[advances in food and nutrition research] advances in food and nutrition research volume 67 volume...

CHAPTER SIX

Bacteriophages for Detection andControl of Bacterial Pathogens inFood and Food-ProcessingEnvironmentLubov Y. Brovko*,1, Hany Anany*,†, Mansel W. Griffiths**Canadian Research Institute for Food Safety, University of Guelph, Guelph, Ontario, Canada†Microbiology Department, Faculty of Science, Ain Shams University, Cairo, Egypt1Corresponding author: e-mail address: [email protected]

Contents

1.

AdvISShttp

Overview of Bacteriophage

ances in Food and Nutrition Research, Volume 67 # 2012 Elsevier Inc.N 1043-4526 All rights reserved.://dx.doi.org/10.1016/B978-0-12-394598-3.00006-X

242
1.1 Bacteriophage discovery 242 1.2 Bacteriophage biology 243 1.3 Life cycle of bacteriophage 245
2.
Using Bacteriophages as Biocontrol Tools for Bacterial Pathogens 248 2.1 Criteria for phage biocontrol in foods 249 2.2 How to select the proper phage for food applications? 251 2.3 Is it a safe tool? 252 2.4 How to apply phages in food? 253 2.5 What are the advantages and disadvantages of using phages as
biocontrol agents?
255 2.6 Commercialization and governmental approvals 256 2.7 Phage biocontrol 257 2.8 Phages for prevention of food spoilage 261 2.9 Phages for surface decontamination and controlling biofilm formation 262 2.10 Phage lysin as an alternative to whole phage application 263
3.
Bacteriophages for Detection of Bacterial Pathogens 264 3.1 Culture methods 265 3.2 Methods based on formation of bacteriophage–host cell complex 266 3.3 Reporter phages 270 3.4 Bacteriophage-mediated lysis as an indicator of the presence of
target pathogens
272 4. Conclusion 276 References 276
241

http://dx.doi.org/10.1016/B978-0-12-394598-3.00006-X

242 Lubov Y. Brovko et al.

Abstract

This chapter presents recent advances in bacteriophage research and their applicationin the area of food safety.

Section 1 describes general facts on phage biology that are relevant to theirapplication for control and detection of bacterial pathogens in food and environmentalsamples. Section 2 summarizes the recently acquired data on application of bacterio-phages to control growth of bacterial pathogens and spoilage organisms in foodand food-processing environment. Section 3 deals with application of bacteriophagesfor detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed.

The chapter is intended for food scientist and food product developers, and peoplein food inspection and health agencies with the ultimate goal to attract their attentionto the new developing technology that has a tremendous potential in providing meansfor producing wholesome and safe food.

1. OVERVIEW OF BACTERIOPHAGE

1.1. Bacteriophage discovery
Bacteriophages (phages) are bacterial viruses that only infect and multiply
within their specific hosts, disrupt bacterial metabolism, and cause the

bacterium to lyse. They were discovered a long time ago but the history

of bacteriophage discovery has been the subject of lengthy debates. Ernest

Hankin, a British bacteriologist, reported in 1896 that the waters of the

Ganges and Jumna rivers in India had marked antibacterial action against

Vibrio cholerae and that ingestion of the water of these rivers prevented spread

of cholera epidemics (Sulakvelidze, Alavidze, &Morris, 2001). He suggested

that an unidentified substance (which passed through fine porcelain filters

and was heat labile) was responsible for this phenomenon. A similar obser-

vation was made 2 years later by the Russian bacteriologist Gamaleya,

while working with Bacillus subtilis (Deresinski, 2009). However, their find-

ings were not explored further until in 1915 Frederick Twort, a British

pathologist, and a French-Canadian bacteriologist Felix d’Herelle working

at the Pasteur Institute in Paris independently reported in 1917 isolating

filterable entities that could destroy bacterial cultures and produce small

clear areas on bacterial lawns. D’Herelle called them “bacteriophages”—

bacteria eaters (Kutter & Sulakvelidze, 2005; Sulakvelidze et al., 2001;

Summers, 2005).

243Bacteriophages for Detection and Control

1.2. Bacteriophage biologyPhages are the largest group of viruses, utilizing species in the Bacteria and

Archaebacteria as hosts. Measuring between 20 and 200 nm (Ackermann &

DuBow, 1987), they are the most abundant form of life on the planet with

estimated 1031 phages in the biosphere (Kutter & Sulakvelidze, 2005).

Like other viruses, phages are infectious particles that have at least two

components: (1) a genome comprising nucleic acid surrounded by (2) protein

subunits that form a protective capsid (Ackermann, 2003). It was suggested

that the capsid plays three important roles in the phage life cycle: protecting

the phage genome (e.g., fromDNA-degrading enzymes); effecting phage ad-

sorption to a susceptible bacterium; and uptake of the phage genome into the

cytoplasm of the infected bacterium (Gill & Abedon, 2003).

The capsid encloses a single copy of the genome,which is usually onemol-

eculeofdouble-strandedDNA, single-strandedDNA,double-strandedRNA,

or single-strandedRNA(Kutter,Raya,&Carlson,2005).Somephageshavean

extremely small genome, for example, Escherichia coli phage R17, which only

contains four genes and has around 3600 bases. Others are relatively large,

for example, E. coli phage PB51 possesses a genome which is around

2.5�105 bases in length and encodes for over 240 genes (Birge, 1994).

Phages may be roughly categorized by shape into tailed, polyhedral

(icosahedral or quasi-icosahedral bodies), filamentous, and pleomorphic

phages (Ackermann, 2012). Figure 6.1 shows the main morphological struc-

tures of bacteriophage.

Of the 5500 phages examined by electron microscopy, about 96.2% are

tailed and only (3.7%) are polyhedral, filamentous, or pleomorphic

(Ackermann, 2007). In the tailed phages, the tail is a hollow tube, through

which the phage nucleic acid passes during infection. The size of the tail can

vary and some phages do not have a tail structure. In some phages, the tail is

surrounded by a contractile sheath, which contracts during infection of the

bacterium. At the end of the tail, the more complex phages, like T4, have a

baseplate and one or more tail fibers attached to it (Fig. 6.2). Tail fibers

contain proteins that recognize molecules on the surface of bacterial cell

walls, which provide the ability to attach only to host cells (Ackermann,

2009; Guttman, Raya, & Kutter, 2005).

The majority of bacteriophages are stable in a wide range of environ-

mental conditions. For instance, most tailed phages are stable in the pH

range from 5 to 9 and are inactivated by heating at 60 �C for 30 min

(Ackermann, 2007). Phages with sheathed tails and large heads are

Figure 6.1 Representative TEM images of different phage main morphological struc-tures. (A) fBC6 of B. cereus with extended tail. (B) g phage of B. anthracis. The capsidof the right particle shows a pentagonal outline indicating an icosahedral shape. (C)P22 of Salmonella Typhimurium. (D) FX174 phage of E. coli. (E) 37–14 phage of Thermusthermophilus showing outer capsid and inner vesicle. One particle at left (arrow) displaysa full, deformed vesicle. (F) MS2 phage of E. coli. (G) X phage of E. coli, showing unusualflexibility. (H) MVL51 phage of Acholeplasma laidlawii. (I) L2 phage of A. laidlawii. Barsindicate 100 nm. Final magnifications are �297,000 (A–C, E, F), �148,000 (D and E),�183,000 (H), and approximately �150,000 (I). Reproduced from Ackermann (2012).


Figure 6.2 Transmission electron microscope image of negatively stained E. coli O157:H7 T4-like phage particle, EcoM-AG2, showing different parts of its morphological struc-ture. (Image was taken by the Microscopy Unit, University of Guelph.)


apparently more sensitive to freezing and thawing than other types. Tailed

phages are best preserved by lyophilization or in liquid nitrogen after

addition of 15–50% glycerol, but some are quickly inactivated under these

conditions. Storage at 4 �C is a good alternative for most phages (Anany,

Chen, Pelton, & Griffiths, 2011; Guttman et al., 2005; Puapermpoonsiri,

Ford, & van der Walle, 2010).

1.3. Life cycle of bacteriophageThe phage life cycle can be one of two types, the productive or virulent

cycle and the temperate or lysogenic cycle (Ackermann, 2012; Guttman

et al., 2005). According to this, phages are classified as lytic (virulent) or

lysogenic (temperate).

Lytic phages infect bacterial cells causing inhibition of host metabolism

and subverting it to the production of phage progeny. The lytic cycle results

in the lysis of the bacterium accompanied by the release of multiple phage

particles. The new progeny phages produced by the host bacterium spread to

infect other cells. The time for the whole cycle is usually within 1–2 h and

the number of phage produced depends upon the phage type (Guttman

et al., 2005).

The typical lytic cycle of phages consists of the following sequential steps:

adsorption, penetration, latent period, maturation, and lysis.


Adsorption. Infection with tailed phages starts when the specialized

adsorption structures, such as fibers or spikes, bind to the specific surface

molecules on their target bacteria. The nature of the bacterial receptor

varies for different bacteria, they may be located on the cell wall, flagella,

pili, capsules, or the plasma membrane (Lindberg, 1973). The environ-

ment is an important factor in the adsorption process and some cofactors

may be required to enhance the adsorption. The most frequently required

cofactors are Caþþ ions, followed by Mgþþ ions (Ackermann & DuBow,

1987; Guttman et al., 2005). In general, phage adsorption to its host is a

function of phage-bacterium chemical and physical interaction (Gill &

Abedon, 2003).

Penetration or injection. For most phage groups, only the phage nucleic acid

enters the host and the shell remains outside. Mechanisms of nucleic acid

injection are different for phages of different morphology.

When the sheathed tailed phages (e.g., T4 phage) contact with the host

outer membrane receptors, conformational changes in the phage structure

are initiated that lead to the contraction of the tail sheath, which in turn

forces the hollow inner tube into the cell. The short-tail fibers help to anchor

the distal portion of the tail and baseplate to the cell surface receptors. At this

time, the whole tail structure shrinks and widens, bringing the internal

pin-like tube in contact with the outer membrane of the bacterial cell

and phage enzymes located on the tail tip degrade the bacterial cell wall.

