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10 Control of
Bacteriophages in
Industrial Fermentations
Sylvain Moineau1,2 and Céline Lévesque1
1Département de Biochimie et de Microbiologie, Faculté des
Sciences et de Génie, Groupe de recherche en écologie
buccale (GREB), Faculté de Médecine Dentaire, Université
Laval, Québec, Canada
2Félix d’Hérelle Reference Center for Bacterial Viruses,

Université Laval, Québec, Canada

CONTENTS

1. Introduction.....................................................................................................285
2. Phages and the Dairy Industry .......................................................................287
2.1. Overview......................................................................................................287
2.2. Phage Detection....................................................................................288
2.3. Sources of Phage Contamination .........................................................289
3. Phage Control Strategies ................................................................................290
3.1. Controlling Phage Proliferation ...........................................................290
3.2. Strain Selection.....................................................................................291
3.3. Development/Selection of Bacteriophage-Insensitive
Bacterial Mutants..................................................................................291
3.4. Genetically Engineering Phage-Resistant
Bacterial Strains....................................................................................293
Acknowledgments..................................................................................................294
References..............................................................................................................295

1. INTRODUCTION
Bacteria are used in a variety of industrial fermentation processes because of their
ability to convert a wide variety of substrates into complex products or specific
molecules. In principle, any industries and technological processes relying on bacte-
rial fermentation are vulnerable to bacteriophage infection, and phages represent a

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286 Bacteriophages: Biology and Applications

constant threat of serious economic losses for those industries. The literature describing
such problems goes back several decades (reviewed by Ackermann and DuBow, 1987;
Ogata, 1980; Wünsche, 1989), and faulty fermentations due to phages also are well
recognized in many modern biotechnological industries commercializing or using
various bacterial fermentation products (Table 10.1). However, many other cases of

TABLE 10.1
Examples of Faulty Fermentations Due to Bacteriophages
Bacterial Species Product

Acetobacter sp. Vinegar

Bacillus colistinus Colistin
Bacillus polymyxa Polymycin
Bacillus subtilis Amylase, protease
Bacillus subtilis var. natto Fermented soy beans
Bacillus thuringiensis Insecticide (BT)
Brevibacterium lactofermentum L-glutamic acid
Clostridium sp. Acetone, butanol
Corynebacterium sp. L-glutamic acid
Escherichia coli Various biotechnology products
Gluconobacter sp. Gluconic acid
Lactobacillus acidophilus Fermented milk
Lactobacillus brevis Lactic acid
Lactobacillus casei Fermented milk
Lactobacillus delbrueckii subsp.
bulgaricus Yogurt
Lactobacillus delbrueckii subsp.
lactis Cheese
Lactobacillus fermentum Sourdough bread
Lactobacillus helveticus Cheese
Lactobacillus plantarum Silage, sauerkraut
Lactococcus lactis Buttermilk, cheese, sour cream
Leuconostoc mesenteroides Sauerkraut, buttermilk, sour cream
Leuconostoc fallax Sauerkraut
Oenococcus oeni Malolactic fermentation
Propionibacterium freudenreichii Cheese
Pseudomonas aeruginosa 2-Ketogluconic acid
Streptococcus thermophilus Cheese, yogurt
Streptomyces aureofaciens Tetracycline
Streptomyces endus Endomycin
Streptomyces griseus Streptomycin
Streptomyces kanamyceticus Kanamycin
Streptomyces venezuelae Chloramphenicol
Tetragenococcus halophila Soy sauce
Xanthomonas campestris Xantham

