Fighting antibiotic resistance in the local management of bovine mastitis

Full text

Biomedicine & Pharmacotherapy 170 (2024) 115967
Available online 2 December 2023
0753-3322/© 2023 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license
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Review
Fighting antibiotic resistance in the local management of bovine mastitis
Lara Touza-Otero
a
,
b
,
c
, Mariana Landin
a
,
b
,
c
, Patricia Diaz-Rodriguez
a
,
b
,
c
,
*
a
Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Grupo I+D Farma (GI-1645), Facultad de Farmacia, Universidade de Santiago de
Compostela, Santiago de Compostela 15782, Spain
b
Instituto de Investigaci´
on Sanitaria de Santiago de Compostela (IDIS), IDIS Research Institute, 15706 Santiago de Compostela, Spain
c
Instituto de Materiais da Universidade de Santiago de Compostela (iMATUS), 15706 Santiago de Compostela, Spain
ARTICLE INFO
Keywords:
Antibacterial
Bovine mastitis
Intramammary
Biomimetic antibiotic alternatives
ABSTRACT
Bovine mastitis is a widespread infectious disease with a signicant economic burden, accounting for 80 % of the
antibiotic usage in dairy animals. In recent years, extensive research has focused on using biomimetic approaches
such as probiotics, bacteriocins, bacteriophages, or phytochemicals as potential alternatives to antibiotics. The
local administration of therapeutic molecules through the intramammary route is one of the most commonly
strategies to manage bovine mastitis. This review highlights the most important ndings in this eld and dis-
cusses their local application in mastitis therapy. In contrast to antibiotics, the proposed alternatives are not
limited to promote bacterial death but consider other factors associated to the host microenvironments. To this
end, the proposed biomimetic strategies can modulate different stages of infection by modifying the local
microbiota, preventing oxidative stress, reducing bacterial adhesion to epithelial cells, modulating the immune
response, or mediating the inammatory process. Numerous in vitro studies support the antimicrobial, anti-
biolm or antioxidant properties of these alternatives. However, in vivo studies incorporating these components
within pharmaceutical formulations with potential clinical application are limited. The development of secure,
stable, and effective drug delivery systems based on the proposed options is necessary to achieve real alternatives
to antibiotics in the clinic.
1. Introduction
The One Health Action Plan against antimicrobial resistance (AMR)
initiated by the European Union (EU) in 2017 highlighted the link be-
tween human and animal health. Therefore, approaches to manage AMR
should consider both equally [1]. On the other hand, according to the
World Health Organisation (WHO) animals consume twice the amount
of antibiotics of humans, making livestock farming a sector that requires
urgent actions [2,3].
The dairy industry is a signicant contributor to the livestock sector.
In 2020, the EU produced 160.1 million tonnes of raw milk, predomi-
nantly sourced from dairy cows. Currently, there are an estimated 1.7
million farms across the EU, collectively housing 23.5 million dairy
cows. Over a lactation period of 10 months, each cow produces an
average of 25Kg of milk per day [4].
Bovine mastitis is a disease characterized by inammation of the
mammary gland and udder tissues due to trauma, chemical irritation, or
infection by various microorganisms, including fungi, viruses, algae and
especially bacteria [5,6]. It is one of the most prevalent diseases in the
dairy industry, affecting 40 % of cows in herds. This condition poses a
signicant economic burden, costing over 185
€
/cow per year in the EU
and rising to $35 billion worldwide [5,7]. These losses can be attributed
to various factors, including reduced milk production, diminished milk
quality, discarded milk, veterinary expenses, and premature culling of
cows [5].
As bacterial infections stand as the primary cause of bovine mastitis,
antibiotics have been the mainstay of treatment for decades [8].
Astonishingly, around 80 % of antibiotics employed in the dairy industry
are dedicated to control and treat mastitis [9]. They are usually
administered through two routes: parenteral and, most commonly,
intramammary (IMM). The local administration of therapeutic mole-
cules through IMM route offers several advantages for mastitis treatment
in dairy cows, allowing high local drug concentrations with minimal
systemic absorption reducing undesired side effects [10]. However,
considering the current scenario, the pressing needs to mitigate the
development of AMR demand a shift away from the excessive antibiotic
* Corresponding author at: Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Grupo I+D Farma (GI-1645), Facultad de Farmacia, Uni-
versidade de Santiago de Compostela, Santiago de Compostela 15782, Spain.
E-mail address: patricia.diaz.rodriguez@usc.es (P. Diaz-Rodriguez).
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
https://doi.org/10.1016/j.biopha.2023.115967
Received 19 September 2023; Received in revised form 15 November 2023; Accepted 27 November 2023

Biomedicine & Pharmacotherapy 170 (2024) 115967
2
usage associated to bovine mastitis management. Therefore, it is
essential to explore innovative non-antibiotic strategies that can effec-
tively prevent or treat bovine mastitis [6].
This review aims at identifying and discussing the main strategies
pointed out to manage bovine mastitis through local administration
avoiding antibiotic use. Several approaches are currently under devel-
opment. These alternatives are not only focused on promoting bacterial
death, but also consider the infection as a broken equilibrium between
the host and the bacteria. Therefore, this review highlights and discusses
the latest ndings, encompassing research conducted from 2018 to the
present. A deep literature review was performed to elucidate the
mechanism of action followed to restore the homeostasis of the identi-
ed strategies. Moreover, clinical efcacy of the developed approaches
and future perspectives of bovine mastitis management have also been
discussed.
