MINI REVIEW

published: 30 November 2021

doi: 10.3389/fmicb.2021.793135

Uses of Bacteriophages as Bacterial

Control Tools and Environmental
Safety Indicators
Paula Rogovski 1 , Rafael Dorighello Cadamuro 1 , Raphael da Silva 1 ,
Estêvão Brasiliense de Souza 1 , Charline Bonatto 2,3 , Aline Viancelli 4 , William Michelon 4 ,
Elmahdy M. Elmahdy 5 , Helen Treichel 3 , David Rodríguez-Lázaro 6,7* and
Gislaine Fongaro 1*
1
Laboratory of Applied Virology, Department of Microbiology, Immunology and Parasitology, Federal University of Santa
Catarina, Florianópolis, Brazil, 2 Department of Chemical and Food Engineering, Federal University of Santa Catarina,
Florianópolis, Brazil, 3 Laboratory of Microbiology and Bioprocesses, Federal University of Fronteira Sul (UFFS), Erechim,
Edited by: Brazil, 4 Contested University (PMPECSA), Concórdia, Brazil, 5 Laboratory of Environmental Virology, Environmental Research
Laurent Dufossé, Division, Department of Water Pollution Research, National Research Centre, Giza, Egypt, 6 Division of Microbiology,
Université de la Réunion, France Department of Biotechnology and Food Science, Universidad de Burgos, Burgos, Spain, 7 Centre for Emerging Pathogens
and Global Health, Universidad de Burgos, Burgos, Spain
Reviewed by:
Diogo Silva,
University of Brighton, Bacteriophages are bacterial-specific viruses and the most abundant biological form
United Kingdom
Hak-Kim Chan, on Earth. Each bacterial species possesses one or multiple bacteriophages and
The University of Sydney, Australia the specificity of infection makes them a promising alternative for bacterial control
*Correspondence: and environmental safety, as a biotechnological tool against pathogenic bacteria,
David Rodríguez-Lázaro
including those resistant to antibiotics. This application can be either directly into foods
[email protected]
Gislaine Fongaro and food-related environments as biocontrol agents of biofilm formation. In addition,
[email protected] bacteriophages are used for microbial source-tracking and as fecal indicators. The
Specialty section:
present review will focus on the uses of bacteriophages like bacterial control tools,
This article was submitted to environmental safety indicators as well as on their contribution to bacterial control in
Food Microbiology, human, animal, and environmental health.
a section of the journal
Frontiers in Microbiology Keywords: bacteriophages, food safety, biocontrol, foodborne pathogens, antimicrobial resistance
Received: 11 October 2021
Accepted: 11 November 2021
Published: 30 November 2021 INTRODUCTION
Citation:
Rogovski P, Cadamuro RD, Bacteriophages, also known as phages, are prokaryotes viruses, being the most abundant life
da Silva R, de Souza EB, Bonatto C, form, present in all environments and the predominant entities in the sea (Boehme, 1993;
Viancelli A, Michelon W, Elmahdy EM, Suttle, 2005). Several studies have demonstrated a 1:5 relative abundance between bacteria and
Treichel H, Rodríguez-Lázaro D and bacteriophage (Fuhrman, 1999; Balter, 2000; Rohwer, 2003). They were discovered independently
Fongaro G (2021) Uses
by Twort (1915), who isolated them from Staphylococcus spp., and from patients with dysentery.
of Bacteriophages as Bacterial
Control Tools and Environmental
D’Herelle (1926) described bacteriophage as a virus that has the capability to parasitize bacteria
Safety Indicators. (Twort, 1915; Delbruck, 1942). Bacteriophages vary greatly in morphology and replicative
Front. Microbiol. 12:793135. characteristics, containing either RNA or DNA, being these parameters currently used by
doi: 10.3389/fmicb.2021.793135 the International Committee on Taxonomy of Viruses (ICTV) for bacteriophage classification

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Rogovski et al. Bacteriophages Uses

