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Phage-Derived Endolysins Against Resistant Staphylococcus spp.: A Review of Features, Antibacterial Activities, and Recent Applications

January 2025
Infectious Diseases and Therapy 14(1):13-57

DOI:10.1007/s40121-024-01069-z

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CC BY-NC 4.0

Authors:

Hossein Kazemian

Medical University of Ilam

Hamid Heidari

Shahid Sadoughi University of Medical Sciences and Health Services

Antimicrobial resistance is a significant global public health issue, and the dissemination of antibiotic resistance in Gram-positive bacterial pathogens has significantly increased morbidity, mortality rates, and healthcare costs. Among them, Staphylococcus, especially methicillin-resistant Staphylococcus aureus (MRSA), causes a wide range of diseases due to its diverse pathogenic factors and infection strategies. These bacteria also present significant issues in veterinary medicine and food safety. Effectively managing staphylococci-related problems necessitates a concerted effort to implement preventive measures, rapidly detect the pathogen, and develop new and safe antimicrobial therapies. In recent years, there has been growing interest in using endolysins to combat bacterial infections. These enzymes, which are also referred to as lysins, are a unique class of hydrolytic enzymes synthesized by double-stranded DNA bacteriophages. They possess glycosidase, lytic transglycosylase, amidase, and endopeptidase activities, effectively destroying the peptidoglycan layer and resulting in bacterial lysis. This unique property makes endolysins powerful antimicrobial agents, particularly against Gram-positive organisms with more accessible peptidoglycan layers. Therefore, considering the potential benefits of endolysins compared to conventional antibiotics, we have endeavored to gather and review the characteristics and uses of endolysins derived from staphylococcal bacteriophages, as well as their antibacterial effectiveness against Staphylococcus spp. based on conducted experiments and trials.

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Characteristics of the engineered endolysins with antibacterial activity

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Characteristics of the phage-encoded endolysins with antibacterial activity

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Infect Dis Ther (2025) 14:13–57

https://doi.org/10.1007/s40121-024-01069-z

REVIEW

Phage‑Derived Endolysins Against Resistant

Staphylococcus spp.: AReview ofFeatures,

Antibacterial Activities, andRecent Applications

MinaGolban· JavadCharostad· HosseinKazemian· HamidHeidari

Received: August 19, 2024 / Accepted: October 22, 2024 / Published online: November 16, 2024

ABSTRACT

Antimicrobial resistance is a signicant global

public health issue, and the dissemination of

antibiotic resistance in Gram-positive bacterial

pathogens has signicantly increased morbid-

ity, mortality rates, and healthcare costs. Among

them, Staphylococcus, especially methicillin-

resistant Staphylococcus aureus (MRSA), causes a

wide range of diseases due to its diverse patho-

genic factors and infection strategies. These bac-

teria also present signicant issues in veterinary

medicine and food safety. Effectively managing

staphylococci-related problems necessitates a

concerted effort to implement preventive meas-

ures, rapidly detect the pathogen, and develop

new and safe antimicrobial therapies. In recent

years, there has been growing interest in using

endolysins to combat bacterial infections. These

enzymes, which are also referred to as lysins, are

a unique class of hydrolytic enzymes synthesized

by double-stranded DNA bacteriophages. They

possess glycosidase, lytic transglycosylase, ami-

dase, and endopeptidase activities, effectively

destroying the peptidoglycan layer and resulting

in bacterial lysis. This unique property makes

endolysins powerful antimicrobial agents, par-

ticularly against Gram-positive organisms with

more accessible peptidoglycan layers. Therefore,

considering the potential benets of endolysins

compared to conventional antibiotics, we have

endeavored to gather and review the charac-

teristics and uses of endolysins derived from

staphylococcal bacteriophages, as well as their

antibacterial effectiveness against Staphylococcus

spp. based on conducted experiments and trials.

Keywords: Endolysin; Staphylococcus; Antimi-

crobial activity

M.Golban· J.Charostad· H.Heidari(*)

Department ofMicrobiology, Faculty ofMedicine,

Shahid Sadoughi University ofMedical Sciences,

Yazd, Iran

e-mail: heidarii.hamid@gmail.com; h.heidari@ssu.

ac.ir

H.Kazemian

Clinical Microbiology Research Center, Ilam

University ofMedical Sciences, Ilam, Iran

Infect Dis Ther (2025) 14:13–57

Key Summary Points

Drug-resistant infections caused by Gram-

positive bacteria, such as Staphylococcus, pose a

major global public health challenge, leading

to a signicant rise in morbidity, mortality

rates, and healthcare expenses.

To effectively address these issues, it is essen-

tial to implement preventive measures, rap-

idly detect the pathogen, and develop new

and safe antimicrobial therapies.

Endolysins, synthesized by double-stranded

DNA bacteriophages, exhibit glycosidase,

lytic transglycosylase, amidase, and endo-

peptidase activities, effectively destroying the

peptidoglycan layer, particularly in Gram-

positive organisms.

These enzymes have unique characteristics,

notable antibacterial properties, and appeal-

ing applications that we have taken into

account.

INTRODUCTION

Antimicrobial resistance is a signicant global

public health issue that requires immediate

attention and intervention [1]. Theemergence

and dissemination of antibiotic resistance in

Gram-positive bacterial pathogens, particu-

larly methicillin-resistant Staphylococcus aureus

(MRSA) and methicillin-resistant coagulase-

negative staphylococci (MRCoNS), have signi-

cantly increased morbidity, mortality rates, and

healthcare costs [2].