2. Current research lines on non-antibiotic alternatives
In recent years, there has been a signicant increase in research
aimed at discovering non-antibiotic alternatives for the management of
bovine mastitis. The literature reveals that most of them are based on
biomimetic approaches, taking inspiration from the resources used by
organisms to maintain homeostasis, defend themselves or survive. As
summarize in Fig. 1, these options include probiotics, bacteriocins,
phytochemicals (plant extracts and essential oils) and bacteriophages. In
addition, recent studies have explored innovative options, such as
nanoparticles and antimicrobial peptides of non-bacterial origin
[11–13].
2.1. Searching for homeostasis: Probiotics
Probiotics are “live microorganisms which, when administered in
adequate amounts, confer a health benet on the host” [14]. They exert
their benecial effects through different mechanisms as the modication
of the local microbiota composition or acting directly on pathogenic
microorganisms. Probiotics can adhere and interact with epithelial cells,
improving their barrier and promoting cell renewal. Moreover, they can
also enhance the host immune system response [15,16].
Probiotics are Generally Recognised as Safe (GRAS) products and
widely used in the food industry as preservatives. Among them, lactic
acid bacteria (LAB) are the best-known and most widely used [17,18].
These Gram-positive bacteria, primarily belonging to the genera Lacto-
bacillus (Lb.), Lactococcus (Lc.), Bidobacterium, and Streptococcus, have
been in vitro and in vivo evaluated for bovine mastitis prevention or
therapy.
Several in vitro studies were performed to assess the interaction be-
tween probiotics and pathogenic microorganisms or mammary epithe-
lial cells, and to examine their impact on the immune response (Table 1).
Species, such as Lb. rhamnosus, Lb. casei, or Lc. lactis, have shown activity
against different bacteria, especially Staphylococcus and Streptococcus
[19–24].
Certain LAB have also demonstrated signicant potential in treating
persistent infections by pathogenic bacteria biolm elimination or
replacement [21,25,26]. As an example, Lb. rhamnosus and Lb. planta-
rum, have been found to replace the biolms of S. aureus, S. xylosus and
S. epidermidis [21]. Additionally, Lb. sakei has demonstrated antibiolm
activity against S. aureus, S. agalactiae and S. dysgalactiae [25].
Additionally, the antimicrobial activity of some LAB as Lb. casei has
been associated with the inhibition of adhesion and internalization of
the pathogen to epithelial cells [22]. Other species also modulate the
immune response and mediate the endothelial cells inammatory pro-
cess. This has been demonstrated with Lb. casei, Lb. acidophilus and Lb.
plantarum on bovine mammary epithelial cells (BMEC) [22,27–29] and
also with Lb. rhamnosus strains in bovine mammary alveolar epithelial
cells (MAC-T). In this case, it was shown that Lb. rhamnosus decreases
apoptosis, reduces cytokine levels, and increases IL-10 mRNA expression
in infections caused by E. coli [19]. Lb. rhamnosus also reduces cell
damage by protecting cell to cell tight junctions and decreases NLRP3
inammasome activation, regulating the inammatory response caused
by Bacillus cereus infection [20]. Furthermore, strains of Lc. lactis, Lb.
plantarum, Lb. brevis, Lb paracasei, Lb. rhamnosus or Lb. perolens have
shown high colonization capacity of BMEC or MAC-T [23,24,26].
In vivo experiments conducted in murine and bovine mastitis models
have employed local administration methods. Lc. and Lb. species have
been the focal points of extensive study (Table 2). Across these in-
vestigations, milk samples were consistently collected at various in-
tervals, analysing SCC and bacteriological counts.
Studies in a murine model have proved that strains of Enterococcus
mundtii can inhibit infection and reduce the inammation in the
Fig. 1. New strategies for local bovine mastitis therapy avoiding AMR.
Table 1
Summary of in vitro studies carried out with probiotics on pathogens of bovine
mastitis or epithelial cells.
Probiotic species Pathogens Results Reference
Lb. * and Lc.
* *strains
- Cell adhesion
Biolm forming ability
[26]
Lb. *casei S. aureus Anti-inammatory
Immunomodulatory
potential
[22]
Lb. *rhamnosus; Lb.
*plantarum; Lb.
*brevis; Lb.
*plantarum; Lb.
*plantarum
S. aureus; S.
xylosus; S.
epidermidis
Antibiolm
Biolm forming ability
(Lb. rhamnosus; Lb.
plantarum 2/37)
[21]
Lc. * * lactis; Lb.
*perolens
S. aureus; S.
dysgalactiae
Cell adhesion
Bacteriostatic
[24]
Lb. *acidophilus E. coli Anti-inammatory
properties
[27]
Lb. * sakei S. aureus; S.
agalactiae; S.
dysgalactiae
Antibacterial
Antibiolm
[25]
Lb. * rhamnosus E. coli Apoptosis improvement
Reduction of
inammation
[19]
Lb. *plantarum E. coli Antibacterial
Reduction of
inammation
[28]
LAB strains S. aureus Cell adhesion
Bacteriostatic
[30]
Lb. *rhamnosus Bacillus cereus Pathogen reduction
Cytoprotective
[20]
Lb. *plantarum E. coli Reduction of
inammation
[29]
Lb. *perolens; Lc. * *
lactis
S. aureus; S.
chromogenes; S.
uberis; E. coli
Reduction of pathogen
adhesion
[23]
*Lactobacillus (Lb.);
* *Lactococcus (Lc.).
L. Touza-Otero et al.

Biomedicine & Pharmacotherapy 170 (2024) 115967
3
mammary gland, preventing mastitis. The efcacy of this intervention
was unequivocally demonstrated through histological evaluation con-
ducted 24 h post-administration [31].