As the tail tube punctures the outer and inner membranes of the cell, the

viral genome is injected through the tail tube into the host cell’s cytoplasm

(Kostyuchenko et al., 2003, 2005).

In sheathless tailed phages (e.g., phage l), the tail sheath does not contractduring DNA injection. However, filamentous DNA phages of E. coli (e.g.,

phage M13) seem to enter the cell by being drawn into the inner membrane

of the cell envelope while being uncoated; the DNA is released intracellu-

larly as the coat protein dissociates into subunits which remain in the

membrane (Gottesman & Oppenheim, 1994).

Latent period. Immediately after the entry of the viral genome, the expres-

sion of early proteins begins, which are needed to replicate the phage genome

and tomodify the cellularmachinery so that the synthetic capacity of the cell is

diverted to phage reproduction. During this stage, the synthesis of a number

of copies of the phage DNAoccurs. Each of these copies can then be used for

transcription and translation of a second set of proteins, called the late proteins

thatmake up the capsomeres and the various components of the tail assembly.

Lysozyme is also a late protein that will be packaged in the tail of the phage to


be used to escape from the host cell during the last step of the replication

process (Birge, 1994; Guttman et al., 2005).

Maturation or morphogenesis is the period during which the new phage

components are assembled into virions. Assembly can occur spontaneously

or with the help of specific enzymes. The DNA is packaged into

preassembled protein shells called procapsids. In most phages, their assembly

involves complex interactions between a specific scaffolding protein and the

major head structural proteins. In tailed phages, the head and tails are assem-

bled by separate pathways and are joined together after DNA encapsidation

(Guttman et al., 2005; Miller et al., 2003).

Lysis or release. Phages are liberated by lysis. Specific enzymes hydrolyse

the cell wall from inside, liberating infectious phages that are capable of

starting the cycle over again and infecting new susceptible host cells

(Ackermann, 2003; Guttman et al., 2005). The number of phages

produced depends on the phage type and the physiology of the host cell.

The tailed phages use two enzymes for the lysis of the host cell: lysin—an

enzyme capable of degrading the cell wall peptidoglycan and holin—

an enzyme that assembles pores in the inner membrane to let the lysin

reach the peptidoglycan layer. These enzymes disrupt the cell membrane

and cell wall causing the cell to burst, and phages are released into the

surrounding medium. The tailless phages encode a variety of single lysis-

precipitating proteins that sabotage the host peptidoglycan-processing

enzymes by different modes of action (Gottesman & Oppenheim, 1994;

Guttman et al., 2005; Maloy, Cronan, & Freifelder, 1994). The number

of new phages released per infected cell is called the “burst size”

(Guttman et al., 2005).

Some phages infect cells and incorporate their nucleic acid into the

genome of the host cell or exist as an episomal element, leading to a perma-

nent association as a prophage with the cell and all its progeny. During ly-

sogeny, phages neither produce virions nor lyse bacteria. These phages are

called temperate, and the cells that harbor a prophage are known as lyso-

genic. The lysogenic relationship between a temperate phage and its

host bacterium provides a safe home to the temperate phage genome, blocks

replication of nonvirulent homologous phages, and has the potential to alter

the phenotype of the host cell (Gill & Abedon, 2003). The lysogenic host

bacterium may carry prophage for many generations until it is reactivated

and produce new copies of phages that lead to lysis and release of progeny

phages. The mechanism of this reactivation varies between phages, but is

usually triggered when the host cell is placed under adverse environmental

Phage DNABacterialchromosome

ProphageCell division

Lysogenicbacterium

Induction of lytic cycle by excision of phage chromosome from bacterial chromosome

Lytic

cyc

le

Lysogenic cycle

Figure 6.3 Life cycle depicting the lytic and lysogenic pathways of a typical bacterio-phage when it infects bacterial cell.


conditions (Strauch, Hammerl, & Hertwig, 2007). The life cycle of bacte-

riophages is illustrated in Fig. 6.3.

2. USING BACTERIOPHAGES AS BIOCONTROL TOOLSFOR BACTERIAL PATHOGENS

The emergence of new antibiotic-resistant bacterial strains alongwith an

increase in consumers’ dislike of chemical preservatives in food has highlighted

the need for adoption of alternative and more natural approaches to mitigate

the effect of these “super-bugs.” Phages can infect and multiply within their

specific hosts even if they are antibiotic resistant. Host specificity is generally

observed at a strain level, species level, or, more rarely, at genus level. This

specificity led to the idea of using phages for directed targeting of dangerous

bacteria (Hagens & Loessner, 2010). Phages have been employed in human

and veterinary medicine to control bacterial infections after Felix d’Herelle

proved their effectiveness in 1919.D’Herelle used phages to treat bacillary dys-

entery and reduce the mortality rate due to cholera in India. D’Herelle also


injected his family aswell as his colleagueswith the phages to evaluate the safety

of this treatment (Sulakvelidze et al., 2001; Summers, 2005).

However, this path was initially abandoned with the discovery of anti-

biotics and as a result of the conflicting results of phage therapy (Summers,

2001). In Eastern Europe and the Soviet Union, however, research and

application of phages in human medicine continued. Phage therapy is

currently used in this region to treat bacterial infections in humans and

is used as a complement to conventional antibiotics (Kutter et al., 2010).

Phages have recently emerged as a novel approach in the food industry to

control bacterial contamination in food in a process called “biocontrol”

(Hagens & Loessner, 2010). As mentioned above, lytic phages have the abil-

ity to attach to bacteria and integrate into their cellular machinery, while

utilizing the host resources to reproduce. The release of new phages leads

to destruction of the bacterial cell. Lysogenic phages, on the other hand,

have the ability to remain dormant within their host and to transfer genes

from one bacterium to another, potentially allowing for the development

of more virulent and resistant pathogens (lysogenic conversions) (Greer,

2005; Guttman et al., 2005). Hence, virulent (strictly lytic) phages are the

obvious choice for food safety applications (Hagens & Loessner, 2010;

Mahony, McAuliffe, Ross, & van Sinderen, 2011).

2.1. Criteria for phage biocontrol in foodsHistorically, most research on phage biocontrol has been done in liquids and

usually with a high concentration of pure target bacteria (Hagens &

Loessner, 2010). In liquid environments, thermal motion-driven particle

diffusion and mixing due to either fluid flow or active swimming (bacterial

motility) increase the likelihood of phages to encounter and infect suscep-

tible host bacteria (Murray & Jackson, 1992). When it comes to food appli-

cations, one might face two major obstacles. First, a significant portion of

targeted foods is solid rather than liquid in nature. Second, bacterial contam-

ination would likely occur at very low numbers due to the expected high

hygiene standards in place (Hagens & Loessner, 2010). So, a high number

of phages is required (threshold of approximately 1�108 plaque-forming

units (PFU)/ml) to ensure sufficiently rapid contact with and infection of

the few targeted bacterial cells present. In other words, low numbers of

phages are unlikely to infect low numbers of bacteria simply because phages

and bacteria are unlikely to come into contact with each other. The bacterial

host concentration is not a limiting factor if the critical concentration of

phage numbers is reached and is able to cover the entire available surface


of the targeted food matrix (Hagens & Loessner, 2010). Experimental

verification of this claim has been achieved when a Salmonella phage (P7)

was incubated with its respective host at 24 �C for up to 2 h in Luria-Bertani

(LB) broth at varying ratios of phage and host cell concentrations, and the

surviving host cells were counted (Bigwood, Hudson, & Billington,

2009). It was observed that inactivation of Salmonella by P7 seemed to be

independent of the host concentration, with nearly complete inactivation

occurring at a phage concentration of around 5�108 PFU/ml. The

requirement of a minimum bacterial density as a prerequisite for successful

phage biocontrol was not accepted (Kasman et al., 2002). This was again

supported by studies on the control of spoilage bacteria on meat surfaces,

which suggest that phages can be effective biocontrol agents when the

population of host cells is as low as 46 colony-forming units (CFU)/cm2

(Greer, 1988).

The exact phage concentration that needs to be used in a given applica-

tion will depend on several factors: surface microstructure, which affects

phage diffusion rates and accessibility of target bacteria; the amount of fluid

that is available, which affects phage diffusion; and the target reduction levels

required (Hagens & Loessner, 2010). The results of phage-mediated inacti-

vation of foodborne pathogens in some reports using high phage concentra-

tions may be due to lysis fromwithout (Delbruck, 1940). Lysis fromwithout

occurs when host cells to which numerous phage particles are adsorbed are

inactivated rapidly in the absence of phage replication. In E. coli phage T4,

this “lysis from without phenomenon” is mediated by a lysozyme on the

baseplate (Abedon, 1999). It occurs when more than 100 phages are

adsorbed on a bacterial cell, which is followed by swelling and bulging of

the membrane within 5–10 min after adsorption. Finally, this results in

the formation of holes in the cell wall, through which cytoplasmic contents

may escape (Tarahovsky, Ivanitsky, & Khusainov, 1994).

In some reports on the use of phage for biocontrol of foodborne path-

ogens, the ratio of phages to host cells is described as multiplicity of infection

(MOI) (O’Flynn, Ross, Fitzgerald, & Coffey, 2004). However, it was

suggested that PFU/CFU ratio can be considered as a more descriptive term

in food applications where there may be physical barriers preventing or

slowing phage adsorption (Bigwood et al., 2009; Whichard, Namalwar,

& William, 2003).

Another important factor that should be considered for potential phage

application is incubation temperature. The efficacy of phages to control

target pathogens should be tested both at higher than normal storage


temperature, which provides good growth conditions for the undesired con-

taminants, as well as under recommended storage conditions (Hagens &

Loessner, 2010; Mahony et al., 2011).

2.2. How to select the proper phage for food applications?Phages intended for use in food have to meet several requirements. Obvi-

ously, only strongly lytic phages should be considered for biocontrol appli-

cations. Their host range should cover all epidemiologically important

strains of the target microorganism, and, of course, safety of the phage

application should be tested very thoroughly. It should be determined if

their DNA carries any genes coding for virulence factors like toxins and/

or lysogenic properties (Hagens & Loessner, 2010; Mahony et al., 2011).

