Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:6±17 (2000)

Review
Bacteriophages and biotechnology: a review
Trevor Marks* and Richard Sharp
Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 OJG, UK

Abstract: Bacteriophages are viruses whose hosts are bacterial cells. Like all viruses, phages are
metabolically inert in their extra-cellular form, reproducing only after infecting suitable host bacteria.
Discovered over 80 years ago, they have played a key role in the development of modern biotechnology.
Their initial isolation appeared to offer the ®rst therapeutic for the control of infectious disease. The
discovery of antibiotics in the 1940s eclipsed bacteriophage-based therapies although, with the increase
in multiply drug-resistant pathogens, bacteriophages are being re-evaluated as the basis of new
therapeutic strategies. Their de®ned host speci®city facilitated their application in the typing and
identi®cation of a wide range of bacteria. Bacteriophage typing schemes were developed for most
groups of pathogenic of bacteria and more recently their host speci®city has been applied to the
development of bacterial detection and diagnostic strategies. The advance in molecular biology over
the past 30 years has been built on the study of phage structure and genetics carried out through the
1950s and 1960s. Restriction endonucleases which form the basis of molecular cloning were developed
following studies of phage infection and many phage enzymes provide tools for the molecular biologist.
The technique of phage display has more recently provided a powerful technique for the identi®cation
and optimisation of ligands for antibodies and other biomolecules. In the environment they have been
widely applied as tracers, as indicators of pollution and in the monitoring and validation of biological
®lters. While providing a valuable resource to the development of modern biotechnology, their ability
to mobilise and transfer toxin genes in the environment is viewed with concern. They also present a
continuing challenge to the fermentation and in particular, the dairy industry, where phage infection
can prove commercially disastrous.
# 2000 Society of Chemical Industry

Keywords: bacteriophage; biotechnology; phage display; phage therapy; gene transfer; diagnostics; biological
tracers

1 INTRODUCTION Bacteriophages, like other viruses, carry their

Bacteriophages were identi®ed independently by
Twort and Lond1 and d'Herelle2 as ®lterable, trans-
missible agents of bacterial lysis. They were initially
thought to be the key to controlling bacterial infections
but early studies proved largely unsuccessful.
Bacteriophages able to infect most procaryotic
groups of organisms have been isolated, and are
readily isolated from soil, water, sewage and, as might
be expected, from most environments colonised by
bacteria. Ecologically, phages are as varied and as
versatile as their hosts with some able to survive
extremes of temperature up to 95 °C and extremes of
pH as low as pH 1.3±5
In the 1940s and 1950s, pioneering studies into the
structure and physiology of host/phage interactions by
DelbruÈck,6±8 Luria,9,10 Doermann,11,12 Hershey,13,14
Lwoff,15 Lederberg16 and others laid the basis for the
development of molecular biology which in turn
became the foundation for a spectrum of new
biotechnologically-based industries.
genetic information in the form of either DNA or
RNA. Most bacteriophages have tails, the tips of which
bind to speci®c receptors such as carbohydrate,
protein and lipo-polysaccharide molecules on the
surface of the host bacterium (Fig 1). The bacterioph-
age injects its nucleic acid into the host where it utilises
the genetic machinery of the host to replicate its
genetic material and to transcribe this to form new
phage capsule material for the formation of new phage
particles. The number of phages produced during a
single cycle of infection (the burst size) varies between
50 and 200 new phage particles. Bacteriophage
resistance can develop through loss or changes in
receptor molecules on the host cell surface. Bacteria
also have speci®c mechanisms to protect themselves
against the invasion of foreign DNA. Host DNA is
modi®ed by methylation at speci®c points on the DNA
sequence; this gives protection to cleavage by host-
speci®c restriction endo-nucleases. Host-mediated
restriction results in the cleavage of all foreign DNA

* Correspondence to: Trevor Marks, Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 OJG, UK
(Received 10 March 1999; accepted 27 July 1999)

# 2000 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/2000/$17.50 6

 Bacteriophages and biotechnology

Figure 1. Electron micrographs of bacteriophages indicating differences in gross morphology. (a) Phage JS025 infecting Bacillus caldotenax, (b) Phage JS017
showing an unusually long tail infecting Bacillus caldotenax, (c) Phage JS027 infecting a thermophilic bacillus, (d) Phage JS017 infecting Bacillus
stearothermophilus strain RS93, (e) Phage 8724/25 infecting B anthracis, (f) Phage infecting Staphylococcus carnosus. All bar markers are 100 nm.

which does not carry the corresponding methylation
pattern. A small number of unmodi®ed phage
genomes avoid host-mediated restriction and, on
being replicated, become modi®ed, enabling them to
evade restriction by that particular host restriction/
modi®cation system in subsequent infective cycles.
Bacteriophages fall into two groups, virulent and
temperate. Virulent phages induce lytic infection,
resulting in the lysis of host cells and producing clear
plaques in lawns of susceptible bacteria. Temperate
phages integrate their DNA within the genome of the
host bacteria, resulting in a lysogenic infection and the
phage genome is passed to all daughter cells at cell
division. Some phage genomes are spontaneously
induced, resulting in transcription and production of
new phage particles which then infect and lysogenise
other uninfected hosts. Induction of a lysogenic micro-
organism (lysogenic induction) can occur through the
action of mutagenic agents such as ultraviolet light and
chemical mutagens. Lysogenic phages generally devel-