Staphylococcus aureus is a common oppor-

tunistic pathogen found in the nasal mucosa

of 20–40% of the human population, playing a

key role in spreading hospital and community-

acquired infections [3]. The spectrum of diseases

caused by this bacterium ranges from mild skin

infections such as impetigo, skin burns, boils,

and abscesses to severe, life-threatening dis-

eases such as bacteremia, osteomyelitis, infec-

tious endocarditis, meningitis, and pneumonia

[4]. The capacity of S. aureus to induce a broad

spectrum of diseases is attributed to its substan-

tial diversity of pathogenic factors and infection

strategies, including evasion of the host immune

system and resilience to antibiotic treatment [5].

Apart from its role as a human infectious agent,

this bacterium is signicant in the food and ani-

mal husbandry industries as a foodborne patho-

gen and the primary cause of bovine mastitis [6].

S. aureus is also capable of producing treatment-

resistant biolms, posing challenges in medi-

cine, veterinary medicine, and food safety [7].

Coagulase-negative staphylococci colonize

the skin and mucous membranes of humans

and animals, but compared to S. aureus, possess

fewer virulence properties and rarely produce

invasive pathogens [8]. However, these species

have pathogenic potential due to their capacity

to form biolms on living and non-living sur-

faces and cause foreign body-related infections,

especially in immunocompromised patients and

premature infants [9]. Meanwhile, Staphylococ-

cus epidermidis and Staphylococcus haemolyticus

species, are regarded as important challenges in

the eld of contemporary medicine due to the

increasing use of indwelling medical equipment

such as intravascular catheters, the rise in the

number of immunocompromised patients and

the spread of drug resistance [10].

Successful management of staphylococci-

induced infections, especially those caused by

antibiotic-resistant strains, requires a concerted

effort to implement preventive protocols, rap-

idly identify the pathogen, and develop new

and safe antimicrobial treatments [11]. One of

the best future solutions to combat bacterial

infections is phage-derived endolysins. These

enzymes effectively cleave the bonds in the

peptidoglycan layer, a critical component of

the bacterial cell wall, resulting in bacterial lysis.

This unique characteristic denes endolysins as

potent antimicrobial agents, particularly against

Gram-positive organisms with more accessi-

ble peptidoglycan layers [12, 13]. The history

of phage-derived endolysins began between

1915 and 1926 when the rst evidence of these

enzymes was discovered and confirmed. In

1971, highly puried lysin was successfully pre-

pared, and in 2001, another research team used

invivo endolysin to treat respiratory streptococ-

cal pathogens. In 2013, the rst clinical trial of

Infect Dis Ther (2025) 14:13–57

the endolysin-based drug SAL-1 entered phase

I, leading to numerous advancements thereafter

[14].

Considering the potential advantages of

endolysins over traditional antibiotics, this com-

prehensive review aims to explore endolysins

derived from staphylococcal bacteriophages and

their applications in various elds.

This article is based on previously conducted

studies and does not contain any new studies

with human participants or animals performed

by any of the authors.

STRUCTURE AND FUNCTION OF

PHAGE‑DERIVED ENDOLYSINS

Phages, or bacteriophages, are viruses that selec-

tively target and infect bacterial cells. In light of

the alarming rise in antibiotic-resistant bacterial

infections, research on phages as potential com-

plementary or alternative treatments is rapidly

advancing [15]. Endolysins, also known as

lysins, are a unique class of hydrolytic enzymes

synthesized by bacteriophages with double-

stranded DNA in the nal phase of the lytic

cycle [16]. These enzymes access the peptidogly-

can through membrane pores created by holins,

bacteriophage-encoded membrane proteins,

and cleave the covalent bonds, initiating bacte-

rial cell lysis and the release of progeny virions

[17]. The structures and mechanism of action of

endolysins targeting Gram-negative and Gram-

positive bacteria differ due to the variations in

their cell wall [18].

Different types of endolysins are classied

into ve categories based on their specic cleav-

age sites in the peptidoglycan. Glycosidases

(acetylmuramidases, glucosaminidases) and

lytic transglycosylases cleave glycosidic bonds

in the peptidoglycan; Amidases are involved in

Fig. 1 Schematic presentation of specic cleavage sites of endolysins. NAM N-acetylmuramic acid, NAG N-acetylglucosa-

mine

Infect Dis Ther (2025) 14:13–57

cleaving amide bonds between N-acetylmuramic

acid and l-alanine, and endopeptidases target

dipeptide-binding motifs consisting of l-lysine

and d-alanine links (Fig.1). Such classifica-

tions reflect the diverse enzymatic activities

of endolysins, underscoring their versatility as

potent antimicrobial agents able to combat a

wide range of bacterial infections [19, 20].