The inhibitory efcacy of Lc. Lactis and Lb. perolens against the
adhesion of S. aureus, S. chromogenes, S. uberis and E. coli to BMEC has
also been demonstrated in vivo. The combined administration of both
strains followed by histological evaluation, conrmed their effective-
ness. However, the effect was less pronounced for S. uberis and E. coli,
compared to in vitro results [23]. In addition, Lc. lactis showed immu-
nomodulatory properties by stimulating defence mechanisms and
enhancing the inammatory response in vivo [32].
Moreover, when Lc. Lactis, Bidobacterium breve or Bacillus velezensis
were administered in a bovine mastitis model, they successfully reduced
pathogen and somatic cell counts (SCC), while promoting an immune
response [33–35].
Overall, these studies point out probiotic strains can inhibit mastitis-
causing pathogens, reduce their adhesion and internalization, and
regulate the hosts immune response. Relatively standardized doses of
probiotics were used, as most trials employ bacteria counts ranging from
10
6
to 10
9
colony forming units (CFU). Although more research is
needed, probiotics hold great potential for the treatment and prevention
of bovine mastitis.
2.2. Killing as bacteria: bacteriocins
Bacteriocins are small antimicrobial proteins or peptides ribosomally
synthesized by Gram-positive and Gram-negative bacteria. They are
often released to kill or inhibit the growth of competing bacteria, fungi
or viruses, providing a selective advantage [36,37]. The mechanism of
action of bacteriocins varies depending on the type and the target bac-
terium. However, most bacteriocins exert their antimicrobial activity by
disrupting cell functions (transcription, translation, replication…) or
altering the cell membrane structure (by channels and/or pores) [38].
Bacteriocins, particularly from Gram-positive bacteria, have been
extensively studied for their potential as alternatives to antibiotics in the
treatment of bacterial infections in the veterinary eld, as well as for
their use as food preservatives [36,38–40]. They present advantages
over other antimicrobials as activity at low concentrations, easily
metabolised and capacity of modication by bioengineering techniques.
However, they also present limitations related to their size as the
incorporation into adequate pharmaceutical formulations and the
identication of suitable administration routes. Nanotechnology is
being considered as a viable option in this regard [36,37,41,42].
These antimicrobial molecules are classied into four main classes
depending on their structure, mode of action, spectrum activity and
other characteristics. Lantibiotics or class I bacteriocins are peptides that
contain post-translationally modied amino acids, such as lanthionine.
They are characterized by a broad-spectrum activity against Gram-
positive bacteria. Class II bacteriocins are small heat-stable non-lanti-
biotic peptides that have a narrow spectrum of activity, targeting spe-
cic bacteria. Class III bacteriocins are large heat-labile proteins that
often have a broad spectrum of activity against Gram-negative bacteria.
Finally, Class IV bacteriocins are cyclic peptides that have a broad
spectrum of activity against both, Gram-positive and Gram-negative
bacteria [31,43].
The most studied bacteriocin is nisin, a lantibiotic produced by Lc.
Lactis. Nisin has been used as a food preservative since the 1960 s and is
currently approved as a food bio-preservative in many countries,
including the United States (US) and the EU. It is also approved as an
udder disinfectant for the prevention of IMM infections in dairy cattle
[44].
Nisin has antimicrobial activity against S. aureus, S. uberis and
S. dysgalactiae by interfering with the cell wall synthesis and altering the
membrane structure through pore formation [36,39,45]. Unfortunately,
several nisin-resistant bacterial strains have already been identied (e.g.
Staphylococcus). This resistance can be intrinsic or acquired, and in-
volves various mechanisms, including the production of modifying en-
zymes, efux pumps, and modications to the bacterial cell membrane
[46]. This fact has led to the search for new bacteriocins or derivatives.
Bioengineered nisin derivatives namely nisin A M17Q, A HTK, A T2L or
PV exhibit antimicrobial activity against resistant strains and biolm
eradication [45]. The results of these studies are shown in Table 3.
Other LAB bacteriocins, such as bactofencin (produced by Lb. sali-
varius) and reuterin (produced by Lb. reuteri) have shown in vitro activity
against Staphylococcus or Streptococcus strains isolated from clinical
cases of bovine mastitis. The antimicrobial activity of bactofencin is
based on the modication of teichoic acids, components of the cell wall,
while reuterin acts by inducing oxidative stress through the modication
of proteins thiol groups [36]. Furthermore, their IMM administration in
an in vivo bovine mastitis model, showed a decrease in the bacterial load,
proving their potential as teat disinfectants [37].
Staphylococcus or Streptococcus strains also produce bacteriocins,
which act against competing bacteria, even from the same genus [38,
47]. Aureocin 4181, for example, a staphylococcin produced by
S. aureus, has been found to be effective against a wide range of
Gram-positive bacteria, including other strains of Staphylococcus and
Streptococcus. It disrupts the bacterial membrane, leading to cell death
[48,49]. On the other hand, Bovicin HC5, produced by Streptococcus
bovis HC5, has been found to be effective against a wide range of
pathogens with a similar mechanism of action than nisin [50].
Ubericin K (produced by Enterococcus faecium) has been found to be
effective against Listeria monocytogenes, Streptococcus pyogenes, and
Table 2
Summary of in vivo studies carried out with IMM administration of probiotics in mastitis animal models.