J Chem Technol Biotechnol 75:6±17 (2000) 7

T Marks, R Sharp
op hazy or turbid plaques on lawns of susceptible host
cells. The lysogenisation of a strain with a particular
phage confers immunity to infection by other related
phage sharing the same immunity group pro®le.
Phages with different immunity pro®les are able to
infect normally and cells can become lysogenised by a
number of different phages belonging to different
immunity groups.17
2 BACTERIOPHAGE THERAPY
Bacteriophage therapy (the use of bacterial viruses to
treat bacterial infections) was a major area of interest
60 years ago in the ®ght to combat bacterial
infections.18±21 The development of penicillin and
other antibiotics during the 1940s provided a more
ef®cient and comprehensive approach to the eradica-
tion of infection and led to the cessation of work in this
area. In Eastern Europe, however, work continued and
a number of strategies were developed to combat
infection using bacteriophages. Hospital studies have
shown that the application of bacteriophages to room
surfaces and objects, such as toilets, prevents the
transfer of Escherichia coli infections to children and
adults.22 In the veterinary ®eld, it has been demon-
strated that E coli infections in calves can be prevented
by spraying the litter in calf pens with aqueous
suspensions of bacteriophages.23
While a considerable degree of success was demon-
strated in many of the early studies, failure to become a
generally accepted therapy has been attributed to
failure to select highly virulent phages and selection of
phages with too narrow strain speci®city. Other factors
included the emergence of phage-resistant strains, the
neutralisation or clearance of phages by defence
mechanisms of the body and the liberation of
endotoxins as a result of widespread bacterial lysis.
The potential for the phage-mediated horizontal
transfer of toxin genes is also a factor which may
restrict their use in the treatment of particular
infections.
A number of studies to re-evaluate bacteriophage-
based therapies have been carried out in the past 20
years for the treatment of human and veterinary
infections, and have been reviewed recently.24±26
Smith and Co-workers23,27±29 published a series of
studies on the treatment of systemic E coli infections in
mice and diarrhoeal disease in young calves. They
demonstrated that both prophylaxis and treatment
were possible using phage titres much lower than the
number of target organisms, indicating in vivo multi-
plication of the bacteriophages.
They demonstrated that injecting 106 cfu (colory
forming unit) of E coli intramuscularly resulted in the
death of 10 test mice, while a simultaneous injection in
the opposite leg of 104 phages, selected against the K1
capsule antigen, gave complete protection.
When presented before the onset of diarrhoea, a
mixture of two phages protected calves against a
potentially lethal oral infection with an enteropatho-
8 J Chem Technol Biotechnol 75:6±17 (2000)
genic strain of E coli (O9:K30,99), a different mixture
of phages resulted in protection after the onset of
symptoms. Bacteria developing phage resistance were
far less virulent due to loss of their capsule. Subse-
quently, Smith demonstrated the ability of phage
suspensions, sprayed onto the litter in calf pens, to
control E coli infections.23
Soothill30 examined the effect of bacteriophages to
control organisms often implicated in the colonisation
of skin burns. Subsequently he demonstrated the use
of phages to protect skin grafts from destruction by
Pseudomonas aeruginosa and Acinetobacter baumanii.
The latter gave protection at the level of 1 pfu (plaque
forming unit) per 106 bacteria and the number of
phages, isolated from the tissues, indicated consider-
able in vivo replication.31
To reduce the elimination of phages by the host
defence mechanisms of the body during systemic
treatment, a serial passage technique in mice was
developed that selected for phages able to survive for
longer periods prior to elimination by the immune
system.32,33
Bacteriophage therapy offers a number of advan-
tages over antibiotic therapy, eg it is active against
drug-resistant organisms and may be used as an
alternative therapy for patients with allergies to
antibiotics. It can be used prophylactically to combat
the spread of infection where the source is identi®ed at
an early stage or where outbreaks occur within a
relatively closed institution, such as old people's
homes or schools. Bacteriophages offer high speci®city
towards the target organism and have no effect on non-
target organisms. They are self-replicating and self-
limiting; when the target organism is present, they
replicate until all target bacteria are infected and lysed.
Likewise when no target organism is present the
phages will be eliminated from the body. Bacterio-
phages mutate to combat host resistance mutations
naturally; or they can also be mutated deliberately in
the laboratory.
With the widespread development of antibiotic
resistance in pathogenic bacteria, the need for new
antibiotics and alternative strategies to control micro-
bial infections is of increasing urgency. Bacteriophages
may yet have a role to play in the treatment of infection
both independently and in combination with antibiotic
therapy.
3 BACTERIOPHAGE-MEDIATED MICROBIAL
CONTROL
Bacteriophages have been evaluated for the control of
bacterial contamination in the food industry, the
control of water-borne pathogens such as Vibrio
cholera34,35 and air-borne pathogens in the hospital
environment. They have been considered for the
control of Campylobacter in the poultry industry and
the control of mycobacteria in the environment.36
A reduction in the numbers of Pseudomonas fragi in
arti®cially inoculated milk was achieved following the
 Bacteriophages and biotechnology