Gram-negative bacteria possess a more com-

plex cell wall structure characterized by a thin

peptidoglycan layer in the periplasmic space and

an outer membrane. Endolysins targeting Gram-

negative bacteria are typically simple globular

proteins with a single enzymatically active

domain (EAD) and a mass ranging from 15 to 20

kDa [21]. They may also contain cell wall-bind-

ing domains (CBDs) that inuence their ability

to interact with and lyse the target Gram-posi-

tive bacteria [22]. In contrast, endolysins that

target Gram-positive bacteria, such as staphy-

lococci, primarily focus on degrading the thick

peptidoglycan layer without the additional chal-

lenge of penetrating an outer membrane. The

simpler cell wall structure in this group of bac-

teria facilitates the access of endolysins to target

and lyse cells effectively. These endolysins com-

monly have a modular conguration with one

or more EADs at the N-terminus linked to a CBD

at the C-terminus [21].

The EAD is responsible for the catalytic func-

tion in endolysins, enabling them to cleave

bonds essential for the structural integrity of

the peptidoglycan, disrupting the osmotic

balance in the cell, and ultimately leading to

bacterial cell lysis [23]. The catalytic domain

of endolysins plays a crucial role in their func-

tion, and even a single residue change in this

domain can signicantly modify their biologi-

cal activity. For instance, a comparative analy-

sis of the staphylococcal endolysins SAL-1 and

LysK highlighted this phenomenon. These

endolysins feature two enzymatically domains

containing cysteine, histidine-dependent ami-

dohydrolase/peptidase (CHAP), and central ami-

dase-2, along with an SH3b cell-binding domain.

SAL-1, despite amino acid sequence similarity

to LysK, exhibited lower Minimum Inhibitory

Concentration (MIC) against S. aureus strains

due to a specic amino acid substitution in the

CHAP domain, enhancing its enzymatic activity

[24]. On the other hand, the CBD in endolysins

plays a crucial role in recognizing and binding

the enzyme to the bacterial cell wall. The abil-

ity of CBD to interact with specic substrates

in the peptidoglycan layer allows endolysins

to target and bind to their bacterial hosts pre-

cisely [23]. A recent study detailed the interac-

tion between the PlyGRCS endolysin domains

and the cold-shock protein C (CspC), which is

known as an RNA chaperone that regulates bac-

terial adaptation to different stress conditions.

PlyGRCS exhibited strong lytic activity against

MRSA, and the PlyGRCS–CspC complex greatly

reduced CspC–nucleic acid binding, indicating a

potential downregulation of CspC function dur-

ing bacterial infection [25].

SPECIFICITY AND EFFICACY

The high specicity of staphylococcal endolysins

is among their most valuable and unique prop-

erties. The structural components of endolysins

play an important role in this special function.

CBDs provide genus-specicity by binding to

different epitopes on the cell wall, facilitating

EAD binding for peptidoglycan hydrolysis [26,

27]. Endolysins also target and break highly

conserved regions of the bacterial cell wall and

reduce the likelihood of mutations that lead

to resistance, as any mutation in these regions

would likely be lethal to the bacteria [28]. This

special binding mechanism enables endolysins

to selectively lyse bacteria such as S. aureus and

MRSA regardless of antibiotic resistance caused

by mutations at the target sites [29]. Due to fur-

ther reasons, resistance to endolysins is improba-

ble. The coevolution of bacteriophages and their

host bacteria reduces the likelihood of resistance

development. Moreover, instead of entering the

target cell, endolysins act on the bacterial cell

wall, and possible resistance mechanisms such

as efux pumps and decreased membrane per-

meability cannot affect them. Many endolysins

also possess two catalytic domains that hydro-

lyze different bonds in the peptidoglycan, fur-

ther reducing the possibility of resistance forma-

tion [30].

Infect Dis Ther (2025) 14:13–57

In a notable 2016 study, the chimeric

endolysin HY-133 exhibited potent lytic activ-

ity with low MIC values against various African

isolates of the S. aureus complex, regardless of

their antibiotic resistance proles and species

type [31]. Subsequent research in 2019 dem-

onstrated the rapid and selective bactericidal

effects of HY-133 against MSSA and MRSA,

with MIC and MBC concentrations ranging

from 0.12 to 1 mg/l across different staphy-

lococcal strains. Moreover, time-kill studies

revealed a swift reduction of ≥ 3-log10 CFU/

ml compared to oxacillin. The mode of action

of HY-133 remained unaffected by the growth

phase, resistance pattern, and phenotype [32].

PlyGRCS is another endolysin that exhibited

dose-dependent antimicrobial activity against

planktonic forms and biofilms of S. aureus,

including MRSA. The lytic spectrum of this

enzyme encompassed all tested strains of S.

aureus and S. epidermidis, while it showed no

activity against other Gram-positive patho-

gens. This endolysin is characterized by a cata-

lytic domain (CHAP) and a binding domain

(SH3-5), which are pivotal in determining its

specicity and activity [33].

Endolysins exhibit bactericidal activity

against species closely related to the phage host

[34]. This targeted antibacterial activity separates

endolysins from broad-spectrum antibiotics and

helps preserve the host’s benecial microbiota

[13]. For instance, the MurNAc-LAA domain

of endolysin Ph28 from S. epidermidis bacte-

riophage PH15, following cloning and expres-

sion in E. coli, displayed enhanced specicity

for multidrug-resistant S. epidermidis over other

staphylococcal spp. in the turbidity reduction

assay [35]. In contrast, the PRF-119 endolysin

effectively targeted methicillin-susceptible and

methicillin-resistant S. aureus strains with an

MIC90 of 0.391 μg/ml, while having no impact

on CoNS strains associated with the natural ora

[36].