(King et al., 2012; Table 1). However, the identification associated with contamination of food and water, is a serious
of bacteriophages is difficult since there are no universally health problem affecting millions of people annually in the world,
conserved markers, unlike e.g., the bacterial 16S rRNA gene (Paul with shigellosis, a disease caused by Shigella spp., resulting in
et al., 2002), with only minor parts of bacteriophage genomes approximately 600,000 deaths each year (Walker et al., 1990;
being used to determine family specific makers, such as the viral World Health Organization [WHO], 2017).
capsid g20 of T4 (Fuller et al., 1998; Marston and Sallee, 2003; Lytic bacteriophages are the main tools for phage therapy, for
Sullivan et al., 2008). their capacity to invade the bacterial cell and kill it. Lysogenic
Bacteriophages can present different life cycles: lytic, bacteriophages could also have an application, the transduction
lysogenic, and chronic (Figure 1A). Lytic bactériophages, such mechanisms could allow the use of bacteriophages as genetic
as T4 and MS2, insert their genetic material inside the bacteria, tools to increase bacterial susceptibility to antibiotics; however,
forcing the cell to produce a large amount of bacteriophage this approach has not been widely studied (Lu and Collins,
copies. After replication the membrane is then ruptured, 2009; Edgar et al., 2012). The use of a bacteriophage cocktail for
releasing the new bacteriophages. Lysogenic bacteriophages the treatment and prophylaxis of intestinal infections caused by
(such as T1) possesses an alternative sub-cycle, in which the Shigella resulted in the patient recovery in 9 days, while antibiotic
virus may integrate its DNA in the bacterial genome, becoming chemotherapy revealed only a clinical improvement after 29 days
non-infectious and replicating together with the bacterial (Kutateladze and Adamia, 2008). Similarly, many other bacterial
chromosome; the bacteriophage then becomes a prophage, infections can be alternatively treated with bacteriophages,
producing new bacteriophage particles under appropriate including chronic otitis, chronic infections of wounds, cystic
conditions. Finally, chronic bacteriophages (such as M13) fibrosis-associated pulmonary infections, osteomyelitis, mastitis,
preserve their genome in the bacterial cell, in which the release chronic infections of the urinary tract, gastrointestinal infections,
from the host occurs gradually with less damage to the cell, dental caries, and endodontic infections (Harada et al., 2018;
preserving it longer (Clokie et al., 2011; Cann, 2016; Janczuk Abedon, 2019).
et al., 2016). There is an intimate relation between bacteriophages There is evidence that bacteriophages can be effectively used
and bacterial cell functions acquisition (Forterre, 1999; Filée against bacterial infections, including those that have proved
et al., 2002, 2003). Bacteriophages can serve as points for to be resistant to treatments with antibiotics (Abedon, 2019).
genomic rearrangements due to their mosaic nature, with Staphylococcus aureus, for example, is reported to be resistant
lysogenic bacteriophages even protecting bacteria from lytic to methicillin (MRSA), vancomycin (VRSA), and vancomycin-
infection in certain conditions (Brüssow et al., 2004; Tree et al., intermediate sensitivity (VISA) (Fadlallah et al., 2015). Some
2014; Penadés et al., 2015). While bacterial hosts can benefit from studies have shown that bacteriophage therapy for the treatment
the presence of bacteriophages (as they can express important of infections caused by such bacteria has been successful.
regulators for adaptation to specific niches by the addition of Fadlallah et al. (2015) reported the treatment of corneal abscess
bacteriophage genes in the cell’s genome) bacteriophages can be caused by VISA using the bacteriophage S. aureus SATA-8505
involved in the transfer of virulence genes, producing proteins (ATCC PTA-9476).
participating in invasion, immune evasion, and toxins related to Although treatment with bacteriophages seems a promising
toxin-mediated diseases (Brüssow et al., 2004; Boyd, 2012; Tree advantage compared to conventional antibiotics and
et al., 2014; Penadés et al., 2015). disinfectants, a major drawback of this approach is the
The specificity of bacteriophage infection allows their need for identification of the specificity range against the
application in several areas such as biotechnology, ecology, pathogenic bacteria prior to starting the bacteriophage treatment
health and environment (bacterial control), and as environmental and the lack of protocols for testing bacterial susceptibility
monitoring agents (Armon and Kott, 1996; Leclerc et al., in vitro (Kutateladze and Adamia, 2010). As with antibiotics,
2000; Arredondo-Hernandez et al., 2017; McMinn et al., 2017; if incomplete bacterial elimination by bacteriophages occurs,
Friedman and Lai, 2018; Vandamme and Mortelmans, 2019). this could result in the pathogen reemergence (Carlton et al.,
In this review, a vast amount of scientific literature has 2005; Bigwood et al., 2008). A probable explanation could be
been reviewed on the application of phage-based products, that bacteria might show a temporal resistance, or that the
discussing the benefits and limitations of the use of bacteriophage infection results in high levels of reduction but not
bacteriophages as bacterial control tools in the health, food, a complete elimination of bacteria (Hoskisson and Smith, 2007;
and environmental fields. Tokman et al., 2016; Moye et al., 2018).
However, contrasting findings of the bacteriophages cocktails
effectiveness (compared to “conventional” treatments such as
BACTERIOPHAGES APPLICATION antibodies) were also achieved, with limitations and advantages
in the use of cocktails to treat bacterial infections being
Fighting Bacterial Infections extensively reviewed (Altamirano and Barr, 2019; Furfaro et al.,
Bacterial infections are a major public health concern worldwide, 2018; Principi et al., 2019). In study conducted by Jault et al.
representing an enormous economical and medical burden with (2019), a cocktail contend 12 bacteriophages was compared
a fatal outcome in a significant proportion of those affected. to antibody treatment in patients with skin infections, in
Dysentery caused by Shigella spp., Salmonella spp., Proteus a randomized control trial. At the end of the study, the
spp. Staphylococcus, Escherichia coli, and Pseudomonas, usually conventional treatment with a 1% sulfadiazine silver emulsion

