Review

Selected Mechanisms of Action of Bacteriophages in Bacterial

Infections in Animals
Renata Urban-Chmiel and Ewelina Pyzik *

Department of Veterinary Prevention and Avian Diseases, Faculty of Veterinary Medicine, University of Life
Sciences in Lublin, 20-033 Lublin, Poland; renata.urban@up.lublin.pl
* Correspondence: ewelina.pyzik@up.lublin.pl

Abstract: Bacteriophages, as ubiquitous bacterial viruses in various natural ecosystems,

play an important role in maintaining the homeostasis of the natural microbiota. For many
years, bacteriophages were not believed to act on eukaryotic cells; however, recent studies
have confirmed their ability to affect eukaryotic cells and interact with the host immune sys-
tem. Due to their complex protein structure, phages can also directly or indirectly modulate
immune processes, including innate immunity, by modulating phagocytosis and cytokine
reactions, as well as acquired immunity, by producing antibodies and activating effector
cells. They can therefore have a profound impact on the course of bacterial infections
by stimulating and at the same time inhibiting the systemic pro-inflammatory response.
This review article presents a characterization of the processes by which bacteriophages
affect selected immune mechanisms in selected animal species. The results of our own
experiments using calves are also presented as examples. The paper contains many new
examples of potential uses of bacteriophages and their effects on eukaryotic cells, especially
in the course of bacterial infections, which are extremely important in experimental treat-
ments exploiting phages as alternatives to antibiotics. The positive results of the effects of
bacteriophages on eukaryotic cells during infections open up promising new prospects for
their use as natural tools in the treatment of bacterial, fungal, and viral diseases in animals
and humans.

Keywords: bacteriophages; anti-phage antibodies; bacteria; infections

Academic Editors: Grażyna
Majkowska-Skrobek and Daria
Augustyniak

Received: 11 December 2024

1. Introduction
Revised: 8 January 2025 Bacteriophages are also known as bacterial viruses, and their life cycles are inextricably
Accepted: 10 January 2025 linked to the bacterial cells serving as their hosts. Due to the specific nature of their
Published: 14 January 2025
functioning, resulting from their lack of specific enzymatic structures, bacteriophages
Citation: Urban-Chmiel, R.; Pyzik, E. cannot exist independently outside the bacterial host cell. Phages are ubiquitous in various
Selected Mechanisms of Action of
natural ecosystems, and their presence has also been confirmed in the bodies of animals
Bacteriophages in Bacterial Infections
in Animals. Viruses 2025, 17, 101.
and humans, e.g., in the mouth, the gastrointestinal tract, and the respiratory system,
https://doi.org/10.3390/v17010101 as well as in urine and serum. Their roles include maintenance of the homeostasis of
natural ecosystems [1].
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
Bacteriophages were first described as separate particles at the beginning of the
This article is an open access article 20th century, at about the same time, by two independent scientists, Félix d’Hérelle [2] and
distributed under the terms and Frederick William Twort [3]. At that time, phages were recognized as being effective in
conditions of the Creative Commons treating bacterial infections. However, due to Alexander Fleming’s discovery of penicillin
Attribution (CC BY) license
in 1928 and the subsequent development of the antibiotic era, work on phage therapy was
(https://creativecommons.org/
abandoned for many years, especially in Western countries. However, research on the
licenses/by/4.0/).

Viruses 2025, 17, 101 https://doi.org/10.3390/v17010101

Viruses 2025, 17, 101 2 of 16

development of bacteriophages continued to be conducted in Eastern European countries,

for instance, at the Eliava Institute of Bacteriophages, Microbiology, and Virology (EIBMV)
of the Georgian Academy of Sciences, in Tbilisi, Georgia, and the Hirszfeld Institute of
Immunology and Experimental Therapy (HIIET) of the Polish Academy of Sciences, in
Wroclaw, Poland [4,5].
Currently, due to the crisis induced by the globally increasing occurrence of drug resis-
tance in bacteria, which poses a threat to humans and animals, bacteriophages may become
one of the key alternatives to antibiotics, with practical potential in controlling bacterial
infections [6]. Moreover, bacteriophages are the most abundant viral components in the
bodies of humans and animals [7,8] and can play a key role in maintaining homeostasis in
eukaryotic organisms [9].
According to the International Committee on Taxonomy of Viruses [10], the class
Caudoviricetes currently comprises four identified orders of bacterial viruses, Crassvirales,
Kirjokansivirales, Methanobavirales, and Thumleimavirales, as well as one order described as
‘unidentified’. Within the four identified orders, 14 families, 11 subfamilies, 59 genera,
and 96 species have been identified. In the order classified as ‘unidentified’, 33 families
have been distinguished, as well as an additional ‘unidentified’ family, 48 subfamilies,
690 genera, and 2156 species [11].
Experimental phage treatments in humans and animals show varied therapeutic
efficacy and are very safe in the treatment of numerous diseases caused by pathogenic
bacteria, from the treatment of burn wounds and infections of the skin and subcutaneous
tissue to urinary tract, lung, and gastrointestinal infections [12].