Despite their species specicity, endolysins

can also target diverse strains within their host

species [26]. One such endolysin, LysMR-5,

exhibited substantial lytic activity against vari-

ous clinical isolates of S. aureus and 95% of S.

epidermidis strains. Importantly, LysMR-5 did

not affect other tested Gram-negative bacteria,

underscoring its specicity towards staphylococ-

cal species [37]. Furthermore, endolysin LysSAP8

demonstrated substantial lytic activity against a

diverse range of staphylococcal strains in con-

trolled experimental environments. Its selectiv-

ity towards staphylococcal species, along with

resistance to divalent metal ions and adapt-

ability to different environmental conditions,

suggested its prospective efcacy as a targeted

biocontrol agent against staphylococcal infec-

tions [38].

While endolysins have a high level of speci-

city, their narrow host range may restrict their

effectiveness in treating infections caused by

diverse pathogens (e.g., sepsis). To address this

limitation, researchers have explored the clon-

ing of broad-spectrum endolysins and employed

molecular engineering to modify their structure,

enhancing specicity and targeting a broader

range of bacterial species [39]. For example,

LysSAP26, a recombinant broad-spectrum

endolysin, successfully suppressed the growth

of various multidrug-resistant (MDR) Gram-

positive and Gram-negative strains in lab tests

and exhibited a 40% survival rate in Acineto-

bacter baumannii-infected mice [40]. Likewise,

the recombinant endolysin LysSA52 exhibited

potent lytic activity against multiple Gram-pos-

itive bacteria, including MRSA and Streptococcus

strains, and efciently reduced S. aureus and S.

epidermidis biolms [41].

Further, by fusing the CBD of S. aureus endoly-

sin Lys87 with the soluble catalytic domains

of Enterococcus faecalis endolysins Lys168 and

Lys170, researchers have successfully created

highly soluble lytic enzymes with broad activity

against various pathogens, including MRSA and

other staphylococcal, streptococcal, and entero-

coccal species [42].

Altogether, the specicity of endolysins allows

for targeted action against pathogens by bind-

ing to specic cell wall components, minimiz-

ing harm to the microbiome. Ongoing research

focuses on developing endolysins with broader

specicity to optimize their antimicrobial prop-

erties against multidrug-resistant pathogens.

Infect Dis Ther (2025) 14:13–57

CLONING, EXPRESSION, AND

PRODUCTION OF ENDOLYSINS

The utilization of bacterial expression systems,

notably Escherichia coli, is a common choice for

the production of staphylococcal endolysins due

to their efciency and well-established protocols

for protein synthesis [6]. However, challenges

such as the necessity to purify recombinant pro-

teins to remove toxic endotoxins present in E.

coli, the complex processes of preparing soluble

and active proteins in a puried and concen-

trated form, and the risk of nal product inac-

tivation due to improper folding are associated

with this approach [43, 44].

To tackle these hurdles, researchers investi-

gated various strategies, including alternative

expression hosts and optimization of growth

and expression conditions. For example, a

study by Chun etal. indicated the capability of

yeast surface display technology in successfully

expressing endolysin LysSA11 on the surface of

Saccharomyces cerevisiae. The surface-displayed

LysSA11 exhibited potent antimicrobial activ-

ity against S. aureus, reducing viable bacteria by

5 logs in 3 h without further purication steps.

Additionally, it demonstrated improved stabil-

ity and lytic activity compared to E. coli-puried

LysSA11 [45].

Therefore, the advantages of this system over

traditional bacterial expression systems, includ-

ing the ability to perform post-translational

modications and proper protein folding, sim-

plicity of cell culture and genetic manipulation,

and production of therapeutic proteins free

from endotoxin, render it an effective, stable,

and simple platform for producing antibacterial

endolysins [46].

Lactic acid bacteria are also utilized as hosts

for expressing active endolysins, especially in

improving food safety. Endolysins produced in

these organisms are commonly employed as pro-

biotics in dairy products and fermented foods to

inhibit the growth of pathogenic bacteria and

toxin production [39]. In this context, Lactococ-

cus lactis, a Generally Recognized as Safe (GRAS)

host, has been introduced as an efcient expres-

sion system for staphylococcal endolysins [47].

In a study by O’Flaherty etal. (2005), Lactococcus

lactis was used for the rst time to produce the

endolysin LysK from Staphylococcus phage K

using a nisin-inducible expression system to

address solubility issues observed in E. coli. The

crude lysates of recombinant endolysin LysK

resulted in a 99% reduction of MRSA strains

within 1 h, showing its potential in eradicat-

ing live antibiotic-resistant staphylococci cells

[48]. Subsequent studies successfully expressed

and secreted staphylococcal endolysins LysH5

and Endo88 using signal peptides SPLcn972

and SPK1, respectively, in Lactococcus lactis.

Unlike the prior study, the utilization of signal

peptides in recent research resulted in the gen-

eration of active extracellular endolysins in the

culture supernatant [49, 50]. Nonetheless, the

efciency of production platforms still needs to

be improved, and further optimization for large-

scale production, while ensuring safety, stability,

and cost-effectiveness, could facilitate the prac-

tical use of endolysins as food preservatives or

therapeutic agents [51].

SAFETY AND TOXICITY PROFILES

Endolysins, large protein molecules originat-

ing from prokaryotes, possess the remarkable

ability to stimulate host immune responses,

leading to the production of IgG antibodies or

inducing IgE-mediated allergic reactions [52].

