Introduction fuelled multiple biotechnological applications in very diverse

Introduction

Everyday
people worldwide buy and consume a diversity of products of animal and plant
origin expecting these products to be safe. However, the current technologies
employed to inactivate bacterial pathogens in foods are not infallible.
Annually, millions of people become ill, are hospitalized, and die due to a
variety of food borne pathogens, such as Salmonella, Staphylococcus aureus, Campylobacter, Escherichia coli, Listeria, transmitted through
foods. The most e?cient means for limiting the growth of microbes are good
production hygiene, a rational running of the process line, and a well-designed
use of biocides and disinfectants. Regardless of modern technologies, good
manufacturing practices, quality control and hygiene contaminating bacteria
still can get access to food during slaughtering, milking, fermentation,
processing, storage or packaging. Moreover, the extensive use of sanitizers and
antibiotics has led to the development of resistant bacteria rendering these
procedures less effective. Thus, to meet the primary goal of any food safety
program, the consumer protection, new food preservation techniques have to be
continually developed to meet current demands, in order to control the emerging
pathogens and their impact at global scale (1).

Research
on phage molecular biology has fuelled multiple biotechnological applications in very diverse
fields including bacterial detection systems, novel antimicrobials against
antibiotic-resistant bacteria, etc. Another promising field of application is
the use of phages as natural antimicrobials in food to inhibit undesirable
bacteria (2).

 

Bacteriophage description

A bacteriophage is a virus that infects and replicates within a bacterium. Bacteriophages are
composed of proteins that encapsulate a DNA or RNA genome. Phages replicate within the bacterium following the injection
of their genome into its cytoplasm. To enter a host
cell, bacteriophages attach to specific receptors on the surface of bacteria.
This specificity means a bacteriophage can infect only certain bacteria bearing
receptors to which they can bind, which in turn determines the phage’s host
range. After making contact with the appropriate receptor, phage injects
genetic material through the bacterial membrane. The host’s normal synthesis of
proteins and nucleic acids is disrupted, and it is forced to manufacture viral
products instead. These products go on to become part of new virions within the
cell, helper proteins that help assemble the new virions, or proteins involved
in cell lysis. Phages may be
released via cell lysis achieved by an enzyme called endolysin, which attacks and
breaks down the cell wall peptidoglycan (3).

Bacteriophages are ubiquitous viruses, found wherever
bacteria exist (soil, drinkable or sea water, even the intestines of animals). The
most outstanding advantage of phages is their harmless interaction to
eukaryotic cells (human, animal, plants).

 

 

Bacteriophage application

Phages
were discovered by Ernest Hankin (1896) and Frederick Twort (1915) who
described their antibacterial activity. However, Felix d’Herelle (1919) was
probably the first scientist who used bacteriophages as a therapy to treat severe
dysentery. At that time, several companies then actively started up the
commercial production of phages against various bacterial pathogens for human
use. However, phage production was quickly displaced by the discovery of
antibiotics. Nowadays, the current threat of antibiotic-resistant bacteria has
renewed the interest in exploring bacteriophages as biocontrol agents (1) (2).

Phages
have a wide range of application: phage therapy, tools for detecting pathogens,
water and food safety, agriculture and animal health. Bacteriophage-based
biocontrol measurements have a great potential to enhance microbiological
safety based on relatively easy handling and their high and specific
antimicrobial activity. The concept of combating pathogens in food can be
addressed at all stages of production in entire food chain. Bacteriophages are
suitable:

(i) to prevent or
reduce colonization and diseases in livestock (phage therapy). Phages have high
specificity to target specific bacteria determined by the cell wall receptors
and leave remaining microbiota untouched.

(ii)
to extend the shelf life of perishable manufactured foods as natural
preservatives. Low phages inherent toxicity is an advantage compered to
antibiotics.

(iii)
to decontaminate carcasses and other raw products, such as fresh fruit and
vegetables, and to disinfect equipment and contact surfaces (phage
biosanitation and biocontrol). Phages can generally withstand food processing
environmental stresses (including food physiochemical conditions) (1) (2).