So, the complete genome sequence of the phage should be known to

fully assess its applicability in food systems as a biocontrol agent. A second

phenomenon that should be kept in mind when selecting candidate

phages is generalized transduction, which is a process whereby host DNA

is packaged into phage heads, rather than phage DNA. This might lead to

introduction of new genes into the recipient bacterium (Ikeda &

Tomizawa, 1965). Distribution of a virulence-associated genome region

via transduced DNA has been reported for several pathogens (Cheetham &

Katz, 1995). So, only phages that display minimal transduction frequencies

should be used for biocontrol purposes.

In addition, selected phages should have a broad host range by infecting a

large number of the target species and/or genus (Hagens & Loessner, 2010).

Phages possessing a narrow host range may present a problem for biocontrol

purposes, as in some species of bacteria there are numerous subtypes that

need to be controlled. Therefore, an effective phage should have a

“Goldilocks” host range, not too narrow and not too broad. Felix O1 is

a perfect example of broad host range phages; it lyses 96–99.5% of Salmonella

serovars. However, the limitation of a narrow host range may be mitigated

by using phage cocktails (McIntyre, Hudson, Billington, & Withers, 2007).

Generally, host range criteria should be assessed based on the final application

and the required effect.

Stability at different storage and application conditions is another

important aspect that should be defined in the selected phage. Indeed, it

is important to test phages for durability within the intended-use environ-

ment, which requires investing resources in phage characterization (Gill &

Hyman, 2010). From the economical point of view, the ability to propagate

in surrogate nonpathogenic hosts for large-scale commercial production is


essential for phages that are considered for biocontrol of pathogens in food

(Hagens & Loessner, 2010).

2.3. Is it a safe tool?Phage preparations should be safe, showing no adverse effect upon oral

feeding (Gill & Hyman, 2010). As they are bacterial viruses, infection of

mammalian cells is unlikely. All available evidence indicates that their oral

consumption is entirely harmless to humans as they represent a normal com-

ponent of an everyday diet. Oral toxicity tests on rats that were given phages

against Listeria monocytogenes at a dose of 2�1012 PFU/kg body weight/day

showed no signs of abnormality with regards to histological changes, mor-

bidity, or mortality (Carlton, Noordman, Biswas, de Meester, & Loessner,

2005). Similar results were found in a human study with E. coli T4 phages

that were added to drinking water (Bruttin & Brussow, 2005). Individuals

with HIV and other immunodeficiency diseases and healthy volunteers have

also been intravenously injected with purified phages (e.g.,FX174) withoutany apparent side effects (Atterbury, 2009). Indeed, early phage therapy

pioneers demonstrated safety by ingesting preparations themselves as we

mentioned earlier in this chapter. Moreover, thousands of people have

received phage therapy in Eastern countries, especially the former Soviet

Union and Poland with great success in treating the causal agents (Kutter

et al., 2010). The phages used not only were administered orally or superfi-

cially, but alsowere injected intramuscularly, intravenously, and even into the

pericardium and carotid artery without any adverse effect being observed.

Phages are natural components of the microflora and are found ubiqui-

tously. They are commonly isolated from soil, water, food, sewage, and from

different environments containing their bacterial hosts (Kutter &

Sulakvelidze, 2005). It was reported that freshwater environments contain

up to 109 phages/ml, and up to 107 phage-like particles/ml were found

in marine surface systems. Similar numbers have been reported for terrestrial

ecosystems such as topsoil (Rohwer & Edwards, 2002). They are detectable

from the farm to the retail outlet and are remarkably stable in all environ-

ments (Greer, 2005). Phages have been isolated from a number of foods, like

lettuce, pork, oysters, mussels, mushrooms, turkey, chicken, cheese, yogurt,

buttermilk, and beef and so they are ingested by everyone every day

(Hudson, Billington, Carey-Smith, & Greening, 2005). E. coli phages have

been isolated from fresh chicken, pork, ground beef, mushrooms, lettuce,

raw vegetables, chicken pie, and delicatessen food, with numbers up to

104 PFU/g (Allwood et al., 2004). Moreover, Campylobacter phages have


been isolated from chicken at levels of 4�106 PFU/g (Atterbury, 2003),

and Brochothrix thermosphacta phages have been reported in beef (Greer,

1983). In addition, fermented foods were found to have high numbers of

those phages infecting the fermentation flora. For example, one study

described 26 different phages isolated from commercial cabbage (Sauerkraut)

fermentation plants (Lu, Breidt, Plengvidhya, & Fleming, 2003). Swiss

Emmental cheese samples yielded phages active against Propionibacterium

freudenreichii at levels ranging from 14 to 7�105 PFU/g (Gautier, Rouault,

Sommer, & Briandet, 1995). Sixty-one phages infecting Streptococcus

thermophilus andLactobacillus delbrueckii subsp. bulgaricus have been isolated from

Argentinean dairy plants at numbers of up to 109 PFU/ml (Suarez,

Quiberoni, Binetti, & Reinheimer, 2002). Moreover, phages are seen as

“green” and environmentally friendly and could be considered as a natural

alternative to chemical preservatives (McIntyre, Hudson, Billington, &

Withers, 2012). Although phages are and will be present forever in foods,

the consumer’s perception of adding viruses to foods will, arguably, be the

most critical hurdle to be overcome in order for phages to be used widely

for biocontrol of bacterial pathogens within food (Strauch et al., 2007).

2.4. How to apply phages in food?Phage biocontrol strategies in food-processing facilities should be conve-

nient, economical, and not affect the process itself. Hagens and Loessner

discussed in detail the current different proposed methods of the industrial

application of phages in food in their review (Hagens & Loessner, 2010).

Phages can be applied at different or even multiple points in the food-

processing facility, where the likelihood of bacterial contamination is

highest, thereby enhancing the killing efficiency and reducing the potential

for bacteria to acquire resistance. Phage application could be useful at all

stages of production in the classic “farm-to-fork” approach throughout

the entire food chain (Garcıa, Martınez, Obeso, & Rodrıguez, 2008).

Phages can be added by dipping, spraying, or as a liquid to a large volume

of food material. These methods may not be ideal as they could be wasteful

and potential decay of the phage particles could happen as a consequence

of inclusion of other materials within the wash fluid such as sanitizer.

Moreover, if the washing fluids themselves support bacterial growth, then

the potential for bacterial evolution of phage resistance might exist.

When phages are added directly to a batch of food, two major problems

may be detected: dilution of phages and evolution of bacterial resistance.


The dilution problem can be overcome by adding large numbers of phages

or by applying phages before the mixing or disruption of foodmaterials, such

as spraying carcasses before processing. However, phage resistance can

be addressed by regular sanitation of the equipment using highly efficient

disinfectant (Hagens & Loessner, 2010). Immobilization of phages on food

packaging materials might be considered as an interesting alternative

approach for application of phages. Encapsulation can be used for phage

immobilization that can be used to broaden application areas. Phages have

been adsorbed onto a solid matrix (such as skim milk powder, soya protein

powder, and whey protein powder) and then dried by heating under vacuum

(Murthy & Rainer, 2008). The adsorbed phages were embedded in a solid

support such as microbeads, cellulose-based material, and carbohydrate-based

material. It was suggested that these immobilized phages might be encapsu-

lated and incorporated into a capsule or tablets to be protected from the

physico-chemical stresses of their environment. The release rate was related

to the material used for encapsulation. It was claimed that this innovation

could be used for phage therapy applications in human and veterinary med-

icine and in aquaculture in addition to agricultural applications. For instance,

encapsulated phages can be used as a feed additive for fish, livestock, birds, and

poultry to aid in reducing the shedding of target pathogens.

The electrospinning process for the encapsulation and immobilization

of T7, T4, and l phages in electrospun polymer nanofibers was demon-

strated as a potential technique for phage immobilization (Salalha, Kuhn,

Dror, & Zussman, 2006). The encapsulated phages managed to survive the

electrospinning process while maintaining their infectivity. These

immobilized phages were able to infect their target bacterial host after

dissolving the polymer fibers, thus releasing them from the nanofibers.

The potential of nanoencapsulating of a broad lytic phage (F-PVP-SE1) in a water-in-oil-in-water multiple emulsion has also been investi-

gated (Costa et al., 2009). Cocktails of E. coli O157:H7 or L. monocytogenes

phages were physically immobilized on cellulose membranes and were

shown to effectively control the growth of their host in raw and ready-

to-eat meats, respectively, under different storage temperatures and

packaging conditions (Anany, Chen, et al., 2011). Recently, attempts have

been made to encapsulate different phages using different chemical formu-

lations in alginate microspheres and gelatin capsules to provide protection

for these phages against the low pH found in the stomach upon oral

delivery (Jiayi et al., 2010; Ma et al., 2008; Stanford et al., 2010;

Yongsheng et al., 2012).


2.5. What are the advantages and disadvantages of usingphages as biocontrol agents?

There are many advantages of phages over traditional antimicrobials such as

antibiotics and sanitizers. The most obvious ones are phages target only

specific bacteria so there is no adverse effect on the natural microflora;

no serious side effects on humans have been detected; simple and low-cost

production; relatively high storage stability under different environmental

conditions; and they are self-replicating, so there is no need to carry out

repeat dosing. However, they have some drawbacks including limited host

range, the risk for the development of resistant mutants, and the potential

for the transduction of virulent characters from one bacterial strain to

another.

In addition, the effectiveness of using phage for bacterial control depends

on the likelihood that phage and susceptible bacteria are in the same spot.

Another important drawback is that most current research on their efficacy

have involved experiments with artificially inoculated foods that do not

necessarily reflect the real commercial environments where phages will be

applied (Coffey, Mills, Coffey, McAuliffe, & Ross, 2010; Greer, 2005;

Hagens & Loessner, 2010; Hanlon, 2007; Mahony et al., 2011).