1.1. Phages and Eukaryotic Cell Interactions

For many years, the prevailing opinion was that bacteriophages did not act on eu-
karyotic cells and exhibited tropisms only for bacteria serving as hosts for a given phage.
Recent research, however, confirms completely new traits of bacteriophages, including the
potential to penetrate mucous membranes and interact with the immune system of humans
and animals [13]. For example, bacteriophage T4 has developed mechanisms of action on
human mucosal immunity [14]. The study cited showed that this mechanism is possible
due to binding of the phage to mucin, which restricts the ability of pathogenic bacteria to
colonize epithelial cells and increases the phage’s capacity for subdiffusive motion on the
mucosal surface and penetration into specific areas of the mucus, making it more effective
in killing bacteria [15].
The effects of phages on the cells of complex organisms include their effects on immune
tolerance, mucosal immunity, and homeostasis of the intestinal microbiome [16].
Apart from the extracellular antibacterial effects of bacteriophages, there have also
been studies confirming that phages can inactivate intracellular bacteria. For example,
a study conducted by Kaur et al. [17] demonstrated that phage MR-5 specific to S. aureus
enhances the potential of phagocytic cells to kill intracellular bacteria.
In the course of bacterial infections, bacteriophages also interfere with eukaryotic cells,
affecting inflammatory response mechanisms and innate immunity and even reducing
transplant rejection [12]. Due to their protein structure, the presence of phages in the
human or animal body can also directly or indirectly modulate immune processes. Phages
can affect both innate immunity, through phagocytosis and cytokine reactions, and ac-
quired immunity, by inducing antibody production and activating effector cells. They can
therefore have a profound impact on the course of bacterial infections by modulating the
immune response [18].
The microbiome of a healthy human being or animal, apart from very numerous
bacteria, also contains a large number of bacterial viruses, i.e., bacteriophages, which form
Viruses 2025, 17, 101 3 of 16

the phageome [19]. Some research [20] indicates that intestinal bacteriophages mainly show
affinity for anaerobic bacteria and that as many as 75% to 99% of sequences from intestinal
phages do not yet fully match any known viral genome. It has also been suggested that
disturbing the homeostasis of the phageome can lead to intestinal dysbiosis, stimulating
the development of type 1 diabetes and intensifying the development of inflammation in
inflammatory bowel disease. However, the presence of phages can also cause changes in
the characteristics of bacteria, especially in the case of formation of intracellular prophages.
Examples include immunity to a superinfection, elimination of competition for bacterial
colonization, the possibility of horizontal transfer of genes determining an increase in drug
resistance in bacteria, and an increase in their virulence [16].
Bacteriophages also exhibit the ability to move from the intestinal environment to
other systems and organs, such as the kidneys, liver, spleen, muscles, lymph nodes, and
the circulatory system [19,21].

1.2. Transport of Phages Through Eukaryotic Cells

The affinity of bacteriophages for mucus activates their ability to penetrate cells and
tissues, usually by endocytosis and transcytosis, e.g., in intestinal epithelial cells or through
dendritic cells [18,22]. Phages can migrate through layers of epithelial cells and then enter
the bloodstream and spread throughout the body, activating immune responses [18]. This
is the result of interactions between phages and eukaryotic cell membranes via transmem-
brane mucins and specific and nonspecific receptors, which enable signal transmission in
epithelial cells. The phenomenon of phage–epithelial transcytosis takes place in various
types of epithelial cell layers (e.g., in the cells of the intestines, lungs, liver, kidneys, and
brain) and is a response to the spread of phages in the body, during bacterial infection as
well as in states of health [23,24].
An example of bacteriophage interaction with eukaryotic cells and their translocation
through them is illustrated in Figure 1.
The mechanisms by which bacteriophages activate B and T lymphocytes are not yet
fully understood, as suggested by the latest research [12]. Earlier research, however [19],
indicates that activation of B lymphocytes as a result of contact between dendritic cells and
phages induces the production and release of anti-phage antibodies in the intestines and
other organ systems. Bacteriophages have also been shown to influence the production
of cytokines, such as IFN-γ and IL-6, due to activation of T cells in Peyer’s patches and
mesenteric lymph nodes.
Bacteriophages are also currently believed to influence the immunity of eukaryotic
organisms. In particular, phages present in the bloodstream have been shown to be able to
modulate the innate and acquired immune response through contact with immune cells. Some
studies have shown that despite the significant role of the intestine, it is not the only source of
bacteriophages in the blood, and translocation of phages from the intestine to the bloodstream
is irregular and weak [21]. Bacteriophages can penetrate the bloodstream at a rate of 98.5% via
injection (i.v., i.m. s.c, or i.p.), 66.7% via inhalation, 50% by topical administration, and 41.1%
through oral administration (oral cavity and gastrointestinal tract) [27].
There are two main aspects of interactions between phages and immune cells. The
first is phage immunogenicity, i.e., the natural ability of phages to induce a specific immune
response, especially the production of antibodies against phage antigens (mainly capsid
proteins). The second aspect is the immunomodulatory activity of bacteriophages, i.e., their
nonspecific effects on various functions of the major immune cell populations involved
in both the innate and acquired immune response. In this case, bacteriophages can affect
a variety of immune functions, such as phagocytosis or respiratory burst of phagocytic cells;
antibody production; and proliferation of B cells, monocytes, and macrophages [28,29].
 then enter the bloodstream and spread throughout the body, activating immune re-
sponses [18]. This is the result of interactions between phages and eukaryotic cell mem-
Viruses 2025, 17, 101 branes via transmembrane mucins and specific and nonspecific receptors, which enable 4 of 16
signal transmission in epithelial cells. The phenomenon of phage–epithelial transcytosis
takes place in various types of epithelial cell layers (e.g., in the cells of the intestines, lungs,
For example,
liver, kidneys,itand
wasbrain)
suggested
and isthat bacteriophages
a response may induce
to the spread phagocytosis
of phages in the body,processes
during
by adhesion and opsonization to bacterial cells, thereby
bacterial infection as well as in states of health [23,24]. increasing the ability of immune
cells An
to recognize
example ofbacteria–phage complexes,with
bacteriophage interaction which increasescells
eukaryotic the and
phagocytic activity of
their translocation
neutrophils and macrophages [30,31].
through them is illustrated in Figure 1.