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Rogovski et al. Bacteriophages Uses

TABLE 1 | Taxonomy, morphological, and molecular characteristics of bacteriophage groups.

Family Genus Nucleic acid Morphology Main host

Leviviridae Levivirus ssRNA Icosahedral E. coli

Cystoviridae Cystovirus dsRNA Icosahedral Pseudomonas spp.
Microviridae Phix174microvirus ssDNA Icosahedral E. coli
Inoviridae Fibrovirus ssDNA Filamentous or rod Vibrio spp.
Podoviridae P22virus dsDNA Tailed S. typhimurium
Plasmaviridae Plasmavirus dsDNA Spherical to pleomorphic Mycoplasma spp.

cream was still more effective than the cocktail. However, more and SalmonelexTM (Hagens and Loessner, 2010). The use of
randomized, placebo-controlled trials must be done in order to a bacteriophage cocktail to inactivate foodborne bacteria like
have some consensus in dealing with the use of bacteriophages to S. enteritidis and S. typhimurium on the chicken breast has also
treat infections. been proposed (Duc et al., 2018). These bacteriophages have
There are some limitations in the use of bacteriophages been isolated from environmental sources such as wastewater,
for treating human infections. Due to the ability of certain sewage, water or food (Pereira et al., 2016). Bacteriophages
bacteriophages to integrate their genome into the host’s genome, may also be applied for biofilm control on the food industry,
care must be taken when selecting isolated bacteriophages. Some such structures form on surfaces that have been improperly
bacteriophages have potential for gene transfer; for instance, sanitized (Jessen and Lammert, 2003). Outbreaks of bacterial
the bacterial acquisition of antibiotic resistance genes (ARGs) pathogens associated to biofilms in food chain have been related
occurs by transduction, with bacteriophages acting as mobile to the presence of Listeria monocytogenes, Yersinia enterocolitica,
genetic elements (MGE). Consequently, bacteriophages have Campylobacter jejuni, Salmonella spp., Staphylococcus spp. and
been studied as possible vehicles of ARGs, not only as a E. coli O157:H7 (Aarnisalo et al., 2007). In this context,
source, but also as propagators in the environment (Gunathilaka bacteriophages have been suggested as a green strategy for biofilm
et al., 2017). Bacteriophages containing ARGs are present in control, as they may provide a natural, highly specific, non-
a wide range of environments; however, some environmental toxic, feasible approach for biofilm formation control (Grant
niches have a greater abundance, such as freshwater or marine et al., 2017). Biofilm control using bacteriophages has also
environments (Lekunberri et al., 2017a,b; Calero-Cáceres and been used to prevent dental caries, where the bacteriophages
Luis, 2019). Bacteriophages can be also found in hospital were first isolated from saliva samples and also in biofilm-
wastewater, yet human-associated viromes rarely charge ARGs mediated illness like endodontic disease, which is caused by
(Figure 1B; Enault et al., 2017; Lekunberri et al., 2017a,b). dental pulp infection (Stevens et al., 2009; Dalmasso et al.,
2015). However, it is important to highlight that each bacterial
serovar could show different degrees of susceptibility to
Control Tools for Food and different bacteriophages (Grant et al., 2017). In addition, it is
Environmental Bacterial Contamination important to highlight that biofilm control by bacteriophages is
Foodborne pathogens are a major food safety threat, in mediated by the chemical composition, environmental factors,
2010 an estimated 2 billion individuals contracted foodborne growth stage and bacteriophage concentration. Additionally,
