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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
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Acta Veterinaria Scandinavica
Bacteriophage-derived endolysins
asinnovative antimicrobials againstbovine
mastitis-causing streptococci andstaphylococci:
astate-of-the-art review
Niels Vander Elst1*
Abstract
Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell
wall, represent a groundbreaking class of novel antimicrobials to revolutionize the veterinary medicine field. Wild-
type endolysins exhibit a modular structure, consisting of enzymatically active and cell wall-binding domains,
that enable genetic engineering strategies for the creation of chimeric fusion proteins or so-called ‘engineered
endolysins’. This biotechnological approach has yielded variants with modified lytic spectrums, introducing new
possibilities in antimicrobial development. However, the discovery of highly similar endolysins by different groups
has occasionally resulted in the assignment of different names that complicate a straightforward comparison. The
aim of this review was to perform a homology-based comparison of the wild-type and engineered endolysins
that have been characterized in the context of bovine mastitis-causing streptococci and staphylococci, grouping
homologous endolysins with ≥ 95.0% protein sequence similarity. Literature is explored by homologous groups
for the wild-type endolysins, followed by a chronological examination of engineered endolysins according to their
year of publication. This review concludes that the wild-type endolysins encountered persistent challenges in raw
milk and in vivo settings, causing a notable shift in the field towards the engineering of endolysins. Lead candidates
that display robust lytic activity are nowadays selected from screening assays that are performed under these
challenging conditions, often utilizing advanced high-throughput protein engineering methods. Overall, these
recent advancements suggest that endolysins will integrate into the antibiotic arsenal over the next decade, thereby
innovating antimicrobial treatment against bovine mastitis-causing streptococci and staphylococci.
Keywords Bacteriophage-derived endolysins, Bovine mastitis, Dairy industry, Endolysins, Gram-positive, Homology-
based, Staphylococcus, Streptococcus, Veterinary medicine
Background
Endolysins are bacteriophage‑encoded peptidoglycan
hydrolases
Bacteriophages, or shortly phages, are viruses that infect
bacteria [1–3]. Phages recognize specific receptors on
the cell wall of bacterial species or strains, which cause
them to adsorb and inject their DNA. A successful phage
infection subsequently can result in two different out-
comes: (i) the phage enters a lysogenic state, in which its
*Correspondence:
Niels Vander Elst
niels.vander.elst@ki.se
1 Department of Neuroscience, Karolinska Institutet, Biomedicum 7D,
Solnavägen 9, 17165 Solna, Stockholm, Sweden
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
genome is inserted into the bacterial genome and repli-
cates together with the bacterium (i.e., prophage), or (ii)
the phage passes a lytic cycle in which it reprograms the
bacterial host cell to synthesize and assemble new viral
particles [2, 3]. ese lysogenic and lytic cycles can trans-
fer into one another, but strictly virulent phages exist
and replicate by a lytic infection cycle only. At the end
of the lytic cycle, endolysins are produced by the phage
to release these newly assembled viral particles from the
infected host. is process is known as ‘lysis-from-within’
(Fig. 1) [4]. In this process, a secondary phage protein
named a holin will first perforate the lipid layer of the
inner bacterial membrane, which then enables endolysins
to hydrolyse the outer peptidoglycan layer [5]. us,
endolysins are peptidoglycan hydrolases that degrade
the bacterial cell wall. In the case of Gram-positive bac-
teria, endolysins are known to retain this functionality
when they are recombinantly produced in the labora-
tory and incubated with Gram-positive bacteria, which
is a process referred to as ‘lysis-from-without’ (Fig. 1)
[6]. Indeed, when endolysins hydrolyse the bacterial cell
wall of Gram-positive bacteria, the high internal osmotic
pressure inside the bacterial cell causes the bacterium to
‘burst’ or ‘lyse’. From that perspective, endolysins have
been proposed and investigated as promising, novel anti-
microbials [3, 4, 7, 8].
Endolysins are modular proteins amenable togenetic
engineering
Endolysins derived from Gram-positive bacteriophages
typically feature a modular structure [4, 7, 9]. ey
consist of one or more so-called enzymatically active
domains [EADs; introduced after (i)] and cell wall-
binding domains [CBDs; introduced after (ii)], which
confer either peptidoglycan hydrolysis or binding
activity, respectively. EADs are sometimes also referred
to as catalytic domains. ese domains are usually,
but not exclusively, coupled by proline- or lysine-rich
regions referred to as linkers [introduced after (iii)] [10,
11]. is modular structure of endolysins allows the
Fig. 1 The action of endolysins in a ‘lysis-from-within’ versus ‘lysis-from-without’ scenario. Bacteriophages employ endolysins at the end of their lytic
replication cycle to release newly assembled viral particles from the infected host, causing ‘lysis-from-within’. In the “lysis-from-without” scenario,
the endolysin gene is cloned in a vector, after which the endolysin is expressed and purified. In the case of Gram-positive pathogens, the purified
endolysin retains its functionality when applied externally to the targeted pathogen (created with https:// biore nder. com/)
Page 3 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
easy generation of chimeric fusion proteins or so-called
‘engineered endolysins’ [4, 7, 9]. Indeed, the EADs or
CBDs of an endolysin can readily be altered by making
changes on DNA level (e.g., PCR followed by restriction/
ligation in a vector and overexpression in a host). is
biotechnological strategy to engineer endolysins has been
extensively pursued and endolysins that combine EADs
and CBDs of different origin have been created after
this concept was introduced to the field. Many different
types of EADs and CBDs have been described, which are
categorized based on the specific position where they
either hydrolyze the peptidoglycan or bind the cell wall,
respectively.
(i) EADs are biochemically categorized based on the
peptidoglycan bond(s) that they hydrolyze (Fig.2)
[4, 7, 9]. e most common types of EADs found
in endolysins from Gram-positive bacteriophages,
are: (1) -acetyl-muramoyl--alanine (ala)
amidases (subtypes 2, 3 and 5), (2) endopeptidases
such as M23 peptidases or cysteine/histidine-
dependent aminohydrolases/peptidases (CHAPs),
(3) -acetyl-β--muramidases, and (4) -acetyl-β-
-glucosaminidases [12–14]. Amidases hydrolyse
the bond between the -acetyl-muramic acid
(MurNAc) and the first -ala in the stem peptide,
whereas endopeptidases can impact different
bonds in the stem peptide, or the interpeptide
bridge, or between those two latter. Muramidases
and glucosaminidases hydrolyze bonds between
the MurNAc and -acetyl--glucosamine
(GlcNAc) repeating sugar units.
(ii) CBDs recognize certain patterns in and bind to
the bacterial peptidoglycan, (lipo)teichoic acids
or other cell wall components [4, 7, 9]. ey
frequently consist of repeated sequences [12].
Multiple studies have shown the binding activity
of these CBDs by either fusing them to green
fluorescent protein (GFP) or by conjugation of the
CBD to a fluorophore (e.g., Alexa Fluor) [15, 16].
In general, an endolysin requires at least one CBD
to be functional for Gram-positive endolysins, as
was shown by deleting the CBD [15–17]. In most
cases, this resulted in loss or inferior activity of
the remaining EAD(s). An exception has been
described for PlyL and PlyK (a.k.a. LysK) [18, 19].
On the other hand, it is also hypothesized that
CBDs play a major role in countering resistance
development [20]. Indeed, if endolysins were
massively released from the infected host at the end
of the lytic cycle, exposure of neighboring Gram-
positive bacteria to the endolysins may select for
resistant variants in the bacterial population. It is
therefore accepted that the CBD plays a major role
in keeping the endolysin bound to the lysed host
cell. As such, removal of the CBD may in some
cases counterintuitively improve the activity of the
endolysin, as it can increase the enzyme’s turnover
by not keeping it bound to the peptidoglycan of
only one bacterial cell [20]. In addition, the iso-
electric point (pI) of the endolysin is altered, which
may result in an improved affinity for the bacterial
cell wall [18, 21]. CBDs are also categorized, but the
exact binding place of the peptidoglycan is often
not yet fully elucidated and remains understudied.
It is reported that LysM recognizes the GlcNAc
moiety, whereas SH3 domains interact with both
the stem peptide and the interpeptide bridge, more
specifically—but not exclusively—the pentaglycine
bridge in staphylococcal peptidoglycan [22–24].
CW_7 is believed to bind MurNAc and the region
of the adjacent stem peptide [25].
(iii) Linkers, usually composed of proline- or lysine-
rich regions, connect the modular domains of
an endolysin. ese linkers may exhibit diverse
properties, such as being short or long, rigid or
flexible, or others. e linker regions impact the
lytic activity of the endolysin, and this can be
improved by selectively editing the linker regions
only [10, 11].
Of note, recent insights have revealed that many
endolysins form multimers [26, 27]. is is specifically
stated to frequently occur in streptococcal endolysins.
Multimeric endolysins either consist of multimers of
the same endolysin, but endolysin genes frequently con-
tain additional ribosomal binding sites where shorter
Fig. 2 Schematic representation of the streptococcal bacterial
peptidoglycan structure, including the endolysin cleavage
sites. The peptidoglycan is composed of repeating sugar units,
n-acetyl-glucosamine (GlcNAc) and n-acetyl-muramic acid (MurNAc),
which are cross-linked via a d-Alanine (d-Ala) interpeptide bridge
between l-Lysine (l-Lys) and d-Ala residues. The chains also contain
l-Ala and d-glutamic acid (d-Glu). The cleaved bonds and major
classifications of enzymatically active domains (EADs) of endolysins
are indicated: (1) n-acetyl-muramoyl-l-ala amidase; (2) various
endopeptidases; (3) n-acetyl-β-d-muramidase; (4) n-acetyl-β-d-glucos
aminidase (created with https:// biore nder. com/)
Page 4 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
sequences of the endolysin are translated [26]. An exam-
ple of a multimeric endolysin is PlyC, which is composed
from two separate gene products [27]. e CBD of PlyC
self-assembles into a circular octamer to which the EAD
is subsequently linked by salt bridges and/or Van der
Waals forces.