Table 1. Phage products and enzymes

Phage product Application Phage

Phage DNA46,47 Generation of molecular weight markers Substrate in restriction enzyme l, f X174
assays
Unmethylated l DNA. Produced in Substrate for restriction enzymes sensitive to DNA methylation l
E coli dam and dcm methylase-
de®cient strain46,47
l insertion vectors46,47 Construction of cDNA libraries and genomic libraries with small size inserts l,
l cloning vectors46,47 Multifunctional cloning vector accepting fragments from 9±23kb l,
DNA ligase46,47 Catalyses the joining of two strands of DNA between 5'-phosphate and T4
3'hydroxyl groups of adjacent nucleotides
DNA polymerase46,47 Catalyses the 5'±3' synthesis of DNA from a primed single stranded DNA T4, T7
template
DNA polynuceotide kinase46,47 Catalyses the transfer of the phosphate from ATP to the 5'-terminus of poly T4
nucleotides or to mononucleotides bearing a 3'-phosphate group
RNA ligase46,47 Catalyses the ATP-dependent inter- and intramolecular ligation of single T4
stranded RNA or DNA
RNA polymerase46,47 DNA-dependent RNA polymerase with high speci®city for T3, T7 or SP6 T3, T7, SP6
promoter sequences

addition of bacteriophages.37 Applying the conditions
used for retail display, Greer38 examined the control of
food spoilage in extracts of beef muscle and refriger-
ated beef using bacteriophages Growth of Pseudomonas
sp was controlled at 7 °C for a period of 3 days.
Treatment of Pseudomonas inoculated steaks with a
high titre of phages resulted in a 1±2 log decrease in
bacteria with a 2 log increase in phage numbers. There
was a signi®cant marked decrease in surface discolora-
tion of the meat and the shelf-life of steaks was
increased from 1.6 to 2.9 days.
In contrast, other studies, using a pool of seven
bacteriophages were unsuccessful in controlling a wide
range of Pseudomonas species in beef.39 The lack of
success was attributed to the narrow range of host
speci®city within the phage pool. Of 1023 strains of
pseudomonads isolated from meat originating from
®ve abattoirs, only 57.2% were sensitive to the phage
Persistence and virulence were not problems with
signi®cant phage titre being maintained after 14 days.
The development of bio®lms through cells directly
attached to surfaces and those indirectly attached to
subsurface bacteria via exopolymeric substances pre-
sents a considerable challenge to their removal and
control. Incorporated within a bio®lm, bacteria
become less accessible to antibiotics and chemical
control and are intrinsically more resistant. They
present a challenge to industry through biofouling,
through the development of protective microbial ®lms
on food processing equipment and in the medical
devices industry through growth on the surfaces of
catheters, prostheses and implants.
Bio®lms of E coli strain 3000 XIII developed on the
surfaces of polyvinylchloride coupons were demon-
strated to be infected and lysed using bacteriophage
T4D.40 Similar studies with phages to Pseudomonas sp
within bio®lms indicated phages were only infecting
the surface organisms. The control of bio®lms was
considered to require phages able to interact with
speci®c cell receptors, and bio®lm-speci®c phages may
exist and infect only cells growing as a bio®lm.41
Bacteriophage SF153b produces a polysaccharide
depolymerase enzyme speci®c for exopolysaccharide
which is able to disrupt bio®lms.42
Listeriaphages were evaluated for the disinfection of
stainless steel and polypropylene surfaces contami-
nated with Listeria monocytogenes. Phage suspensions
at concentrations up to 3.5  108 pfu cmÿ3 were found
to have a comparable activity with 20 ppm solution of a
quaternary ammonium compound (QUATAL). Two
phages used in combination, or in combination with
QUATAL, produced a synergistic affect; they were
resistant to 4 h exposure to QUATAL at concentra-
tions up to 50 ppm.43
Streptococcus sanguis is one of the ®rst colonisers of
newly cleaned teeth and initiates plaque formation,
enabling other microorganisms to attach and grow.
Norris44 proposed the incorporation of bacteriophages
into toothpaste, dental ¯oss, chewing gum and sweets
to control the development of dental plaque induced
by S sanguis. Similarly the use of phages to control
acid-producing bacteria such as Lactobacillus acidophi-
lus to combat the development of dental caries was
proposed.45
4 BACTERIOPHAGE ENZYMES
Coliphage T4 falls into the group of larger T phages
with a genome of 170kb. Over 200 genes have been
identi®ed and 43 phage-encoded proteins identi®ed.
These are essential for replication of the phage DNA
and the formation of new phage particles. Bacterioph-
age genomes have been developed as cloning vectors
and phage-encoded promoters utilised as components
in the construction of expression vectors. Bacterioph-
age enzymes are widely exploited as tools for molecular