illnesses, resulting in 1 million deaths around the world bacteriophage-biofilm interactions depend on the susceptibility
(Kirk et al., 2015). Food safety is regarded by the World of the biofilm cells and availability of receptor sites, where
Health Organization [WHO] (2015) as a major obstacle in bacteriophage production of polysaccharide-degrading enzymes
human development, especially in developing countries that combined with effective cell lysis may rapidly destroy the biofilm
lack infrastructure and proper environmental health practices (Simões et al., 2010).
to counter the issue. The application of bacteriophages has Bacteriophages also show significant potential in the animal
been proposed as an alternative tool to disinfect food and production chain such as fish, poultry, shrimps, oysters, sheep,
food-related environments (Pang et al., 2017). The advantage pork and also as additives in food products such as poultry
of this method is that bacteriophages kill their bacterial hosts meat and eggs (Moye et al., 2018). They can prevent foodborne
without changing food organoleptic properties (Loc-Carrillo and pathogens such as Campylobacter spp., E. coli, L. monocytogenes,
Abedon, 2011; Perera et al., 2015). Also, bacteriophage low- Salmonella enterica, and Shigella spp., that are the top five
cost large scale production, self-replicating nature, and low foodborne public health threatening pathogens (Figueiredo and
toxicity provide a cheap and safe disinfecting agent for low- Almeida, 2017; Harada et al., 2018; Kim et al., 2020).
income communities, being employed in the former Soviet Union Bacteriophages have shown very effective to control
for over 100 years (Skurnik et al., 2007; Abedon et al., 2011; L. monocytogenes by the commercial product based on
Wójcik et al., 2020). bacteriophages LISTEXPTM 100 reported a better efficacy
The United States Department of Agriculture (USDA) against L. monocytogenes than nisin and sodium lactate in
approved some products based on bacteriophages as food ready-to-eat (RTE) sliced pork ham (Figueiredo and Almeida,
sanitizers, such as ListShieldTM , Listex P-100TM , SalmoFreshTM , 2017). Chibeu et al. (2013) used a Listeria-specific bacteriophage

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Rogovski et al. Bacteriophages Uses

FIGURE 1 | (A) Bacteriophage life cycle. Bacteriophages can replicate by lytic, lysogenic or remain in a chronic state. In the lytic cycle of replication the viruses are
released from the host after completing their replication. Lysogenic cycle involves the inclusion of genetic material into the genome of host cells, this way phages can
contribute to the transferring of tetracycline resistance plasmids to bacterial cells (Pratama and Van Elsas, 2017). This is evidenced considering bacteriophages
genomes, which can represent up to 13 and 5% of the Escherichia coli O157:H7 and Salmonella Newport genomes, respectively (Bobay et al., 2013). (B) Phage’s
journey through different cell lines in different environments: (1) Bacteriophage adhering to the resistant bacterial host. (2) Insertion of the phage genetic material. (3)
New phages carrying resistance genes derived from the infected bacteria, being released into the environment. (4) Adhesion of the new phage on non-resistant
bacteria. (5) Passage of resistance genes inserted into the phages genome to the bacteria. (6) After the genes insertion, the bacteria can enjoy the resistance genes
acquired while new bacteriophages can be released keeping the journey of resistance genes on the environment.