In vitro assays toevaluate endolysin activity
e invitro assays to evaluate endolysin activity consti-
tute the spot-on-plate and -lawn assay, the zymogram, the
turbidity reduction assay, the time kill assay and the deter-
mination of the minimal inhibitory (MIC) and minimal
bacteriocidic (MBC) concentration (Fig.3).
In a spot-on-plate assay, a drop of endolysin is placed
on top of a nutrient-poor agar in which the bacterial tar-
get is embedded and incubated at 37°C (Fig.3A). If the
endolysin is active, a clearing zone or ‘halo’ will become
visible [9]. A similar assay is the spot-on-lawn, in which
the bacteria are plated on top of a nutrient-rich agar
together with a drop of endolysin [28, 29]. If the bacteria
are killed by the endolysin, they will not grow, and a halo
will become visible. e spot-on-plate assay is regarded
more stringent than the spot-on-lawn assay. Of note,
the diameter of the halo does not correlate with the lytic
activity of the endolysin [4, 9, 30, 31]. It is rather depend-
ent on the size of the endolysin that diffuses through the
agar. Indeed, endolysins with a low molecular weight are
believed to cause a bigger halo than those with a high
molecular weight. e zymogram is not further dis-
cussed, as it is not relevant for the reviewed literature in
this work, but can be consulted in O’Flaherty etal. [29].
Overall, the spot-on-plate, spot-on-lawn and zymogram
assays are highly sensitive to detect enzymatic endoly-
sin activity, as there is a long contact time between the
endolysin and the bacteria which are statically embedded
in an agar or gel [9]. erefore, they are regarded qualita-
tive rather than quantitative assays [16].
e turbidity reduction assay (TRA) is a method to
evaluate the biochemical activity of an enzyme (Fig.3B)
[16, 31–37]. It measures a decrease of the optical density
(OD) over time using a spectrophotometer, typically
monitored at a wavelength of 600 nm (OD600nm). e
TRA measures the hydrolysis of peptidoglycan caused by
the endolysin, which results in osmotic lysis and killing
of the bacteria [16]. It is a very powerful and useful assay
to quantify the biochemical activity of an endolysin
compared to a negative control at the end of the TRA
(i.e., ΔOD600nm), or to calculate the rate at which the
bacterial cells are lysed [i.e., (ΔOD600nm/min)/µM] [33]. It
is also important to note that bacteria at early and mid-
exponential phases are much more susceptible than those
in the late exponential or stationary phase. For instance,
one study showed that the turbidity of a solution of
bacteria in the exponential phase decreased by 50%
in 15 min, whereas cells harvested in the stationary
phase decreased by only 5% at equimolar endolysin
concentrations [37].
To quantify bacterial killing caused by the endolysin,
a time killing assay (TKA) is more appropriate (Fig.3C).
A TKA challenges a certain number of target bacteria,
typically lower than the TRA, during a predetermined
time interval [16, 31, 34, 35]. As a standard for
endolysins, 106 colony forming units per milliliter (CFU/
mL) of target bacteria are usually challenged during a
fixed time interval [16, 31]. ereafter, the bacteria are
serially diluted and plated on an agar. is agar is then
incubated overnight at the appropriate temperature to
determine the surviving CFU/mL. e number of killed
bacteria is calculated in comparison with a negative
control, typically expressed logarithmically (i.e., Δlog10).
In the context of mastitis research, a kinetic TKA in both
ultra-high temperature treated (UHT) whole milk as well
as in (mastitic) raw cow’s milk can also be performed [31,
38, 39]. Note that a milk test setting cannot be used for a
TRA given its turbidity.
Alternative antibacterial assays that can be performed
to evaluate the antibacterial activity of an endolysin, are
the determination of the MIC or MBC (Fig. 3D) [40,
Fig. 3 Assays in place to determine the lytic activity of endolysins. A In a spot-on-plate or -lawn assay, a droplet of endolysin is brought on top
of an agar that contains the target bacteria, after which a lysis zone or ‘halo’ becomes visible. B The turbidity reduction assay measures the OD600nm
over time to observe a reduction in the turbidity of the target bacteria, which correlates with hydrolysis of the peptidoglycan and osmotic lysis
caused by the endolysin. C In a time kill assay, triplicates of the target bacteria are incubated at approximately 106 CFU/mL with either an endolysin
or a negative control, after which they are serially diluted and plated on an agar. After incubation of the plate at 37 °C during 18 h, CFUs are
counted, and the bacterial killing is calculated in comparison with the negative control (difference expressed as Δlog10). D The target bacteria
are challenged with decreasing concentrations of the endolysin in broth and incubated at 37 °C during 18 h. The lowest concentration at which
no visible growth in liquid broth is observed, is defined as the minimal inhibitory concentration (MIC). The conditions that show no growth
are subsequently plated on an agar to determine if surviving bacteria are present. The lowest concentration at which no surviving bacteria are
observed is defined as the minimal bacteriocidic concentration (MBC). E In the mouse mastitis model, bovine mastitis-causing streptococci
(or other pathogens) are inoculated in the fourth mammary gland pair with a blunted 32G paediatric needle while the mice are under general
anaesthesia (created with https:// biore nder. com/)
(See figure on next page.)
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
Fig. 3 (See legend on previous page.)
Page 6 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
41]. e MIC is defined as the lowest concentration of
an antibacterial agent which, under strictly controlled
in vitro conditions, completely prevents visible growth
of the target bacteria. A MIC assay thus differs from a
TKA as it measures the capacity to keep a low number
of bacteria under control and to inhibit their growth,
whereas a TKA effectively quantifies cell number
reduction. To perform a MIC assay, the target bacteria
are incubated in broth with decreasing concentrations
of the antimicrobial and incubated at 37 °C. To
subsequently determine the MBC thereof, the contents of
the wells that visibly fully inhibit the growth are plated
on an agar. is whole is then incubated and checked
for bacterial growth. e MBC is defined as the lowest
concentration of an antibacterial agent required to kill all
bacteria over a certain period. When the MBC ≤ 4 × MIC,
the antibacterial is called bactericidal.
To bridge the gap between in vitro characterization
of endolysin activity and subsequent validation in
(pre)clinical models, cell culture models have been
employed as a useful tool. Furthermore, cell cultures
have specifically been used in the context of endolysins
to: (i) study cytotoxicity (e.g., PlyC on bovine
polymorphonuclear leukocytes (PMN) [42]), and (ii)
evaluate intracellular killing (e.g., engineered endolysins
carrying so-called cell penetrating peptides (CPPs) [30,
31, 39, 43]). ree major bovine mammary epithelial
cell lines (boMECs) are mainly used throughout the
scientific community: MAC-T (established in 1991) [44],
BME-UV1 (established in 2001) [45], and PS (established
in 2015) [46]. It is important to understand that these
established boMECs are inherently different from
primary cells and from each other, especially the MAC-T
and PS vs. BME-UV1 cells [47, 48]. In fact, the bovine
mammary alveolar and ductal epithelium consists of two
layers of epithelial cells, a luminal and a basal layer, where
in between so-called mammary ductal macrophages are
situated [49]. MAC-T and PS cells originate from the
basal mammary gland epithelial layer, whereas BME-UV
originate from the luminal layer [48].
Preclinical mouse models toevaluate endolysin activity
Mouse models have been used as preclinical tools to
provide proof-of-concept of new antimicrobial agents.
A specific example thereof is the mouse mastitis model,
in which mastitis pathogens are inoculated through the
teat orifice with a blunted pediatric needle into the mam-
mary ducts (Fig. 3E) [50]. e use of these preclinical
models has many advantages in comparison with dairy
cows [50, 51]. First of all, mice are cost-effective, easy to
house and have a short generation time. Secondly, there
are many anatomical similarities between the fourth
inguinal mammary gland pair of mice and the udder of
cows. Indeed, both species contain functionally and ana-
tomically separated glands that consist of one main milk
duct ending on the top of the nipple in one teat orifice.
In addition, this inguinal gland pair in mice also con-
tains lymph nodes that similarly occur in cows. irdly,
there are many analytical tools and immunological rea-
gents available for mice, which is rather limited for cows.
Despite these advantages and similarities between the
mammary glands of mice and cows, there are obviously
also some fundamental differences [50, 51]. e composi-
tion of the milk is different and the cow’s udder contains
more phagocytic cells under physiological conditions
than the mammary gland of mice. Furthermore, mice
undergo ‘forced weaning’ 1h before administration of the
intraductal inoculation, which means that the pups are
permanently removed. is initiates the involution of the
lactating mammary gland, accompanied by a physiologi-
cal influx of macrophages in the alveolar lumen. Whereas
dairy cows are still milked throughout mastitis, this is not
done in mice. Mice are small in comparison to cows and
their mammary gland tissue is sensitive to an intraductal
inoculation, which can result in sepsis caused by the mas-
titis pathogens [51]. Taken together, the mouse mastitis
model is a valid tool to deliver preclinical proof-of-con-
cept of new antimicrobials—including endolysins—that
target bovine mastitis pathogens in the lactating bovine
mammary gland. is mouse model comes with certain
limitations which should be considered when data from
mice are translated towards the target species (i.e., the
dairy cow).
Search strategy
is state-of-the-art review conducted an exhaustive
exploration through PubMed (http:// www. ncbi. nlm. nih.
gov/ pubmed) and Google Scholar (https:// schol ar. google.
com/) utilizing a targeted search strategy with keywords
such as “endolysin”, “bovine mastitis”, and additional
relevant terms. e search aimed to identify articles
focusing on endolysins characterized or engineered in the
context of Gram-positive bovine mastitis. e evaluation
process involved a thorough examination of the title and
abstract of the obtained hits, with a subsequent retrieval
and detailed assessment of articles meeting the specified
criteria. Amino acid sequences were sourced either from
the NCBI GenBank (https:// www. ncbi. nlm. nih. gov/
genba nk/) or extracted from supplementary materials
provided in the selected articles.