Figure
Figure 1.1. Schematic representationof
Schematic representation ofvarious
variousinteractions
interactionsand
andtranslocation
translocationofofphages
phages with
with epithe-
epithelial
lial
cellscells adapted
adapted in from
in part part from [18,22,23,25]
[18,22,23,25] with
with our ourmodifications.
own own modifications.
Legend:Legend: Bacteriophages
Bacteriophages present
on mucosal surfaces can be taken up by dendritic cells or epithelial cells by means of transcytosis
or can diffuse through damaged barriers to reach internal tissues, including the bloodstream [18].
Contact between phages and eukaryotic cells is usually possible through extracellular factors such as
glycoproteins and glycolipids forming a mucin layer which protects the cells. Adherence of phage
particles to this layer creates an antimicrobial barrier, which reduces colonization by bacteria and
epithelial cell death [18]. Beyond this layer, phages can be adsorbed (in just 30 s) directly to integrin
and other T cell receptors (TCRs), or, alternatively, can bind directly to sialic acid residues [25].
Intensification of phage adsorption from the eukaryotic cell surface can be enhanced by modulating
electrostatic interactions between the phage and eukaryotic cells using a nano-cap strategy, as
demonstrated by [26]. Next, phage particles are taken up by epithelial cells and can then be degraded,
leading to intracellular release of phage components and free phage DNA. Following endocytosis,
phage nucleic acids can trigger TLR (Toll-like receptor) pathways, particularly those dependent on
TLR9 receptors, thereby stimulating the acquired immune response [22]. Bacteriophages can also
penetrate eukaryotic cells, which allows them to spread through the body, including the bloodstream.
This is known as ‘phage transcytosis’. They can also move through the body through a ‘leaky
gut’, which allows them to bypass the barrier of epithelial cells at damaged sites or sites of blood
vessel perforation [18].

Differences in the mechanisms of bacteriophage action on eukaryotic cells may also

result from their modes of translocation. As shown by Sun et al. [32], bacteriophage translo-
cation is related to their morphological structure, e.g., Caudovirales phages with large
tails, the best-known representative of which is phage T4, use for this purpose a contractile
envelope surrounding the tail tube, which contracts after detecting the host cell surface,
causing puncture of the outer cell membrane. On the other hand, small, filamentous, tailless
Viruses 2025, 17, 101 5 of 16

icosahedral bacteriophages, with the exception of microviruses, use channels encoded by

host cells for translocation. Microviruses, a well-known representative of which is phage
ΦX174, have quite specific conditions. In the studies of Jaźwiński et al. [33], it was sug-
gested that specific phage H proteins may create a channel for DNA translocation through
the cell wall. However, the authors indicated that this is only a hypothesis, and the exact
mechanisms of translocation have not been identified.
The latest studies [34], which analyzed the translocation patterns of phages T4,
ΦX174, and M13 across the intestinal barrier, showed that the highest level of associa-
tion with eukaryotic cells and the highest translocation were exhibited by the M13 phage;
in second place was phage T4, and the lowest translocation was observed in the case of
phage ΦX174. However, no statistically significant differences were observed in the rate of
translocation of the tested phages across the endothelial barrier [34].
The observed differences in the translocation of the tested phages resulting from
their structure may affect the route of their translocation. Most phages with a filamentous
structure, like M13, can migrate through mammary cells by macropinocytosis. In contrast,
T4 and ΦX174 phages may use different endocytosis pathways. The least-understood
mechanism is characteristic of microphages [35,36].

1.3. Anti-Inflammatory Effect of Bacteriophages

Bacteriophages can also exhibit anti-inflammatory activity, taking part in controlling
inflammatory responses, e.g., in the following ways:
• Inducing an increase in the expression of the anti-inflammatory interleukin-1 receptor
antagonist (IL-1RA);
• Stimulating the production and release of IL-10; blocking the expression of pro-
inflammatory cytokines (IL-1α and -β and IL-6); and inhibiting the activity of Th1
cells, NK cells, and macrophages [1];
• Inhibiting the release of TNF-α (the main inflammatory cytokine), nuclear factor-κB
(NF-κB), and C-X-C motif chemokine ligand 12a (CXCL12a);
• Inhibiting the activity of Toll-like receptor 4 (TLR4), whose activation stimulates the
production and release of pro-inflammatory factors;
• Inhibiting the development of oxidative stress, e.g., by reducing overproduction of
reactive oxygen species (ROS) in phagocytic cells—macrophages and neutrophils [12];
• Increasing the expression of anti-inflammatory factors, such as suppressors of cytokine
signaling (SOCS3) or IL-1 (IL1RN) and IL-10 receptor antagonists [12];
• Inhibiting migration of neutrophils and granulocyte–macrophage colony-stimulating
factor (GM-CSF) [37,38].
Examples of the pro- and anti-inflammatory mechanisms of action of bacteriophages
on eukaryotic cells are presented in Figure 2.