However, the advantages of phages for food applications outweigh their

disadvantages; for instance, spontaneously occurring phage-resistant mutants

are not likely to significantly influence treatment efficacy and the complex

phage resistance mechanisms common in bacteria can be overcome by

screening for broad host range phages and/or use of phage cocktails

(Hagens & Loessner, 2010). From another perspective, it has been noted that

a phage-resistant strain of E. coli O157:H7 had a smaller, more coccoid cel-

lular morphology than the parental strain and it reverted to phage sensitivity

within 50 generations (O’Flynn et al., 2004). Likewise, phage-resistant

mutant strains of Salmonella Enteritidis lost the O-polysaccharide layer,

which is required for phage adsorption, and as a result became avirulent

(Santander & Robeson, 2007).

In addition, increasing the concentration of the applied phages will

increase the likelihood for phages meeting target bacteria (Greer, 2005).

Another possible way to overcome the possibility of phages to mediate

horizontal transfer of virulence genes is to use phages with no genetic

material, termed “ghost phages” or severely damaged nucleic acid or

lysis-deficient phages as biocontrol agents (Hudson et al., 2010; Paul

et al., 2011).


2.6. Commercialization and governmental approvalsBased on the recent extensive scientific research with promising results that

support using phages as biocontrol agents in foods, several companies

throughout North America and Europe are adopting this new technology

and starting to release commercial products to the market. For instance,

ListShieldTM (formerly LMP-102TM) is a bacteriophage mixture produced

by Intralytix Inc. (USA) that targets L. monocytogenes in ready-to-eat meat

products, it has no preservatives or allergens and most importantly, does

not change the taste, color, odor, or quality of the food. This preparation

has gained the approval of the FDA to be used as a safe food additive on

ready-to-eat meat and poultry food products prior to packaging (www.

intralytix.com, www.fda.gov). The same company also produces

EcoShieldTM that is used to control E. coliO157. Another phage preparation

comprising a single lytic Listeria phage—LISTEXTM P100 (www.

micreosfoodsafety.com)—has received the highly desirable GRAS (gener-

ally recognized as safe) status for its use in all food products. E. coli and Sal-

monella phage preparations are also available (www.omnilytics.com) and

have been approved as a spray on cattle and chickens, respectively, prior

to slaughter of the animals to decrease pathogen transfer to meat (Monk,

Rees, Barrow, Hagens, & Harper, 2010; Sulakvelidze & Barrow, 2005).

Moreover, phage preparations active against Pseudomonas putida that were

developed for treatment of tomato and pepper against bacterial spot

diseases (www.omnilytics.com) have been approved for use by the U.S.

Environmental Protection Agency (EPA) (Balogh, Jones, Iriarte, &

Momol, 2010). The U.S. EPA has also recently approved the use of an

anti-E. coli O157:H7 phage product to be sprayed or used as a wash on

cattle hides prior to slaughter (Hagens & Loessner, 2010). Considering

the extent of the research being performed in this area, it is likely that

more phage products will emerge in the near to mid-term future.

Companies who seek permission for commercial use of phages for bio-

control should consider some safety issues. For instance, phage preparations

have to be tested for the absence of the pathogen, toxins, and/or virulence

factors. Also, a monitoring system for development of phage-resistant bac-

terial cells would be sensible way to ensure the effectiveness of the phage

preparation. Genome analysis and bioinformatic studies should be done

to ensure the absence of virulence genes and any genes that might lead to

mutation of the virulent phages to be temperate variants and lysogenize a

pathogen by horizontal gene transfer (Strauch et al., 2007).

http://www.intralytix.com

http://www.intralytix.com

http://www.fda.gov

http://www.micreosfoodsafety.com)

http://www.micreosfoodsafety.com)

http://www.omnilytics.com

http://www.omnilytics.com


It is important to mention that phages do not represent a “magic bullet”

and it is unlikely that they will replace chemical preservatives and cleaning

agents. Phage application should be considered as one of the approaches in

the so-called hurdle technology in combination with existing methods or

other natural antimicrobial agents, such as bacteriocins, to enhance food

safety (Leverentz et al., 2003; Martınez, Obeso, Rodrıguez, & Garcıa,

2008; Roy, Ackermann, Pandian, Picard, & Goulet, 1993).

2.7. Phage biocontrolOver the past two decades, concentrated research efforts have been devoted

to phage biology to enhance our knowledge of these interesting organisms

and their possible applications. Foodborne diseases and outbreaks are costly

in any country, and recent estimates showed that they cost the U.S. econ-

omy from about $51.0 to $77.7 billion (Scharff, 2012) and cost Canada

around $1.33 billion CAD a year (Snowdon, Buzby, & Roberts, 2002).

The application of phages to reduce pathogenic bacteria during the pre-

and postharvest stages of food production has shown promise (Strauch

et al., 2007). Moreover, the recent FDA approval of phage preparations

as food additives for preservation has also triggered the search for new

applications for these natural bacterial killers. In this context, we provide

below examples of studies that investigate biocontrol ability of phages against

some common foodborne pathogens.

2.7.1 Phages to control bacteria in food of plant originMany studies aimed at assessing the ability of phage to eliminate bacterial

pathogens from food of plant origin and to control plant diseases have been

carried out. In the latter approach, there were several promising trials to con-

trol plant diseases such as bacterial blotch, bacterial spot, and fire blight in

cultivated mushrooms, tomato, and apple, respectively, by using phages

(Greer, 2005). Moreover, a mixture of two phages of Xanthomonas campestris

pv. vesicatoria and Pseudomonas syringae pv. tomato has received approval from

the U.S. EPA to be used commercially to control bacterial spot on tomatoes

and peppers and bacterial speck on tomatoes (Maura & Debarbieux, 2011).

However, the application of phages in an open field is associated with some

difficulties such as uncontrolled environmental factors including tempera-

ture, sun exposure and humidity, uneven phage distribution and allocation,

and rapid inactivation of the applied phages (Maura & Debarbieux, 2011).

These problems may be overcome by using phages in greenhouses and by


using special formulae during phage preparation that could reduce the rate of

phage inactivation by sunlight and rain washout (Balogh et al., 2010).

Listeria phage cocktails alone and in combination with nisin were tested

on honeydew melon and apple slices (Leverentz, Conway, Janisiewicz, &

Camp, 2004; Leverentz et al., 2003). In honeydew melon, a reduction of

2–4.6 log units of bacteria was detected using phage alone and a

synergistic effect was detected when nisin was added. Reduced activity

and phage decay were noticed in the apple slices, which might be due to

the phage sensitivity to low pH or presence of inhibitory apple-derived

components (Guenther, Huwyler, Richard, & Loessner, 2009). When

the same experiment was repeated using a cocktail of four Salmonella lytic

phages to control Salmonella Enteritidis, reductions of approximately 3.5

log units on honeydew melon slices stored at 5 and 10 �C and

approximately 2.5 log units on slices stored at 20 �C were achieved

(Leverentz et al., 2001). It was reported that P100 and A511 Listeria

phages resulted in up to a 4-log unit reduction in their host count on

cabbage and iceberg lettuce stored for 6 days at 6 �C (Guenther et al., 2009).

Another study evaluated the ability of two broad host range Salmonella

bacteriophages (SSP5 and SSP6) to control Salmonella Oranienburg in vitro

and on experimentally contaminated alfalfa seeds. There was an incomplete

lysis during the in vitro treatment and no significant reduction in the viable

Salmonella population in treated seed (Kocharunchitt, Ross, & McNeil,

2009). The use of a low MOI (�70) might be the reason for these negative

results. A bacteriophage cocktail (ECP-100) containing three Myoviridae

phages lytic for E. coli O157:H7 was used to control the pathogen on con-

taminated fresh-cut iceberg lettuce and cantaloupe. Significant reductions of

the target cells were observed on both treated food products after incubation

at 4 �C for 2 and 7 days, respectively (Sharma, Patel, Conway, Ferguson, &

Sulakvelidze, 2009).

2.7.2 Phage to control bacteria in food of animal originUsing phages as biocontrol agents in various food products of animal origin

and to control the growth of pathogenic bacteria in diverse species of food-

producing animals has shown very promising results. Phage treatment of

food-producing animals reduces the probability of contamination of the

resulting food products during processing. Risk assessment models indicate

that a 1- or 2-log reduction in the amount of pathogens shed in feces of the

slaughtered animal could reduce the risks to the consumers by 45% and 75%,

respectively (Havelaar et al., 2007).


In another example, a broad host range lytic Listeria phage P100 was used

to control L. monocytogenes in soft cheese (Carlton et al., 2005). The efficacy

of the phage in controlling artificial contamination during manufacture was

evaluated; it was found that no cells were able to regrow at higher phage

doses. It was also reported that P100 and A511 Listeria phages were able

to cause rapid reduction of L. monocytogenes counts below the level of direct

detection in chocolate milk andmozzarella cheese brine, after storing at 6 �Cfor 6 days and by up to 5 log units in hot dogs, sliced turkey meat, smoked

salmon, and seafood (Guenther et al., 2009). In another study, Listeria phages

were used in combination with a proactive culture (Lactobacillus sakei TH1)

to reduce L. monocytogenes on sliced cooked ham (Holck & Berg, 2009).

Phages alone caused a 10-fold reduction of L. monocytogenes in the tested

samples, while using phages and proactive culture resulted in a 100-fold

reduction after 14–28 days of storage.

A significant reductionwas reported in thenumberofSalmonellaEnteritidis

PT4 recovered from artificially contaminated chicken skin samples after

immersion in a suspension containing a cocktail of three lyticSalmonella phages

and stored for 9 days at 5 �C (Fiorentin, Vieira, & Barioni Junior, 2005). In a

similar experiment, chicken carcasses artificially contaminated with Salmonella

were used as a model for surface disinfection using phages (Atterbury, 2009).