Review
To facilitate a rigorous comparative analysis, a multiple
sequence alignment was executed using Clustal Omega
and the MAFFT algorithm, generating a Pearson/
FASTA output [52]. e percentage values presented
Page 7 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
in this review were consistently derived from the per-
centage identity matrix of the aligned amino acid
sequences. A schematic representation of the primary
protein structures has been created to cluster and vis-
ualize the homologous wild-type endolysins that have
≥ 95.0% protein sequence similarity, that also shows
pairwise homology of their individual subdomains
(Fig. 4). Literature will be discussed per homologous
group to allow straightforward comparison of the
wild-type endolysins, going from the top to the bottom
of this schematic representation. ereafter, the engi-
neered endolysins are discussed in chronological order
according to their year of publication. A general over-
view is provided (Table1).
To improve uniformization of the endolysin nomen-
clature that is currently lacking in the field [53] (e.g.,
LysK, PlyK and phage K endolysin all refer to the same
endolysin), a clear distinction was made between the
Fig. 4 Schematic representation of primary protein structures with annotated enzymatically active domains and cell wall-binding domains.
Percentages of pairwise identities between subdomains are presented by means of a grey scale with a 20% interval. Pairwise identities were
determined by multiple sequence alignments in Clustal Omaga (MAFFT algorithm) analyses on the protein sequences derived from the NCBI
GenBank, or sequences extracted from the supplementary materials provided in the articles that were selected. PlyC is a multimeric endolysin
that is composed of the PlyCA and PlyCB subdomains, which are translated from separate genes
Page 8 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
Table 1 Overview of the endolysins that have been characterized in the context of Gram-positive bovine mastitis
S. uberis S. dysgalactiae
Qualitative TRA
(% ΔOD600)TKA
(Δlog10 CFU/mL) MIC
(µM) In vivo
(Δlog10 CFU/g) Qualitative TRA
(% ΔOD600)TKA
(Δlog10 CFU/mL) MIC
(µM) In vivo
(Δlog10 CFU/g)
Homologous wild‑type endolysins
PlyC ND ND ND ND ND ND 40–50 [55] ND ND ND
Ply0643 ND 60–80 [63] ND ND ND ND 60–80 [63] ND ND ND
PlySK1249 & PlySs9 + [16] ± 60 [16] ± 2.50 [16] ± 0.50 [16] ND ND 15–65 [37] ± 2.00 [37] ND ND
PlyLambdaSA2 + [35] ND > 4.00 [35] ND ± 1.50 [35] + [35] ND ± 3.50 [35] ND ± 2.20 [35]
Ply700 ND + [32] < 1.00 [32] ND ND ND 20–90 [32] ND ND ND
PlyGBS & PlyB30 + [35] ND ± 0.50 [35] ND ± 2.10 [35] + [35] ND ± 1,50 [35] ND ± 0.00 [35]
PlySs2 (a.k.a. CF-301) + [16] ± 60 [16] ± 1.10 [16] > 5.00 [16] ND ND ND ± 2.70 [31] ND ND
Plyphi12 & PlyA72 ND ND ND ND ND ND ND ND ND ND
PlyPhi11 & PlyPhiH5 ND ND ND ND ND ND ND ND ND ND
PlyRODI & PlytrxSA-1 & PlyK ND ND ND ND ND − [89] ND ND ND ND
Engineered endolysins
ClyR ND 10–40 [55] ND ND ND + [55] 80–90 [55] > 4.00 [55] ND ND
ClyNC5 + [31] 80–90 [31] > 4.00 [31] ND > 4.00 [95] ND 70–80 [31] ± 1.70 [31] ND ND
ClyλSA2-PlyK-SH3 & ClyλSA2-Lyso-SH3 + [20] 10–20 [20] ND ND ND ND ND ND ND ND
ClyK-L(-PTD) & ClyL-K(-PTD) ND ND ND ND ND ND ND ND ND ND
Cly109 ND ND ND ND ND ND ND ND ND ND
ClyCHAPK_CWT-LST & ClyM23LST(L)_
SH3b2638 ND ND ND ND ND ND ND ND ND ND
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
A tabular overview of homologous wild-type and engineered endolysins characterized against bovine mastitis-causing pathogens, including Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae and
Staphylococcus aureus, which should be interpreted cautiously due to variations in testing conditions between the dierent works from which these results were derived, all of which are cited. Qualitative assays involve
spot-on-plate, spot-on-lawn, and zymogram assays; quantitative assays include turbidity reduction assay (TRA), time kill assay (TKA), and determination of minimal inhibitory or bactericidal concentration (MIC/MBC).
The invivo condition reects results obtained in the mouse mastitis model only. ‘ + ’ and ‘−’ denote positive and negative results for qualitativeassays, whereas ‘ ± ’ and ‘ND’ signify approximate and unknown results (not
determined), respectively
Table 1 (continued)
S. agalactiae S. aureus
Qualitative TRA
(% ΔOD600)TKA
(Δlog10 CFU/mL) MIC
(µM) In vivo
(Δlog10 CFU/g) Qualitative TRA
(% ΔOD600)TKA
(Δlog10 CFU/mL) MIC
(µM) In vivo
(Δlog10 CFU/g)
Homologous wild type endolysins
PlyC ND < 10 [55] ND ND ND ND ND ND ND ND
Ply0643 ND 60–80 [63] ND ND ± 3.30 [63] ND ND ND ND ND
PlySK1249 & PlySs9 ND 15–65 [37] ± 1.50 [37] ND ND ND < 10 [37] ND ND ND
PlyLambdaSA2 + [35] ND > 1.50 [35] ND ± 2.00 [35] − [28] ND ND ND ND
Ply700 ND ± 10 [32] ND ND ND ND < 10 [32] ND ND ND
PlyGBS & PlyB30 + [35] ND ± 2.00 [69] ND ± 4.50 [35] ND < 10 [69] ND ND ND
PlySs2 (a.k.a. CF-301) ND 20–40 [70] > 4.00 [70] 9.50 [70] ND + [31] 0–30 [70] > 4.00 [70] 0.59 [70] ND
Plyphi12 & PlyA72 ND ND ND ND ND ND ND ND 1.47 [41] ND
PlyPhi11 & PlyPhiH5 ND < 10 [84] ND ND ND ND 80–90 [36] > 4.00 [36] ND ND
PlyRODI & PlytrxSA-1 & PlyK − [89] ND ND ND ND + [89] ± 30 [29] > 4.00 [41] 0.57 [41] > 3.00 [41]
Engineered endolysins
ClyR ND 30–40 [55] < 3.00 [55] ND ND ND 5–30 [55] ND ND ND
ClyNC5 ND 40–50 [31] ± 1.50 [31] ND ND + [31] < 10 [31] < 0.50 [31] ND ND
ClyλSA2-PlyK-SH3 & ClyλSA2-Lyso-SH3 ND 10–20 [20] ND ND ND + [20] 80–90 [20] ± 1.50 [28] ND ± 3.30 [28]
ClyK-L(-PTD) & ClyL-K(-PTD) ND ND ND ND ND + [43] 40–70 [43] ND ≤ 0.2 [43] ≥ 3.40 [43]
Cly109 ND ND ND ND ND + [40] ND > 4.00 [40] ≤ 0.4 [40] ND
ClyCHAPK_CWT-LST & ClyM23LST(L)_
SH3b2638 ND ND ND ND ND ND ND ≤ 3.00 [38] < 1.0 [38] ND
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
wild-type and engineered endolysins in this work. Wild-
type endolysins will always be preceded by Ply (phage
endolysin), whereas engineered endolysins will be pre-
ceded with Cly (chimeric endolysin). is nomenclature
has been proposed and used by most research groups in
the USA and Asia [16, 27, 54–57].
Wild‑type endolysins evaluated againstbovine
mastitis‑causing streptococci
PlyC—discovered in1957 byRichard M. Krause (USA)
PlyC (a.k.a. C1 lysin) was discovered in the Streptococcus
dysgalactiae infecting C1 bacteriophage [58]. It is a
multimeric endolysin consisting of the two gene products
PlyCA and PlyCB that are linked by salt bridges and/
or Van der Waals forces [27, 59]. PlyCA serves as the
catalytic domain (containing a GyH and CHAP domain),
whereas PlyCB forms an octamer which binds the
streptococcal peptidoglycan. PlyC showed promising
in vitro activity against Streptococcus pyogenes by
eliminating 106CFU in 5s using only 10ng enzyme [60].
It was also found effective in mice to protect the oral
cavity from colonization by Streptococcus pyogenes [60].
Furthermore, PlyC could eradicate streptococci from the
oral cavity of mice within 2h if the oral mucosae were
infected a few days before with 107 CFU S. pyogenes.
Besides these observations made for S. pyogenes, activity
against Streptococcus uberis and S. dysgalactiae has also
been reported [42, 61]. e lysis speed was evaluated by
TRA for 250 U (units) enzyme and was in the range of
0.6–1.0 ΔOD600nm/min for S. pyogenes, whereas this was
reduced for S. dysgalactiae with an observed ΔOD600nm/
min of 0.1–0.2. In a follow-up toxicity study, bovine
PMN were isolated from blood samples of 12 healthy,
mid-lactation, primiparous cows and incubated with
increasing concentrations of maximally 50µg/mL PlyC
during up to 2h. PlyC was non-toxic for bovine blood
PMN as evaluated by unchanged lactate dehydrogenase
levels [42]. Furthermore, the PMN oxidative burst was
also not affected. Both in vitro observations indicate
that PlyC may not interfere with the function of PMN
during the inflammatory response in the bovine udder.
Another remarkable feature of PlyC is the ability to cross
the cell barrier of epithelial cells (i.e., shown for human
adenocarcinoma A549 cells) due to interactions of PlyCB
with phosphatidylserine [62]. PlyC can lyse S. pyogenes
inside these latter mentioned cells in a dose-dependent
manner. Of note, phosphatidylserine is usually only
present on the inside of the epithelial cell membrane,
but it is believed that these interactions can occur on
the outer cell membrane through a cellular recycling
mechanism.