1.4. Examples of Experimental Studies with Bacteriophage-Caused Immunomodulatory Effects

The effect of bacteriophages on eukaryotic cells has been the subject of numerous
studies. For example, a study by Przerwa et al. [39] showed that bacteriophage T4 inhibits
phagocyte activity and reduces ROS production in response to infection with pathogenic
Escherichia coli strains. This phenomenon seems to depend on specific phage–bacteria
interactions, but, according to the authors, the precise mechanism is not fully understood.
A study on zebrafish (Danio rerio) exposed exclusively to bacteriophages specific
to strains of P. aeruginosa showed that the anti-inflammatory mechanisms of bacterio-
phages are not dependent on their bactericidal activity. Moreover, the induction of an
anti-inflammatory effect depends on the identification of phage capsid proteins through
activation of the TLR receptor pathway [40,41]. An anti-inflammatory effect has been
Viruses 2025, 17, 101 6 of 16

confirmed following injection of a cocktail of two phages of the family Podoviridae

(GenBank vB_PaeP_PYO2, MF490236, and vB_PaeP_DEV, MF490238) and two of the fam-
ily Myoviridae (vB_PaeM_E215, MF490241, and vB_PaeM_E217, MF490240), at titers of
5 × 108 pfu/mL. Reduced expression of the IL-1-beta and TNF-alpha genes was observed
in fish embryos, which translated into a significant reduction in neutrophil migration to
Viruses 2025, 17, x FOR PEER REVIEW 6 of 16
the inflamed site [41]. The authors suggest that elucidating the regulatory mechanisms of
bacteriophages will enable their use in preventing chronic inflammatory diseases.

Figure 2.
Figure Pro- and
2. Pro- and anti-inflammatory
anti-inflammatory mechanisms
mechanisms of of action
action of
of bacteriophages
bacteriophageson on eukaryotic
eukaryoticcells.
cells.
Legend: Upon contact with immune cells, various pro- or anti-inflammatory cytokines
Legend: Upon contact with immune cells, various pro- or anti-inflammatory cytokines are induced, are induced,
which allows phages to influence the immune response. The interaction of bacteriophages and
which allows phages to influence the immune response. The interaction of bacteriophages and an-
antigen-presenting cells (AP cells, e.g., dendritic cells) activates B lymphocytes to produce specific
tigen-presenting cells (AP cells, e.g., dendritic cells) activates B lymphocytes to produce specific anti-
anti-phage antibodies. Antigen-specific T cells can then activate B cells to produce anti-phage
phage antibodies. Antigen-specific T cells can then activate B cells to produce anti-phage antibodies.
antibodies. When cells involved in the immune response detect phages, they release cytokines, such as
When cells(IFN)-β,
interferon involved in the immune
interleukin response
(IL) 6, IL-10, and detect phages,
IL-12, which they release
activate T and Bcytokines, such
cells. Phages asinduce
can inter-
feron (IFN)-β, interleukin (IL) 6, IL-10, and IL-12, which activate T and B cells. Phages
a cytokine response, but exactly which cells and pathways are responsible for these mechanisms is can induce a
cytokine
yet unknownresponse, but exactly
[12]. Phages which
can also cells and
increase pathwaysofare
phagocytosis responsible
bacteria for these mechanisms
by macrophages, especially asisa
yet unknown
result [12]. Phages
of opsonization can alsowhich
of bacteria, increase
makesphagocytosis
them moreofrecognizable
bacteria by macrophages,
to the immuneespecially as
system [17].
aPhages indirectly
result of modulate
opsonization phagocyte
of bacteria, which damage, whichmore
makes them reduces ROS production
recognizable by phagocytic
to the immune system
cells,Phages
[17]. in partindirectly
through absorption of bacteriadamage,
modulate phagocyte by phage particles
which [39].ROS
reduces Theproduction
mechanismby reducing
phagocyticthe
destructive effect of ROS by eliminating bacterial infections is not yet fully understood
cells, in part through absorption of bacteria by phage particles [39]. The mechanism reducing the [12]. The
anti-inflammatory
destructive effect ofeffect
ROSofbyphages may bebacterial
eliminating associated with theisdirect
infections not yetinterference of phage[12].
fully understood proteins
The
in cell receptors, such as binding to cell integrins and TRL-like receptors, which activates cellular
anti-inflammatory effect of phages may be associated with the direct interference of phage proteins
metabolism cascades, thus inhibiting the regulatory activity of integrin receptors present on cells
in cell receptors, such as binding to cell integrins and TRL-like receptors, which activates cellular
involved in the host immune response and interfering with their natural inflammatory response [12].
metabolism cascades, thus inhibiting the regulatory activity of integrin receptors present on cells
involved in the
Other host immune
authors response
[42] have shownand
thatinterfering with their
the properties natural inflammatory
of bacteriophages response
open the door
[12].
to their application in diseases with symptoms of inflammation, such as Clostridium sp.
infections and Crohn’s disease.
1.4. Examples of Experimental Studies with Bacteriophage-Caused Immunomodulatory Effects
The effect of bacteriophages on eukaryotic cells has been the subject of numerous
studies. For example, a study by Przerwa et al. [39] showed that bacteriophage T4 inhibits
phagocyte activity and reduces ROS production in response to infection with pathogenic
Escherichia coli strains. This phenomenon seems to depend on specific phage–bacteria in-
teractions, but, according to the authors, the precise mechanism is not fully understood.
Viruses 2025, 17, 101 7 of 16