This dual functionality of endolysins has raised

concerns about their development as antimicro-

bials because immune responses can compro-

mise their efcacy by antibody neutralization,

alterations in pharmacokinetics, and the risk of

adverse effects such as hypersensitivity reactions

and septic shock [13]. However, the favorable

results of safety, immunogenicity, and toxicity

studies conducted on staphylococcal endolysins

support their broad therapeutic application in

the near future. For example, endolysins like

MV-L, ClyS, and LysGH15 were examined to

assess immune responses in S. aureus infections,

focusing on resistant strains. These enzymes

exhibited strong antimicrobial activity in lab-

oratory and invivo settings, providing a pro-

tective effect in infected mice without adverse

Infect Dis Ther (2025) 14:13–57

reactions. Notably, although these endolysins

elicited measurable humoral responses in mouse

infection models, the resulting antibodies did

not hinder their bacteriolytic activity. In the case

of LysGH15, a signicant reduction in the num-

ber of bacteria and pro-inammatory cytokine

levels was observed in mice infected with lethal

MRSA, suggesting the potential of endolysins

to combat resistant bacterial strains and miti-

gate the detrimental effects of the host immune

response [53–56].

Overall, various strategies can be employed

to deal with endolysins’ immunogenicity. One

effective method involves modifying their struc-

ture to create chimeric endolysins with reduced

immunogenic epitopes [57]. Encapsulation of

endolysins in nanoparticles can also reduce

immune recognition upon systemic administra-

tion [58]. Lastly, the combined use of endolysins

and other antimicrobial agents, such as antibi-

otics or antimicrobial peptides may lower the

required dosage, thereby minimizing immuno-

genicity [23].

ENGINEERING OF ENDOLYSINS

The modular architecture of Gram-positive

endolysins with distinct functional domains

(cell wall-binding domain, linker, and enzymatic

activity domain), provides a versatile framework

for molecular engineering. This structure facili-

tates the creation of new fused enzymes with a

wider lytic spectrum, improved specicity, and

increased stability and solubility [59, 60]. Fur-

thermore, advancements in endolysin design

offer opportunities for developing commercial

products with various applications, including

disinfection, food safety, and healthcare [51, 61,

62]. One approach to molecular modication of

staphylococcal endolysins is through the swap-

ping and recombining of domains from various

endolysins to yield chimeric enzymes (known as

chimeolysins) with favorable characteristics [63].

For example, chimeric endolysins SA.100 and

XZ.700 with similar domain architecture were

created by replacing the low-activity (M23)

endopeptidase domain of Staphylococcus Ply2638

endolysin with lysostaphin’s endopeptidase

domain [64]. Lysostaphin is a bacteriocin that

specically targets the pentaglycine bridge in the

peptidoglycan of S. aureus [65]. The structural

variance between XZ.700 and SA100 endolysins

stems from removing a 44-amino-acid linker

region at the N-terminus of the Ply2638 ami-

dase domain in XZ.700. This modication in

XZ.700 enhanced its antibacterial activity,

showing how optimization of linker regions in

endolysins can signicantly improve their ef-

cacy [64]. It is worth mentioning that the results

of several invitro and invivo studies, empha-

sizing the anti-biolm properties, preservation

of the microbiome, and absence of resistance

induction by these chimeolysins, have shown

their therapeutic potential, especially in combat-

ing inammatory skin conditions caused by S.

aureus [66–70].

Researchers have developed more effective

and stable antibacterial agents against various

forms of S. aureus, including resistant strains and

biolms, by fusing the cell wall-binding domain

of lysostaphin with different catalytic domains

(CDs). Chimeric fusion proteins like HY-133,

λSA2-E-Lyso-SH3b, PRF-119, CHAPSH3b, and

P128 are the result of combining these compo-

nents [36, 71–74].

Staphylococcal endolysin Ply187 has also

been widely utilized in creating chimeric

endolysins due to its unique modular structure.

This endolysin lacks the C-terminal cell wall-

binding domain, which is typically found in

Gram-positive bacterial endolysins; instead, it

features a CHAP-type endopeptidase domain at

the N-terminus and a glucosaminidase domain

at the C-terminus [75]. Ply187AN-KSH3b,

Ply187N-V12C, ClyH, and ClyF are chimeolysins

that merge the CHAP domain of Ply187 with

the CBD domains of LysK, enterococcal endoly-

sin PlyV12, staphylococcal endolysin phiNM3,

and streptococcal endolysin PlySs2, respec-

tively. The chimeric endolysin Ply187N-KSH3b

demonstrated improved performance against S.

aureus in multiple functional assays compared

to the truncated Ply187AN enzyme [75]. Besides,

Ply187AN-V12C, in contrast to its parental

enzyme, had a broader lytic spectrum, effec-

tively targeting various staphylococcal, strep-

tococcal, and enterococcal species [76]. ClyH

exhibited superior antibacterial activity against

Infect Dis Ther (2025) 14:13–57

MRSA clinical isolates compared to lysostaphin

and protected mice from MRSA-induced mortal-

ity without adverse effects when administered

intraperitoneally [77]. Finally, ClyF displayed

remarkable thermostability and effectiveness

against diverse staphylococcal strains, success-

fully eradicating MRSA biolm in both invitro

and invivo settings [78].