Ply0643—discovered in2022 bytheHuochon group (China)
Ply0643 was discovered in the Streptococcus agalactiae
prophage S. a 04 [63]. It consists of a N-terminal
glucosaminidase, a C-terminal LysM CBD and a middle
position amidase_3, the latter two sharing 61% protein
sequence similarity with that of PlySK1249 and PlySs9
(Fig.4). is endolysin was found enzymatically effective
in a TRA by reducing the OD600nm of 20 bovine S.
agalactiae strains, as well as one strain of S. uberis and
S. dysgalactiae with 60 to 80% at 30µg/mL after 1h [63].
Moreover, the same concentration of Ply0643 reduced
the OD600nm of S. agalactiae in 1h by 80% at pH 6.0 and
7.0, which is consistent with the pH of raw milk. e
physiological pH of milk ranges from 6.5 to 6.7 and can
be > 7.0 in mastitic cows. Ply0643 was further evaluated
in vivo in a murine mouse model for S. agalactiae
mastitis and showed a Δlog10 of 2.4 × 102 CFU/g tissue
in the murine mammary glands after 24 h, as well as
reduced levels of interleukin (IL)-1β, IL-6 and mouse
IL-8 [63]. ese results are consistent with a reduced
immune cell influx evaluated on hematoxylin & eosin
(H&E) stained mammary gland sections. Treatment was
given intramammarily, but soon (i.e., 1h) post-infecting
the third and fourth gland pairs with 1.75 × 104 CFU S.
agalactiae. Glands on the left side received endolysin
treatment (i.e., 100µg/gland), whereas glands on the right
side received buffer as placebo treatment. e anatomical
relevance of using the third gland pair of mice as a model
for mastitis in dairy cows can be questioned, given their
axillary instead of inguinal position [50, 51].
PlySK1249 andPlySs9—discovered in2013 and2020
bytheResch (USA) andNelson (USA) groups, respectively.
PlySK1249 was discovered in the S. dysgalactiae
prophage SK1249 and contains an N-terminal amidase_3,
a middle LysM and a C-terminal CHAP [37]. It has
been shown that the amidase_3 and CHAP domains
synergize for peptidoglycan digestion and bacteriolysis
[64]. PlySK1249 was lytic for one S. dysgalactiae and four
S. agalactiae isolates in a TRA, as OD600nm reductions
were obtained in the range of 15 to 65% with 3.3U/mL
within 15min. Interestingly, S. uberis and Streptococcus
suis were also susceptible under the same conditions,
with 20% and 30% decreases in OD600nm, respectively.
Two Staphylococcus aureus strains were additionally
challenged, albeit found non-susceptible (i.e., ΔOD600nm
of < 10%). e optimal pH was found to be between pH
7 and 8.5, with a 50% decrease in OD600nm achieved
at these pH values in 15 min with 3.3 U/mL and S.
dysgalactiae SK1249 as the target. e bactericidal
activity was further evaluated in a kinetic TKA with
3.3 U/mL against one isolate of S. agalactiae and S.
dysgalactiae, which caused 1.5 and 2.0 log10 reductions
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
within 15min, respectively. Subsequently, the activity of
PlySK1249 was further investigated in a mouse model of
S. agalactiae bacteremia. e animals were challenged
intraperitoneally with 106CFU of S. agalactiae and 20%
survived within 72h, whereas this was increased by 60%
if the infected mice received daily injections of 45 mg/
kg PlySK1249. Another endolysin, named PlySs9, shares
96.5% protein sequence similarity with PlySK1249 and
was discovered in a prophage element from a S. suis
isolate belonging to serotype 9 (Fig.4) [16]. e activity
of the wild-type endolysin, as well as of the individual
domains, was evaluated against S. uberis and S. suis in
comparison with a second S. suis-derived endolysin
PlySs2. PlySs9 was found more potent than PlySs2 against
reference isolate S. uberis 0140J, as 0.5 µM PlySs9 in
comparison with an equimolar amount of PlySs2 showed:
(i) a slightly higher ΔOD600nm by TRA (i.e., 0.05 after
1h), one additional Δlog10 by TKA (i.e., 2.54 ± 0.08 vs.
1.09 ± 0.37 after 2h), and (iii) a lower MIC (i.e., 0.48 ± 0.16
vs. > 5µM). Five clinical and subclinical bovine mastitis-
derived S. uberis strains were similarly challenged by
TRA, which showed that all strains were susceptible to
both endolysins, but PlySs9 could again decrease the
OD600nm more. Molecular dissection of these endolysins
in EAD and CBD subdomains resulted in nearly complete
loss of killing and binding activity, respectively.
PlyλSA2—discovered in2007 bytheBaker group (USA)
PlyλSA2 has been discovered in the S. agalactiae
prophage LambdaSA2 [65]. PlyλSA2 consists of an
N-terminal amidase_5, a middle CW_7 and a C-terminal
glucosaminidase. e activity of PlyλSA2 has been
comparatively characterized with the non-homologous
PlyB30 (Fig.4) [35]. Both endolysins were qualitatively
evaluated by spot-on-lawn, in which 0.12 ± 0.01 and
0.26 ± 0.01 µg caused a halo against S. dysgalactiae and
S. uberis, respectively. An approximate tenfold dose
was needed for PlyB30 to observe a similar halo against
these strains, which made the authors conclude that
PlyλSA2 was the most potent endolysin. S. agalactiae
was challenged in similar fashion with both endolysins
but was found less susceptible, given that 1.72 ± 0.58
and 4.69 ± 1.12 µg were needed to observe halos. e
optimal pH was found to be consistent with that of
mastitic and healthy raw cow’s milk, i.e. in the range
of 7.0 to 7.5 and around 6.5 for PlyλSA2 and PlyB30,
respectively. Increasing concentrations of Ca2+ (i.e.,
0.1, 1.0 & 10.0mM) inhibited the activity of PlyλSA2 as
was evaluated by TRA against S. dysgalactiae, whereas
that of PlyB30 was enhanced. More specifically, a ± 50%
reduction in residual activity was observed at 10 mM
Ca2+ for 25µg/mL PlyλSA2. In contrast, this was a ± 50%
increase at 1mM Ca2+ for 100µg/mL PlyB30. Of note,
the most abundant divalent cation in cow milk is calcium
with a concentration of approximately 3.0mM unbound
or ‘free’ Ca2+. A kinetic TKA was performed in UHT-
milk during 3h and 100µg/mL of both endolysins, which
caused log10 reductions of all three streptococcal species
at the end of the assay, although the reduced activity of
PlyB30 vs. PlyλSA2 was again observed. CFU levels were
increased by 1.5 to 2.0 log10 after 3h in these UHT-milk
conditions in the wells that received no treatment (the
negative control). At an inoculum dose of 103CFU/mL,
PlyλSA2 and PlyB30 caused approximately 3.5 and 2.5
Δlog10 at a concentration of 100µg/mL of S. dysgalactiae,
respectively. is was > 4.0 and > 1.5 for PlyλSA2, and,
approximately 2.0 and 0.5 for PlyB30 against S. uberis
and S. agalactiae, respectively. us, the reduced activity
that was observed against S. agalactiae in the spot-
on-lawn assay was likewise visible in the kinetic TKA.
Given that both endolysins attack different bonds in the
streptococcal peptidoglycan, their potential synergy was
further investigated. PlyλSA2 and PlyB30 showed synergy
(i.e., Σ 0.42 ± 0.09) against S. dysgalactiae in an invitro
checkerboard-based assay. Σ represents the fractional
inhibitory concentration index value, which indicates
synergy if Σ < 0.50. Synergy of both endolysins against the
S. uberis and S. agalactiae strains was not determined.
Both endolysins were subsequently tested individually
and as a potentially synergistic combination in a mouse
mastitis model side-by-side against all three streptococcal
pathogens. e doses used per gland consisted of 25
and 250 µg for either PlyλSA2 or PlyB30, respectively,
whereas the synergistic combination consisted of 12.5
and 125µg PlyλSA2 and PlyB30. It should be noted that
intramammary therapy was given as soon as 45 min
after intraductally inoculating only 102CFU pathogenic
bacteria in the murine mammary glands. For all three
streptococcal species, bacterial concentrations in the
mammary glands were significantly reduced 24 h after
bacterial challenge in the range of 1.5 to 4.5 Δlog10,
except for PlyB30 against S. dysgalactiae that showed no
log10 reduction. Indeed, the latter contrasted the invitro
results of PlyB30 for S. dysgalactiae, which thus could not
be retrieved in vivo. Furthermore, PlyB30 unexpectedly
caused higher log10 reductions for S. agalactiae than for
S. uberis, of approximately 4.5 vs. 2.1 Δlog10, respectively.
e most surprising finding, however, was that the
synergistic action of both lysins was lost in the murine
mammary gland.