on the surface of deli meats; a single bacteriophage strain effective approach against a unique species of bacteria, ensuring
was effective in reducing the numbers of Listeria cells (ATCC that resistant bacteria are not selected. The application of the
19115). The evaluation of LISTEXTM P100 as a bacteria controller cocktail ListShieldTM including six L. monocytogenes specific
measured the bacteriophage inactivation using black tea extract bacteriophages efficiently reduces this pathogen in cheese,
and ferrous sulfate and isolation of regrowth bacteria and their smoked salmon, apple slices, and frozen entrees (reduction
control. The result was the reduction of 1.5–2.1 log10 CFU/cm2 of 91, 82, 90, and 99%, respectively), without changing the
on RTE meat samples by application of 100 µl LISTEXPTM 100 food organoleptic properties (Perera et al., 2015). Similarly,
covering 10 cm2 area during 28 days, resulting on 107 PFU/cm2 reductions of up to 5 logs of L. monocytogenes were observed
final concentration. A cocktail of bacteriophages can be a more in various solid foods, such as smoked salmon, iceberg lettuce

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Rogovski et al. Bacteriophages Uses

leaves, sliced cabbage, hot dogs, mixed seafood, turkey meat, adsorb to solid particles in the environment, and also due to
and mozzarella cheese brine (Guenther et al., 2009). In fact, the some limitations of traditional indicators for public health such
use of a lytic bacteriophage on soft cheese was able to reduce as fecal coliforms, E. coli and enterococci (Armon and Kott, 1993;
2 logs of the Listeria contamination while maintaining the Ashbolt et al., 2001; Jofre et al., 2016; McMinn et al., 2017).
natural microbial community of the cheese, reinforcing the host Somatic coliphages are more persistent than traditional
specificity of bacteriophages, and in this case the bacteriophage indicators, being also more resistant to sludge treatments,
A511 (Guenther and Loessner, 2011). particularly when adsorbed to surfaces (Martín-Díaz et al.,
The number of commercial solutions containing 2020). Many authors highlight the use of bacteriophages as
bacteriophages is increasing worldwide, being an emerging indicators not only for enteric pathogenic bacteria, but also for
industry and field of research (Sulakvelidze, 2013; Vikram enteric viruses such as human noroviruses, adenoviruses, and
et al., 2020). Different examples of bacteriophage applications rotaviruses (Guelin, 1948; Dutka, 1990; Cornax et al., 1991; Kott,
on food industry are already available: a three-bacteriophage 1992; Armon and Kott, 1996; Leclerc et al., 2000; Arredondo-
cocktail (containing EC6, EC9, and EC11) was able to reduce Hernandez et al., 2017; McMinn et al., 2017). This characteristic
E. coli contamination; E. coli ATCC 25922 and E. coli O127:H6 is due to the wide stability of phages in waste, water, soils and
in Ultra High Temperature (UHT) milk at 25◦ C and under residues, with F-specific phages and somatic coliphages being the
refrigeration temperatures (5–9◦ C) (McLean et al., 2013). The most used for monitoring water quality (Grabow, 2001; Sinton
cocktail EcoShieldTM was able to reduce 2 logs of E. coli O157:H7, et al., 2002).
30 min after administration on leafy greens under packaging One of the challenges with bacteriophage application is related
storage (Boyacioglu et al., 2013). Magnone et al. (2013) verified to bacteriophage-host interaction, which could vary depending
the disinfection of E. coli, Salmonella and Shigella from broccoli, on exposition temperature, where greater bacterial reductions are
cantaloupe and strawberries, with the use of commercial associated with higher temperatures (Tomat et al., 2013). The
bacteriophage cocktails (EcoShieldTM , SalmoFreshTM , and use of bacteriophages on wastewater treatment systems is based