Updated from (Ackermann and DuBow, 1987; Jones, et al., 2000; Ogata, 1980; Wünsche,
1989)
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Control of Bacteriophages in Industrial Fermentations 287

phage contamination are not reported in scientific publications, and microbiologists

learn of them primarily via confidential reports and personal communications (Ack-
ermann and Moineau, unpublished data).
One industry that has openly acknowledged phage infection of their bacterial
strains is the dairy industry. In fact, dairy microbiologists have attempted for more
than 70 years to eliminate, or at least to bring under better control, the bacterioph-
ages that interfere with the manufacture of many fermented milk products (Moineau
et al., 2002). As a result, faulty milk fermentations are, undoubtedly, the best-
documented examples of virulent bacteriophage infections in the industrial settings.
The purpose of this chapter is to present an overview of the current strategies used
by the dairy industry to curtail bacteriophage attacks. The general approaches
described here are likely to be effective in many other similar fermentation pro-
cesses (Table 10.1). In addition, more specific information about the ecology of
industrial phages, including phages relevant to the dairy industry and to various
non-dairy food fermentation processes (e.g., the preparation of sauerkraut) is pre-
sented in Chapter 6. Also, comprehensive reviews about dairy phages and their
control strategies have recently been published (Boucher and Moineau, 2001;
Coffey and Ross, 2002).

2. PHAGES AND THE DAIRY INDUSTRY

2.1. OVERVIEW
Nonsporulating, Gram-positive microorganisms known as the lactic acid bacteria
(LAB) are used in the manufacture of yogurt and various cheeses. The LAB include
members of several bacterial genera and species; e.g., Lactococcus lactis, Lacto-
bacillus sp., Leuconostoc sp., and Streptococcus thermophilus (Table 10.1). Large-
scale milk fermentations typically begin after inoculation with carefully selected
cultures containing a blend of a few LAB strains at a level of 107 cells per ml of
milk (Moineau et al., 2002). These commercial fermentative processes are vulner-
able to virulent bacteriophages ubiquitous in the dairy environment, which can lyse
the LAB, thereby delaying lactic acid production and even stopping the fermentation
process. Typically, milk fermentation is compromised when phage titers are higher
than a critical threshold of 104 plaque-forming units (PFU)/ml. When the titer rises
beyond 105–106 PFU/ml, a fermentation failure is the likely consequence. The milk
inoculated with the starter will not coagulate and the product’s desired properties
are altered or entirely lost. The production line must then be carefully cleaned to
eliminate the phages. It should be noted that no level of bacteriophage contamina-
tion is acceptable in other biotechnology industries because of the potential for
process and product adulteration (G. Bogosian, unpublished data). Dairy virulent
phages may spread very rapidly (they have a latent period and a burst size of
approximately 30 min and 100, respectively) within a dairy plant. Their exponential
multiplication is of even more concern in any process that allows the bacteria to
multiply for several generations, such as cheese manufacture. Based on the authors
experiences, between 0.1% and 10% of milk fermentations within any given dairy
factory may be negatively affected by phages.
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288 Bacteriophages: Biology and Applications