The anti-inflammatory properties of phages have been significant enough to reduce

inflammation in lung and urinary tract infections in mice and even reduce the skin-graft
rejection rate in mice [43].
In another study [44], the use of a phage cocktail containing phages specific to
Salmonella typhimurium in drinking water for chicken broilers had a significant anti-
inflammatory effect just one day after the birds had been infected with S. typhimurium. The
anti-inflammatory effect and simultaneous antibacterial effect were manifested as inhibi-
tion of the increase in levels of pro-inflammatory cytokines, including IL-1β, IL-6, IFN-γ,
IL-8, and IL-12, as well as stimulation of the production and release of anti-inflammatory
cytokines (IL-10 and IL-4). Furthermore, the authors showed that the use of the experi-
mental phage therapy in chickens had no adverse effect on the number and activity of
lymphocyte subpopulations crucial to immune function. This suggests that phage therapy
can be used in veterinary medicine without disturbing immune homeostasis, expressed
as cytokine imbalance, abnormal percentages of key subpopulations of immune cells, and
hyperactivity of the hypothalamic–pituitary–adrenal axis, which are common side effects
of antibiotic treatment [44].
A recent study [45] carried out on seven-day-old chicks infected with Salmonella enterica
showed that an antiviral response was initially induced in chickens that received 1 mL of a
cocktail composed of two phages (vB_SenM-2 and vB_Sen-TO17) at a titer of 109 pfu/mL
per os for two weeks. However, the antiviral response was subsequently impaired via a
blockade of one of the main pathways of innate antiviral immunity, i.e., cyclic GMP-AMP
synthase (cGAS) and stimulator of interferon genes (STING), known as the cGAS–STING
pathway, at the stage of phosphorylation of transcription factor IRF3. According to the
authors, this reaction was caused by the inability of RNA polymerase III to recognize the
phage DNA and produce dsRNA molecules crucial to stimulating the large protein complex
necessary for phosphorylation of IRF3.
The number of phages adhering to the intestinal mucosa enables efficient selection of
a specific microbiota, reducing the colonization capacity of pathogenic bacteria by limiting
their adhesion to the mucosa [46]. The role of intestinal bacteriophages is believed to
involve not only efficient regulation of the bacterial population colonizing the gut, but also
regulation of local mucosal defense mechanisms (GALT and MALT) and enhancement of
the activity of the probiotic microbiome [1].
The properties of bacteriophages open the door to their potential application in dis-
eases in which intestinal inflammation plays a major role, such as infections caused by
anaerobic bacteria of the genus Clostridium [1]. For instance, transfer of sterile fecal filtrate to
the intestine or transplantation of purified fecal microbiota in people or mice can positively
influence their health status by improving clinical parameters in Clostridium infections for
up to seven months [47].
In another study [48], oral application of a cocktail of phages specific to E. coli strains
K88, K99, and F41; S. typhimurium; S. enteritidis; and C. perfringens types A and C in
weaned piglets resulted in anti-inflammatory and antioxidant effects by reducing the
concentrations of IL-1β, IL-6, TNF-α, and myeloperoxidase. It is also worth noting that the
treatment significantly reduced the concentrations of selected pathogens in the digestive
tract and feces.
The pro-inflammatory effects of bacteriophages may be associated in part with stim-
ulation of T cell proliferation, increased IL-6 production, and blast transformation of
B lymphocytes and other cells involved in the immune response [49]. The pro-inflammatory
effects of bacteriophages may depend on their individual properties, the route of adminis-
tration, and the exposure time, as well as on the type of infection. For example, in the case of
infections caused by Gram-negative bacteria, their breakdown results in an increase in the
Viruses 2025, 17, 101 8 of 16

level of LPS—the main pro-inflammatory endotoxin. Higher doses of phages in treatment

can also induce a proportional pro-inflammatory effect, as certain inflammatory cytokines
have been detected only at high phage titers (109 pfu/mL) [49]. The pro-inflammatory
activity of bacteriophages stimulates the production of anti-phage antibodies, leading to
the induction of the humoral immune response (Table 1).

Table 1. Anti-phage antibodies in selected animal species following application of experimental

phage formulations [50] with our own modifications.

Animal
Bacteriophage Host Species Administration Route Identified Antibodies Material Tested References
Species
IgG,
GACP Enterococcus faecalis Intraperitoneal Mice Blood [51]
IgM
φ26, φ27, φ29 E. coli Rectal (suppositories) IgG, IgA, IgM Calves Serum [52]
Whole phages:
M. haemolytica
Φ25, φA2, φA5 Nasal IgG, IgA, IgM Calves Serum [53]
Extracted proteins M. haemolytica
specific to E. coli E. coli Subcutaneous Not identified Rabbits Blood [54]
AbArmy ϕ1, AbNavy ϕ1,
Acinetobacter
AbNavy ϕ2, AbNavy ϕ3, Intraperitoneal IgG2a, IgG2b Mice Serum [55]
baumannii
AbNavy ϕ4
A3R, 676Z Staphylococcus aureus Per os IgM, IgG Mice Plasma [56]
Serum
Bacteriophages specific to Streptococcus lactis Subcutaneous Cows
IgG Milk whey [57]
Streptococcus lactis: AM3 and ML1 AM 3 and ML 1 w Intravenous Rabbits Serum
Phages f1 and f2 E. coli Intradermal IgG Sheep Serum [58]
Phages ΦX 174 and T 2 E. coli Intracardiac IgG Piglets Serum [59]
Subcutaneous injection
C2, phages p335 and p013 on
in the neck Colostrum
L. lactis ssp. L. lactis spp. lactis IgG Cows [60]
and intramuscular Serum
lactis F7/2, phage kh on Lac
injection in the rump
Phage SE-W109 Salmonella Subcutaneous Polyclonal IgG Rabbits Serum [61]

