.
Model 2: ‘Biological weapon’ or ‘kill the relative’ model refers
to the ability of lysogenic bacteria to induce the lytic cycle.
The progeny thus produced can attack susceptible nonlysogenic
relatives or other competitors.
Model 3: ‘Community shuffling’ model involves environmental
factors that may result in the induction of prophages in lysogenic
bacteria. This is in contrast to rare spontaneous induction events.
Model 4: ‘Phages and emergence of new bacterial model’ which
involves the introduction of foreign bacterial genes in a new host
via transduction.
It is expected that continued research on gut phage–bacteria
interactions will lead to a better insight into what extent the bac-
teriophages play a role in human health and disease. This hope-
fully will lead to the development of novel phage-based or phage
cocktail-based treatments for dysbiosis and other gut-associated
diseases.67,68
BACTERIOPHAGES: DANGEROUS FRIENDS OR
FRIENDLY ENEMIES?
Finally, the question remains whether phages are dangerous
friends or friendly enemies? They can only replicate and survive
at the expense of another living bacterial host cell and do not actu-
ally live. In 1925, the famous Delft University microbiologist Albert
Jan Kluyver stated that bacterial viruses form a shroud of life, refer-
ring to the difficult definition of life.138,254–256 Bacteriophages con-
tain DNA or RNA, not both nucleic acids as living cells do. They lack
their own ATP generation, do not have a cytoplasmic membrane,
and are rather complex macromolecules of nucleic acids and
proteins. They are metabolically inert in their extracellular form,
replicating only after contacting/recognizing and infecting a suit-
able bacterial host cell.
J Chem Technol Biotechnol (2018)
A century of bacteriophage research and applications
Current basic studies of bacteriophages are focused on: (i)
theoretical modelling of the evolution of such replicators
(viruses/phages and related mobile selfish genetic elements
like plasmids) versus reproducers (cells); (ii) understanding the
role of lateral gene transfers; (iii) geochemical analysis; and (iv)
molecular phylogenetics and comparative genomics. These stud-
ies can offer new insights and also controversies, in the cradles of
virus, membrane and cell evolution, and in the universal Tree of
Life. Recent science-based opinions and views now point to the
inclusion of viruses and phages in the Tree of Life as well.62,254,257,258
As target-directed macromolecules, phages offer complemen-
tary potential to the current use of antibiotics, antivirulence
drugs,259 antiseptics, disinfectants, photoantimicrobials (photody-
namic therapy, use of photosensitizers)260 and other antimicrobial
agents such as antibacterial vaccines, immune stimulants, adju-
vants, probiotics and lysozymes. In addition, they can be used in
the medical and veterinary fields, and in the hygiene, sanitation,
food, agricultural and environmental sectors. This potential has so
far been understudied by scientists and neglected by the biotech
and pharma industries.
In view of the rising ineffectiveness of antibiotic treatments
against once readily treatable bacterial infections, and the often
negative health and environmental effects caused by chemical
pesticides in the crop and agricultural sectors, it is long overdue
to revisit phage therapy and nonmedical phage-based control
and phage bioprocessing applications, both from fundamental
and application points of view.17 Considering the potential that
phage-based technologies is offering in the medical, environ-
mental and industrial sectors, it may be argued that in the long
run phages will become neither a dangerous friend nor a friendly
enemy, but a trusted source for applications that will be of benefit
to humankind.
ACKNOWLEDGEMENTS
Author EJV is indebted to:
• Dr R. Miedzybrodzki, Ludwik Hirszfeld Institute of Immunol-
ogy and Experimental Therapy of the Polish Academy of Sci-
ences (HIIET PAS), Wroclaw, Poland, for the kind invitation to
visit the Phage Therapy Unit at the HIIET PAS and fruitful
discussions.
• Prof. D. De Vos, Laboratory for Molecular and Cellular Technol-
ogy (LabMCT), Queen Astrid Military Hospital, Brussels, Belgium,
for his valuable comments, providing recent phage therapy
information and for his critical reading of the overall manuscript.
• Prof. M. Vaneechoutte, Laboratory for Bacteriology Research,
Faculty Medicine & Health Sciences, Ghent University, Ghent,
Belgium, for help with establishing fruitful contacts and
discussions.
• Prof. A. Billiau, Rega Institute, University Leuven, Leuven, Bel-
gium, for providing relevant information on the forgotten
impact of Belgian pioneers on early phage research.
• Med. Dr G. Laire, Vice-Chief Medical Military Staff, Ministry of
Defence, Belgium, for providing relevant contacts and infor-
mation on ongoing phage therapy research projects at the
Laboratory for Molecular and Cellular Technology (LabMCT),
Queen Astrid Military Hospital, Brussels, Belgium, headed by
Prof. Jean-Paul Pirnay.
• Prof. Dr ir. Inge Van Bogaert, Bioport Group, Centre for Synthetic
Biology, Faculty of Bioscience Engineering, Ghent University,
Ghent, Belgium, for help with literature searches.
J Chem Technol Biotechnol (2018) © 2018 Society of Chemical Industry
www.soci.org
Author KM is indebted to
• Ms Caroline Graham for formatting the references according to
JCTB style.
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