Probiotic species Animal
model
Dose Sample
evaluated
Evaluation time Pathogens Results Reference
Lc. *lactis Bovine 1 ×10
9
CFU
**
Milk 0, 6, 24, 48, 72,
120 and 168 h
- SCC
***
reduction
Anti-inammatory properties
[33]
Bidobacterium
breve
Bovine 3 ×10
9
CFU
**
Milk 0 h and 14 days S. aureus; S. uberis; S.
dysgalactiae; E. coli
SCC
***
reduction
Pathogen reduction
Immune response promotion
[34]
Bacillus velezensis Bovine 1 ×10
3
or
1×10
9
CFU
**
Milk Every 24 h for 4 or
14 days
S. aureus SCC
***
reduction
Pathogen reduction
[35]
Enterococcus mundtii Murine 1 ×10
8
CFU
**
Mammary
tissue
24 h S. aureus Reduction of neutrophil inltration
Reduction of inammation
Protection of the mammary gland
[31]
Lb. *
***
perolens; Lc.
*lactis
Bovine 1.5 ×10
6
CFU
**
of each strain
Mammary
tissue
48 h S. aureus, S. chromogenes;
S. uberis; E. coli
Reduction of pathogen adhesion [23]
Lc. *lactis Bovine 1 ×10
9
CFU
**
Milk 0, 6, 24, 48, 72,
120 and 168 h
S. aureus; S. uberis; S.
dysgalactiae
SCC
***
reduction
Pathogen reduction
Immunomodulatory potential
[32]
*Lactococcus (Lc.);
**
Colony forming units (CFU);
***
Somatic cell counts (SCC);
****
Lactobacillus (Lb.).
L. Touza-Otero et al.

Biomedicine & Pharmacotherapy 170 (2024) 115967
4
S. aureus. As aureocin 4181, it works by disrupting the bacterial cell
membrane [51]. Finally, Hycin 4244 (produced by Streptomyces hygro-
scopicus) was effective against a variety of Gram-positive and
Gram-negative bacteria, including several drug-resistant strains such as
methicillin-resistant S. aureus (MRSA) and vancomycin-resistant
Enterococcus (VRE). It works by inhibiting bacterial protein synthesis
[52].
2.3. Protective power of plants: extracts and essential oils
Plants are a rich source of compounds with antimicrobial, antioxi-
dant, and/or anti-inammatory properties, which can potentially
benet animal health. These phytochemicals are secondary metabolites
implicated in plant defence mechanisms. Alkaloids, terpenes, avonoids
or polyphenols are among the most well-known and studied. They are
primarily obtained in the form of extracts (PEs) or essential oils (EOs)
[56–59].
PEs are mixtures obtained by extraction with solvents. EOs are
lipophilic and aromatic liquids made up of low molecular weight vola-
tile compounds. Both are obtained from different parts of plants (fruits,
roots, seeds, leaves) or from a combination of them, such as pomace or
marc. PEs and EOs are rich in a wide variety of components with a po-
tential antibacterial activity, being capable of acting as bacteriostatics
and/or bactericides [58,60,61]. In addition, its anti-antioxidant prop-
erties may contribute to the prevention of oxidative stress that occurs in
the mammary glands during mastitis [5].
Although the antimicrobial mechanism of action is not fully eluci-
dated, it is believed to be related to bacterial cell rupture [62].
Gram-positive bacteria are highly susceptible to the effects of PEs and
EOs, primarily due to their thinner lipopolysaccharide layer that covers
the cell wall [57,63].
Table 4 summarizes the main in vitro assays studying PEs and EOs for
bovine mastitis. Some of them, such as those extracted from Kalanchoe
or Datura stramonium, have shown antimicrobial activity comparable to
commercial antibiotics such as gentamicin [61,64]. Furthermore, PEs
derived from Salvinia auriculata, Larrea tridentata and Moringa oleifera
[65–67], as well as EOs from Cuminum cyminum, Melissa ofcinalis or
Menthae piperitae have shown potential against antibiotic-resistant
S. aureus strains, targeting both bacteria and biolms [60,64–66,
68–71]. In addition, synergistic effects have been observed when PEs or
EOs (e.g. EOs from Melaleuca armillaris) are administered combined with
antibiotics, potentially allowing to reduce their doses while maintaining
the same bactericidal effects [60]. Also, the combination of EOs
(carvacrol, thymol, trans-cinnamaldehyde, eugenol, p-cymene, menthol,
linalool and citral) with medium-chain fatty acids has shown additive
antibacterial activity, warranting further investigation [72,73].
A limited number of in vitro studies have explored the effects of
phytochemicals on epithelial cell lines in an infectious environment. For
instance, EOs from Malaleuca alternifolia have shown to enhance BMEC
proliferation and reduce the inammatory response triggered by
S. aureus [70]. Additionally, moringa extract has demonstrated anti-
bacterial properties by reducing the invasion and adhesion of E. coli,
Enterococcus faecalis and S. simulans to MAC-T cells [74].
The number of in vivo trials on PEs and EOs-based intramammary
formulations is relatively limited compared to pure PEs and EOs in vitro
investigations. However, these studies validate the antimicrobial po-
tential exhibited by certain PEs and EOs in vitro (Table 5). For those
trials, milk samples were collected at different times after administra-
tion, and SCC and bacteriological count were evaluated to conclude
their antibacterial activity.
An IMM formulation containing EOs from Thymus vulgaris L., Thymus
serpylum L., Origanum vulgare L. and Satureia montana L., has demon-
strated activity against Staphylococcus spp., Streptococcus spp., Klebsiella
spp., Proteus mirabilis, E. coli, S. uberis and Serratia marcenses. This
formulation showed higher effectiveness against Gram-positive bacteria
[62]. The synergistic effect between phytochemicals and antibiotics
have also been proven by combining Aloe vera PE with low concentra-
tions of cloxacillin or ceftiofur in a IMM formulation [86].
Based on these ndings, phytochemicals, whether used alone or in
combination with other antimicrobials, show great potential as anti-
microbial agents for the prevention and treatment of bovine mastitis.