Itwas found that a cocktail of phages could reduceSalmonella recovery bymore

than 1000-fold compared with untreated controls. The broad host range Felix

O1 Salmonella phage and a related phage variant reduced the growth of Salmo-

nella Typhimurium DT104 on chicken frankfurters by about 2 log units

(Whichardetal., 2003). Ina recent study, theapplicationof thebroadhost range

virulent Salmonella Typhimurium phage FO1-E2 resulted in about a 3-log

reduction in bacterial count in different RTE foods stored at 8 �C(Guenther, Herzig, Fieseler, Klumpp, & Loessner, 2012).

O’Flynn et al. reported that treatment with a cocktail of three E. coli

O157:H7-specific phages eliminated the organism from seven of nine

artificially contaminated beef surfaces after incubation at 37 �C for 1 h,

and the two remaining samples had a very low bacterial count. A 5-log

reduction in cell number was observed when the experiment was performed

in broth culture (O’Flynn et al., 2004). This might illustrate the influence

of food matrices on the efficiency of phage biocontrol applications and that

culture broth studies are of limited use to indicate the efficacy of phage

applications in food matrices (Rees & Dodd, 2006a).

It was reported that j 29C phage significantly reducedCampylobacter cell

numbers on chicken skins that were contaminated with this pathogen


(Goode, Allen, & Barrow, 2003). Moreover, other Campylobacter phages jCP8 and j CP34 were used to control the growth of their host in exper-

imentally inoculated chicken (Loc Carrillo et al., 2005). Both phages were

able to survive ingestion; however, j CP34 was more efficient in reducing

Campylobacter jejuni numbers in ceca of the chicken. Interestingly, the lytic

broad host range campyphage j71 was able to delay the colonization of

C. jejuni in 10-day-old chicks when it was used as a prophylactic agent,

while it caused a 3-log reduction in the counts of this pathogen when used

as a therapeutic agent (Wagenaar, Bergen, Mueller, Wassenaar, & Carlton,

2005). More examples for targeted pathogens and food products are pres-

ented in Table 6.1.

Table 6.1 Examples of using bacteriophages as biocontrol agents for themost commonfoodborne pathogen and spoilage bacteria in foodTargetedbacterialpathogen Type of food Reference(s)

Campylobacter

jejuni

Poultry

products

Atterbury, Connerton, Dodd, Rees, and

Connerton (2003a, 2003b), El-Shibiny,

Connerton, and Connerton (2005), Havelaar et al.

(2007), and Wagenaar et al. (2005)

Cronobacter

sakazakii

Infant milk

formula

Kim, Klumpp, and Loessner (2007), Young-Duck

and Jong-Hyun (2011), and Zuber et al. (2008)

E. coli Beef/poultry

products

Abuladze et al. (2008), Anany, Chen, et al. (2011),

Dini and de Urraza (2010), Echeverry et al. (2009),

and O’Flynn et al. (2004)

Plant

products

Abuladze et al. (2008), Jassim, Abdulamir, and Abu

Bakar (2012), and Viazis, Akhtar, Feirtag, and

Diez-Gonzalez (2011)

Listeria

monocytogenes

Produce/

fruits

Leverentz et al. (2003, 2004, 2006)

Dairy

products

Carlton et al. (2005) and Guenther and Loessner

(2006)

Raw and

RTE meat

Anany, Chen, et al. (2011), Bigot et al. (2011),

Dykes and Moorhead (2002), Holck and Berg

(2009), Soni, Nannapaneni, and Hagens (2010),

and Zhu, Du, Cordray, and Ahn (2005)

Table 6.1 Examples of using bacteriophages as biocontrol agents for the mostcommon foodborne pathogen and spoilage bacteria in food—cont'dTargetedbacterialpathogen Type of food Reference(s)

Pseudomonas spp. Beef Greer (1986)

Mushrooms Kim, Park, and Kim (2011)

Fish Sanmukh, Meshram, Paunikar, and Swaminathan

(2012)

Dairy

products

Sillankorva, Neubauer, and Azeredo (2008a)

Salmonella spp. Beef/poultry

products

Atterbury (2007), Echeverry et al. (2009),

Fiorentin et al. (2005), Goode et al. (2003),

Guenther et al. (2012), Higgins et al. (2005), and

Whichard et al. (2003)

Plant

products

Kocharunchitt et al. (2009) and Leverentz et al.

(2001, 2006)

Cheese Modi, Hirvi, Hill, and Griffiths (2001)

Compost Heringa, Kim, Jiang, Doyle, and Erickson (2010)

Waste water Turki, Ouzari, Mehri, Ben Ammar, and Hassen

(2012)

Vibrio harveyi Shrimps Karunasagar, Shivu, Girisha, and Krohne (2007)


2.8. Phages for prevention of food spoilagePhage was considered as a promising agent for the suppression of growth of

spoilage bacteria in different beverage and food matrices to extend shelf life

of these products. In this context, lytic phage, SA-C12, was found to be stable

in beer and controlled the growth of 56 strains ofLactobacillus brevis in commer-

cial beer (Deasy,Mahony,Neve,Heller, & van Sinderen, 2011). Additionally,

the retail shelf life of raw chilled beef was extended significantly from1.6 to 2.9

days after Pseudomonas-specific lytic phage application (Greer, 1988). How-

ever, when similar work was carried out using naturally contaminated beef

samples and Pseudomonas phage mixture, the shelf life was not significantly

affected (Greer & Dilts, 1990). This may be due to the narrow specificity of

the used phages that were unable to infect all the spoilage bacteria present.

Another attempthasbeenmade to investigate theabilityofB. thermosphacta lytic


phage to control the growth of its host and extend the shelf life of pork adipose

tissue (Greer & Dilts, 2002). It was found that bacterial counts were reduced

after 2 days of storage at both 2 and 6 �C but the growth of phage-sensitive

and -resistant strains was detected after this period. However, phage treatment

increased the shelf life from 4 days in the control samples to at least 8 days.

2.9. Phages for surface decontamination and controllingbiofilm formation

Biofilm formation is an important problem in the food industry because itmay

represent an important source of contamination for food materials coming

into contact with biofilm-containing areas and causing food spoilage or trans-

mission of diseases (Bonaventura et al., 2008). Once formed, biofilm allows

pathogens to persist in the food environment for prolonged periods and to

resist treatment with antimicrobial and sanitizing agents (Folsom & Frank,

2006). Several studies have described the use of phages for surface decontam-

ination and to control formation of biofilms by various pathogens. The effec-

tiveness of different phages to remove Listeria from stainless steel and

polypropylene surfaces was investigated (Roy et al., 1993). It was found that

phage treatment alone was able to achieve approximately a 3-log cycle

decrease in cell number. In the same study, theusedphageswere also evaluated

for their ability to tolerate inactivationby a quaternary ammoniumcompound

(QUATAL) used for cleaning, and it was found that theywere not inactivated

by QUATAL at concentrations up to 50 ppm. A combination of phage and

40 ppmQUATAL resulted in a 5-log reduction in levels ofListeria attached to

the surface. Hibma, Jassim, and Griffiths (1997) isolated a phage that was spe-

cific for L-forms ofListeria, inwhich the cellwall structure is either deficient or

absent, and used this phage to control biofilm formation by this bacterium.

The phage was as successful as lactic acid (130 ppm) at inactivating preformed

L-form biofilms on stainless steel; both reduced viable cell numbers by 3-log

cycles over a 6-h period. In a more recent study, Listeria phage P100 was

able to control biofilm formation of L. monocytogenes on stainless steel surfaces

with a mean reduction of 5.29 log CFU/cm2 (Montanez-Izquierdo,

Salas-Vazquez, &Rodrıguez-Jerez, 2012). There are many promising studies

on the use of phages to control biofilm formation by bacteria such as Pseudo-

monas spp. (Knezevic & Petrovic, 2008; Knezevic et al., 2011; Pires,

Sillankorva, & Azeredo, 2011; Sillankorva, Neubauer, & Azeredo, 2008b),

C. jejuni (Siringan, Connerton, Payne, & Connerton, 2011), and

Staphylococcus epidermidis (Curtin & Donlan, 2006).

The phage mixture BEC8 was investigated to control the growth of

enterohemorrhagic E. coliO157:H7 on some food-processing surfaces such


as stainless steel and ceramic tiles (Viazis et al., 2011). The growth on both

surfaces was below the detection limit following BEC8 phage mixture treat-

ment for 10 min at 37 �C or 1 h at 23 �C. Enterococcus faecalis-specific lyticphage jSUT1 produced a significant reduction in bacterial cell number

on hard and porous surfaces contaminated with enterococci (McLean,

Dunn, & Palombo, 2011).

2.10. Phage lysin as an alternative to whole phage applicationLysins are enzymes produced by lytic phages, which play a role in degrada-

tion of the bacterial cell wall through targeting its various peptidoglycan

bonds to allow the newly formed progeny phages to be released from the

host cell (Borysowski, Weber-Dabrowska, & Gorski, 2006). As lysin

enzymes attack the cell wall peptidoglycan, they are highly effective against

Gram-positive bacteria when added externally and may be used as biocon-

trol agents to enhance food safety (Fischetti, 2008). In this context, lysin

could be added as a purified protein directly to food or feed, or via lysin-

secreting recombinant bacteria (Borysowski et al., 2006). An example for

the latter case was demonstrated using recombinant Lactococcus lactis cells

containing listerial lysin encoding genes to lyse L. monocytogenes in the sur-

rounding medium (Gaeng, Scherer, Neve, & Loessner, 2000). This study

also showed that the expression of functional lysin by L. lactis was detected

in the presence of lactose that is used in milk fermentation. These promising

results suggested the possibility of using these recombinant starter lactococcal

cultures to selectively protect dairy products against L. monocytogenes

contamination. Growth of Staphylococcus aureus in pasteurized milk was

controlled by addition of purified lysin at 37 �C (Obeso, Martınez,

Rodrıguez, & Garcıa, 2008). Forty-eight strains of Clostridium perfringens

were lysed by murein hydrolase (lysin) enzyme that is produced by

C. perfringens phage j3626 (Zimmer, Vukov, Scherer, & Loessner, 2002).

Similar approaches for lysin application were investigated to control the

growth of phytopathogenic bacteria. It was shown that when recombinant

lysozyme of Erwinia amylovora phage Ea1h was applied on immature pears

after inoculationwithE. amylovora, disease symptoms such as ooze formation

and necrosis were retarded or inhibited (Kim, Salm, & Geider, 2004).