Some bacteriophages used in therapy may exhibit both pro- and anti-inflammatory
activity at the same time, as confirmed in research by Zhang et al. [62]. In that study,
intramammary application of phages specific to S. aureus in cattle resulted in a reduction in
the levels of cytokines TNF-α, IL-1β, IL-6, and IL-8 in mammary epithelial cells (MAC-Ts)
stimulated with LPS, as well as a reduction in the levels of inflammatory mediators in the
absence of LPS.
Apart from therapeutic uses of bacteriophages, owing to their immunomodulatory
properties they can also be used in immunoprophylaxis. For instance, phage phi X174
specific to E. coli strains has been used for more than 30 years as an antigen to assess the
humoral immune response in patients with primary and secondary immune deficiencies,
such as severe combined immunodeficiency (SCID), X-linked agammaglobulinemia (XLA),
X-linked hyper IgM syndrome, and Wiskott–Aldrich syndrome, as well as in bone-marrow
recipients, people infected with HIV, and patients treated with immunosuppressants such
as CTLA4-Ig [63]. Phage particles can also be used as carriers for vaccine antigens; how-
ever, the potential interactions between a given bacteriophage and immune cells must
be investigated [64].
Apart from regulating the intestinal immune response against bacteria and viruses,
bacteriophages can also play a role in the anti-tumor response. They have been shown
to be capable of exerting a targeted effect on specific molecular determinants of cancer
cells and inhibiting tumor growth by accumulating in cancer tissue. Some studies indicate
that phages can be used as anti-cancer agents and carriers of imaging molecules and
therapeutic agents [65].
For example, in the studies of Hajitou et al. [66], it was shown that the use of hybrid
vectors containing fragments of a eukaryotic virus in combination with a bacteriophage as
a representative of a prokaryotic virus in the form of a chimera (AAVP—adeno-associated
virus/targeted M13 phage) was characterized by high efficiency in terms of affinity for
various types of human malignant tumors, such as Kaposi’s sarcoma (KS1767), bladder
cancer (UC3), prostate cancer (DU145), and mammary tumors (EF43-FGF4), induced in
Viruses 2025, 17, 101 9 of 16

a mouse model. In order to confirm the obtained results, the authors used the cyclic
ligand Arg-Gly-Asp (RGD-4C) located on the bacteriophage capsid, showing affinity for
αv integrin receptors specifically expressed on the blood vessels of the tumors studied. The
obtained results confirmed the possibility of delivering transgenic genes by the developed
vectors only to the areas affected by the cancer process, thus sparing healthy organs, which
is a very good prognostic factor in the diagnosis and treatment of cancer [67].
It should be emphasized that the forerunners of research using phage display were the
Nobel Prize winners from 2018, Prof. Smith, G.P., and Prof. Winter, G.P., who developed the
possibility of creating fusion proteins with antibodies or peptides [68,69]. The developed
technique involves cloning the sequence of interest at the N- or C-terminus of the capsid
protein, using the nonlytic filamentous phage M13. Currently, bacteriophage M13 is the
most common and highly effective phage used in cancer therapies. Genetically modified
bacteriophages used in these studies constitute a matrix (vector) for stable transport of
genes, inducing cancer cell death [70].
A special role is ascribed to temperate phages, i.e., those that multiply through a
lysogenic cycle. It is these bacteriophages that can be a source of transfer of genes involved
in the metabolism of toxins, polysaccharides, and carbohydrates for bacteria, and in rare
cases they can be a source of antibiotic resistance [28,43,71]. Some phages can modulate
the antigenicity of bacteria by producing enzymes capable of modifying LPS O-antigens in
microorganisms such as Escherichia coli, Salmonella sp., Shigella sp., and Vibrio cholerae, thus
enhancing their virulence traits.
Another example of the effect of temperate phages on bacteria is the formation of
prophages, which can directly affect the immune system of people and animals. For
example, prophage SF370.1 encodes extracellular DNase Spd1, leading to DNA degradation
in neutrophil extracellular traps (NETs), thereby increasing the invasiveness of M1 strains
of Streptococcus pyogenes [72].
Bacteriophages can also be induced naturally from lysogenic bacteria. An elevated
level of phage antibodies was observed more than 50 years ago in patients infected with
Staphylococcus spp. between days 6 and 14 of the course of the disease [73]. It was suggested
that this may be the effect of bacteriophage production by lysogenic Staphylococcus strains,
leading to an increase in levels of anti-phage antibodies.
Previous research [4] has shown that anti-phage antibodies can be some of the most
important factors limiting the therapeutic efficacy of phages. However, many recent
studies evaluating the effect of phage antibodies indicate that the efficacy of experimental
treatments can be varied.
‘Natural’ anti-phage antibodies have been shown to be present in sera from peo-
ple and animals that have not been treated with bacteriophages, although the level of
phage-neutralizing antibodies was low [28]. In another study, the presence of anti-phage
antibodies against the most popular bacteriophage, T4, was confirmed in 80% of healthy
human subjects who had never received phage therapy [74].
The presence of ‘natural’ anti-phage antibodies specific to E. coli and M. haemolytica
bacteriophages was also observed in our own study [53] in calves which did not receive
experimental phage preparations, in both prophylaxis and treatment (Table 2). It should be
noted that the study concerned not only preparations containing whole phages but also
selected phage proteins exhibiting immunogenic properties.
Viruses 2025, 17, 101 10 of 16

Table 2. Average concentrations of anti-phage antibodies, IgG, IgM, and IgA, in mg/L, in calf sera for
bacteriophages specific to E. coli and M. haemolytica [53].