ShigActiveTM ) being as effective or even more than chlorine on their lytic capacity, which is a useful tool for the removal
wash. Salmonella is a major threat for the food industry and of human and animal pathogenic bacteria from wastewater or
the most common zoonotic foodborne pathogen isolated from applied as an indicator for the presence of bacteria in wastewater
livestock (Jajere, 2019). The bacteriophage FO1-E2 was able treatment systems (Stefanakis et al., 2019). MS2 bacteriophages
to reduce the levels of Salmonella contamination on milk and have been proposed to be suitable as operational monitoring
mixed seafood for 24 h, remaining undetectable at 8 and 15◦ C indicators as established by guidelines of Australia, due to
(Guenther et al., 2012). Similarly, bacteriophage wksl3 was also resistance to variation of pH and temperature (Amarasiri et al.,
able to decrease by 3 logs Salmonella contaminations on chicken 2017). Other applications of bacteriophages in the improvement
skin (Kang et al., 2013). Likewise, some bacteriophage cocktails of environmental quality are based on their survival in the
for Salmonella control are also available. The commercial environment, and soil percolation to control pathogenic bacteria
formulation SalmoFreshTM was able to reduce 2–3 logs of in underground water (Ye et al., 2019). However, there are still
Salmonella on lettuce and sprouts, showing greater reduction some challenges for the use of bacteriophages in wastewater
(2.7–3.8 logs) when associated with chlorinated water (Zhang treatment: a high bacteriophage dosing must be used for a
et al., 2013). An outstanding biocontrol activity was seen with successful application and the use of polyvalent bacteriophages
the use of a bacteriophage cocktail composed by LPSTLL, with a wider host variety could result in the reduction of
LPST94, and LPST153, being able to reduce >5.23 log viable beneficial bacteria. The bacterial analysis of the system is a
Salmonella counts on biofilm grow in microtiter plates and basic requirement for bacteriophage application, as the bacterial
steel chips, for 72 h. The same bacteriophages combination population can change in the wastewater treatment plant
was also able to completely reduce the Salmonella inoculum (Jassim et al., 2016).
on chicken breast and milk (Islam et al., 2019). Table 2
summarizes the bacteriophages commercially available and the
conditions for its applicability on treatment against different
bacterial agents.
CHALLENGES, CONCERNS AND
TRENDS IN THE USE OF
BACTERIOPHAGES FOR
Indicators of the Presence of Foodborne ENVIRONMENTAL HEALTH PURPOSES
Pathogens
Bacteriophages have been suggested as an alarm system in food Although a worldwide acceptance of bacteriophages as
and environmental microbiology and epidemiology since they environmental agents is not yet achieved, bacteriophage-based
generally fit the indicator criteria of pollution. Bacteriophages technologies in the environmental field are still being developed.
can be used as fecal indicators or microbial water quality Besides being employed as monitoring agents, or by directly
bioindicators as an early warning of contamination by sewage, controlling pathogens, bacteriophages have demonstrated
and as an efficiency marker of water or wastewater treatment promising results in agricultural microbiome modulation,
(Yahya et al., 2015). This can be attributed to the bacteriophage increasing crop production by infecting crop detrimental
response to the presence of pollutants, they are characteristic to bacteria in leaves and soil (Jones et al., 2012; Ye et al., 2019).

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Rogovski et al. Bacteriophages Uses

TABLE 2 | Current commercial products containing bacteriophages and conditions for use.