Alternatively, transgenic plants able to produce lysin enzyme at the inter-

cellular spaces of the plant to kill bacteria at a very early stage of infection

could be developed (During, Porsch, Fladung, & Lorz, 1993; Hanke,

Norelli, Aldwinckle, & During, 1999).

The absence of bacterial resistance against lysin is considered as a major

advantage of using phage lysins (Fischetti, 2010), as the bacterial cell would


have to modify the structure of its cell wall to avoid enzymatic action.

It was found that exposing bacteria to a particular lysin for 40 reproductive

cycles did not give any resistant strains (Fischetti, 2010). However, the pro-

duction of lysin is expensive and, moreover, they are relatively unstable

large proteins that are prone to proteolysis and lose its activity in some foods

(Coffey et al., 2010).

Given the aforementioned data and information, it can be concluded

that phages and their lysin enzymes may be applied along the farm-to-fork

continuum to enhance food safety.

3. BACTERIOPHAGES FOR DETECTION OF BACTERIALPATHOGENS

The availability of rapid and sensitive methods for detection and iden-

tification of bacterial pathogens in food and food-processing environments is

essential for both the food industry and food inspection agencies all over the

world. Rapid methods often lack the required sensitivity and, hence, require

long pre-enrichment steps, which make them more time- and labor inten-

sive. The ability of bacteriophages to only infect and propagate in specific

bacteria makes them an ideal tool to use for the detection of pathogens.

The abundance of bacteriophages in nature and the possibility to isolate

phages with the required specificity allow the development of assays with

a tailored specificity toward the target bacterium. In addition, their ability

to reproduce within the host cell provides an opportunity to increase the

sensitivity of the assay using this “built-in” amplification step. The entire

infection process starting from sensing/finding the specific cell in a complex

environment and ending with bacterial cell lysis and reproduction of mul-

tiple progeny phages takes only 1–2 h, which allows development of rapid

assays. A variety of bacteriophage-mediated assays to detect bacteria based

either on the entire infection process or on its separate stage(s) has been

suggested. The techniques used in these phage-mediated protocols include

visual, optical, and electrochemical detection, or the phages themselves

can be used as the signal through the development of immunoassays with

antibodies raised against the phage in question, or by the use of molecular

techniques such as PCR.

The use of bacteriophage for bacterial detection has been the subject of

many reviews (Goodridge & Griffiths, 2002; Griffiths, 2010; Hagens &

Loessner, 2007; Loessner, 2005; Mandeville, Griffiths, Goodridge,

McIntyre, & Ilenchuk, 2003; Marks & Sharp, 2000; Petty, Evans,


Fineran, & Salmond, 2007; Rees & Loessner, 2005; Schmelcher & Loessner,

2008; Smartt & Ripp, 2011). Recent advances in the area, newmethods and

their experimental setup, analytical characteristics such as specificity,

sensitivity, time of analysis, and their pros and cons are discussed in this

chapter.

3.1. Culture methodsThe classic technique for phage-mediated bacterial identification/detection

is denoted as phage typing. This approach relies on observation of bacterial

lysis following the introduction of a specific bacteriophage to the tested cul-

ture. If the bacterium is susceptible to the phage, it will be lysed, and there-

fore will not grow. The lack of growth results in formation of a clear zone if

solid nutrient medium is used for cultivation of the bacterium, or in absence

of turbidity in the case of liquid medium. Visual observation or simple tur-

bidimeters (spectrophotometers) can be used to record the results. Digital

cameras and/or automated turbidimeters are used for high-throughput

assays (Anany, Lingohr, et al., 2011). Phage sets that provide unique lysis

patterns for almost all epidemiologically important strains of bacteria have

been developed. This method is very specific and simple; however, it

requires a significant number of target cells to be present at the moment

of infection; as a result, the analysis generally includes pre-enrichment

and isolation steps and is labor- and time consuming.

A number of methodologies are available to monitor metabolic activity

and microbial growth of microbial populations. Inhibition of microbial

growth in the presence of phage that is indicative of the presence of a target

bacterium can be detected in a variety of ways.Whenmicroorganisms grow,

they convert large molecules into small, more highly charged compounds

and this causes changes in the electrical properties of the growth medium.

These changes can be detected either as changes in impedance or conduc-

tance. The retardation in the development of these changes in the presence

of phage is indicative that the target organism is present. Chang, Ding, and

Chen (2002) used an E. coliO157:H7-specific phage (AR1) in conjunction

with a conductance method for detection of the organism. The multiplica-

tion of E. coli O157:H7 was inhibited by ARI resulting in no change in

conductance in MacConkey-sorbitol medium over a 24-h period. Of the

41 strains of E. coli O157:H7 tested, all produced positive reactions (i.e.,

no change in conductance within 24 h). Fourteen of 155 strains of non-

O157:H7 E. coli also did not produce a change in conductance within

24 h. However, only one of these strains did not utilize sorbitol and, hence,


would not grow on the medium even in the absence of phage. The sensi-

tivity and specificity of the method was 100% (41 of 41) and 99.4% (154 of

155), respectively. McIntyre and Griffiths (1997) showed that a number of

bacterial pathogens could be detected in dairy products by monitoring

changes in the impedance of selective media in the presence and absence

of host-specific bacteriophage. The application of these methods for path-

ogen detection in foods is limited because they rely on differential or selec-

tive media to identify pathogens and they can be affected by the growth of

contaminating microorganisms associated with the background microflora

of foods (Mandeville et al., 2003).

Another approach to detect bacteria by bacteriophage was proposed by

Jassim and Griffiths (2007). In this method, the sample containing target

pathogen Pseudomonas aeruginosawas treated by a low number of the specific

bacteriophage NCIMB 10116, and after at least one infection cycle (�2 h),

the “helper cells” (bacteria known to be infected by this phage) were added

to the sample and allowed to be infected by the phage providing further

propagation of the phage. After removal of bacteria from the sample by

filtration, the Live/Dead BacLight Bacterial Viability stain was applied.

It was shown that the ratio of green (intact cells) to red (damaged cells) fluo-

rescence in the helper cell population was proportional to the initial number

of target cells present in the original sample. As a result, approximately

100 CFU/ml of P. aeruginosa could be detected within 4 h without the need

for enrichment.

3.2. Methods based on formation of bacteriophage–host cellcomplex

The first stage of the phage infection process is its attachment to specific

receptors on the surface of host bacterium. Due to the multivalent nature

of this attachment (high affinity/avidity), the resulting complex is both very

specific and extremely strong. Thus, phages can be used as efficient capturing

and/or staining agents. The bacteriophages themselves or their sensing

receptors can be used as the biorecognition component of biosensors.

Experimental formats used in this type of phage-based assays are similar to

antibody-based methods.

3.2.1 Phages as staining agentsFluorescently stained bacteriophages adsorbed to the host bacterium allowed

for simple identification of target cells in mixed culture using flow cytometry

or the modified direct epifluorescent filter technique (Goodridge, Chen, &


Griffiths, 1999; Hennes & Suttle, 1995; Hennes, Suttle, & Chan, 1995; Lee,

Onuki, Satoh, & Mino, 2006). The average sensitivity was around

104 CFU/ml for flow cytometric detection and 102–103 CFU/ml for

epifluorescent microscopy.

To improve the sensitivity of this approach, fluorescent quantum dots

(QDs) were used to tag bacteriophages (Edgar et al., 2006; Yim et al.,

2009). QDs provide a very intense and stable fluorescent signal that could

be monitored by both flow cytometry and epifluorescence microscopy.

An E. coli bacteriophage T7 was engineered to carry a small biotin-

binding peptide on the head, which was biotinylated in vivo during phage

propagation in the host. The QDs were coated with streptavidin, which

enabled them to bind strongly to the biotinylated phage. The

fluorescence signal from as few as 10 E. coli with attached bacteriophage

T7–QD conjugate was significantly different from the background and

from the signal originating from non-E. coli strains. The method allowed

the detection of at least 20 E. coli cells in 1-ml water samples within 1 h.

3.2.2 Phage-based biosorbentsBacteriophages immobilized on solid surfaces were shown to be effective

capturing agents for target bacterial cells. Binding of the host cell to bacte-

riophage could bemonitored in real time using various optical, electrochem-

ical, and/or MEMS (microelectromechanical systems) devices.

Surface plasmon resonance (SPR) was employed for monitoring the

attachment of E. coli and S. aureus to the respective bacteriophage

immobilized on a gold surface (Arya, Singh, et al., 2011; Balasubramanian,

Sorokulova, Vodyanoy, & Simonian, 2007). This approach was fast,

specific, and had a detection limit of 7�102 and 104 CFU/ml for E. coli

K-12 and S. aureus, respectively. SPR is a label-free real-time method of

binding event monitoring; portable instruments are available that could be

employed under field conditions. However, the small volume of the

sample that may be sufficient for clinical testing makes it difficult to apply

for food and environmental testing, where analysis of large volume samples

is required. Thus, a pre-enrichment/preconcentration step should be

introduced, which can lengthen the time of analysis significantly.

Another recentlyproposedmethod todetectbacterial attachment tophage-

based biosorbents involvesmagnetoelastic biosensors. Advantages of these bio-

sensors are that they can be interrogatedwirelessly through the use ofmagnetic

fields, can be easily miniaturized, and can be multiplexed for simultaneous

detection of multiple agents (Grimes, Roy, Rani, & Cai, 2011).


Bryan Chin and his coauthors demonstrated that Salmonella Typ-

himurium can be detected in situ on the surface of contaminated tomatoes

and egg shell using a magnetoelastic biosensor with immobilized E2 bacte-

riophages (Chai et al., 2012; Horikawa et al., 2011; Mi-Kyung et al., 2012;

Park, Oh, & Chin, 2011). The observed detection limit was around

102 CFU/cm2, with an analysis time of 15–30 min.

The major problem in the development of phage-based biosorbents is

the method of immobilization of the bacteriophage on the surface of the

sensor. The employedmethod should ensure that the surface is covered with

a high density of phage particles and that phage has access to the receptors on

the target bacterium. Several ways of oriented bacteriophage immobilization

have been proposed.