Calves Untreated Calves Treated

Estimated Parameters FBS
with Bacteriophages with Bacteriophages
E. coli phages
IgG 0.0032 ± 0.001 0.06 ± 0.009 * 0.74 ± 0.16 *
IgM 0.014 ± 0.008 0.096 ± 0.038 * 0.211 ± 0.052 *
IgA 0.001 ± 0.0008 0.02 ± 0.007 * 0.19 ± 0.09 *
M. haemolytica phages
IgG 0.005 ± 0.0008 0.72 ± 0.017 * 0.95 ± 0.08 *
IgM 0.004 ± 0.0001 0.08 ± 0.03 * 0.99 ± 0.11 *
IgA 0.002 ± 0.0005 0.03 ± 0.001 * 0.23 ± 0.005 *
E. coli phage extracted
proteins
IgG 0.004 ± 0.0003 0.062 ± 0.001 * 0.83 ± 0.032 *
IgM 0.002 ± 0.0009 0.042 ± 0.002 * 0.21 ± 0.004 *
IgA 0.001 ± 0.0001 0.02 ± 0.008 * 0.201 ± 0.002 *
M. haemolytica phage
extracted proteins
IgG 0.007 ± 0.0003 0.071 ± 0.04 * 0.77 ± 0.13 *
IgM 0.004 ± 0.0002 0.05 ± 0.02 * 0.3 ± 0.04 *
IgA 0.001 ± 0.0001 0.03 ± 0.01 * 0.15 ± 0.03 *
Legend: FBS—fetal bovine serum; Ig—immunoglobulin; * significant differences (p ≤ 0.05).

The presence of natural phage antibodies produced by immune cells in people and
animals is ascribed to the ubiquitous occurrence of bacteriophages in the environment and
constant contact with various phages. For example, bacteriophages have been detected in
wastewater, water bodies, soil, food, and animal feed, as well as in the oral cavities (dental
plaque and saliva) and gastrointestinal tracts of people and animals. They are also present
in commercially available sera and vaccines [75,76].
The presence of anti-phage antibodies shown in our own research in sera from healthy
calves which had had no physical contact with phage preparations indicates the presence
of commensal bacteriophages in the natural microbiome (phageome) of the gastrointestinal
and respiratory tracts of calves. This can be assumed to be due to the presence of sapro-
phytic strains of M. haemolytica colonizing the nasal cavity and E. coli colonizing the gut, as
part of the natural intestinal microbiome [53,77].
According to Archana et al. [54], the presence of natural anti-phage antibodies against
E. coli indicates that bacterial strains making up the commensal microbiota in animals can
also induce the development of other specific bacteriophages making up the intestinal
phageome, which influences the production of phage-neutralizing antibodies. On the other
hand, the absence of anti-phage antibodies for other microbes, such as Klebsiella pneumoniae,
Salmonella typhi, Pseudomonas aeruginosa, and Staphylococcus aureus, may suggest that these
bacteria should not be considered part of the commensal microbiota but pathogenic mi-
crobes [54].
The results of our own research confirm the suggestions of Nguyen et al. [23], who
found that about 31 billion bacteriophage particles migrate into the human body every
day via transcytosis through intestinal epithelial cells, translocation through a damaged
epithelial barrier, or direct uptake from the intestinal lumen by intestinal dendritic cells,
as well as cells of the lungs, liver, kidneys, and brain, influencing mechanisms of the host
immune response [78].
It has also been demonstrated that commensal intestinal bacteria, including E. coli,
containing prophage genetic material are capable of producing bacteriophages, which
enables them to compete with other bacterial species colonizing the intestines [79]. This
underscores the importance of studies showing that the intensity of the humoral anti-phage
response can also vary depending on the route of administration of phage preparations.
For example, Srivastava et al. [80] found that following intravenous administration of
bacteriophage T7, despite stimulation of the humoral response, T lymphocytes were not
involved in the cellular immune response. In a very early study in 1970 [57], intramuscular
Viruses 2025, 17, 101 11 of 16

administration of a cocktail of bacteriophages specific to Streptococcus lactis strains AM3

and ML1 resulted in a negligible immune response, expressed as the IgG level, in both
sera and milk whey. Somewhat higher immune parameters were observed following
immunization of animals with a bacteriophage cocktail with Freund’s adjuvant. Following
intramammary administration of a bacteriophage suspension, the authors demonstrated the
induction of inflammatory reactions in the udder, resulting in changes in the structure and
organoleptic properties of milk. For this reason, they did not recommend the administration
of bacteriophages directly into the udder.
In our own research on calves, no statistically significant differences (p ≤ 0.05)
were shown in the obtained results depending on the route of administration, i.e., nasal
(M. haemolytica phages) or rectal (E. coli phages). The values were similar in both cases [53].
It has also been confirmed that irrespective of the route of administration (oral, nasal,
or intraperitoneal) of bacteriophages to animals (mice, rats, and rabbits) and humans, they
appear very rapidly in the bloodstream and in internal organs involved in immune pro-
cesses (the spleen, liver, and thymus) [81,82]. The presence of bacteriophages is especially
quickly confirmed in the blood, due to their ability to penetrate endothelial cell barri-
ers. Rectal application in chinchillas of bacteriophages specific to Pseudomonas aeruginosa,
Salmonella enteritidis, and Escherichia coli bacteria, in the form of suppositories, resulted in
a significant concentration in the blood in just 30 min, while a similar concentration was
noted in the urine within an hour [83]. Kawato and Nakai [82] confirmed the ability of
some phages, particularly phage PPpW-4 against Pseudomonas plecoglossicida in goldfish, to
penetrate the intestinal wall to the bloodstream within just 10 min after oral administration.
Some studies have shown that induction of an immune response also depends on the
duration of treatment, the titer of phages, and their individual immunomodulatory prop-
erties [78]. For example, Łusiak-Szelachowska et al. [84] showed the strongest anti-phage
activity (K > 18) following the topical application of a phage cocktail to the skin or mucous
membranes and following simultaneous topical and oral administration. In addition, sig-
nificant differences were confirmed in immunogenicity between bacteriophages, with the
greatest activity noted for the phage specific to S. aureus, 676/Z.
Geller et al. [60], in a study on dairy cows, found that, following subcutaneous and
intramuscular injection of bacteriophages specific to Lactococcus lactis strains, serum anti-
bodies had a low neutralizing effect (k = 6–8) against small bacteriophages with isometric
head structures. Much stronger bacteriophage neutralization was observed in the colostrum
in comparison with sera. The titers in the colostrum were as much as 30 times as high as
those obtained in the sera (Table 3).