Like ClyH, the engineered lysin ClyS uses

the phiNM3 endolysin as a source of its CBD,

while the catalytic domain is derived from the

S. aureus Twort phage lysin. This chimeric con-

struct provided high solubility, rapid lysis of

drug-sensitive and resistant staphylococci, syn-

ergistic effects with antibiotics, and protective

activity in murine models of colonization and

bacteremia [54, 79].

Researchers have also generated libraries of

chimeric endolysins by randomly shufing the

domains of natural staphylococcal endolysins.

This approach has resulted in screening novel

chimeras like Lys109 and ClyC, which possess

potent anti-staphylococcal activity compared to

the original endolysins in diverse matrices such

as milk, blood, and buffer solutions [62, 80].

In addition to the above, chimeric endolysins

have demonstrated the ability to target intra-

cellular pathogens, which are often challenging

due to the evasion of the host’s immune sys-

tem and conventional antibiotics. To face this

issue, researchers have facilitated the importa-

tion into various cell lines by fusing different

protein transduction domains with engineered

constructs such as K-L (LysK lytic domains + lys-

ostaphin) [81].

Another modication strategy to enhance the

versatility of staphylococcal endolysins involves

truncating or deleting specic domains from

the endolysin structure [82]. Research suggests

that while maintaining all domains is generally

essential for optimal lytic activity, removing the

cell wall-binding domain can expand the lytic

range by overcoming binding limitations. Addi-

tionally, reducing the size of the endolysin not

only improves solubility and stability but also

simplies production processes and boosts pen-

etration into the bacterial cell wall [83, 84].

In this context, certain invitro and invivo

studies have shown that the truncated lysin

CHAPk outperformed LysK in terms of lytic

activity, exhibiting potent anti-biolm effects

and successfully eradicating S. aureus from the

nares of mice [84–86]. In another instance, the

endolysins CHAPLysGH15 and LysGH15B were

modified by eliminating the CBD and EAD

domains from the Staphylococcal endolysin

LysGH15, respectively. When tested in whole

and skimmed milk, CHAPlysGH15 demon-

strated strong control against S. aureus, suggest-

ing its potential for dairy product preservation.

Moreover, the fusion of LysGH15B with green

fluorescent protein exhibited selective bind-

ing to staphylococcal isolates, especially MRSA,

which was attributed to the LysGH15B part

[87, 88]. However, the ndings of Becker etal.’s

2009 study demonstrated that although the

endopeptidase domain alone leads to effective

lysis of S. aureus, its activity is enhanced when

fused with the cell wall-binding domain [89].

The results of this study were conrmed in 2018

by Fujiki etal., revealing the interdependence

between the CHAP domain of endolysin Lys-

phiSA012 and the SH3b domain to achieve peak

lytic efcacy in deletion mutants [90]. Further-

more, in a recent study by Behera etal., evalu-

ations against planktonic and biolm-forming

MRSA revealed a higher lytic efcacy of CHAPk-

SH3bk over the CHAPk domain, emphasizing

the CBD’s contribution in increasing the endoly-

sin’s activity [91].

Interestingly, removing the amidase catalytic

domain from the full-length endolysin in some

cases indicated a limited role of this domain in

the lytic activity of endolysin [89, 90, 92, 93],

while others, illustrated the signicant effec-

tiveness of this deletion in reducing bacterial

contamination caused by Staphylococcus species

[94–97]. It has been proposed that the varying

reports on the activity levels of endolysin deriva-

tives in the literature may be linked to factors

such as bacterial strains tested, assay conditions,

and quantication techniques [97].

Table1 shows the properties of the engineered

or chimeric endolysins with the outcomes fol-

lowing the experiments.

Infect Dis Ther (2025) 14:13–57

Table 1 Characteristics of the engineered endolysins with antibacterial activity

Engineered endolysin Origin of phage Structural composition/EAD

donor + CBD donor

Target Outcome Refs.

XZ.700 S. simulans

S. aureus

Lysostaphin, endopeptidase

(M23) + Ply2638, ami-

dase + Ply2638, S H3b

S. aureus Signicant reduction of MRSA

biolms without toxicity to

human osteocyte-like cells com-

pared with povidone-iodine and

gentamicin

[66]

S. aureus e inhibition of skin colonization

by patient-derived S. aureus and

the suppression of malignant T

cell activation in CTCL

[67]

S. aureus Selective elimination of S. aureus

without harming common skin

bacteria like S. epidermidis

[64]

S. aureus Selective inhibition of S. aureus in

pig skin, preservation of micro-

biota, and acceleration of wound

healing

[68]

Staphefekt SA.100 S. simulans

S. aureus

Lysostaphin, endopeptidase

(M23) + Ply2638, ami-

dase + Ply2638, S H3b

S. aureus Ecient and dose-dependent

response, without inducing resist-

ance against MSSA and MRSA

strains

[69]

S. aureus ree cases of successful treat-

ment of chronic S. aureus-related

dermatoses without development

of resistance

[70]

CHAPk-SH3bk S. aureus LysK, CHAP + LysK, SH3b S. aureus Eective reduction of MRSA

biolms from both human and

animal sources

[91]

Infect Dis Ther (2025) 14:13–57

Table 1 continu ed

Engineered endolysin Origin of phage Structural composition/EAD

donor + CBD donor

Target Outcome Refs.