Highly organized phage monolayers prepared on glass using the

Langmuir Blodgett technique were shown to effectively and specifically

bind methicillin-resistant S. aureus. Binding can be monitored in real time

directly on a microscope slide without prior incubation of the bacteria

(Guntupalli et al., 2008). The phage attachment sites remain fully accessible

and fully functional due to the high levels of elasticity of the monolayer. This

method does not require culturing and/or labeling and could prove useful

for rapid screening and analysis of environmental and food samples.

Oriented immobilization of bacteriophages on surfaces could be

achieved also by introducing affinity tags to the phage head, thus providing

the opportunity to orient the phage particle on the surface and leave the

receptor site accessible to the host bacterium. Chemically biotinylated

Salmonella bacteriophage was used for construction of a biosorbent based

on streptavidin-coated magnetic beads (Sun, Brovko, & Griffiths, 2000).

The capturing efficiency of the sorbent was superior to that of the sorbent

based on the Salmonella phage passively adsorbed on polystyrene; however,

it was not greater than that of commercial immunomagnetic sorbents for

Salmonella. Targeted introduction of biotin-binding peptide (BCCP) or

cellulose-binding module (CBM) on the capsid protein of T4 phage by a

phage display technique resulted in construction of biosorbents based on

streptavidin magnetic beads and microcrystalline cellulose particles, respec-

tively (Singh et al., 2009; Tolba, Minikh, Brovko, Evoy, & Griffiths, 2010).

The BCCP-T4 phage-based biosorbent showed high capturing efficiency of

E. coli. At E. coli concentrations in the range from 10 to 105 CFU/ml, about

70–90% of cells were captured within 10 min. On the contrary, the

biosorbent based on the CBM-T4 phage was not effective for bacterial

capture. This was explained by the fact that, despite the availability of


cellulose affinity tags on the phage head, there was still a possibility for phage

tails to interact with sugar moieties on the surface of microcrystalline

cellulose similar to the interaction with sugars on the surface of the

bacterial cell wall thus preventing interaction with host cells.

Though the phage display technique is a very powerful tool for affinity

tag introduction on the phage head, it requires extensive knowledge of the

phage genome, which is not always available. A more generic approach

based on electrostatic interactions of phage head with the solid support

was proposed recently to circumvent this problem (Cademartiri et al.,

2010). Since the majority of bacteria carry a slightly negative charge on their

surface, it was hypothesized that the receptor sites on bacteriophages gener-

ally are positively charged thus making their heads negatively charged.

Hence, positively charged surfaces attract bacteriophages through their

heads allowing the receptors to interact with the cell wall of bacteria.

Chemical modification of the surface and composition of the phage

preparation were shown to greatly affect density of bacteriophage immobi-

lization and, respectively, capture of target bacteria by the phage-modified

surface (Arya, Amit, et al., 2011; Gervals et al., 2007; Ravendra Naidoo

et al., 2012; Singh et al., 2009).

Currently, no general approach has been proposed for the efficient

immobilization of bacteriophage on a surface that ensures high density

and, hence, high capturing efficiency; further studies are needed to under-

stand in greater detail the complex nature of bacteriophage interaction with

the support material.

3.2.3 Bacteriophage receptors as capturing agentsIn order to simplify the biosorbent construction process, it was proposed

to use isolated bacteriophage receptors as capturing agents instead of intact

bacteriophages. Bacteriophage receptors were shown to have high affinity

toward the specific domains of the bacterial cell wall. Recombinant phage

proteins derived from tail fibers are used in the same way as antibodies for

bacterial capture and identification. The cell wall-binding domains (CBDs)

of bacteriophage-encoded peptidoglycan hydrolases (endolysins) have also

been immobilized and used to capture bacteria (Kretzer et al., 2007;

Loessner, 2005). These proteins have high affinity toward the specific

ligands on the cell wall of Gram-positive bacteria. Paramagnetic beads

coated with recombinant endolysin derived from Listeria phage were

shown to capture more than 90% of viable L. monocytogenes cells from

diluted suspensions within 20–40 min. Bacillus cereus, C. perfringens, and


Bacillus anthracis (Fujinami, Hirai, Sakai, Yoshino, & Yasuda, 2007) could

also be captured using specific phage-encoded CBDs for these targets.

This technology is marketed by bioMerieux, France, as part of the

VIDAS system, which can detect E. coli O157 and Salmonella in enriched

cultures within less than 1 h.

Receptor-binding protein (RBP) of Campylobacter bacteriophage

NCTC 12673 was used for the specific capture of C. jejuni bacteria using

RBP-derivatized capturing surfaces (Amit, Arutyunov, McDermott,

Szymanski, & Evoy, 2011). The binding process was monitored by SPR,

and it was shown that the Campylobacter detection limit for this biosensor

was 102 CFU/cm2. The RBP was also immobilized onto magnetic beads

that were successfully used to capture and preconcentrate the host pathogen

from suspension.

3.3. Reporter phagesThe second step in bacteriophage infection involves injection of the bacte-

riophage genetic material into the host cell. After injection of nucleic acid

into the host cell, bacteriophage takes over the whole metabolic process

in the bacterium immediately (within seconds). Phages can be genetically

modified to carry reporter genes, the products of which can be easily detected

after their expression following infection. This method was proposed in late

1980s byUlitzur and Kuhn (Ulitzur &Kuhn, 1989; Ulitzur, Suissa, & Kuhn,

1989). They introduced bioluminescent genes luxAB fromVibrio fischeri into

E. coli phage lCharon 30. Since phages themselves do not have the required

machinery to express proteins encoded in their genome, they remain “dark.”

Once the reporter gene has been introduced into the bacterium following

infection, it was expressed and produced bioluminescence. Therefore, the

observed bioluminescence was indicative of the presence of the infected

cell. Using this luxþ phage, it was possible to detect 10–100 E. coli cells

in milk and urine within 1 h.

Since then, bioluminescent reporter phage (BRP) assays have been

developed for detection of several pathogens such as E. coli O157:H7

(Pagotto, Brovko, & Griffiths, 1996; Waddell & Poppe, 2000), Salmonella

spp. (Chen & Griffiths, 1996; Kuhn, 2007; Stewart, Smith, & Denyer,

1989; Thouand, Vachon, Liu, Dayre, & Griffiths, 2008), L. monocytogenes

(Loessner, Rees, Stewart, & Scherer, 1996), S. aureus (Pagotto et al., 1996),

B. anthracis (Schofield & Westwater, 2009), Yersinia pestis (Schofield,

Molineux, & Westwater, 2009), Mycobacterium tuberculosis (Carriere et al.,

1997; Jacobs et al., 1993), and Mycobacterium avium subsp. paratuberculosis


(MAP) (Sasahara & Boor, 2001). Bioluminescent genes from both bacteria

(lux) and firefly (luc) can be used for BRP engineering. The reported

sensitivity was in the range 102–106 cells/ml. A short pre-enrichment step

(2–6 h) allowed the detection of lower cell concentrations. The time of

analysis was significantly shorter than in conventional methods, being

1–3 h for fast growing bacteria and a few days for slow growing organisms

such as Mycobacteria.

Several other reporter genes were proposed for use in phage-mediated

bacteria assays, including the ice nucleation gene (inaW) (Wolber & Green,

1990), b-galactosidase gene (lacZ) (Goodridge & Griffiths, 2002; Willford

& Goodridge, 2008), and green fluorescent protein gene (gfp) (Miyanaga,

Hijikata, Furukawa, Unno, & Tanji, 2006; Namura, Hijikata, Miyanaga, &

Tanji, 2008; Oda, Morita, Unno, & Tanji, 2004; Tanji et al., 2004). The

products of these genes could be detected by colorimetric, fluorescent, or

luminescent techniques. In general, luminescent detection is considered to

be the most sensitive as there is practically no background bioluminescent

signal in the majority of samples and, hence, the signal-to-noise ratio is the

highest. Fluorescent detection relies on monitoring of fluorescent signal

from the reporter, which could be masked by the strong autofluorescence

originating from the sample. In case of green fluorescent protein (GFP)

reporter, it should be also taken into consideration that formation of the

fluorophore in GFP is a posttranslational oxidation process that requires

time and sufficient oxygen. Maturation time and fluorescence intensity

differ widely for GFP variants (Cormack, Valdivia, & Falkow, 2000).

Advantages of using b-galactosidase as a reporter include the possibility to

measure the signal using different methods including colorimetry,

fluorimetry, and luminometry with the sensitivity of detection increasing in

this order (Nazarenko, Dertinger, & Gasiewicz, 2001). However, the

inherent presence of the lacZ gene in many bacteria may compromise the

selectivity of the assay providing false positive results.

Recently, a novel phage-triggered luminescent signal for bacteria detec-

tion has been proposed (Ripp et al., 2006). This approach is based on the fact

that the bioluminescence inV. fischeri is controlled by the so-called quorum-

sensing molecules, acyl homoserine lactones (AHLs), which allow coordi-

nation of certain behaviors based on the local density of the bacterial

population. These signaling molecules are produced by an AHL synthase

encoded by the luxI gene. AHLs are secreted by bacteria into the environ-

ment at a constant rate, and when the concentration of AHL in the medium

reaches a threshold, it activates the transcription of the entire lux operon in


V. fischeri making them bioluminescent. The luxI gene was incorporated

into the l phage genome, which resulted in production of a large amount

of AHL in the medium following E. coli infection. The synthesized AHL

molecules in turn induced bioluminescence in V. fischeri bioreporter cells

added to the sample, thus announcing the infection event. This two-

component reporter system was able to detect the presence of E. coli cells

at counts ranging from 1 to 1�108 CFU/ml within 10.3 and 1.5 h, respec-

tively. The same approach was used to develop a method for E. coli O157:

H7 detection (Brigati et al., 2007). In this case, the luxI gene was incorpo-

rated into the genome of PP01 bacteriophage that is specific to the pathogen.