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T Marks, R Sharp
biology including DNA polymerase, RNA polymerase,
DNA ligase, RNA ligase and polynucleotide kinase
(Table 1).46,47 These are now produced as recombi-
nant products from E coli. Thermophilic phages have
been examined as a source of thermostable DNA
polymerase.48 The controlled production of phage-
encoded lysozyme is coordinated with maturation of
new phage particles, enabling their release from the
host cell.
Phages infecting exopolysaccharide-producing bac-
teria in bio®lms have been shown to induce the
synthesis of enzymes able to degrade these polymers.
These enzymes appear to act as endo-glucanohydro-
lases42 and may ®nd application in the control of
bio®lm formation.
The lysins of bacteriophages have been examined
for their potential to hydrolyse the cell walls of
organisms resistant to the lysozyme of hens egg
white.49 In cheese manufacture ¯avour maturation is
dependent upon the generation of proteolytic enzymes
such as peptidases being released from the starter cells
as they spontaneously lyse. Recombinant phage lysins
and autolysins have been used for the development of
controlled starter cell lysis to promote intracellular
enzyme release.50 This was ®rst demonstrated by
Shearman et al.51 who expressed f ML3 lysin in
lactococci introducing the gene on a plasmid. The
recombinant strain grew normally through the ex-
ponential phase and lysed spontaneously in the
stationary phase.
5 PHAGE DISPLAY
Phage M13 and other ®lamentous phages have been
used as expression vectors in which foreign gene
products are fused to the phage coat proteins and
displayed on the surface of the phage particle.
The power of phage display centres on the batch
cloning of millions of variants of particular ligands into
the phage genome as a fusion to the gene encoding one
of the coat proteins (pIII, pVI, pVIII). When expressed
the coat protein fusion is incorporated into new phage
particles that are assembled in the bacteria. The ligand
is then expressed at the surface of the mature phage
particles, while its genetic material remains encoded
within the phage genome. A wide range of proteins,
peptides and random peptide libraries have been
displayed on the surface of phages. Phage particles
displaying the desired property can be separated using
af®nity chromatography or panning on immobilised
selector molecules. The selected phage is used to re-
infect bacteria and further enriched. The success of
ligand phage display is dependent upon the combina-
tion of the display and enrichment strategy with the
synthesis of large combinatorial repertoires of phages.
The display of peptides on the surface of a phage
presents a powerful tool for the identi®cation or
optimisation of ligands for antibodies and other
biomolecules. Smith52 constructed the ®rst phage
display library in 1985. Since then the procedure has
10
been developed and widely exploited and new
strategies have been widely reviewed.52±59
6 BACTERIOPHAGE TYPING
One of the ®rst practical applications of bacterio-
phages took advantage of the speci®city of many
phages for their host. The analysis of patterns of
sensitivity to a speci®c panel of bacteriophages gave a
precise technique for the identi®cation of bacteria. A
set of phages for the epidemiological typing of
Staphylococcus aureus was described in 1942.60 The
importance of using a routine test dilution (RTD),
which was de®ned as the concentration of phages to
produce a semi-con¯uent lysis on the propagating
strain, was recognised. In 1953, The International
Subcommittee on Phage Typing was formed to co-
ordinate the selection of basic sets of phages and to
develop the methodology. The Central Public Health
Laboratory in London maintains and distributes the
international Basic Set of phages and their propagation
strains.61 Phage typing systems have also been
established for many other groups of micro-organisms
including Salmonella,62 E coli 0157,63 Mycobacterium
tuberculosis,64 Listeria65 and Campylobacter.66
7 THE USE OF BACTERIOPHAGES AS
BIOLOGICAL TRACERS
Bacteriophages have been utilised as tracers of both
air-borne and water movement.67 Their use in the
study of hydrology of aquifers began in the 1970s by
demonstrating their effectiveness as groundwater
tracers for the delineation of ¯ow paths over consider-
able distances. Coliphage T4 was successfully used to
trace a groundwater ¯ow for 1.6 km in the Karst terrain
of southern Missouri in the USA.68
The advantages offered by bacteriophages as
groundwater tracers include their small size, negligible
impact on water quality, the potential to use high
concentrations and the ability to detect low numbers
using ®ltration recovery strategies. The potential to
add phages at concentrations up to 1013 pfu (much
higher than would be possible with bacterial tracers)
enables signi®cant dilution of phages throughout the
water system under study.
Phages were used by Noonan and McNabb69 to
demonstrate the spread of faecal contamination from a
sewage disposal site in New Zealand using an injection
of coliphage T4 into an alluvial aquifer. The phage was
traced to an observation well 900 m downhill. Simi-
larly, the dispersion of coliphage f2 was monitored,
following continuous addition over 7 days into a rapid
in®ltration sewage disposal site.70
The movement of groundwater beneath a coal
spoilage heap was traced over 680 m using an
Aerobacter aerogenose phage, con®rming results pre-
viously derived from hyrogeological studies. Unlike
other tracers such as organic dyes, this phage was not
absorbed to certain media in the substrata.71
J Chem Technol Biotechnol 75:6±17 (2000)