Lys109 S. aureus LysSA12, CHAP + LysSA97, ami-

dase + LysSA97, CBD

S. aureus

S. saprophyticus

S. hominis

S. haemolyticus

S. capitis

S. warneri

S. xylosus

S. epidermidis

Stronger lytic activity against

biolm and planktonic forms

of Staphylococcus than parent

endolysins

Decrease in S. aureus cell numbers

in milk and stainless steel

[62]

ClyC S. aureus LysSA12, CHAP + LysPALS1,

SH3b

S. aureus

S. epidermidis

S. haemolyticus

S. saprophyticus

S. intermedius

S. xylosus

S. hominis

Rearrangement of the domains

of 12 native staphylococcal

endolysins

Robust antibacterial activity against

S. aureus in buer, milk, and

blood

Protection against MRSA infection

in mice without any toxic eects

[80]

Infect Dis Ther (2025) 14:13–57

Table 1 continu ed

Engineered endolysin Origin of phage Structural composition/EAD

donor + CBD donor

Target Outcome Refs.

HY-133 S. aureus

S. simulans

LysK, CHAP + lysostaphin, SH3b S. aureus High antibacterial activity against

MSSA and MRSA isolates

compared to daptomycin and

mupirocin

[71]

S. aureus Rapid and selective bactericidal

activity against various staphylo-

coccal strains

[32]

S. aureus (SH1000) Moderate bactericidal activity

against S. aureus biolm in vascu-

lar gra infections compared to

daptomycin and rifampin

[98]

S. aureus Signicant lytic activity against a

variety of African isolates of the S.

aureus complex

[31]

LA-MRSA High lytic activity against geneti-

cally diverse LA-MRSA isolates

[99]

ClyS S. aureus PlyTW (S. aureus phage Twort),

endopeptidase + phiNM3, non-

SH3b CBD

S. aureus

S. epidermidis

S. simulans

S. sciuri

Lytic activity against drug-sensitive

and resistant staphylococci and

synergistic eects with vancomy-

cin and oxacillin invitro

[79]

S. aureus More eective than mupirocin

against S. aureus in the epidermis

with no inhibition of lytic activity

by antibodies

[54]

Infect Dis Ther (2025) 14:13–57

Table 1 continu ed

Engineered endolysin Origin of phage Structural composition/EAD

donor + CBD donor

Target Outcome Refs.

λ SA2-E-Lyso-SH3b S. agalactiae

S. simulans

λSa2lys(S. agalactiae prophage

λSa2), endopeptidase

+ lysostaphin, SH3b

S. aureus

S. agalactiae

S. uberis

Expanding the lytic spectrum

through domain fusion

Synergistic activity with lysostaphin

against S. aureus in a mouse

model of bovine mastitis

[72,

100]

λ SA2-E-LysK-SH3b S. agalactiae

S. aureus

λSa2lys(S. agalactiae prophage

λSa2), endopeptidase

+ LysK, SH3b

Lys168-87 E. faecalis

S. aureus

F168/08 lysine (E. faecalis phage),

CHAP + Lys87, CB D

S. aureus

S. epidermidis

S. haemolyticus

S. saprophyticus

E. faecalis

E. faecium

S. pyogenes

Broad lytic activity against MRSA

strains and other Gram-positive

pathogens tested

[42]

Lys170-87 F170/08 lysine (E. faecalis phage),

amidase + Lys87, CBD

PRF-119 S. aureus

S. simulans

LysK, CHAP + lysostaphin, SH3b S. aureus Lysis of all tested S. aureus isolates

without aecting the normal ora

[36]

Ply187AN-KSH3b S. aureus Ply187, CHAP +

LysK, SH3b

S. aureus

S. chromogenes

S. epidermidis

S. hyicus

S. simulans

S. warneri

S. xylosus

S. dysgalactiae

Increasing the lytic activity of

parental endolysins in milk

[75]

S. aureus Improving endophthalmitis

outcomes and preserving visual

function with endolysin intravit-

real injection

[101]

Infect Dis Ther (2025) 14:13–57

Table 1 continu ed

Engineered endolysin Origin of phage Structural composition/EAD

donor + CBD donor

Target Outcome Refs.

Ply187N-V12C S. aureus

faecalisstrain V12.Φ1

Ply187, CH AP + PlyV12 (E. faeca-

lis bacteriophage Φ1), SH3b

S. aureus

S. dysgalactiae

S. agalactiae

S. pyogenes

E. faecium

E. faecalis

Broader lytic spectrum compared

to the parental enzyme

[76]

CHAPSH3b S. aureus

S. simulans

HydH5, CHAP + lysostaphin,

SH3b

S. aureus ermal stability, reduction of

biolm, removal of S. aureus to

undetectable levels in milk, and

reduction of expression of autoly-

sin genes such as AtlA

[73]

P128 S. aureus

S. simulans

Phage K, tail-associated muralytic

enzyme (TAME or Orf56) of

phage + lysostaphin, SH3b

S. aureus

S. epidermidis

S. intermedius

S. haemolyticus

S. lugdunensis

S. delphini

S. auricularis

Rapid and selective antistaphylo-

coccal activity to clear nasal colo-

nization caused by a wide variety

of clinical isolates

[74]

S. aureus

S. epidermidis

S. carnosus

Anti-staphylococcal activity, espe-

cially against clinical isolates of S.

aureus like MRSA

Decolonization of MRSA from rat

nares in an experimental model

[102]

S. aureus Signicant anti-biolm activity

against sinus-derived methicillin-

susceptible and methicillin-resist-

ant S. aureus

[103]

Infect Dis Ther (2025) 14:13–57

Table 1 continu ed

Engineered endolysin Origin of phage Structural composition/EAD

donor + CBD donor

Target Outcome Refs.