The constructed reporter phage was able to induce bioluminescence in the

bioreporterVibriowithin 1 h after exposure to 104 CFU/ml of E. coliO157:

H7. The demonstrated detection limit of the method was 10 CFU/ml,

when a short 4-h preincubation step was included. The method was tested

for the analysis of artificially contaminated apple juice, tap water, spinach leaf

rinses, andminced beef (Ripp, Jegier, Johnson, Brigati, & Sayler, 2008). The

assay was successful in detection of low numbers (1 CFU/ml) of pathogen in

water, juice, and spinach rinses after 1–16 h preincubation. However, the

assay was unable to detect E. coli O157:H7 meat, which was explained by

the AHL naturally present in the sample generating false positive signals.

The described reporter bacteriophages demonstrated high potential for

rapid and sensitive detection of target pathogen in complex media such as

food and environmental samples. The lack of commercially available

reporter phages probably could be explained by the problems in the design

and mass propagation of these phages due to insufficient knowledge of their

genetic structure and physiology.

3.4. Bacteriophage-mediated lysis as an indicator of thepresence of target pathogens

The final result of the infection process is an assembly of phage particles

inside the cell followed by cell lysis and the release of progeny phages in

the environment. These events enable detection by monitoring the release

of intracellular materials (both molecular components of the cell and prog-

eny phages) from the target.

3.4.1 Release of intracellular metabolitesThe intracellular component most widely used for phage-mediated lysis

monitoring is adenosine-50-triphosphate (ATP). ATP is present in all live

cells at a relatively high concentration and can be easily detected using


the bioluminescent ATP assay (Griffiths, 1996; Sanders, 2003). The intensity

of light emitted by the firefly luciferin–luciferase reaction is proportional to

the ATP concentration and hence, the number of bacteria present in the

sample lysed by the specific bacteriophage. The reported detection limit

for this assay is about 105 CFU/ml. The use of a biosorbent based on T4

bacteriophage immobilized on a DisruptorTM filter coupled with a

bioluminescent ATP assay allowed simultaneous concentration and

detection of as low as 6�103 CFU/ml of E. coli in the sample within 2 h

with high accuracy (CV¼1–5% in log scale). An excess of nontarget

microflora at levels 60-fold greater than the target organism did not affect

the results when bacteriophage was immobilized on the filter prior to

concentration of bacterial cells (Minikh, Tolba, Brovko, & Griffiths, 2010).

In order to improve sensitivity, it was proposed to measure the activity of

released adenylate kinase, which produces ATP in the presence of excess

of ADP (Squirrel & Murphy, 1995). This approach allowed the detection

of approximately 103 cells of E. coli and Salmonella within 1–2 h (Blasco,

Murphy, Sanders, & Squirrell, 1998; Wu, Brovko, & Griffiths, 2001).

The specificity of the assay could be improved by removing the target

bacterium using immunomagnetic separation (IMS) prior to its lysis by

the specific bacteriophage (Squirrel, Price, & Murphy, 2002). This

method was commercialized by Alaska Food Diagnostics (UK) under the

name FastrAK.

The technique of combining conductance measurement and phage lysis

has also been shown to be an effective way of screening for the presence of

Salmonella in skimmed milk powder, egg powder, cocoa powder, and var-

ious chocolate products (Pugh, Griffiths, Arnott, & Gutteridge, 1988).

3.4.2 Phage amplification assaysDetection of progeny bacteriophages released in the medium during infec-

tion of a target pathogen is a very promising approach for the rapid and

specific detection of bacteria in food and environmental samples. Consider-

ing that the burst size of each particular phage under certain conditions is

constant and in the presence of an excess of phage particles, all the cells

of the host bacterium present in the sample will be infected, then the number

of released phages is proportional to the initial number of target pathogen

cells present. The number of phage particles in the sample can be detected

by using either the plaque assay technique or by detecting specific phage

proteins and/or nucleic acid sequences. In general, the assay is comprised

of four main steps: (i) bacteriophage attachment to target bacterium followed


by the injection of phage genetic material into the cell; (ii) removal/destruc-

tion of exogenous phage remaining in the sample; (iii) synthesis, assembly,

and release of progeny phages into the solution; and (iv) enumeration of

phage particles released. The crucial step to achieve the highest sensitivity

in this assay is the removal of the exogenous phage particles. Most often,

it has been done by the addition of virucidal agents derived from pomegran-

ate rind (Stewart et al., 1998) or from tea infusions (Almeida et al., 2003).

The following phage enumeration was done first by plaque assay technique

using a “helper” bacteria culture. The method was applied for detection of

P. aeruginosa, SalmonellaTyphimurium, and S. aureus. A detection limit of 40

and 600 bacterial cells/ml was demonstrated for P. aeruginosa and Salmonella

Typhimurium, respectively, and the assay could be completed within 4 h.

This approach was used to detect Salmonella in poultry meat (da Siqueira,

Dodd, & Rees, 2006) and Listeria in milk (Almeida et al., 2003).

Themost progress in commercialization of this approach has been for the

detection of the slow growingM. tuberculosis. It is marketed under the name

FASTPlaque TB by Biotech Laboratories Inc. (UK). When compared with

other rapid TB diagnostic tests, the FASTPlaque TB showed high sensitivity

(76.7%), which was better than the L-J culture method (73.3%) and acid fast

staining (60%), but was below the sensitivity demonstrated for PCR (90%)

(Singh, Saluja, Kaur, & Khilnani, 2008).

The FASTPlaque TB assay has been modified to detect other

mycobacteria such as MAP (Stanley et al., 2007). The bacteriophage used

in this assay is not specific for MAP, so to add specificity to the method,

a PCR was performed to amplify the specific DNA sequences of MAP

allowing rapid (48 h) and specific detection of viable MAP in milk samples.

Foddai, Elliott, and Grant (2010) proposed to use IMS prior to phage

amplification assay to improve the specificity of MAP detection in milk

samples with results available within 48 h.

An alternative to virucide treatment for exogenous phage removal was

proposed by Favrin, Jassim, and Griffiths (2001); they used IMS to isolate

infected host cells from the rest of the sample. The detection limit of this

assay was<104 CFU/ml, which can be detected within 4–5 h. The method

was applied for detection of Salmonella Enteritidis and E. coli O157:H7 in

skim milk powder and ground beef, respectively (Favrin, Jassim, & Griffiths,

2003). The total assay time was about 10 h, and the detection limit was

approximately 3 CFU/g or ml of food tested.

Another interesting approach to overcome the deficiencies of exogenous

phage removal was proposed by Ulitzur and Ulitzur (2006). A pair of


complementing phage mutants was used in this assay. Mutants themselves

cannot complete the infection cycle and form plaques on their bacterial host,

while when the two mutants of a given phage coinfect their host, comple-

tion of the infection cycle may be achieved under certain conditions by

complementation, in which each phage provides the essential element of

the genome missing in the other. The method showed good correlation

between the number of plaques formed following the infection of Salmonella

by the mutants of Felix-O1 phage and the number of host cells present in the

tested sample (in the range from 5 to 106 cells).

Several other methods have been proposed recently for phage enumer-

ation in a phage amplification assay, including matrix-assisted laser desorp-

tion/ionization mass spectrometry, where virus capsid protein was detected

after isolation of target bacteria by IMS and phage infection/amplification

(Madonna, Van Cuyk, & Voorhees, 2003). The presence of E. coli in broth

at a concentration of 5�104 cells/ml was detected in less than 2 h total anal-

ysis time. This approach was further extended for detection of E. coli and

Salmonella (Rees & Voorhees, 2005) and of S. aureus (Pierce, Rees,

Fernandez, & Barr, 2011, 2012). Amplification of metabolically labeled

N15 bacteriophage coupled with LC–MS/MS detection offers speed

(3 h total analysis time), sensitivity (LOD <5.0�104 CFU/ml), high

accuracy, and precision for quantification of S. aureus.

Another way to detect the number of progeny phages is by nucleic

acid amplification technique such as PCR, RT-PCR, and qPCR as well

as isothermal nucleic acid amplification. Advantages of this approach are

that, in this case, the inherent phage amplification step is complemented

by amplification of the unique bacteriophage nucleotide sequence provid-

ing a higher overall sensitivity of the assay (Reiman, Atchley, &

Voorhees, 2007). This method detected a starting B. anthracis concentra-

tion of 207 CFU/ml, equivalent to less than one cell in 20 ml, in less than

5 h. The approach was also used for detection of the plant pathogen

Ralstonia solanacearum with LOD down to approximately 102 CFU/g of

soil after 1 h incubation of the sample with M_DS1 bacteriophage

(Kutin, Borthakur, Alvarez, & Jenkins, 2008). Bacteriophage f A1122-

specific qPCR enabled the detection of an initial bacterial concentration

of 103 CFU/ml of Y. pestis (equivalent to as few as one Y. pestis cell per

1-ml sample) in 4 h (Sergueev, He, Borschel, Nikolich, & Filippov, 2010).

Combination of a phage-based biosorbent with real-time PCR detection

of a unique target sequence of biotinylated T4 bacteriophage (fusion of

biotin carboxyl carrier protein gene (bccp) fused to soc gene encoding outer


capsid protein) allowed detection of as few as 800 E. coli cells within 2 h

(Tolba et al., 2010).

4. CONCLUSION

The research results described in this chapter show a variety of ways in

which bacteriophages can be used for detection and control of bacterial path-

ogens providing unprecedented selectivity, sensitivity, and speed of action.

Widespread commercialization of bacteriophage-based technologies for

control and detection of bacterial pathogens is still in the future. Bacterio-

phages were created by nature to combat bacteria around us. It is intended

through this review to encourage those in need of rapid and effectivemethods

to control and/or detect bacterial pathogens to become familiar with the

possibilities that bacteriophages offer and to join efforts in developing novel

strategies and technologies to further ensure a safer future for all of us.

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