However, their full potential remains largely unexplored. The develop-
ment of pharmaceutical formulations utilizing these natural products
could serve as a promising alternative to conventional antibiotic use,
with a future-oriented approach. Their implementation is expected to
contribute to the reduction of antibiotic usage in mastitis management.
2.4. Using the enemies: bacteriophages and endolysins
Bacteriophages or phages, the most abundant entities on Earth, are
viruses that attach to specic receptors on the surface of host bacteria,
injecting their genetic material, and replicating within the cell [88]. This
replication process ultimately leads to the destruction of the bacteria
and the consequent release of the newly formed phages to infect other
Table 3
Summary of in vitro studies evaluating the potential of bacteriocins against
bovine mastitis pathogens.
Bacteriocin Pathogens Results /
mechanisms of
action
Reference
Hycin 4244 Staphylococcus spp.
including MRSA and
VRE
Antibacterial and
antibiolm
[52]
Bacteriocins produced
by 206
Staphylococcal
strains
Staphylococcus spp. Bacteriostatic [53]
S. uberis bacteriocins S. aureus;
Enterococcus.
Faecalis; S.
agalactiae; S.
dysgalactiae; E. coli;
Corynebacterium spp.
Antibacterial [47]
Cationic nisin/
dioctadecyl-
dimethylammonium
bromide
nanoparticles
Staphylococcus spp.
resistant strains
Bactericidal [41]
Bovicin HC5 S. aureus; S.
agalactiae
Antibacterial [50]
Aureocin 4181 Staphylococcus;
Streptococcus
Antibacterial by
cell membrane
disruption
[48,49]
Nisin derivates Staphylococcus spp.
resistant strains
Antibacterial [45]
Nisin PV S. uberis Antibiolm [45]
Ubericin K Enterococcus;
Streptococcus;
Listeria; Lactococcus
Antibacterial by
cell membrane
disruption
[51]
Staphylococcus agnetis
4244 bacteriocins
44 strains of
antibiotic-resistant
Gram-positive
bacteria
Antibacterial
against resistant
strains
[54]
non-aureus
staphylococci
bacteriocins
S. aureus Effective in
modulating
virulence genes
expression
[55]
Bactofencin and
reuterin
S. aureus; S.
dysgalactiae; S. uberis
Antibacterial by
modication of
teichoic acids of
the cell wall
(bactofencin) or
inducing
oxidative stress
(reuterin)
[36]
S. agalactiae strains S. pyogenes; S.
agalactiae; S.
porcinus; S. uberis
Antibacterial [38]
L. Touza-Otero et al.

Biomedicine & Pharmacotherapy 170 (2024) 115967
5
bacteria [89,90].
Phages can be classied based on various factors, including infection
strategies (lytic or temperate), morphological traits and genomic as-
pects, leading to a diverse categorization within different families such
as Myoviridae, Siphoviridae and Podoviridae [91,92].
In terms of infection strategies, lytic phages exclusively follow the
lytic cycle in bacteria. On the other hand, temperate phages can inte-
grate their genetic content into the hosts chromosomes and replicate as
part of their genome, ultimately leading to host cell lysis. For this reason,
it is crucial to characterize phages based on this aspect, selecting
appropriate non-temperate lytic phages for therapeutic use [90].
The phages lytic capacity depends to a large extent on their pro-
duction of endolysins or lysins, enzymes that play a crucial role in the
breakdown of the host cell wall at the end of the phage replication cycle
[93]. Endolysins, unlike antibiotics, have remarkable specicity, being a
promising alternative to combat antibiotic-resistant bacterial strains.
Furthermore, they minimize adverse effects to bovine hosts when used
to treat conditions such as bovine mastitis [94,95].
Tables 6 and 7 provide a comprehensive summary of the ndings
from in vitro and in vivo tests evaluating the activity of bacteriophages
and endolysins against bovine mastitis causing bacteria.
The lytic capacity of phages and/or lysins has been demonstrated in
different bacteria such as Staphylococcus [90,91,94,96–100], Strepto-
coccus [101,102] or Klebsiella [103]. Moreover, they have shown the
capacity to modify the biolm structure of antibiotic-resistant bacterial
strains [91,97,102,104].
In vivo studies investigating the therapeutic potential of phages or
lysins for mastitis treatment have been primarily assessed in murine
models through post-administration histological evaluation of the
mammary glands. In this regard, the administration of Aerococcus vir-
idans phage, led to bacterial count reduction and mammary tissue
damage alleviation [89]. Likewise, the administration of
vB_EcoM-UFV13 phage decreased the E. coli concentration and the
proinammatory cytokines expression together with the prevention of
the biolm formation [104]. Similar results were obtained by Teng et al.
(2022) in a mastitis murine model caused by S. aureus [105]. Finally, an
Table 4
Summary of in vitro assays evaluating the potential of EO* and PE
**
as alterna-
tives in the treatment of bovine mastitis.
EOs/PEs extracts Pathogens tested Results Reference
Moringa oleifera
seed
S. aureus Antibacterial [65]
Moringa E. coli; E. faecalis; S.
simulans; Serratia
liquefaciens
Antibacterial
Pathogen adhesion
reduction
[74]
Malaleuca
alternifolia EO*
S. aureus Anti- inammatory
response
Immunomodulatory
potential
[70]
Nanoemulsions
with Achyrocline
satureioides
S. aureus Antibacterial
Antibiolm
[71]
Thymus serpyllum L.;
Thymus vulgaris L.