Bacteriophages MS2 and PRD1 were used in
trench-to-trench lateral groundwater ¯ow studies in a
weathered and fractured clay-rich till. The phages
added at 105±106 pfu cm3 were detected in water
seepage collection points set in a trench 4 m from the
source trench. Peak phage titres were in the region of
102±104 pfu cmÿ3 and were detectable for up to 5 days.
Phage attenuation was considered to be due to
inactivation and either attachment to the fracture
walls or diffusion into larger pores. In contrast, similar
studies using bromide as a tracer in place of phages
took up to several months.72
Phage PRD1 was selected for transport and recovery
studies in sand and gravel aquifers and to assess the
affect of sewage-derived organic material on virus
attachment. In the uncontaminated zone, 83% of the
PRD1 was attenuated over the ®rst metre by attach-
ment to aquifer grains. In the contaminated zone, 42%
of the phages were attenuated over the ®rst metre.
Sewage-derived organic material reduced the attenua-
tion of the phages by blocking attachment sites in the
contaminated zone. The results indicated the signi®-
cance of contaminating organic material in the
ef®ciency of virus particle attenuation.72
Pathogens are easily introduced into groundwater
via septic tanks, land®ll, crop irrigation, and treated
sewage ef¯uent. Some of the more common water
borne viral diseases result from the dissemination of
Norwalk virus, Rota virus, and Hepatitis A. Rapid
monitoring and detection of contamination with faecal
material is essentially based on the detection of
bacteria normally considered to be of human origin
such as total faecal coliforms. The potential for
contamination with enteric viruses is therefore de-
duced rather than con®rmed through direct analysis.
Snowdon and Oliver73 proposed the development of
strategies to detect small round RNA coliphages as
surrogates for the con®rmation of enteric viruses.
These phage were considered to survive and behave in
groundwater and under geological conditions in a
manner similar to human small round enteric viruses.
8 THE USE OF BACTERIOPHAGES FOR
MONITORING AND VALIDATION
The removal of viral particles by a wide range of
®ltration systems has been evaluated and validated
using a number of different bacteriophages. A model
system for testing viral barrier properties of simple
®lters was evaluated using a bacteriophage active
against Serratia marcescens.74 Benbough and Bennett75
used air-borne challenges with coliphage T1 to model
the ability of Filta-therm and Filta-guard ®ltration
systems to protect equipment and patients from
human viruses such as Hepatitis B. The removal
ef®ciency of the Pall Ultipor Breathing system ®lter for
particles in the size range of Hepatitis A and HIV was
determined using a mono-dispersed aerosol challenge
of the coliphage MS2.76 To avoid the introduction of
bacteriophages into fermentation systems with the
J Chem Technol Biotechnol 75:6±17 (2000)
Bacteriophages and biotechnology
large amounts of air required for microbial growth,
®ltration systems which eliminate the ingress of
contaminating bacteriophages are required. Rapp et
al 77 carried out short and long-term tests on air-
sterilising ®lters using phage fX174. The model
demonstrated a 108-fold reduction in the numbers of
bacteriophages. Similar studies were reported by
Vandenbroucke-Grauls et al 78 using phage T1.
Tests developed to assess the virucidal activity of a
range of skin disinfectants utilised both poliovirus
(vaccine strain Sabin lan) and coliphages MS2 and K1-
K5. The advantages provided by the use of bacter-
iophages as test organisms precluded the need for
complex recovery systems and could be applied by any
bacteriology laboratory.79
9 PHAGE-BASED DIAGNOSTICS
The speci®city of bacteriophages for their bacterial
hosts has made them the object of study in order to
provide rapid and accurate detection of selected target
organisms. A number of diagnostics based upon the
use of bacteriophages will also only detect metab-
olically-active target organisms. In order for phage-
based diagnostic tests to be readily acceptable, they
must be simple and economical to use. In comparison
to antibodies, phages can be produced in large
quantities at low cost, offering the prospect of
inexpensive, accurate and sensitive diagnostics. How-
ever, one of the principal problems in phage-based
diagnostics is the visualisation of the phage.
One of the methods used for the visualisation of a
positive identi®cation is the cloning of the luciferase
gene (lux) into the phage. Upon infection into the host
bacterium, transcription of the phage DNA results in
production of luciferase which can then be detected by
virtue of the resultant light emission. This technique was
used by Kodikara et al 80 for the detection of enteric
indicator bacteria in food. Without recovery or enrich-
ment, levels in excess of 104 gÿ1 or cmÿ2 could be
detected in 50 min. Levels of 10 gÿ1 or cmÿ2 could be
detected following a 4 h enrichment period. Similarly,
the use of the lux gene in Listeria-speci®c bacteriophages
was evaluated for the detection of L monocytogenes in a
range of food and environmental samples.81 With pre-
enrichment steps of 20 h, one organism per gram could
be detected in foods such as ricotta cheese, chocolate
pudding and cabbage. In foods with a larger and more
diverse microbial ¯ora, such as minced meat and soft
cheese, at least 10 cells per gram were necessary for
detection. The recombinant phages detected 55 listeria
positives from a range of 348 potentially contaminated
food and environmental samples. This compared with
57 positives derived from standard plating procedures.
The lux-phage method appeared to detect more positive
samples in dairy products and environmental samples,
whereas the standard plating method showed more
positives with meat and poultry products. However, the
recombinant phage method gave results in 24 h, whilst
the plating method required 4 days to show positives.