2.2. PHAGE DETECTION

Virulent phages can proliferate quickly; therefore, constant monitoring is critical. A
dependable phage assay should be available for any fermentation process that relies
on bacteria to convert a substrate into a commercial product. The presence of virulent
phages in dairy samples is usually detected using conventional microbiological
methods such as plaque assays (see the Appendix section of this book for a detailed
methodology). Even if starter cultures are composed of a blend of strains, plaque
assays should be performed with pure cultures whenever possible. The presence of
plaques at various sample dilutions confirms the presence of phages. However, it
should be remembered that plaques formed by some phages may be difficult to
observe on the bacterial lawn. In such cases, increasing or decreasing the incubation
temperature, varying the multiplicity of infection, adding co-factors (e.g., Ca++ or
Mg++), or adding a bacterial cell wall-weakening agent (e.g., 0.25% glycine) may
increase plaque size (Lillehaug, 1997).
If a reliable plaque assay is not available, tests assessing bacterial activity may
be used to monitor the effect of fermentation by-products on the metabolic activity
of bacteria. In this context, possibly the most common assay is the milk activity test.
Briefly, 10 ml of milk (in each of two test tubes) are inoculated with aliquots (ca. 1 ml)
of a starter culture. One tube is also inoculated (2%, final concentration) with a
filtered (0.45 µm) sample to be tested for phages. After incubation (the temperature
and time depend on the starter and process), the pH of the milk in the tubes is
measured. If the pH of the milk containing the filtrate is 0.2 unit higher than that of
the milk in the other tube (indicating reduced production of lactic acid), phage
contamination is suspected. A pH indicator; e.g., bromocresol purple (100 µg/ml)
may also be added to the milk, to avoid direct pH measurements. In such an assay,
when the milk’s pH drops below 5.4, the indicator turns from purple to yellow.
Thus, the absence of a color change during incubation indicates the presence of
an agent (e.g., phages) that interferes with lactic acid production in the inoculated
milk. If many samples need to be processed daily, the availability of a pH indicator
is particularly useful for the development of a miniaturized assay enabling simul-
taneous testing of multiple samples in, for example, 96-well microtiter plates.
Another approach for detecting bacteriophages in dairy products is to use an
immunochemical assay (i.e., an ELISA). The ELISA technique uses antibodies raised
against specific phage structural proteins and is usually fairly specific; thus, although
that type of assay can be useful for detecting specific phages, it may not detect
bacteriophages having different structural proteins. Also, the sensitivity of an ELISA
for phage detection in dairy samples is usually low (Chibani Azaiez et al., 1998;
Moineau et al., 1993). The availability of complete phage genome sequences has
increased the popularity of using molecular DNA techniques (e.g., PCR and DNA
hybridizations) to detect bacteriophages rapidly (Labrie and Moineau, 2000; Moineau
et al., 1992). However, under some circumstances, the high sensitivity of DNA-based
methods may yield false-positive results (Labrie and Moineau, 2000; Moineau et al.,
1992). Thus, the choice of the phage detection method will depend on several factors,
including the type of laboratory equipment available and the required detection
sensitivity. Although of limited interest to most industries, additional information
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Control of Bacteriophages in Industrial Fermentations 289

about the detected phages may be provided by electron microscopic observations,

and the determination of host ranges, protein profiles, and genomic restriction pat-
terns. The dairy industry is far ahead of other industries in that regard, as hundreds
(if not thousands) of LAB phages have been characterized around the world
(Ackermann, 2001; Brüssow, 2001; Brüssow and Desiere, 2001).

2.3. SOURCES OF PHAGE CONTAMINATION

After the presence of phages is confirmed, the next step consists of identifying the
source of contamination. This natural biological threat is either endogenous (i.e., a
prophage) or exogenous (it is present in the substrate or environment). The use
of a lysogenic strain is seldom warranted in any industrial process, because
prophage(s) may be induced by the manufacturing conditions. Moreover, serially
passaging a temperate phage may result in its replacement by a virulent, derivative
deletion mutant (Bruttin and Brüssow, 1996). Accordingly, in order to eliminate
strains containing inducible prophages, bacterial strain selection programs should
include a screening assay for lysogeny. Genome sequencing projects have revealed
that prophages are abundant in many bacterial species (Canchaya et al., 2003).
Although the presence of prophages may be, at least in theory, beneficial in
protecting the bacteria against closely related phages, or in allowing their timely
lysis (e.g., during cheese ripening), the benefit of this superinfection immunity is
limited because of the natural diversity of phage populations.
In the dairy industry, the most probable sources of virulent phages are the
substrate and the factory environment (McIntyre et al., 1991). Indeed, most dairy
fermentations are susceptible to phage infections because raw milk naturally contains
lytic phages, albeit at low titers (between 101 and 104 PFU/ml). Two basic types of
ecological situations are distinguishable in the industrial milk fermentation envi-
ronment: the yogurt factory and the cheese factory. Milk for yogurt production
undergoes treatment at 90°C, which usually kills all phages (Quiberoni et al., 1999);
however, raw or pasteurized milk is used in cheese fermentation, and many phages
remain viable after pasteurization (Chopin, 1980). Also, yogurt production is a
relatively aseptic process, and the fermented product is minimally exposed to the
factory environment. In contrast, cheese factories daily experience aerosol contam-
inations during whey separation in the open vats (Budde-Niekiel et al., 1985).
Although the presence of phages is not a regular observation during yogurt produc-
tion, it is sufficiently frequent to make phages the primary source of fermentation
problems during that process. The problem usually manifests itself as fermentation
delays or product alterations, and, occasionally, as a complete loss of product. In
contrast, the potential problems that may occur during the fermentation process
in cheese factories result from the persistent coexistence of phages and bacterial
starter cultures in milk.
The diversity of phages in the milk used to manufacture fermented dairy products
probably results from its being a mixture of milk obtained from several farms, each
with its own agricultural management practices. Moreover, the fluid nature of milk
and fermentation by-products (e.g., whey) ensures good phage dispersion in the
substrate. In contrast, phages are usually trapped and localized in cheese. Also, if
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290 Bacteriophages: Biology and Applications