Staphylococcus spp.;
Streptococcus spp.;
E. coli; Klebsiella
oxytoca
Antioxidant
Antibacterial
[75]
Salvinia auriculata
root
S. aureus Antibacterial
Antibiolm
[66]
Panax ginseng PE
**
+Cephalexin
S. aureus Bactericidal [69]
Sorghum E. coli; Salmonella
Typhimurium;
Campylobacter
jejuni;
Campylobacter coli;
Pseudomona
aeruginosa;
Klebsiella
pneumoniae;
Klebsiella oxytoca;
S. aureus; E. faecalis
Antibacterial [63]
Cinnamon EO* +
silver
nanoparticles
S. agalactiae Bactericidal
Antibiolm
[76]
Cuminum cyminum
L. EO*
S. aureus Antibacterial against
resistant strains
[68]
Salvia ofcinalis;
Satureja hortensis
EO*
S. aureus; S.
agalactiae; E. coli
Antibacterial [77]
Larrea tridentata S. aureus; Listeria
monocytogenes; E.
coli; Bacillus cereus;
Klebsiella
pneumoniae;
Pasteurella
multocida
Antibacterial [67]
Quercus robur
+Calluna vulgaris
L.
E. coli; S. agalactiae;
S. uberis; Serratia
liquefaciens; S.
aureus
Antibacterial [78]
Ricinus communis
leaf
S. aureus; S.
agalactiae; S.
pyogenes; E. coli;
Klebsiella
pneumoniae;
Pseudomona
aeruginosa
Antibacterial [79]
Kalanchoe
densiora;
Kalanchoe
marmorata;
Datura
stramonium;
Clerodandrum
myricoidos
S. aureus Higher antibacterial
activity than
gentamicin
[64]
EO* and PE
**
of
Mentha pulegium
L.; Nepeta cataria
L.; Melissa
ofcinalis L.
S. aureus; E. coli Higher antibacterial
activity than some
antibiotics
[61]
Melaleuca armillaris
EO*
S. aureus Synergistic bactericidal
effect with
erythromycin
[60]
Table 4 (continued )
EOs/PEs extracts Pathogens tested Results Reference
EO* +medium-
chain fatty acids
S. aureus, E. coli,
Klebsiella
pneumoniae; S.
agalactiae
Antibacterial activity
Carvacrol and octanoic
acid showed the most
synergistic effect
[72,73]
Malaleuca
alternifolia EO*
Staphylococcus spp.;
Streptococcus spp.;
E. coli; Klebsiella
pneumoniae;
Candida albicans
Bactericidal and
fungicidal
[80]
Linalool S. agalactiae Antibacterial [81]
EO*: Thymus
vulgaris L.;
Thymus serpyllum
L.; Origanum
vulgare L.
Serratia spp. and
Proteus spp.
Antibacterial [82]
Origanum vulgare L.;
Satureja montana
L. EO*
Staphylococcus spp;
Streptococcus spp.;
E. coli
Antibacterial
Antioxidant
[83]
Melissa ofcinales;
Menthae piperitae
EO*
E. coli,
Staphylococcus spp.,
Streptococcus spp.,
Enterobacter
sakasakii and
K. oxytoca
Antibacterial
Antioxidant
[84]
Nano-emulsions of
Eugenia
caryophyllata;
Origanum vulgare;
Cinnamomum
cassia EO*
S. aureus; E. coli;
Candida albicans
Antibacterial,
fungicidal
[85]
*Essential oil (EO);
**
Plant extract (PE).
L. Touza-Otero et al.

Biomedicine & Pharmacotherapy 170 (2024) 115967
6
E. coli-induced bovine mastitis was used to evaluate the efcacy of a
three-phage cocktail. The results obtained after milk analysis concluded
a signicant reduction in bacteria counts, SSC and inammatory factors
[88].
While results from studies utilizing bacteriophages and endolysins
demonstrate promising potential for mastitis control, it is imperative to
conduct further trials to fully ascertain their efcacy, particularly in
bovine hosts.
2.5. Other alternatives
Other less explored approaches hold potential for the control of
bovine mastitis. Inorganic nanoparticles (NPs), including gold-silver
NPs, have been investigated for eradicating S. aureus and Pseudomonas
aeruginosa bacterial biolms associated with mastitis. These NPs can be
easily synthetized and have demonstrated in vitro antimicrobial effects,
reducing microbial growth [108].
Also, silver and copper NPs, alone or in combination, have exhibited
the ability to in vitro inhibit the formation of biolms of different
mastitis-causing bacteria (Stpahylococcus, Streptococcus, E. coli, Klebsi-
ella) [109]. These NPs did not show toxic effects on human or bovine
mammary cells [110]. The effectiveness of copper NPs in treating
mastitis was validated in murine mastitis models caused by S. aureus,
where their IMM application resulted in a reduction of the bacterial load
and improved oxidative stress levels [111]. Furthermore, the potential
of polymeric NPs based on chitosan has been investigated. These NPs
exhibited bactericidal activity against Pseudomonas, inhibited biolm
formation, and eradicated pre-existing biolms in vitro [112].
Antimicrobial peptides (AMPs) of other origins than bacteria, can
also present promising potential as alternatives to antibiotics. For
instance, Polybia MP-1 obtained from the venom Polybia paulista wasp
has demonstrated antimicrobial activity against S. aureus, E. coli, Kleb-
siella pneumoniae and Pseudomonas aeruginosa strains isolated from
mastitic cow milk [113,114].
On the other hand, caffeic acid is a phenolic compound that has been
recently evaluated in vitro. This versatile compound exhibits notable
antibacterial and antibiolm properties, effectively limiting bacterial
proliferation. Furthermore, caffeic acid has demonstrated its capacity to
not only diminish E. coli cell adhesion and alleviate inammation but
also reduce oxidative stress in BMEC [119,120].