11
T Marks, R Sharp
With the increased incidence of multi-drug resistant
strains of tuberculosis, the need for a rapid screen of
drug susceptibility of a given strain is crucial to the
early commencement of drug therapy. The use of
recombinant Mycobacterium bacteriophage (TM4)
carrying a luciferase reporter gene has been tested as
a tool for rapid assessment of drug susceptibilities.82
When strains of Mycobacterium, some of which were
drug-resistant, were grown for 24 h, and in the
presence of selected antibiotics, light was only
produced in those strains that were resistant to those
antibiotics under test. However, due to poor expres-
sion of the luciferase, in the order of 104±105 cells were
required to produce a signi®cant signal. Use of a
different cloning strategy and phage (L5) resulted in
high levels of luciferase being expressed in integrated
prophages.83 This was shown to be able to detect
rifampicin-resistant strains (RifR) of M smegmatis in a
mixed population at a level of 1:1000 with rifampicin-
sensitive strains (RifS), which is below the accepted
detection level of 1:100.84 In addition, the phage was
able to detect as few as 700 colony forming units (cfu)
of M smegmatis after 20 h incubation, falling to 70 cfu
following 40 h incubation.
A recent development in phage-based diagnostics
has been the `phage ampli®cation assay'.85 This is
based upon the phage lytic cycle and relies on plaque
visualisation as the assay end point. The assay
comprises essentially four stages. Firstly the phages
are used to infect the target bacterium. Next,
exogenous phages are destroyed by a virucidal agent,
pomegranate rind extract. Ampli®cation of the phages
within the host, which have been protected from the
action of the virucide, results in subsequent formation
of visible plaques on lawns of helper bacteria. The use
of the virucide ensures that only those phages which
have been internalised into the target bacteria are able
to form plaques. Using this method, P aeruginosa has
been detected at levels of 40 cmÿ3 and Salmonella
typhimurium at 600 cmÿ3 within 4 h. One signi®cant
advantage of this method of detection is that naturally
occurring phages are used, without the need for
genetic modi®cation, thus reducing the costs required
to develop the test for other organisms.
10 LARGE-SCALE PRODUCTION OF
BACTERIOPHAGES
The production of bacteriophages in large quantities
became essential for their use as tracers, the production
of phage-encoded enzymes and their use as anti-
microbial agents. Much of the work on large-scale
production of phages was undertaken in the 1950s and
1960s. Sergeant and Yeo86 produced E coli bacterioph-
age m2 at the 150 dm3 scale in stirred tank fermenters
during the mid-1960s. Phage titres in excess of 1013 pfu/
cmÿ3 were obtained, resulting in yields of phage of
75 mg/dmÿ3 of culture. The authors found that aeration
of the culture and timing of the addition of bacterio-
phages was critical to obtaining maximum yields. For
12
the best yield of phages per unit volume of culture, the
optimum time for phage infection was such that
bacterial lysis just prevented the carbon dioxide evolu-
tion rate reaching its potential maximum. Cultures
grown and infected under conditions of low aeration
gave poor yields of phages. Inorder to prevent premature
infection of bacterial cultures, separate facilities were
used to prepare bacterial seed cultures and phage
preparations. If cultures are left too long after phage
infection, the cultures can become overgrown by
resistant bacterial cells. Earlier work by Strauss and
Sinsheimer,87 and by Seigel and Singer88 gave yields of
phages of up to 200 mg per dm3 of culture, as calculated
by Sergeant and Yeo,86 albeit at smaller volumes than
those authors.
11 PROCESS CONTAMINATION
The contamination of a fermentation production plant
can lead to signi®cant losses through increased `down-
time' and cause considerable problems in eradication
of the infection. Bacteriophages can effectively shut
down a fermentation plant, the source is often
impossible to identify and the prevention of subse-
quent infection is dif®cult. Certain processes and
production strains appear more susceptible than
others. Phages may enter from external sources such
as the air supply and raw materials, or as lysogens
within the culture itself.
Those fermentations conducted within the food
industry, often with non-sterile raw materials and with
exposed operating processes, are particularly prone to
infection. Industrial fermentations producing anti-
biotics, enzymes or recombinant products generally
only employ pre-sterilised feed stocks, and micro-
organisms are generally cultivated using sterile media
and carefully controlled operating procedures.
The introduction of lysogenic phages via the use of
new seed cultures or new production strains needs
careful control. Prior to the introduction of new seed
or starter cultures to a process they must be intensively
screened for the presence and induction of lysogenic
phages. Once introduced into a plant it may take many
weeks to ensure that the process environment is free
from potential sources of re-contamination. It may be
bene®cial to switch the plant to the growth of
alternative cultures not sensitive to the contaminating
phages. A small pilot plant, growing a wide range of
micro-organisms, had no problem with phage infec-
tion in its processes for a period of 25 years until it
replaced an expensive, electrically operated hot air
sterilisation unit by inlet depth ®lters to supply sterile
air to the plant. Within 6 months, a series of phage
infections almost closed the plant and caused con-
siderable disruption. The plant was situated in
proximity to a farm where slurry spreading was
widespread in the autumn. While it was impossible
to con®rm, the assumption was that coliphages present
within the slurry were aerosolised during spreading
and were taken up in the air inlets to the plant.
J Chem Technol Biotechnol 75:6±17 (2000)