Host Bacteriophage Dose Treatment time Matrix Reduction log References

Listeria LISTEXTM P100 107 PFU/cm2 30 min, 1, 2, 3, 7, Roast beef and cooked 2 log10 CFU/cm2 Chibeu et al. (2013)
monocytogenes 10, 14, 20, and turkey
28 days
FWLLm1 2.5 × 107 PFU/cm2 24 h Ready-to-eat chicken 2.5 log10 CFU/cm2 Bigot et al. (2011)
breast roll
P100/A511 3 × 108 PFU/g 6 days Hot dogs (sausages), 1–3 log10 CFU/cm2 Guenther et al.
cooked and sliced turkey (2009)
breast meat (cold cuts),
smoked salmon, mixed
seafood (cooked and
chilled cocktail of shrimp,
mussels, and calamari),
chocolate milk (pasteurized,
3.5% fat), mozzarella
cheese brine (unsalted
pasteurized whey from
plastic bag containers
containing fresh mozzarella
cheese), iceberg lettuce
(leaves), and cabbage
(sliced fresh leaves)
ListShieldTM 109 PFU/mL 0, 2, 5, and 7 days Fresh-cut melons and 3.5 log10 CFU/cm2 Leverentz et al.
apples (2001)
Salmonella SJ2 108 PFU/mL 24 h Raw and pasteurized milk 1–2 log10 CFU/cm2 Modi et al. (2001)
nteritidis cheeses
PHL 4 1010 PFU/mL 24 h Poultry carcass 3 log10 CFU/mL Higgins et al. (2005)
Salmonella Felix-O1 5.25 × 106 PFU 24 h Chicken frankfurters 2 log10 CFU/g Whichard et al.
typhimurium (2003)
SalmoFresh 108 PFU/mL 5h Ready-to-eat chicken 2 log10 CFU/mL
products
S. enteritidis wksl3 2.2 × 108 PFU/mL 1, 2, 3, 5, and Chicken skin 3 log10 CFU/mL Kang et al. (2013)
and 7 days
S. typhimurium
SalmoFREE 108 PFU/mL 36 days In vivo–chicken production 3 log10 CFU Clavijo et al. (2019)
SalmonelexTM 109 PFU/mL 24 h Ground beef and ground 1.1 and 0.9 Yeh et al. (2017)
pork log10 CFU/g

Plant-soil microbiome modulation by bacteriophages was even and fitness by transferring toxin and environmental resistance
related to an increase in ammonium concentration, likely encoding genes to nearby bacteria, essentially creating genetic
through lysis of certain bacteria and overall community shifting hazards in the area of application (Colomer-Lluch et al., 2011;
(Braga et al., 2020). The use of bacteriophages on plant soil was Feiner et al., 2015). Besides bacteriophage-induced resistance,
referred to as a safer and more reliable antibacterial agent than the bacteria may also become resistant to the virus activity
antibiotics, in which the exaggerated use of these chemicals through spontaneous mutations or through adaptive immunity
was related to the development of ARGs and inhibition of soil via the CRISPR system (Labrie et al., 2010). Another possible
phosphatase activity (Liu et al., 2009; Zhang et al., 2017; Sun major drawback in bacteriophage application is the potential
et al., 2019). disruption of the local microbiome, consequently favoring the
Similar to soil applications, bacteriophages appears to development of harmful bacteria or health problems associated
have a low environmental impact in fish farming plants to a microbiome disbalance. Bacteriophage application has
compared to “traditional” methods such as antibodies, as it is been tied to microbiome dysbiosis in humans, and can be
necessary a continuous application since seawater is considered related to the subsequent development of intestinal and mental
a reservoir of antibiotic resistance bacteria (Almeida et al., diseases (Norman et al., 2015; Tetz et al., 2018). Microbiome
2009; Alves et al., 2014; Hatosy and Martyiny, 2015). Even disruption was also related to the development of diseases in
though bacteriophages can be considered as highly flexible both livestock and plants, therefore an improper bacteriophage-
and cheap tools, some drawbacks concerning the safety and based product (i.e., bacteriophages that may infect healthy
overall effectiveness of the phage product may hinder their microbiome) may also potentially harm animal and plant farming
implementation as a widely accepted technology (Payne and production (Meaden and Koskella, 2013; Zeineldin et al., 2018;
Jansen, 2003). Bacteriophages can increase bacteria pathogenicity Lei, 2020).