Mesenchymal stem cells (MSCs), as well as their secretome,
including extracellular vesicles (EVs), have garnered signicant atten-
tion in bovine mastitis therapy. MSCs have demonstrated their capacity
to modify the tissue homeostasis in S. aureus or S. uberis infections,
including methicillin-resistant strains, exhibiting antimicrobial proper-
ties, and promoting tissue repair and regeneration. These effects have
been observed both in vitro and in vivo [115,116]. Furthermore,
inammation was considerably improved when MSCs overexpressed
angiotensin-converting enzyme 2 (ACE2) [117]. In a bovine model, the
intramammary inoculation of both MSCs and their secreted EVs has
been demonstrated to contribute to a decrease in SCC and a simulta-
neous increase in the expression of anti-inammatory cytokines [118].
Other options under evaluation for mastitis treatment or prevention
include antibody therapy [121]; vaccines [122] and nitric
oxide-releasing formulations [123].
3. Conclusions and future perspectives
The development of AMR is a major challenge for global health,
closely related to the excessive use of antibiotics in cattle, with promi-
nence for bovine mastitis. Better protocols are needed to address this
problem in practice and to explore new non-antibiotic alternatives for
prevention and treatment of mastitis.
Biomimetic approaches are trending topic. The use of probiotics,
bacteriocins, phytochemicals or bacteriophages gives a universe of
possibilities. As illustrated in Fig. 2, the suggested alternatives, unlike
traditional antibiotics, go beyond the simple goals of promoting
Table 5
Summary of in vivo assays evaluating the potential of EO* and PE
**
as alternatives in the treatment of bovine mastitis.
EOs/PEs Animal
model
Pathogens tested Results Reference
Brewers Gold; Perle hops; Propolis; Lum lichen; Common mallow;
Marigold, Absinthe wormwood; Black poplar buds; Lemon balm;
oregano; Lavender; Rosemary
Bovine Different gram-positive and gram-negative
strains
Bactericidal [87]
Panax ginseng PE
**
+Cephalexin Bovine Non-aureus staphylococci; S. aureus; S. uberis; S.
dysgalactiae; Streptococcus spp.
Bactericidal [69]
Aloe vera L. Bovine S. aureus Antibacterial combined
with low doses of
antibiotics
[86]
Thymus vulgaris L.; Thymus serpyllum L., Origanum vulgare L. and
Satureja montana L. EO*
Bovine Staphylococcus spp.; Streptococcus spp.; Klebsiella
spp.; Proteus mirabilis; E. coli; S. uberis; Serratia
marcenses
Antibacterial [62]
*Essential oil (EO);
**
Plant extract (PE).
Table 6
Summary of in vitro assays evaluating the potential of different bacteriophages
and endolysins as alternatives in the treatment of bovine mastitis.
Bacteriophage / Lysin Pathogens tested Results Reference
Lytic phages of Klebsiella
oxytoca
Klebsiella oxytoca Bacteriostatic [103]
vB_EcoM-UFV13 phage E. coli; Trueperella
pyogenes
Bacteriostatic
Reduction of
cytokines
expression
Antibiolm
activity
[104]
Lytic phages (SAJK-IND;
MSP)
S. aureus Lytic activity [100]
Isolated phages S. aureus Lytic activity [99]
Cocktail of phages (STA1.
ST29; EB1. ST11; EB1.
ST27)
S. aureus Pathogen
reduction
[96]
Lytic phages (SYGD1,
SYGE1; SYGMH1)
E. coli Pathogen
reduction
SCC* reduction
Inammation
reduction
[88]
vB_SauM_SDQ phage S. aureus Antibiolm [97]
Phage 24A2 S. aureus Antibacterial [94]
Staphylococcus M8; B4
phages (Podovidirae
familiy)
S. aureus resistant
strains
Antibacterial and
antibiolm
[91]
S. aureus phages
(B_UFSM4; B_UFSM5)
Staphylococcus
spp.
Lytic activity [90]
PlySs2; PlySs9 endolysin S. uberis Antibacterial [101]
LysGH15 endolysin S. aureus Bactericidal [98]
Lysin CHAPk endolysin S. agalactiae Antibacterial and
antibiolm
[102]
*
Somatic cell counts (SCC).
L. Touza-Otero et al.

Biomedicine & Pharmacotherapy 170 (2024) 115967
7
bacterial death and reducing bacterial growth. Instead, they consider a
spectrum of factors associated to host microenvironments. These bio-
mimetic strategies present a comprehensive approach with the potential
of modulating various stages of infection, encompassing the modica-
tion of the local microbiota and the immune response, preventing
oxidative stress, interacting with epithelial cells, and controlling the
inammatory process. However, these strategies have only been
partially explored in the veterinary eld for the treatment of bovine
mastitis.
Probiotics have emerged as the most researched option, showing
great potential as possible alternative to antibiotics. Both in vitro and in
vivo studies have demonstrated their antimicrobial effects, with the ca-
pacity to combat mastitis-causing pathogens, even in persistent in-
fections, due to their antibiolm activity. In addition, their ability to
interact with the host’s microbiota and epithelial cells, preventing the
pathogen adhesion, makes them a good alternative for prophylaxis.
Probiotics are already approved for use in the food industry, which
supports their low-toxicity and safety for consumption. This eliminates
concerns about potential residues in milk, a problem with the use of
antibiotics. The fact that different in vivo trials have administered pro-
biotics by IMM and achieved sufcient antibacterial effect, suggests that
an extremely advanced technology for administration may not be
necessary. The identication of the most effective probiotic strains and
the appropriate development of IMM formulations, represent the next
relevant steps towards addressing bovine mastitis prevention, without
requiring antibiotics.