Many phages require the presence of cations such as
Ca2‡ and Mg2‡ to promote adsorption to bacteria.
The use of media incorporating cation chelators such
as phosphates, citrate or oxalate remove free cations
and inhibit phage adsorption. Other medium compo-
nents inhibiting phages include non-ionic detergents,
ascorbic acid, phytic acid and spermine.89 Good
sanitation is critical in controlling phage infections
with readily cleaned equipment, no obscure or hidden
ledges or open drainage channels within the plant, the
use of chlorine-based disinfectants and the sterilisation
of all raw materials used within the process. It is
imperative that, if contamination does occur (often
®rst observed by foaming within the fermentation
vessel), sampling ceases immediately and all samples
are immediately heat sterilised. In addition sampling
areas must be disinfected and the contents of the
fermentation vessel sterilised prior to disposal. The
harvesting of batches contaminated with phage can
lead to the generation of phage aerosols throughout
the process plant leading to major subsequent decon-
tamination problems. In any plant where phage
contamination is a recurrent problem, routine surface
and air-borne monitoring should be carried out.
12 THE DAIRY INDUSTRY AND THE
DEVELOPMENT OF PHAGE-RESISTANT STRAINS
While the details of phage infections of industrial scale
processes are not generally published, the food
industry is generally the most affected, in particular
the fermented dairy products industry producing
cheese and yoghurt.
Fermented dairy products were one of the ®rst
applications of microbiology to the production of food.
Early cheese-makers utilised complex mixed cultures
developed over years of sub-culturing. With an increas-
ing understanding of microbiology, starter cultures were
developed which often-comprised single or paired
microbial cultures. This together with the increasing
scale of modern dairy fermentations enhanced the
ability of a phage to wipe out a single strain resulting in
a considerable ®nancial loss. The dairy industry has thus
taken the lead in the development of strains resistant to
phage infection and much of the recent research centres
around organisms used in the dairy industry.
Dairy starter cultures are predominantly composed
of sub-species and strains of Lactococcus lactis. Com-
ponents of a starter culture are selected for their fast
acid-producing ability, high phage insensitivity and
differing phage susceptibilities. Speci®c combinations
are used continuously until they become phage-
sensitive or alternatively they may be rotated to
minimise phage build-up by limiting exposure of any
one combination to potential phage exposure.90
The harnessing of naturally occurring mechanisms
of phage resistance together with the development of
novel strategies developed through genetic manipula-
tion are the subject of considerable research.91
Adsorption inhibition results through the develop-
J Chem Technol Biotechnol 75:6±17 (2000)
Bacteriophages and biotechnology
ment of bacteriophage-insensitive mutants (BIMs)
which are generally derived from the isolation of
spontaneously arising phage-resistant strains. These
mutations are generally considered to be due to
chromosomal mutations resulting in loss of phage
receptors on the cell coat. At least seven different
lactococci plasmids have been found to encode genes
that inhibit phage adsorption to the host cell. Plasmid
pME0030 48kb from L lactis ssp lactis ME2 reduced
phage adsorption by 80±90% to 20±40% when carried
in the host strain.92 Plasmids pSK11 (54kb) from L
lactis ssp lactis SK110 and pCI528 (46kb) from L lactis
ssp cremoris UC503, appeared to prevent phage
adsorption by masking the phage receptor sites.93±95
pSK11 appears to produce a galactose-containing
lipoteichoic acid moiety which is implicated in the
masking of the receptor.96,97 pCI528 is involved in the
formation of a cell wall-associated hydrophilic polymer
containing rhamnose and galactose which is respon-
sible for phage inhibition.98
There is evidence that after successful phage adsorp-
tion to the host, injection of phage DNA can be
prevented. Inhibition of DNA injection to the host was
demonstrated in Lactococcus carrying a lactococcal
plasmid, pNP40.99±101 Speci®c host-cell membrane
proteins appear to be required for successful phage
infection and it is considered that these may be the target
for a mechanism interfering with DNA penetration.91
The mechanism of host Restriction and Modi®ca-
tion (RM) has been discussed earlier. The RM systems
of many lactococci spp have been characterised and
enzymes cloned.91 Modi®cation or incorporation of
these into susceptible hosts presents an opportunity to
prevent phage multiplication.
The inhibition of the intracellular development of
bacteriophages has also been observed and, although
the mechanisms acting are not clearly understood,
they appear to be the result of abortive infection (Abi)
plasmids.91 The 64kb plasmid, pNP40, has been
demonstrated to inhibit infection by phage c2 follow-
ing conjugal transfer of the plasmid to the plasmid-free
recipient L lactis ssp lactis LM2301.102 The plasmid
appears to carry three independent phage insensitivity
traits, two operating as abortive infection mechanisms
interfering with the lytic cycle and the other interrupt-