the same starter cultures are continuously used, they may provide permanent hosts
for phage proliferation (Boucher and Moineau, 2001). Thus, the source of phage
contamination will dictate the appropriate phage control strategies.

3. PHAGE CONTROL STRATEGIES

3.1. CONTROLLING PHAGE PROLIFERATION
Phages are omnipresent in the dairy environment; therefore, during the last several
decades, considerable efforts were aimed at controlling their proliferation rather than
trying to eradicate them (Moineau, 1999). The control of phage proliferation relies
on the development and implementation of a variety of practical approaches at both
the factory and laboratory levels (Table 10.2). Controlling phage proliferation in
production facilities primarily depends on the risks and sources of phage infection
associated with the manufacturing process used, as illustrated earlier for yogurt and
cheese production. These control strategies include adequate factory hygiene, an
adapted manufactory design, regular cleaning and disinfecting of the equipment and
clothes, and an appropriate airflow. More specifically, cross-contamination should be
avoided between the bacterial medium, the fermentation by-products and the factory
equipment. Sodium hypochlorite and peracetic acid should be used in the cleaning
and disinfecting procedures because they both rapidly eliminate phage particles

TABLE 10.2
Various Measures to Control Phages within a Dairy Food Factory
• Use starter cultures that do not contain strains with similar phage sensitivity patterns
• Perform daily tests for phage detection
• Aseptically prepare the starter cultures
• Use an anti-phage starter medium
• Directly inoculate milk with concentrated cultures
• Rotate bacterial starter strains
• Air filtration (HEPA) and positive pressure from starter room to product packaging
• Use footbaths (cleaned daily) containing disinfecting agent at the entry of rooms
• Limit the production of aerosols
• Clean and chlorinate vats between fills
• Use separate rooms for starter production and cheese manufacture
• Proper planning of the production pipelines to avoid contact between substrate (milk)
and wastes (water, whey)
• Avoid using the same equipment (e.g., centrifuges) for milk and whey (which may
contain high levels of phages)
• Clean the floor drains in the starter and fermentation rooms
• Regularly inspect equipments’ surfaces to remove biofilms and milkstone deposits
• Spray the environment with adequate disinfecting agents (e.g., chlorine-containing
compounds)
• Rinse the vats with water and not with by-products such as whey
• Rotate the manufacturing processes
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Control of Bacteriophages in Industrial Fermentations 291

(Binetti and Reinheimer, 2000). Phages are also usually sensitive to acid (pH 4 and
lower) and alkaline (pH 11 and above) conditions (Adams, 1959). The air should
be filtered and the efficiency of the air filters should be checked regularly because
phage infections may arise from damaged filters. In that regard, phages have been
detected (105 PFU/m3) in the air of a dairy plant (Neve et al., 1994). Thus, to prevent
contamination by phages from the outside environment, bacterial starter cultures
also should be prepared in a positively pressurized room. In addition, plant personnel
should be aware of the importance of phage control procedures and trained in their
application. Other phage control measures are available at the factory level, and they
will vary according to the type and size of the production facilities (e.g., yogurt
production facilities are usually smaller than their cheese counterparts). These and
other approaches have been reviewed by Champagne and Moineau (1998).