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Rogovski et al. Bacteriophages Uses

In sight that bacteriophages may persist in food production characterization of the virus and host synergy is highly advisable
plants due the virus high stability, potentially creating a for easy escalation of the phage product (García et al., 2019).
genetic hazard in such facilities, the adoption of strategies With advances in molecular biology the engineering of
for the use and manipulation of bacteriophages are required bacteriophage particles allows a selected virus (favorited due
to counter bacteria resistance and achieve successful pathogen desirable characteristics to the target therapy, such as host range
control (Hungaro et al., 2013; Chaturongakul and Ounjai, and replicative potential) to be further enhanced through genetic
2014; Fister et al., 2016, 2019). In this regard, practices that modifications, removing undesirable viral properties that could
reduce the probability of bacteriophage resistance occurrence hinder the application of the bacteriophage product as a safe
must be preferred, such as a two-stage self-cycling or a and reliable object (Górski et al., 2018). Genetic engineering of
cellstat process (García et al., 2019). Bacteriophage cocktails phage products was able to remove toxin encoding genes and
have been also been employed as a way to counter bacterial increment the virus stability in low pH environments, enhancing
bacteriophage resistance, in this strategy the bacteria would the functionality and removing safety hazards of the final viral
be unable to adapt (or have their viability greatly reduced) product without requiring the selection of new bacteriophage
to the different infective dynamics of each virus, however, strains (Nobrega et al., 2016; Park et al., 2017).
knowledge about the cocktail pharmacodynamic is required to Although bacteriophages present certain safety drawbacks,
achieve a multi-targeting system against the same bacterial strain largely due to negligence of mapping the product properties,
(Abedon et al., 2021). bacteriophages are still considered safer than chemical treatments
Aside from ARGs screening and mapped host targeting, in environmental and food processing plants treatments
the phage product must be suitable to the external factors applications (Meaden and Koskella, 2013; Zaczek et al., 2014).
present in the area of application, being resistant to the Bacteriophages stand as cheap and highly flexible structures,
pH, temperature, UV radiation, salinity and ionic profile of being able to be selected and edited for different approaches
the environment (Jończyk et al., 2011; Zaczek-Moczydłowska (Farr et al., 2014; Sunderland et al., 2017). Most of the research
et al., 2020). In addition, the criteria for bacteriophage use on bacteriophages has highlighted the potential for in vitro
in food and the environment, such as minimum exposure applications, and the number of scientific publications has
time, minimum effective dosage and characterization of animal increased in the last decades due to the potential use of
local application must be established to achieve the expected bacteriophages in a broad spectrum of applications. In health
therapy result while avoiding potentials drawbacks such as the sciences, bacteriophages are a promising approach in the fight
presence of inhibitory compounds like antibodies, whey proteins against antibiotic-resistant bacteria, and, in the food chain, they
or bacteriocins (Abedon, 2012; Vongkamjan et al., 2013; Ly- could be a safe alternative for the control of foodborne pathogens.
Chatain, 2014). However, to guarantee effectiveness, a detailed understanding of
Special regards covering the bacteriophage properties are the interaction between bacteriophages and the hosts is needed,
also advisable for an optimal and highly scalable confection of considering restrictive criteria for their use to minimize their
the final viral product, being of special relevance in extensive negative impact on food and food-related environments.
environmental applications. Bacteriophage production is directly
related to the characteristics of the bacterial host (e.g., metabolic
activity, growth rate, stage in cell life cycle, and abundance of AUTHOR CONTRIBUTIONS
bacteriophage receptors on cell surface), and the bacteriophage
attributes (e.g., lysis time, burst size, and adsorption rate) PR directs the first version of the manuscript. GF and DR-L
(Agboluaje and Sauvageau, 2018). In addition, the initial revised the first version of the manuscript and wrote the
multiplicity of infection (MOI), pH, aeration rate, presence of final version of the manuscript. The rest of the authors
ions or cofactors, agitation and medium composition may also gave fundamental contributions to the first version of the
influence the outcome of infections, thus affecting bacteriophage manuscript. All authors contributed to the article and approved
production (Agboluaje and Sauvageau, 2018). Therefore, a full the submitted version.

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Yeh, Y., Purushothaman, P., Gupta, N., Ragnone, M., Verma, S. C., de Mello, A. S., potential conflict of interest.
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Myoviridae bacteriophages inhibits the growth of Pectobacterium spp. under Michelon, Elmahdy, Treichel, Rodríguez-Lázaro and Fongaro. This is an open-access
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