Ply700—discovered in2008 bytheKerr group (USA)
Ply700 was discovered after inducing a prophage in
the genome of S. uberis ATCC 700407 with mitomycin
C [32]. It consists of an N-terminal amidase_5 and
C-terminal SH3_4, of which the latter has approximately
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
70% protein sequence similarity with the CBD of PlyGBS
and PlyB30 (Fig. 4). is wild-type endolysin has an
optimal activity against S. uberis at a pH of 6.0 to 7.0,
which is consistent with that of raw cow’s milk. At these
pH values, a 40% decrease in turbidity was observed by
TRA with 20 µg/mL enzyme in 25 min. In addition, it
was also described that a 10mM Ca2+ concentration is
required to exploit its maximal activity. As observed by
TRA using the same endolysin concentration, Ply700
caused a ± 60% decrease in turbidity of S. uberis in the
presence of 10 mM Ca2+, whereas this was only ± 25%
at a tenfold lower Ca2+ concentration. Ply700 was found
effective by TRA against one S. dysgalactiae and ten
S. uberis strains, in comparison with one S. agalactiae
strain that only showed minor activity. More specifically,
the ΔOD600nm was in the range of 20 to 90% for S. uberis
and S. dysgalactiae, whereas this was only ± 10% for
the S. agalactiae isolate, all evaluated with 50 µg/mL
enzyme during 30 min. e authors also determined
the activity of Ply700 against S. uberis in UHT whole
milk with a non-kinetic TKA. ey found that spiking
the milk with 50 µg/mL Ply700 could kill 30% of a S.
uberis strain within 15 min, if 4.5 × 103 CFU/mL were
added. Additionally, the C-terminal SH3_4 domain was
fused to GFP and binding to S. uberis was investigated
in the presence and absence of Ca2+. is revealed that
binding of the SH3_4 domain was calcium-dependent,
as addition of 10.0 mM Ca2+ increased the binding by
approximately 4 × in comparison to the condition where
Ca2+ was not supplemented. e latter phenomenon is
similarly reported for PlyB30 [66].
PlyB30 andPlyGBS—discovered in2004 and2005
bytheBaker (USA) andFischetti (USA) groups, respectively
PlyB30 has been derived from the virulent S. agalactiae
phage B30 [66]. It consists of an N-terminal CHAP, a
central muramidase and a C-terminal SH3_5 domain,
of which the former has been characterized as the
major contributor of the lytic activity observed with
S. agalactiae [67]. e activity of PlyB30 has been
comparatively characterized with the non-homologous
PlyλSA2, as already discussed. A homologous endolysin,
PlyGBS, has likewise been discovered in the virulent S.
agalactiae phage NCTC 11261 [68]. PlyGBS dropped
the OD600nm in a TRA to the baseline within 10min at
40 U of a S. agalactiae isolate. is rapid rate of cell lysis
led to a ± 2.0 log10 decrease of the same S. agalactiae
isolate, observed in a subsequent TKA. Furthermore, the
lytic spectrum of PlyGBS was evaluated at 15 U during
60min by TRA against six S. agalactiae isolates, as well
as one isolate of S. dysgalactiae and S. aureus. Like the
first observation, PlyGBS lysed all tested S. agalactiae
serotypes and the S. dysgalactiae isolate invitro, but not
S. aureus. To further evaluate the potency of PlyGBS
in vivo, mice were challenged vaginally with 106 CFU
S. agalactiae [68]. After 24 h, mice received vaginal
treatment with either endolysin buffer or 10 U PlyGBS
and were swabbed 2 and 4h post-treatment. e treated
animals showed a Δlog10 of 3.0 at both the 2 and 4h time
intervals compared to the buffer control. In addition,
mice were also challenged orally and nasally with
108CFU S. agalactiae to determine if PlyGBS can be used
to reduce colonization of the upper respiratory tract.
Mice treated with 10 U PlyGBS exhibited a reduction
in S. agalactiae colonization of approximately 0.5 and
1.5 Δlog10 at both the 2 and 24 h swabbing intervals,
in comparison with the buffer control. In a follow-up
study, DNA mutagenesis methods were explored to
produce hyperactive PlyGBS mutants with 18- to 28-fold
increased lytic activity against S. agalactiae, evaluated by
TRA [69]. Using the same mouse vaginal model, one of
these hyperactive mutants (i.e., PlyGBS90-1) reduced S.
agalactiae with ± 4.0 Δlog10 at 4h post-treatment using a
single dose of only 30nmol.
Wild‑type endolysins evaluated againstbovine
mastitis‑causing staphylococci
PlySs2—discovered in2013 bytheFischetti group (USA)
PlySs2, a.k.a. CF-301 or Exebacase, is by far the best
characterized Gram-positive endolysin with in vivo
confirmed broad lytic activity against a large variety of
both streptococci and staphylococci [16, 70–79]. It was
considered a lead therapeutic candidate in the endolysin
field, evaluated in clinical trials by ContraFect. is
company aimed a therapeutic application in humans
suffering from sepsis and right-heart endocarditis caused
by methicillin-resistant S. aureus (MRSA), as well as
chronic prosthetic joint infections of the knee [73].
However, the company recently filed for bankruptcy.
Nevertheless, PlySs2 reached breakthrough therapy in
phase III clinical trials for the former application that
were discontinued due to a lack of statistical power [74].
ContraFect stated this was possibly due to an imbalance
in the baseline disease severity of patients in the
PlySs2 vs. placebo group, with more patients having an
extremely poor prognosis (i.e., APACHE II score above
15) and increased risk of mortality in the PlySs2 treated
group. Of note, PlySs2 was only administered to patients
as an add-on to the currently used standard-of-care
antibiotics [74].
PlySs2 was discovered in a prophage element of a S.
suis isolate belonging to serotype 2 [16, 75]. Multiple
studies report that relatively low concentrations of
PlySs2 can efficiently lyse S. aureus, S. agalactiae, S.
dysgalactiae and S. uberis below detection limits in
different kinds of qualitative and quantitative assays,
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
including TRAs, TKAs, MIC/MBCs and in vivo studies
[16, 70–73]. is endolysin consists of an N-terminal
CHAP and a C-terminal SH3_5. Two studies performed
a molecular dissection of PlySs2 to study the EAD and
CBD separately [15, 16]. e EAD alone lost its catalytic
activity completely, as was observed by combining TRAs
and TKAs. is indicates the necessity for the SH3_5
domain to be present to exploit the full functionality of
PlySs2. On the other hand, the SH3_5 domain was fused
to either GFP or conjugated with Alexa Fluor 555 to
study binding to several streptococcal and staphylococcal
isolates. It was qualitatively shown by fluorescence
microscopy that the SH3_5 domain can bind S.
agalactiae, S. dysgalactiae and S. suis, but remarkably not
S. uberis and S. aureus. e authors state that this is not a
surprising finding, but rather underscores the importance
of the EAD to be present for full binding functionality of
PlySs2 [16]. For completion, PlySs2 SH3_5 has also been
attributed several eukaryotic cell penetrating properties
[76]. To proceed, PlySs2 has been characterized with
potent biofilm eradicating activity [77, 78]. Minimum
biofilm-eradicating concentration (MBEC) assays on
95 S. aureus strains revealed a 90% MBEC (MBEC90)
value of ≤ 0.25 μg/mL. is was evaluated on biofilms
formed on polystyrene, glass, surgical mesh, catheters
and in human synovial fluid. Of note, one Streptococcus
agalactiaebiofilm was also found sensitive to disruption,
with an MBEC90 value of 0.50 μg/mL. PlySs2 also
exhibited substantially increased potency (32 to ≥ 100-
fold) in human blood vs. laboratory testing media in
three complementary microbiological testing formats
(i.e., MICs, TKAs and checkerboard synergy) [79].
More specifically, PlySs2 acts synergistically with two
key human blood factors: serum lysozyme and albumin.
For lysozyme, it is reasoned that the synergistic effect is
obtained through initial disruption of the staphylococcal
peptidoglycan which makes it sensitive to the subsequent
action of lysozyme, whereas the synergistic action of
albumin occurs through high affinity. Indeed, both
S. aureus and PlySs2 have high affinity for albumin,
causing them to colocalize in blood. e latter might be
interesting from a bovine mastitis point of view, as raw
milk contains lactalbumin which might exert a similar
synergistic effect.
PlyPhi12 andPlyA72—described in2004 and2021
bytheBierbaum (Germany) andGarcia (Spain) groups,
respectively
PlyA72 was discovered in the virulent S. aureus
phiIPLA35 phage and shares approximately 98.0%
protein sequence similarity to PlyPhi12 (Fig.4) [80]. Both
endolysins consist of an N-terminal CHAP, a middle
amidase_3 and a C-terminal SH3_5. Although PlyPhi12
was bio-informatically discovered 17 years before
PlyA72, it was not further characterized because the
endolysin auto-aggregated in the assays performed [80].
us, antibacterial assays were only performed later for
the homologous endolysin PlyA72 in comparison with
PlyRODI [41]. PlyA72 had inferior staphylococcal activity
compared to the latter endolysin. More specifically,
LysRODI displayed lower MIC values in comparison
with PlyA72 of 0.57 vs. 1.47 µM, respectively, against
two bovine mastitis-derived S. aureus strains. e
biofilm eradicating potential of PlyA72 was also reduced
if compared to PlyRODI, as 7 µM of these endolysins
reduced 17% and 94% of a S. aureus biofilm in 5 and 1h,
respectively.
PlyPhi11 andPlyPhiH5—discovered in2006 and2008
bytheDonovan (USA) andGarcia (Spain) groups,
respectively
PlyPhi11 was discovered in the virulent phage phiIPLA88
infecting S. aureus [36]. It consists of an N-terminal
CHAP, a middle amidase_2 and C-terminal SH3_5.
It was shown that the middle amidase_2 has minor
contributions to PlyPhi11’s activity, as its deletion did not
result in loss of activity against S. aureus in a TRA [81].
e same has been reported for PlyK, which has an equal
molecular architecture [82]. In fact, it has been shown
that the amidase_2 or _3 in staphylococcal wild-type
endolysins has an auxiliary role and mainly possesses
a binding function [83]. A PlyPhi11 dose of 25.0 µg
was able to lyse mastitis-causing staphylococci in a 1 h
pending TRA, such as one S. aureus, one S. chromogenes
and one Staphylococcus simulans isolate [84]. e
activity was similarly tested against one mastitis-causing
S. agalactiae isolate, but no activity was observed. e
authors therefore claimed PlyPhi11 to be a staphylococcal
endolysin. e activity of PlyPhi11 was evaluated by TRA
under the same conditions at the pH (i.e., 6.7) and Ca2+
concentration (i.e., 3mM) of milk, at which peak activity
of PlyPhi11 was observed. No subsequent assays in UHT-
milk or raw milk were conducted. However, another lysin
named PlyPhiH5 that shares > 97.0% protein sequence
similarity to PlyPhi11 (Fig. 4) has been reported to
reduce S. aureus in UHT whole milk from 106CFU/mL
to undetectable levels after 4h starting at a concentration
of 1.6 µM [36]. Similarly to PlyPhi11, the lytic activity
of PlyPhiH5 was found optimal around the pH of milk
and no activity was observed against streptococci, all
evaluated by TRA using 15 U/mL enzyme. Interestingly,
the authors reported increased activity of PlyPhi11 on
bovine mastitis S. aureus isolates in comparison with
those from human origin, more specifically a specific
activity of 11.3 ± 1.7 vs. 7.5 ± 2.9, respe ctively.