3.2. STRAIN SELECTION

The use of effective bacterial starter cultures is critical to obtain quality products.
Strain selection is a long and expensive operation based on several microbiological,
biochemical, and technical criteria (Moineau, 1999). The nature of these criteria will
depend primarily on the product. Whenever possible, a mixture of two or three well-
characterized bacterial strains should be used to prevent the possible phage infection
of the entire starter culture and subsequent fermentation or process failures. In this
context, one successful anti-phage strategy that is widely applied by cheese-makers
involves rotating bacterial strains. A basic operation is to determine the phage host
range with a collection of potential starter bacteria. This knowledge then allows the
rational design of a starter rotation system. The impact of phages on the fermentation
process can be limited if a starter strain with a susceptibility pattern A is alternated
with a starter possessing a non-overlapping susceptibility pattern B. This strategy
usually will maintain phage titers at levels that do not interfere with the fermentation
process. However, the rotation is based on the availability of a sufficient number of
performing strains. Difficulties in identifying phage-insensitive strains, and the vari-
able fermentations caused by strain differences, are among the problems associated
with rotation schemes (Moineau, 1999). Also, from an evolutionary point of view,
the use of different strains may also favor the emergence of a heterogeneous phage
population, which may enhance the potential for genetic recombination within the
phage gene pool. Thus, if possible, it may be worthwhile to rotate the type of
fermented dairy products manufactured in the same facility. For example, the man-
ufacture of a cheddar cheese (by means of mesophilic bacteria) could be followed
by the production of a mozzarella cheese, which uses thermophilic bacteria. If the
population of a specific mesophilic phage has amplified during the cheddar cheese
making, it is likely to be diluted out after the mozzarella production process.

3.3. DEVELOPMENT/SELECTION OF BACTERIOPHAGE-INSENSITIVE

BACTERIAL MUTANTS
When an industrial bacterial strain is found sensitive to a phage, the first step
consists of replacing it with a phage-resistant strain possessing the same functional
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292 Bacteriophages: Biology and Applications