ing phage injection.99,100 In bacteriophage lambda,
phage exclusion mechanisms include the Rex proteins
which appear to be encoded by plasmids or prophages
and promote the death of the host cell following phage
infection. Phage infection triggers early cells death of
the host before the phage infection cycle can be
completed and progeny phage released and hence
prevents their spread to nearby cells.103
13 BACTERIOPHAGE-MEDIATED GENE
TRANSFER
Many bacterial virulence determinants are encoded by
accessory element-borne genes, rather than those that
are chromosomally located. In the 1950s, the b
13
T Marks, R Sharp
prophage of Corynebacterium diptheriae was implicated
in the increased virulence of strains harbouring this
phage.104 Many such virulence determinants are now
believed to be phage-encoded, for example, the genes
coding for shiga-like toxins (SLT) in strains of E coli
0157 are located on bacteriophage genomes.105,106 The
conversion of non-toxigenic strains of E coli to toxigenic
strains has also been demonstrated. A phage, H19A,
isolated from E coli serotype O26:H19, encoding SLT I,
has been used to convert the enteroadherent E coli
RDEC-1, which is a rabbit pathogen, into a signi®cantly
more virulent strain, RDEC-H19A.107 It has been
proposed that the new strain, RDEC-H19A, may form
the basis of an animal model for shiga-like toxin
production.108 More recently, VT genes have been
identi®ed in other bacterial strains including Citrobacter
freundii109 and Aeromonas sp.110
Similarly, in Vibrio cholerae the structural genes for
cholera enterotoxin are located on the genome of a
bacteriophage, CTXΦ, which is related to the E coli
phage M13. For full virulence, V cholerae requires two
factors for virulence, cholera toxin and toxin-co-
regulated pili, which are required for colonisation.
Phage CTXΦ uses the pili as its receptor. Infection by
CTXΦ was reported to occur more ef®ciently in the
gastrointestinal tract of mice than in vitro.111
Other bacterial toxins that are encoded on bacter-
iophage genomes include type A exotoxin in Strepto-
coccus pyogenes112±114 coded on bacteriophage T12,
staphylokinase and enterotoxin A115 in S aureus116 and
cytotoxins in P. aeruginosa.117 The transfer of neuro-
toxin type E from strains of Clostridium butyricum to
non-toxigenic strains of Cl botulinum has been shown
to be mediated by bacteriophage.118 Other virulence
determinants coded on bacteriophages include capsule
production by Streptococcus pnuemoniae119 and lipopo-
lysaccharide acetylase.120 Cheethan and Katz121 pre-
sent a more detailed review of the transfer of bacterial
virulence determinants by bacteriophages.
Where phages do encode such virulence factors, the
repercussions of the possibility of horizontal transfer of
these factors cannot be ignored. Whilst the factors
alone generally do not confer pathogenicity to the
recipient strain, if the recipient already has a number
of virulence determinants, phage-mediated transfer of
additional factors can only increase the subsequent
virulence of the strain. This transfer may play a role in
the emergence of seemingly new pathogens.122
Careful consideration should be given to the potential
for increased pathogenicity following phage transfer of
virulence components when designing regimes for
phage-related microbial control techniques (such as
phage therapy). Widespread, indiscriminate use of
bacteriophages may exacerbate problems with bacterial
pathogens rather than control them.
CONCLUSIONS
Since their discovery at the start of the 20th century,
bacteriophages have helped shape the development of
14
modern biotechnology. From the early hopes of
developing methods for the control of microbial
infections, later eclipsed by the discovery of anti-
biotics, to the latest developments in phage display
technology, bacteriophages have played key roles in
biotechnological advancements. Their usefulness is
underlined by the diversity of their uses in biotechnol-
ogy. Phages have been used as tracers in the environ-
ment, as challenge agents for monitoring and
validation, and as bacterial typing systems. In the ®eld
of molecular biology they have been used as sources of
enzymes and have been invaluable as cloning vectors.
As we enter the next millennium, the wheel has turned
full circle, with re-awakened interest in phage therapy,
sparked by concerns over the emergence of drug-
resistant micro-organisms, and the re-emergence of
pathogens believed to have been eradicated from the
developed world.
The continuing struggle between phages and their
hosts resulting in the infection of microbial fermenta-
tions will continue to present a challenge to the
biotechnology industry. Increased concerns over the
ability of phages to transfer genes, especially virulence
factors, amongst microbiol populations, potentially
resulting in the creation of pathogens with enhanced
virulence, have ensured that fundamental research on
the biology of phages is likely to continue unabated.
At the end of the 20th century, the emergence of the
powerful technique of phage display, with its potential
to revolutionise screening in the biotechnology and
pharmaceutical industries, provides ample testament
to the pioneering work and beliefs of d'Herelle, Twort
and other workers, who achieved so much at its
beginning.
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