Table 3. Examples of the effects of bacteriophage-neutralizing antibody titers obtained in

various studies.

Degree of Antibody Neutralization (K) or

Type of Bacteriophage Bacterial Host Animal Species References
Serum Neutralization Titer (Dilution)
φ c2, ml3, sk1;
φ p335, p013; Lactococcus lactis Cattle 3.8 [60]
φ kh, φ16–18
φ25, φA2, φA5 M. haemolytica 5.053–5.57
Calves [53]
φ26, φ27, φ29 E. coli 6.74–7.28
E. coli
1/10; 1/100
Klebsiella pneumoniae
10x dilution >300 to complete neutralization
Wild-type phage P. aeruginosa Rabbits [54]
at 28d10x, >300 to complete neutralization at
Salmonella typhi
21d10x, >300 to complete neutralization at 28d
S. aureus
φX 174 E. coli Piglets 1/5 [59]
Cows 1/400
Wild-type phage Streptococcus lactis [57]
Rabbits 1/80
Legend: nd—not detectable.
Viruses 2025, 17, 101 12 of 16

Production of anti-phage antibodies can also be stimulated by some bacterial cell

components present in phage preparations, such as lipopolysaccharides (LPSs). However,
Łusiak-Szelachowska et al. [84] showed no negative effect of phage antibodies on the
therapeutic or prophylactic effects of phages, and the levels of anti-phage antibodies varied.
In several cases, the titers of antibodies against the phages studied were negligible, while
in other patients the differences were statistically significant compared to the controls.
According to the authors, the differences observed in antibody levels may be due to the
varied immunogenicity of phages, even those infecting the same host, which may in part
be explained by high variation in protein sequences in the structural proteins of phages.
Studies presented by many research centers indicate the significant role of bacterio-
phages present in the intestines of both humans and animals in protecting the host against
numerous pathogens, including E. coli, Salmonella enteritidis, Pseudomonas aeruginosa, and
Clostridium sp. [1].
Our own research [53] also showed that despite the presence of anti-phage antibodies
in phage-treated calves, there was no negative effect on the experimental treatments using
phage preparations specific to E. coli and M. haemolytica strains. The absence of a significant
suppressive effect of antibodies induced by phages was confirmed in part by the lytic
activity of bacteriophages remaining at a similar level, improved health parameters in the
sick calves, and a protective effect lasting for about three weeks, i.e., the absence of cases of
disease in calves.
Positive results of phage therapy have also been obtained in the treatment of udder
diseases in dairy cattle, both clinical and subclinical. Application of a suspension of bacterio-
phages specific to various pathogens responsible for these infections not only did not cause
a significant immune response but also reduced inflammation and clinical symptoms [85].
However, particular caution is required when considering the use of bacteriophages to
prevent mastitis in dairy cattle. Application of a bacteriophage suspension to healthy udder
quarters during lactation was shown to cause a significant increase in the somatic cell
count in the milk from the quarters in which phages were applied. This also confirms that
bacteriophages induce an immune response in udder tissue [86]. Data from in vivo studies
and positive results of the application of bacteriophages to the udders of dairy cows are
currently lacking.

2. Conclusions
The results of various experimental studies, including our own, demonstrate that
specific anti-phage antibodies can be induced in humans and animals; however, in many
cases, this has no significant effect on the efficacy of experimental phage treatments.
In order to prevent an overly intensive immune response against phages, it can be
helpful to purify them to eliminate any unnecessary protein ballast and thereby limit the
induction of an immune response that could disrupt the therapy process.
The immunomodulatory effect of bacteriophages on eukaryotic cells, especially in
the course of infection, opens up promising prospects for the use of phages as natural
tools in the treatment of bacterial, fungal, and viral diseases, especially immune diseases,
particularly autoimmune diseases, including cancers.
The few published studies on the effects of bacteriophages on eukaryotic cells in the
course of bacterial infections, particularly in farm animals and pets, show that there is an
unquestionable need to continue this research.

Author Contributions: Conceptualization, R.U.-C. and E.P.; writing—original draft preparation

R.U.-C.; writing—review and editing, R.U.-C. and E.P.; visualization and figure conception and
preparation, E.P.; table conception and preparation, R.U.-C. All authors have read and agreed to the
published version of the manuscript.
Viruses 2025, 17, 101 13 of 16

Funding: This research received no external funding.

Conflicts of Interest: The authors declare no conflicts of interest.

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