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
PlyK, PlytrxSA‑1 andPlyRODI—discovered in2005, 2016
and2021 bytheRoss (Ireland), Jingyun (China) andGarcia
(Spain) groups, respectively
PlyK, PlytrxSA-1 and PlyRODI were discovered in
the virulent S. aureus phages K [85–88], IME-SA1
[89] and phiIPLA-RODI [90, 91], respectively. ese
endolysins consist of an N-terminal CHAP, a middle
amidase_2 and a C-terminal SH3_5. PlyK is one of
the earliest discovered endolysins with confirmed
qualitative activity against three bovine mastitis S.
aureus in a spot-on-plate assay [29]. PlytrxSA-1 has
likewise been evaluated in a spot-on-lawn assay at 6µg/
µL against 100 S. aureus isolates, of which 43 showed
a halo. is included bovine mastitis-derived strains.
Also, one isolate of bovine mastitis S. agalactiae and S.
dysgalactiae were similarly challenged, but no activity
was observed [89]. Interestingly, PlytrxSA-1 has been
used to treat three bovine udder quarters infected
with S. aureus (i.e., milk with a positive culture for S.
aureus and somatic cell count (SCC) > 500,000 cells/
mL). Each udder quarter received a daily inoculation of
20.0mg PlytrxSA-1 during three consecutive days. By
day 3, S. aureus CFU counts as well as milk SCC were
reduced to the limit of detection and milk consistency
appeared back to normal. Unfortunately, this study
did not include an appropriate negative control (i.e.,
untreated S. aureus infected udder quarters). e
latter experimental design makes it hard to state
that the observed clinical improvement is (at least
partially) due to the PlytrxSA-1 treatment. PlyRODI
has been discovered and comparatively studied with
PlyA72 [41]. Both endolysins showed optimal activity
in a TRA against a bovine S. aureus isolate at the pH
and calcium concentration of milk and their lytic
activity increased by a twofold at higher calcium
concentrations, in line with PlyK for PlyRODI [92].
Both endolysins were further compared in a MIC assay
against two bovine mastitis-derived S. aureus isolates,
in which PlyRODI yielded a lower MIC value (0.57µM)
than PlyA72 (1.47 µM). e results corroborated a
subsequent 1 h pending kinetic TKA, in which both
enzymes were challenged at 0.1 µM against a bovine
mastitis-derived S. aureus isolate. PlyRODI eradicated
107CFU/mL within 1h, in contrast to PlyA72 which
killed 103CFU/mL. Moreover, PlyRODI could remove
94% of an S. aureus biofilm within 1h, compared to
17% after 5h for PlyA72. erefore, only PlyRODI was
selected for further invivo preclinical characterization
(i.e., in a zebrafish and mouse mastitis model). In the
zebrafish model, PlyRODI was found to be non-toxic
and 1.0µM increased the survival rate with 44.4% at
72 h after intraperitoneal infection with 105 CFU S.
aureus. In the mouse model for bovine mastitis, murine
mammary glands were pre-treated with 24µg PlyRODI
and then challenged with 104CFU S. aureus. A short
time interval of 1.5h was used between the preventive
treatment and the S. aureus challenge. Mice were
euthanatized at 18h post-S. aureus challenge to harvest
the mammary glands. is pre-treatment prevented
the occurrence of mastitis lesions and reduced the
outgrowth of S. aureus by 103–4 Δlog10. ese data show
the potential of PlyRODI as a preventive treatment for
S. aureus mastitis.
Engineered endolysins evaluated againstbovine
mastitis‑causing streptococci
ClyR—engineered in2015 bytheNelson (USA) andHongping
Wei (China) groups
ClyR was selected from a library consisting of 21
engineered endolysins (i.e., constructed out of seven
EADs and three CBDs) against a bovine mastitis S.
dysgalactiae isolate [55]. is engineered endolysin
combines the CHAP of PlyC with the SH3_5 of PlySs2.
It caused a ΔOD600nm of ± 0.8 after 30 min against
one S. dysgalactiae isolate at 25 µg/mL in a TRA,
which corresponded to a 4.0 log10 reduction in a TKA.
Moreover, the same concentration of ClyR also showed
activity in a TRA against seven S. uberis, six S. agalactiae
and two additional S. dysgalactiae isolates with a
ΔOD600nm in the range of 0.1–0.4, 0.3–0.6 and around
0.5, respectively, after 20 min using a dose of 25 µg/
mL. Moreover, this activity was found to be calcium
independent as there was no reduction observed with
the addition of 50mM ethylenediaminetetraacetic acid
(EDTA). ClyR’s activity was also evaluated against S.
agalactiae and S. dysgalactiae in pasteurized raw cow’s
milk from both healthy and mastitic cows at 40µg/mL,
causing Δlog10 between 1.0 and 3.0 after 1h. Interestingly,
this activity was found to show a 2.0-log10 fold increase
in the mastitic vs. healthy pasteurized milk. e latter
is most likely caused by a pH-related effect, as ClyR
displayed its highest lytic activity around pH 8.0. When
compared to the wild-type endolysin PlyC in a TRA, the
activity of ClyR was found to be slightly increased against
S. dysgalactiae (i.e., additional ΔOD600nm of ± 0.05), but
greatly increased against one S. agalactiae isolate (i.e.,
additional ΔOD600nm of ± 0.3). In addition, a broader lytic
spectrum was present because several staphylococcal
species such as S. aureus could be lysed, in contrast to
PlyC (i.e., additional ΔOD600nm in the range of ± 0.15–
0.35 for 3 S. aureus isolates). It has also been shown that
ClyR is able to tackle streptococci intracellularly, due to
certain cell penetrating properties of PlySs2 SH3_5 [76].
e intracellular uptake of ClyR is believed to happen
through caveolin-dependent endocytosis and was shown
by confocal laser microscopy, after exposing human
Page 15 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
adenocarcinoma A549 cells with 100µg/mL Alexa Fluor
488-labeled ClyR during 40min.
ClyNC5—engineered in2023 bytheBriers, Lavigne
andMeyer (all Belgium) groups
e engineered endolysin ClyNC5 was selected from
an extensive library comprising over 80,000 theoreti-
cal endolysin variants [31]. is library was crafted
through the high-throughput DNA assembly platform
known as VersaTile [93, 94]. e library underwent a
systematic screening process by employing a halo-based
assay, wherein bacteriolytic activity was serially assessed
through overexpression in individual E. coli colonies
on agars embedded with either the reference isolate S.
aureus N305 or S. uberis 0140J. is innovative approach
not only facilitated the identification of design rules, but
also involved statistical analyses for the robust validation
thereof. It was found that CPPs are ideally fused to the
N-terminal site, and the engineered construct preferably
has a pI in the range 9.05 to 9.65. e standout candidate
arising from this screening process was named ClyNC5
and consisted of (from N- to C-terminus) the trans-acti-
vator of transcription (TAT) peptide of human immu-
nodeficiency virus (HIV)-1 as CPP, the PlySs2 CHAP, a
repeated CW_7 as CBD, and the PlySs9 amidase. ClyNC5
was further evaluated against Gram-positive isolates
sourced from (sub)clinically affected cows and had an
impressive killing efficacy of 4.05 ± 0.07 Δlog10 against S.
uberis at 0.3µM and demonstrated comparable activity
of 1.50 ± 0.02 and 1.77 ± 0.43 Δlog10 against S. agalactiae
and S. dysgalactiae, respectively. In addition, ClyNC5
effectively eradicated approximately 70% of a S. uberis
biofilm at 1.5 µM, corresponding to 1.17 ± 0.39 Δlog10,
and displayed intracellular activity at 2.5 µM within
MAC-T and PS of 1.62 ± 0.05 and 2.12 ± 0.08 Δlog10,
respectively. e intracellular presence of ClyNC5 was
verified in MAC-T by confocal microscopy. Furthermore,
the lead candidate potentiated cloxacillin in raw cow’s
mastitic milk at 0.5µM, which is a beta-lactam penicillin
commonly employed for the intramammary treatment
ofGram-positive bovine mastitis.
To validate ClyNC5’s therapeutic potential as a
supplemental therapy to cloxacillin, a preclinical study
was conducted using a mouse model for S. uberis mastitis
[95]. is study involved intramammary infection with a
bovine S. uberis field isolate of the pathotype GCC ST-5,
followed by treatment with cloxacillin combined with
varying doses of ClyNC5 (i.e., 23.5 and 235.0 µg) that
was administered 12 h post-infection. e hallmarks
of mastitis were evaluated mid-treatment, being 16 h
post-infection. e results unveiled distinct responder
profiles, categorizing mice as either fast (n = 17) or slow
(n = 10) responders. In the fast responders, the high-dose
combination therapy (i.e., 235.0 µg ClyNC5 + 30.0 µg
cloxacillin): (i) reduced the bacterial load by 13,000-
fold, (ii) mitigated the intramammary neutrophil influx,
and (iii) reduced the pro-inflammatory chemokine
IL-8 13-fold. Furthermore, the intramammary immune
profile was further complemented by the evaluation of
other pro-inflammatory cytokines, chemokines, growth
factors and metabolites (i.e., TNF-α, MCP-1, M- &
G-CSF as well as IL-1α, -1β, & -6). is similarly revealed
an overall dose-dependent reduction of the evaluated
markers caused by the supplementation of endolysin
ClyNC5 to cloxacillin. Together, both studies provide
compelling evidence of ClyNC5’s efficacy as an adjunct to
intramammary cloxacillin treatment.