Some bacteriocins, such as nisin, are already marketed as a teat
disinfectant. They are effective at low concentrations, but also vulner-
able to resistances development. The main advantages of these products
are their antibacterial activity at low concentrations, their easier
metabolization compared to classical antibiotics and the possibility of
modication by bioengineering. On the other hand, its administration is
hampered by the lack of stability associated to the protein nature, which
makes impossible to use certain administration routes. This explains the
limited number of in vivo studies. To overcome this limitation, nano-
technology is proposed as a promising formulation strategy.
Plant-derived products offer a wide array of options for obtaining
antimicrobials. PEs and EOs can be used as holistic systems with anti-
bacterial activity, alongside with antioxidant and anti-inammatory
properties, effectively addressing the concomitant oxidative stress and
inammation in bovine mastitis. Plant-derived products offer numerous
advantages over other alternatives in the short-term development of
pharmaceutical formulations for the management of bovine mastitis.
They are easily accessible, require less specic production and have
greater stability compared to probiotics, bacteriocins or bacteriophages.
Specic species can be cultivated for their production, nevertheless
phytochemicals can also be obtained as by-products of other industries.
By establishing a connection between the waste-chain and livestock
industries, it is possible to promote the circular economy while simul-
taneously enhancing animal health and welfare, and without resorting
to antibiotics.
Bacteriophages and their endolysins also constitute a wide spectrum
of possibilities, not only in the therapy of bovine mastitis, but also in
many other pathologies. They are abundant in nature and highly spe-
cic, limiting the possibility of developing resistance and avoiding
adverse side effects.
Several bacteriophages and endolysins have been isolated and suc-
cessfully characterized in vitro as antibacterials for bovine mastitis
pathogens. Despite their promising prospects, the number of in vivo as-
says is much more limited and, moreover, studies have mainly been
carried out in murine models, thus further investigation in this eld is
necessary. The development of pharmaceutical formulations for use in
bovine mastitis therapy should also be addressed.
Overall, only few studies have nally developed formulations with
potential clinical applications for these alternatives. While numerous in
vitro studies support their properties, there is a lack of in vivo research,
Table 7
Summary of in vivo assays evaluating the potential of different bacteriophages and endolysins as alternatives in the treatment of bovine mastitis.
Bacteriophage/Lysin Animal
model
Pathogens Sample
evaluated
Evaluation
time
Results and conclusions Reference
vB_EcoM-UFV13 phage Murine E. coli Mammary
tissue
48 h Reduction of cytokines
expression
[104]
S. aureus phages (vBSM A1; vBSP-A2) Murine S. aureus Mammary
tissue
24 h Pathogen reduction
Symptoms improvement
[106]
Aerococcus viridans phage Murine Aerococcus viridans Mammary
tissue
24 h Symptoms improvement [89]
vB_PaeS_PAJD-1 phage Murine Pseudomona
aeruginosa
Mammary
tissue
24 h Pathogen reduction
Symptoms improvement
[93]
CM8–1 phage Murine Klebsiella
pneumoniae
Mammary
tissue
12, 24 and
36 h
Pathogen reduction [107]
S. aureus phages (4086–1; 4086–2, 4086–3; 4086–4;
4086–6)
Murine S. aureus Mammary
tissue
- Bacteriostatic
Anti-inammatory
[105]
Lytic phages (SYGD1; SYGE1; SYGMH1) Bovine E. coli Milk 24 h Pathogen reduction
SCC* reduction
Anti-inammatory
[88]
LysGH15 endolysin Murine S. aureus Mammary
tissue
48 h Symptoms improvement [98]
*
Somatic cell counts (SCC).
Fig. 2. Different points targeted by biomimetic approaches under
consideration.
L. Touza-Otero et al.

Biomedicine & Pharmacotherapy 170 (2024) 115967
8
particularly, in bovine hosts to conrm their efcacy and security.
Ultimately, achieving signicant progress in the clinic requires the
obtention of stable pharmaceutical formulations containing the pro-
posed alternatives. In the pursuit of this objective, bacteriocins seems to
be the active ingredients with most notable limitations due to their
instability. Although probiotics and bacteriophages have been tested in
vivo, their incorporation into pharmaceutical formulations remains
underexplored. However, plant extracts and essential oils have a sig-
nicant advantage in this context.
Future steps ought to be focused on developing formulations with
suitable physicochemical properties for their administration into the
mammary gland. Leveraging pharmaceutical development would
ensure precise local drug delivery, optimal retention time at the site of
action and consequently, the enhancement of the therapeutic efcacy.
Given the current scenario, plant extracts and essential oils emerge as
the most promising short-term alternatives.
CRediT authorship contribution statement
Lara Touza-Otero: Conceptualization, Investigation, Formal anal-
ysis, Writing – original draft. Mariana Landin: Conceptualization,
Writing – review & editing, Supervision. Patricia Diaz-Rodriguez:
Conceptualization, Writing – review & editing, Supervision, Funding
acquisition.
Declaration of Competing Interest
The authors declare the following nancial interests/personal re-
lationships which may be considered as potential competing interests:
Patricia Diaz-Rodriguez reports nancial support was provided by Eu-
ropean Union’s Horizon 2020 research and innovation program under
grant agreement No. 101036768 (NeoGiANT project), Mariana Landin
reports nancial support was provided by Xunta de Galicia.
Data availability
No data was used for the research described in the article.
Acknowledgements
This study was funded by the European Union’s Horizon 2020
Research and Innovation Program under grant agreement No.
101036768 (NeoGiANT project) and by Project ED431C 2020/17
(Galician Competitive Research Groups, Xunta de Galicia).
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