characteristics during the fermentation process. If such a strain is not readily avail-
able, the entire strain selection procedure must be repeated. This strategy is time-
consuming and costly; therefore, the preferred approach may be to develop/select
for phage-resistant bacterial mutants. The development of bacteriophage-insensitive
mutants (BIMs) is generally the first approach used to transform a phage-sensitive
strain into a phage-resistant mutant. At the present time, BIMs are obtained in the labo-
ratory by a protocol which is very similar to the classical Delbrück-Luria phage
challenge experiment (1943). After long-term exposure to lytic phages, a few out-
growing cells that have lost the capacity to adsorb the challenge phage, most likely
because of a spontaneous mutation in the chromosomal gene encoding the phage
receptor, are selected for further characterization. Phage-resistant mutants are not
selected for in the factory because the fermentation process is restarted each time
with a frozen standard culture. BIMs may also result from mutations that alter the
bacterium’s carbohydrate composition, protein profile, or concentration of lipote-
ichoic acid, which lead to modifications in its cell surface characteristics (Forde and
Fitzgerald, 1999). Although the process for obtaining BIMs is relatively simple, it
may have substantial disadvantages; e.g., a high frequency of phenotype reversion,
and changes in bacterial physiology (e.g., a reduced growth rate). Nonetheless, many
BIMs have been isolated and are being used successfully under industrial conditions.
Another strategy to develop a phage-resistant strain relies on the so-called phage
defense mechanisms. Bacteria possess natural defense mechanisms enabling them to
resist bacteriophage attacks. For example, more than 50 naturally occurring L. lactis
phage-resistance systems have been characterized, which are divided into four
groups according to the time at which they act during the lytic cycle (for a recent
review, see Coffey and Ross, 2002). They include (i) inhibition of phage adsorption,
(ii) blocking of DNA injection, (iii) restriction/modification systems (R/M), and (iv)
abortive infection mechanisms (Abi). The genes encoding these natural defense
mechanisms may be plasmid- or chromosomally encoded. One useful feature is that
several plasmids are transferable by conjugation, enabling the development of phage-
resistant strains without affecting their technologically useful properties. Moreover,
these natural anti-phage systems are usually effective against several phage groups
(Moineau, 1999). Anti-phage barriers have important commercial value, and many
of them have been patented worldwide.
The above-noted four mechanisms of phage resistance have been studied to
various degrees. Interference with phage adsorption is most likely achieved via the
masking of bacterial receptors. This phenomenon has been attributed to the production
of various substances, including galactosyl-containing lipoteichoic acid, a galactose/
rhamnose or galactose/glucuronic acid polymer, and cell wall proteins (Coffey and
Ross, 2002). However, the genetic determinants responsible for this natural defense
mechanism remain to be determined. The second defense mechanism (blocking of
DNA injection) is still poorly understood. It prevents the translocation of phage
DNA into the bacterial cell without affecting phage adsorption. The genetic loci
responsible for this bacterial phenotype have yet to be determined. The R/M systems
have been identified in various bacterial groups, including LAB. They are charac-
terized by the presence of two types of enzymes, a site-specific restriction endonu-
clease and a methylase. R/Ms eliminate a wide variety of phages by cutting their
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Control of Bacteriophages in Industrial Fermentations 293

genomes. Finally, the Abi systems include all of the resistance mechanisms that act
after phage adsorption/nucleic acid injection and lead to phage-elicited bacterial
death. They include defense mechanisms that interfere with phage genome replication,
transcription, translation, packaging, or phage particle assembly. These systems are
more widespread than originally thought; e.g., more than 20 distinct Abi systems
have been already characterized in lactococci (Coffey and Ross, 2002). Each system
generally requires one or two genes for expression, and few similarities have been
observed between the genes. However, little is known about the exact mode of action
of the Abi proteins.
The potential of an anti-phage protective mechanism needs to be thoroughly
evaluated because not all systems may be able to withstand phage attacks in industrial
settings. Ideally, a system’s efficacy of protection (EOP = the phage titer obtained with
the resistant host containing the anti-phage system divided by the titer obtained with
the sensitive host) should be tested against the main phage groups. A strong phage
resistance mechanism will give EOPs in the range of 10−7 to 10−9, with 10−9 being the
maximum, technically measurable strength. An EOP in the range of 10−4 to 10−6 is
considered to be a medium potency system, whereas a weak anti-phage barrier has an
EOP of 10−1 to 10−3. Strong and medium systems are sought by the dairy industry
because weak mechanisms may not provide sufficient protection during fermentation
processes (Moineau, 1999). Another way of measuring the effectiveness of an anti-
phage system is via a milk-based assay. Briefly, an improved strain will go through
(in the presence of phages) a time-temperature curve that mimics a fermentation
process. If its anti-phage system protects the strain against a high initial concentration
of phages (>106 PFU/ml), it is likely to be effective in a factory environment. Inter-
estingly, the efficacy of anti-phage mechanisms may be influenced by temperature,
with some mechanisms being less efficient at higher temperatures (>37°C). Therefore,
the type of dairy fermentation for which the modified strain is designed will also
influence the overall evaluation of a resistance barrier.
Multiple, natural anti-phage defense mechanisms may be combined to strengthen
the overall phage resistance of a particular strain. In addition, the rotation of an iso-
genic strain’s derivatives containing various phage resistance mechanisms has been
proposed (Sing and Klaenhammer, 1993). However, because of their remarkable
evolutionary capacity (via point mutations or by exchanging modules with other
phages or the host DNA), phages can overcome these host defense mechanisms
(Bouchard and Moineau, 2000). Hence, novel phage defense strategies are needed to
provide long-term phage resistance in agriculturally important bacterial strains.