Engineered endolysins evaluated againstbovine
mastitis‑causing staphylococci
ClylSA2‑PlyK‑SH3 & ClylSA2‑Lyso‑SH3—Engineered in2012
bytheDonovan group (USA)
ClylSA2-LysK-SH3 and ClylSA2-Lyso-SH3 are two
rationally created engineered endolysins that com-
bine the N-terminal streptococcal amidase_5 domain
of PlyλSA2 with the C-terminal staphylococcal SH3
domains of either the wild-type endolysin PlyK or lys-
ostaphin, respectively [28]. Of note, lysostaphin is not an
endolysin but a bacteriocin. Both engineered endolysins
showed qualitative lytic activity in a spot-on-lawn assay
against 16 bovine mastitis S. aureus isolates, includ-
ing the bovine mastitis reference strain S. aureus N305.
e activity of both engineered endolysins has also been
evaluated comparatively between S. aureus N305 and
one isolate of S. uberis and S. agalactiae, albeit only bio-
chemically in a spot-on-plate assay and via TRA [20].
A concentration of 100µg/mL had lytic activity against
all strains, although the relative specific activity was at
least fivefold reduced against both streptococcal iso-
lates. A kinetic TKA was also performed in UHT-milk
and 100 µg/mL of both engineered endolysins showed
a reduction of the 106CFU/mL S. aureus spiked UHT-
milk with a Δlog10 of 1.0 to 1.5 after only 1h. is Δlog10
continued to increase for ClylSA2-Lyso-SH3 till 3 h,
resulting in a 3.0 Δlog10 [28]. Compensatory growth was
observed after 1h for ClylSA2-LysK-SH3, which caused
log10 values to increase to 6.5 resulting in a difference of
only 1.0 Δlog10 compared to the negative control. Subse-
quently, both engineered endolysins were tested invivo
in a murine model of bovine S. aureus mastitis using
the N305 strain and compared with the natural bacte-
riocin lysostaphin as a positive control. Treatment with
25µg/gland was given either only 30min or 6h after the
inoculation of 104 and 102CFU S. aureus, respectively.
e outcome was, however, found to be independent of
these variables. More specifically, ClylSA2-LysK-SH3 and
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
ClylSA2-Lyso-SH3 caused a Δlog10 of 0.63 and 0.81 at
24h after the invivo challenge, respectively. Both engi-
neered endolysins had poor lytic activity in comparison
with lysostaphin, which caused a 2.82 Δlog10 at 24 h.
Given the promising latter result, the authors determined
invitro synergy of lysostaphin in a checkerboard-based
assay with both engineered endolysins and found it to
be strong (i.e., Σ of 0.46 ± 0.07 and 0.42 ± 0.07 for lys-
ostaphin with ClylSA2-LysK-SH3 and ClylSA2-Lyso-SH3,
respectively). Furthermore, the lysostaphin/ClylSA2-
LysK-SH3 combination caused a Δlog10 of 3.36 in the
murine S. aureus mastitis model, which was increased
compared to lysostaphin and ClylSA2-LysK-SH3 sepa-
rately (i.e., Δlog10 of 2.14 and 0.86, respectively). It should
be noted that both experiments cannot directly be com-
pared, as the authors changed some of the parameters
of the latter invivo experiment. Indeed, the inoculation
dose (103CFU), the treatment dose (1:2 diluted) and the
endpoint of the experiment (18h) were altered. Tumor
necrosis factor (TNF)-α concentrations were also deter-
mined on mammary gland lysates, which followed a
decreasing trend correlating with the number of CFU/
mL.
Triple acting enzymes ClyK‑L(‑PTD) & ClyL‑K(‑PTD)—
engineered in2015 bytheDonovan group (USA)
ClyK-L was engineered by altering the M23 EAD of the
bacteriocin lysostaphin by the CHAP and amidase_2
EADs of the wild-type endolysin PlyK, whereas in the
ClyL-K variant these latter mentioned EADs were inte-
grated in between the M23 and SH3b domains of lys-
ostaphin [43]. Both rationally engineered endolysins
showed similar biochemical activity in a TRA against
one S. aureus isolate, but this was increased with ± 0.2
ΔOD600nm if compared to lysostaphin and PlyK. How-
ever, an increased MIC-value was sometimes observed
for both variants in comparison with one or both paren-
tal enzymes, illustrating again the low or inexistent cor-
relation between both assays. For example, lysostaphin
yielded a MIC of 0.77 µg/mL against S. aureus New-
man, whereas this was ≥ 7.0µg/mL for both engineered
endolysins. In addition, ClyK-L was also tested against
a biofilm-forming MRSA strain at 100 µg/mL, which
reduced the intra-biofilm viability with 24%. ClyK-L and
ClyL-K were then further engineered by adding sev-
eral C-terminal protein transduction domains (PTDs),
a.k.a. cell penetrating peptides (CPPs), resulting in
ClyK-L-PTD & ClyL-K-PTD variants. Subsequently,
their intracellular eradicating capacity was evaluated in
MAC-T against S. aureus ISP479C at 25.0µg/mL during
2.5h. Surprisingly, this revealed that addition of a PTD
decreased the intracellular killing capacity of the ClyL-
K-PTD variant, whereas that of ClyK-L-PTD was not
improved. However, the addition of a PTD could reduce
the intra-biofilm viability by an additional 16%. One of
the ClyK-L-PTD variants was shown to colocalize with
S. aureus intracellularly as was observed by confocal
microscopy. One ClyK-L-PTD variant was selected for
invivo validation in a mouse model for bovine S. aureus
mastitis, in which mice were intramammarily infected
with 100CFU S. aureus and side-by-side treated 30min
thereafter with 25.0 µM of either lysostaphin, ClyK-L
or the ClyK-L-PTD variant. Up to six glands per animal
were used and animals were euthanized 18h post-treat-
ment to harvest the mammary glands. e authors found
that the ClyK-L-PTD variant, which previously showed
intra-biofilm killing activity, caused an additional killing
of 2.7 log10 in comparison with ClyK-L. Of note, this lat-
ter mentioned non-PTD-containing variant only caused a
Δlog10 of 0.7 in comparison with the phosphate buffered
saline (PBS) negative control. e ClyK-L-PTD variant
did not perform better than lysostaphin and even had a
reduced killing of 0.5 log10 in comparison with the latter.
TNF-α levels were also determined in mammary gland
lysates and followed a correlating trend with the CFUs at
18h post-treatment.
Cly109—engineered in2020 bytheSangryeol group (South
Korea)
Cly109 was hit-to-lead selected from 480 theoretical
combinations after high-throughput assembly of various
S. aureus EADs and CBDs, which were subsequently
screened for lytic activity against S. aureus, including one
bovine mastitis-derived isolate [40]. Cly109 combines
the N-terminal CHAP of PlySA12 with the middle
amidase_3 and C-terminal CBD of PlySA97 [83, 96].
PlySA12 bears > 96.0% protein sequence similarity to the
previously described wild-type endolysins PlyPhi11 and
PlyPhiH5. is engineered endolysin was found to be
superior to both parental endolysins against S. aureus
if tested at an equimolar concentration of 300 nM,
showing a: (i) > twofold increase in activity evaluated
by TRA, (ii) > twofold lower MIC value of 0.37 nM,
and (iii) > threefold efficacy in eradicating S. aureus
biofilm. It had an optimal activity at the pH of milk
and a concentration of 0.9µM eliminated 105CFU/mL
from UHT-milk in only 45min. e authors reasoned
that altering the wild-type CBD from PlySA12 with the
amidase_3 + CBD from PlySA97 through their domain
swapping method could increase the binding capabilities
of the PlySA12 CHAP. Indeed, it has been proposed for
amidases in staphylococcal endolysins that these mainly
constitute a binding function [83].
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VanderElst Acta Veterinaria Scandinavica (2024) 66:20
ClyCHAPK_CWT‑LST & ClyM23LST(L)_SH3b2638—
engineered in2018 and2022 bytheLoessner group
(Switzerland), respectively
ClyCHAPK_CWT-LST was discovered by high-
throughput assembling approximately 170 endolysins
from staphylococcal EADs and CBDs, followed by hit-
to-lead selection of the most promising endolysins
with activity against S. aureus N305 in UHT-milk
[38]. e latter was done by challenging 103 CFU/mL
S. aureus N305-spiked UHT-milk with E. coli lysates
that contained the expressed endolysins. e selected
top candidate (i.e., ClyCHAPK_CWT-LST) combines
the CHAP domain of PlyK with a SH3b CBD of which
the origin is undefined. Interestingly, the activity of the
selected engineered endolysin was equal to that of the
positive control lysostaphin. Both were able to eradicate
103 CFU/mL S. aureus N305 from UHT whole milk
to undetectable levels in 3 h. erefore, ClyCHAPK_
CWT-LST was combined with lysostaphin to exploit
synergy that was observed in broth and UHT whole
milk (i.e., Σ of 0.46 ± 0.04 and 0.38 ± 0.03, respectively).
Different bovine mastitis-derived staphylococcal
isolates, including 23 S. aureus isolates and one isolate of
Staphylococcus chromogenes and S. simulans, were found
susceptible with MBC values < 1.0 µM to ClyCHAPK_
CWT-LST, lysostaphin and the synergistic combination.
An exception to the latter was S. chromogenes, which
yielded a MBC > 2.5 µM of the individual enzymes,
but not for the synergistic combination that still had
an MBC < 1.0 µM. To investigate the potential of
ClyCHAPK_CWT-LST and the synergistic combination
with lysostaphin, a kinetic TKA was performed in
UHT-milk with 103 and 106 CFU/mL S. aureus N305.