 

Biocontrol of Escherichia coli using
bacteriophages

Escherichia coli
is a gram-negative bacterium. SerotypeO157:H7 in particular, classi?ed as Shiga
toxin-producing E. coli, is a well-known food poisoning pathogen. This
microorganism is highly virulent and a public health threat because ingestion
of a concentration as low as 10-100 cells is able to cause infection (4) {2>15} Ruminants comprise
the principal reservoir for this strain and contamination of animal products
occurs during milking or slaughtering (1). The most common route of E. coli transmission to humans is
via undercooked contaminated food. To avoid food safety problems
bacteriophages therapy could be applied during preharvest or postharvest
period.

Postharvest
interventions have been e?ective in reducing E. coli levels from fresh produce.
Efficacy of bacteriophages against E.
coli O157:H7 strain was tested on fresh cut lettuce surface by spay and
immerse application methods. Both methods provided a degree of protection from
introduction of E. coli O157:H7 to fresh cut lettuce. However,
spray application of lytic bacteriophages was reported to be more effective in
immediately reducing E. coli O157:H7 populations on lettuce
surfaces compared with immersion of lettuce in phage solutions (EcoShield™) (5). {9}

A
cocktail of three lytic phages specific for E. coli O157:H7
(EcoShield™) or a control (phosphate buffered saline, PBS) was applied to
lettuce in different ways. 

(i)
immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml
for up to 2 min before inoculation with E. coli O157:H7.
Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed
for E. coli O157:H7 populations for up to 7 d. Immersion of
lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. (ii) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 ?g/ml chlorine for 30 sec. Spraying technique was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2) (5).  {9} Preharvest application of phages to poultry by aerosol spraying and intramuscular injection methods has been successful to prevent fatal respiratory infections in broiler chickens (6). {2>16} However, phage administration via addition to bird drinking water proved to be ine?cient in protecting the birds from fatal E. coli respiratory infections (7).
{2>17}The main speculated causes for the failure of oral
treatments have been reported to be nonspeci?c binding of phages to food particles and other debris in the rumen and gastrointestinal tract (8). {2>18}

 

Biocontrol of Staphylococcus aureus using bacteriophages

Staphylococcus aureus
is a gram-positive bacterium, is considered to be a major threat to food
safety.  S. aureus can cause
toxin-mediated diseases within 1 to 6 h after consumption of contaminated
foods. Staphylococcus aureus also is
the most common agent of mastitis in dairy cows (1). {2} Mastitis caused by Staphylococcus aureus is
a major concern to the dairy industry due to its resistance to antibiotic
treatment and its propensity to recur chronically (9)
{10}.

The ability of lytic S. aureus bacteriophage
K to eliminate bovine S. aureus intramammary
infection during lactation was evaluated in a placebo-controlled, multisite
trial. Results revealed that phage treatment was not effective against
preexisting subclinical S. aureus mastitis.
The efficacy of bacteriophage in the treatment appeared to be limited under the
treatment conditions studied. Natural inhibitory effect of raw milk caused
phage inhibition and treatment failure. Moreover, in healthy lactating cows
phage therapy even elicited a large increase in the milk somatic cell count (9). {10}

Phage
K inactivation was also reported in raw milk. The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw
bovine whey and serum. Incubation of S. aureus with
phage in whey showed that the bacteria is more resistant to phage lysis when
grown in whey or bovine serum. The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage
attachment and thereby hindered lysis (10).
{11}

However,
a cocktail of two lytic phages of dairy origin produced a complete elimination of the pathogen in
ultra-high-temperature (UHT) whole milk at 37 °C.
This indicates that lytic bacteriophages could be
used as biopreservatives when natural inhibitory milk properties
are removed (11).
{12}

 

Biocontrol of Salmonella using bacteriophages

Salmonella,
is a genus of gram-negative facultative intra-cellular species, is considered
to be one of the principal causes of zoonotic diseases reported worldwide. Salmonella serovars can colonize and
persist within the gastrointestinal tract, and so human salmonellosis is
commonly associated with consumption of contaminated foods of animal origin or
transmitted via contaminated water. Salmonella
is also a known spoilage bacterium in processed foods. Once ingested, this
microorganism can cause fever, diarrhea, abdominal cramps, and even
life-threatening infections (1)
{2}.