3.4. GENETICALLY ENGINEERING PHAGE-RESISTANT

BACTERIAL STRAINS
If the use of BIMs or of LAB possessing natural phage resistance mechanisms is
not satisfactory, it is now possible to construct novel anti-phage systems. For exam-
ple, during the last decade, some phage genetic elements have become a source of
resistance traits (Moineau, 1999). One such type of system, called Per (phage-
encoded resistance), involves the presence of the origins of replication of phages
supplied on a high copy number plasmid in the bacterial cell (Hill et al., 1990;
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294 Bacteriophages: Biology and Applications

O’Sullivan et al., 1993). These origins of replication interfere with phage multipli-
cation by competing for replication factors, thus providing false replication signals.
However, the Per systems are only effective against the phage group from which
the origin is derived.
Gene silencing by antisense RNA technology is an alternative means of gener-
ating engineered phage-resistant strains (Walker and Klaenhammer, 2000). This
technology has rapidly progressed because of the emergence of complete nucleotide
sequences of phage genomes and the characterization of individual phage genes. A
variety of phage genes have been targeted by antisense RNAs, including genes
encoding a (i) putative subunit of DNA polymerase, (ii) single-stranded binding
protein, (iii) putative transcription factor, (iv) major tail protein, (v) terminase, (vi)
major capsid protein, and (vii) putative helicase. Since they are highly conserved
among bacteriophages, the genes involved in DNA replication functions are, perhaps,
the most obvious targets for antisense technology.
Triggered suicide is another bacteriophage defense strategy that has been
designed as a genetically engineered form of an abortive infection (Djordjevic et al.,
1997). This approach uses a bacteriophage-inducible promoter to trigger the expres-
sion of a suicide system that simultaneously kills the bacterial cell and the infecting
phages. Lethal genes made of restriction gene cassettes are cloned on a high copy
plasmid without their corresponding methylase gene downstream of an inducible
promoter.
Unfortunately, these engineered resistance mechanisms are only effective against
the phage group from which the targeted genetic element was derived. Thus, the
systems are only likely to find an application with specific strains of high commercial
value. It is also noteworthy that many industrially important strains are refractory
to genetic manipulation. Therefore, there is a need for optimizing gene transfer
methods and developing novel expression vectors that will be suitable for practical
approaches.
In conclusion, several phage control measures are currently available, and their
use largely depends on the type and size of the manufacturing facilities. These
procedures include an adapted factory design, cleaning and disinfection, and appro-
priate air control. Although process failure is not always caused by phages, an efficient
phage-detection method should be available. In addition to characterizing the con-
taminating phages, effective phage control measures often require that the source of
contamination must be identified. The bacterial strain selection program also should
include an assay for lysogeny as well as provide several phage-resistant strains.
Fortunately, there are now many options for transforming a phage-sensitive strain
possessing desirable fermentation properties into a phage-resistant derivative. Finally,
the phage population within a factory is likely to change over time; therefore, control
measures should be periodically revisited.

ACKNOWLEDGMENTS
We thank H.-W. Ackermann, H. Brüssow, S. Labrie, and D. Tremblay for critical
reading of the manuscript. S.M. is grateful to the following organizations for funding
his bacteriophage research program: Natural Sciences and Engineering Research
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Control of Bacteriophages in Industrial Fermentations 295

Council of Canada, Fonds de recherche sur la nature et les technologies du Québec,

Agropur, Novalait, Inc., and Rhodia Food.

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