After 3 h, lysostaphin and ClyCHAPK_CWT-LST
reduced bacterial concentrations by 2.64 and 2.37
log10 compared to the 103 CFU/mL control at ± 1.0 µM,
respectively. eir synergistic combination reduced
the CFU count by 4.74 log10 after 3h below the limit of
detection in comparison with the 106CFU/mL control.
However, when repeating the same assay in raw cow’s
milk, ClyCHAPK_CWT-LST completely lost its activity
which was retrieved by diluting the raw milk, clearly
showing that raw milk components inhibit the activity
of ClyCHAPK_CWT-LST. e authors repeated the
screening a couple of years later but now used raw cow’s
milk instead for UHT-milk and a novel engineered
enzyme, named ClyM23LST(L)_SH3b2638, was hit-
to-lead selected for staphylolytic activity [39]. is
enzyme consists of the M23 EAD of lysostaphin and
the C-terminal CBD of the staphylococcal phage 2638A
endolysin [97]. eir selected ClyM23LST(L)_SH3b2638
had, however, still a few relevant limitations: (i) it did not
perform better than the natural bacteriocin lysostaphin
in raw cow’s milk (i.e., at least a twofold increase of the
MBC against S. aureus N305 was present), (ii) it did
not show synergy in UHT-milk with other endolysins
that target different bonds in the staphylococcal
peptidoglycan, and (iii) resistance against the M23 EAD
of lysostaphin was mentioned. e reason for the latter is
that lysostaphin’s M23 EAD hydrolyses the pentaglycine
bridge in the staphylococcal peptidoglycan, which can
be altered [98]. Interestingly, ClyM23LST(L)_SH3b2638
was further rationally engineered by fusion of the TAT
peptide of HIV-1 to the C-terminus, which increased
the construct’s capacity to kill S. aureus intracellularly in
MAC-T. Cells treated with non-TAT engineered enzymes
had approximately 105 CFU/mL intracellular S. aureus
N305 (i.e., approximately 5CFU/cell), whereas this was
reduced in boMECs that were treated with 1.0µM of the
TAT engineered variant during 3h (i.e., ± 1.0 additional
Δlog10). Noteworthy, the TAT fusion to ClyM23LST(L)_
SH3b2638 decreased the antibacterial activity by ± 1.0
log10, observed with S. aureus N305 in buffer and diluted
raw cow’s milk. is has also been reported for ClyK-L-
PTD in comparison with ClyK-L, of which the former
also contains the C-terminal TAT [43].
Overall discussion andconclusion
Gram-positive bovine mastitis is a prevalent disease with
significant economic implications for the dairy industry
[99, 100]. While preventive measures such as hygiene
practices, teat sealants, vaccination and probiotics have
proven effective in controlling the disease, they do not
result in a complete elimination [101, 102]. Consequently,
there is a need for effective therapy. Antibiotics
currently provide therapeutic relief, but their use is
increasingly questioned, particularly antibiotics that
are regarded critical for human health care [103, 104].
In this context, bacteriophage-derived endolysins have
emerged as promising antimicrobials to either replace or
complement existing treatments against Gram-positive
bovine mastitis pathogens [3].
e first endolysin that came in focus of this review,
PlyC, was discovered in 1957 by Krause [58]. After
that, the endolysin technology gained increasing
attention from the early 2000s on. is resulted in the
rapid discovery of new and sometimes highly similar
endolysins by different groups, as well as different
nomenclatures [53]. While the initially discovered
wild-type endolysins exhibited high promise under
simplistic invitro conditions, such as laboratory buffers
or pasteurized milk, challenges persisted in raw milk
and in vivo scenarios [35, 38]. For example, multiple
studies reported a matrix inhibitory effect caused by
raw milk, even though the endolysin initially displayed
potent antimicrobial activity in pasteurized milk [31,
Page 18 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
38]. Another study demonstrated synergy between two
endolysins but reported that this synergistic action was
lost in the murine mammary gland [35]. It is important
to understand that endolysins are evolutionarily designed
to release the bacteriophage from the infected host cell
and cause ‘lysis-from-within’. us, their efficacy is not
necessarily guaranteed when causing ‘lysis-from-without’,
certainly not in the demanding conditions that they
encounter in the infected mammary gland [6, 105]. is
limitation has posed an initial drawback for the wild-
type endolysins to achieve therapeutic breakthrough in
veterinary medicine.
From 2012 onward, the engineering of endolysins has
predominantly transpired, which is possible due to their
modular structure [9]. is also enabled the addition
of novel modules or peptides to expand their working
spectrum beyond their conventional antimicrobial
capabilities, such as incorporating CPPs to target mastitis
pathogens within the bovine mammary epithelium [31,
39, 43]. In the meantime, endolysins also have been
reported to target other virulence factors of Gram-
positive bovine mastitis pathogens, such as eradicating
biofilms and killing antibiotic resistant strains [31, 41,
106]. In addition, recent protein engineering methods
have evolved from rational assembly to creating complex
libraries, allowing for an extensive screening and hit-to-
lead selection of candidates that display robust ‘lysis-
from-without’ activity under the specific conditions
encountered in the infected mammary gland [31, 93].
Indeed, the first library only consisted of a few dozen
variants [55], which were later expanded to a few
hundred variants [38, 40], and most recently, to tens of
thousands of variants [31]. It must be emphasized that
screening these libraries under the end-user conditions is
crucial, as multiple groups have reported that screening
in pasteurized milk does not guarantee activity in raw
milk [31, 38, 39]. However, it became clear that these
high-throughput protein engineering methods yield lead
candidates that offer great therapeutic potential for the
treatment of bovine mastitis [95].
e integration of endolysins as stand-alone products
or in combination with non-critical antibiotics should
be a focal point for future research. Isoxazolyl penicillins
(e.g., oxacillin, cloxacillin) have been reported to be
promising candidates for supplementation, potentially
exhibiting synergy or a potentiating effect when
used together with engineered endolysins [56, 95].
Considering that mastitis treatment typically occurs
at milking with a 12-h interval, integration as a stand-
alone product may be impractical due to the endolysin’s
short half-life, although sufficient pharmacokinetic and
-dynamic data about endolysins in a mastitis context are
currently lacking [105]. Exploring their application as dry
cow treatment in a slow-release formulation can also be
proposed, as was recently reported for another veterinary
application [107].
In conclusion, recent advances in modular protein
engineering methods present novel opportunities for
endolysins to simulate horizontal gene transfer in a
high-throughput manner and select for lead candidates
under the end user conditions. Identifying endolysin
candidates capable of withstanding the demands of the
lactating bovine mammary gland is now highly feasible.
This review asserts that endolysin therapy, either as
stand-alone or as a supplementation to antibiotics,
is on the verge of a transformative breakthrough
in veterinary medicine. Expectations are high that
bacteriophage-derived endolysins will integrate into
the antibiotic armamentarium over the next decade,
revolutionizing the approach to combating bovine
mastitis-causing streptococci and staphylococci. A
promising era marked by innovative antimicrobial
treatment strategies is about to begin, that will further
shape the future of veterinary medicine.
Abbreviations
BME-UV1 Bovine mammary epithelial cell line BME-UV1
BoMECs Bovine mammary epithelial cell lines
CBD Cell wall-binding domain
CFU Colony forming units
CHAP Cysteine/Histidine-dependent Aminohydrolase/Peptidase
Cly Chimeric endolysin
CPP Cell penetrating peptide
EAD Enzymatic active domain
GFP Green fluorescent protein
H&E Hematoxylin & eosin
HIV-1 Human immunodefficency virus 1
IL Interleukin
Lys Phage endolysin
MAC-T Bovine mammary alveolar cell line stable transfected with SV-40
large T-antigen
MBC Minimal bacteriocidic concentration
MBEC Minimum biofilm eradicating concentration
MIC Minimal inhibitory concentration
MRSA Methicillin-resistant Staphylococcus aureus
OD600nm Optical density measured at 600 nm
PBS Phosphate buffered saline
pH Potential of hydrogen
Phages Bacteriophages
pI Iso-electric point
Ply Phage endolysin
PMN Polymorphonuclear leukocytes
PS Penn state bovine mammary epithelial cell line
SCC Somatic cell count
TAT Trans-activator of transcription
TKA Time kill assay
TLR Toll-like receptor
TNF Tumor necrosis factor
TRA Turbidity reduction assay
UHT Ultra-high temperature treated
Acknowledgements
The author expresses sincere gratitude to Evelyne Meyer, Yves Briers, Rob
Lavigne, Daniel C. Nelson and Lone Brøndsted for their valuable feedback.
Page 19 of 21
VanderElst Acta Veterinaria Scandinavica (2024) 66:20
Author contributions
The author confirms sole responsibility for the following: study conception
and design, data collection, analysis and interpretation of results, and
manuscript preparation. The author has read and approved the final version of
the manuscript.
Funding
Open access funding provided by Karolinska Institute. This research was
funded by the Research Foundation of Flanders (FWO Vlaanderen) grant
number 1.S.236.20N, and the author is grateful for his current postdoctoral
scholarship received from the Kronprinsessan Lovisas Förening För
Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond.
Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This study did not require official or institutional ethical approval.
Consent for publication
Not applicable.
Competing interests
The engineered endolysin NC5 in this work is the subject of an issued and
pending patent submitted at the European Patent Office with application
number EP 23164960.9 to which the author has inventorship.
Received: 6 January 2024 Accepted: 5 April 2024
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Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Niels Vander Elst is a postdoctoral researcher affiliated to the
Karolinska Institute, Sweden. He specializes in engineering endolysin
variants with applications in (veterinary) medicine and beyond. Dr.
Vander Elst earned a dual PhD in Biotechnology and Veterinary Medi-
cine that was jointly awarded by Ghent University and KU Leuven,
Belgium. During his doctoral research, he spent a significant amount
of time working on endolysins at the Institute for Bioscience and Bio-
technology Research (IBBR) associated to the University of Maryland,
Rockville, MD, USA. Dr. Vander Elst has received recognition through
scholarships and fellowships, also holding inventorship to one provi-
sional and one full patent application.