Salmonella biocontrol
efficacy of bacteriophage FO1-E2 was evaluated. Collected data showed that virulent
phages such as FO1-E2 offer an effective biocontrol measure for Salmonella in
foods. Experiment conducted at 8 °C storage temperature reported no viable
bacteria detection after treatment. However, at 15 °C storage
temperature, Salmonella growth was detected but suppressed
by at least 2 and up to 5 log units on different foods. Results prove that
bacteriophage FO1-E2 is an effective biocontrol agent for Salmonella (12). {3}

 

Materials
and methods:

Hot
dogs, cooked and sliced turkey breast, mixed seafood, chocolate milk, and egg
yolk (pasteurized) were used for experiment. After screening for contamination
with Salmonella, foods were stored frozen at – 80 °C until use
(except egg yolk, which was used fresh).

Three
food samples were required for each experiment: (i) negative control containing
no bacteria, (ii) positive control containing Salmonella Typhimurium only, and (iii) sample
containing Salmonella Typhimurium
and bacteriophage FO1-E2. The concentration of Salmonella in the food sample were based on inoculum corresponding
to approximately 1% of the total sample size. Before addition of phage, food
samples were pre-incubated at 8 °C or 15 °C for 1–2 h to allow
the bacteria to adapt to the conditions. Phage FO1-E2 was then added at a
concentration of 3 × 108 pfu/g or ml. Samples were
further incubated at 8 °C or 15 °C, for up to 6 days. Bacterial
and phage counts were determined by duplicate plating, and all food experiments
were independently performed at least twice. As a result, determination of
viable counts (cfu) from the food samples was straightforward (12). {3}

Food treated with phage
and incubated at 8 °C showed no viable cells could be recovered by direct
plating after 1 day of incubation, corresponding to a reduction of
approximately 3 logs (12).
{3}

During incubation at 15 °C
application of FO1-E2 resulted in a Salmonella viable count
decrease of at least 2 logs in the first two days, which was followed by
some regrowth during the remaining incubation period. After six days, viable
counts were more than five orders of magnitude lower in chocolate milk and on
sliced cooked turkey breast (p < 0.05). On hot dogs, Salmonella numbers were reduced by 3.0 logs (p < 0.05), and on mixed seafood by 1.9 logs (p < 0.05). In egg yolk, a significant difference to the control samples was observed after two days (2.6 log units, p < 0.05), but not after five or six days of incubation (p > 0.05) (12).
{3}

More
examples of successful phage application against Salmonella were reported. The activity
of the Salmonella phage SJ2 was tested in cheddar cheese
manufacturing (13).  {13} In the presence of phages (MOI 104), Salmonella did
not survive in the pasteurized cheeses after 89 days. In another study,
Salmonella phage cocktails have been assayed on fruits. Phage numbers remained
relatively stable on melon and gave a significant reduction of target bacteria.
On the contrary, a quick decline of infective phage particles was observed on
apples due to the lower pH of this fruit (14).
{14}

 

Conclusion

Phages
could be successfully applicated in food industry to solve major food safety
problems. Phages are harmless to human cells and do not secrete toxic
compounds, expose only targeted bacteria, thus, natural microbiota is not
exterminated. Phage therapy could be used to both preharvest and postharvest
application.

Control
of E. coli, Salmonella and S. aureus was explored in this review. E. coli control by bacteriophages on
fresh cut lettuce surface and respiratory infections in broiler chickens
proved to be advantageous. However, phages treatment has not succeeded to heal
mastitis caused by S. aureus.
Interaction with other molecules could reduce phage performance. On the other
hand, Salmonella growth rate was
significantly reduced in most of tested ready to eat (RTE) foods both at 8 °C
and 15 °C incubation temperatures. Cheddar cheese
manufacturing control was successful using phages and Salmonella was not detected. In conclusion, phage therapy
has a great potential to solve food safety problems caused by microbiological
contamination.