S. aureus

S. simulans

Higher thermal stability than

lysostaphin

Lytic activity against S. simulans

unlike lysostaphin

[104]

S. epidermidis

S. haemolyticus

S. lugdunensis

Strong bactericidal activity against

planktonic and biolms of CoNS

and potential synergy with anti-

biotics

[105]

S. aureus Rapid bactericidal eect as mono-

therapy or in combination with

vancomycin and daptomycin in

MRSA/VRSA bacteremia models

[106]

ClyF S. aureus

S. suis

Ply187, CH AP + PlySs2 (S. suis

prophage), SH3b

S. aureus

S. saprophyticus

S. equorum

S. sciuri

S. chromogenes

S. haemolyticus S. epidermidis

S. capitis

S. albus

Extensive lytic activity against

various staphylococcal strains and

elimination of MRSA biolm

invitro and invivo

[78]

ClyH S. aureus Ply187, endopeptidase + phiNM3,

non-SH3b CBD

S. sobrinus

S. aureus

Greater antibacterial ecacy

against all MRSA clinical isolates

tested than lysostaphin and the

parent enzyme

Protection of mice from MRSA-

induced mortality

[77]

Infect Dis Ther (2025) 14:13–57

ENDOLYSIN APPLICATIONS

Here we highlight the studies that have explored

various applications of endolysins;

Anti‑biolm Activity

Staphylococci, especially S. aureus, are known for

their capacity to develop resistance to antibiot-

ics. The formation of biolms by staphylococci

leads to a 10–1000-fold increase in antibiotic

resistance, making them highly resistant to

treatment compared to planktonic cells [107].

Biolms are adhesive biological structures con-

sisting of bacterial cells, proteins, polysaccha-

rides, and DNA, held together by a matrix of

extracellular polymeric substances (EPS) pro-

duced by the microorganisms themselves [108].

This matrix provides cohesion and strength to

the biolm, shielding the bacteria from various

threats such as mechanical and chemical attacks,

desiccation, high pressure, antibiotics, disinfect-

ants, radiation, and the host’s immune system

[109]. The biolm structure also facilitates the

horizontal transfer of genetic materials, includ-

ing antibiotic-resistance genes, between bacterial

cells, leading to the spread of resistance within

the biolm community [110]. Additionally, the

limited penetration of antimicrobials in the

deep layers of the biolm and the presence of

persister cells lead to the resistance of the bio-

lm against these factors and help the continu-

ation of biolm-related infections [111].

Addressing these issues requires the develop-

ment of therapeutic perspectives aimed at over-

coming infections associated with staphylococ-

cal biolms and improving treatment outcomes.

Endolysins have become an effective strategy to

counter biolm formation in recent years. The

mechanism of action of endolysins involves

the enzymatic degradation of the bacterial cell

wall peptidoglycan, leading to biolm destruc-

tion [112]. In contrast to antibiotics, endolysins

can penetrate the biolm matrix and eliminate

viable bacterial remnants [113]. They can also

effectively inhibit metabolically inactive per-

sister cells [114]. Endolysins carry a low risk

of resistance development and are efcacious

against multidrug-resistant pathogens [13].

Several studies have investigated the use of

endolysins for treating staphylococcal biolm

infections. The LM12 endolysin proved effective

in reducing biolm biomass, particularly against

stationary and exponential phase cells. The cell-

wall-degrading enzyme SAL-2 exhibited a wider

range of activity in eradicating S. aureus biolms

compared to its own bacteriophage [115, 116].

The Lys84 endolysin and its domains also exhib-

ited a broader bactericidal spectrum compared

to its parental phage. Lys84 eradicated approxi-

mately 90% of S. aureus biolms at concentra-

tions above 10 μM. Also, the CHAP and Ami-

dase_2 domains retained about 84% and 70%

of endolysin anti-biolm activity, respectively

[117]. The recombinant φ11 endolysin efciently

degraded staphylococcal biofilms and killed

heat-exposed staphylococci [93]. Furthermore,

the endolysin LysSAP33, distinct from LysK-like

endolysins, exhibited biolm removal capabil-

ity and sustained lytic activity across a broader

range of conditions than LysK-like endolysins

[118].

A 2018 study demonstrated that the endoly-

sin LysGH15 was able to reduce bacterial counts

of various Staphylococcus species, including mul-

tidrug-resistant ones, by approximately 4 log

units within 30 min after treatment with 20 μg/

ml of LysGH15 (MICs between 8 to 32 μg/ml).

This endolysin was also effective in preventing

biolm formation by the four staphylococcal

species at a dose of 50 μg/ml and disrupting 24-h

and 72-h biolms at a higher dose of 100 μg/ml

[119]. Besides, recombinant SAL200, containing

the SAL-1 endolysin, has demonstrated efcacy

in eradicating planktonic cells and S. aureus bio-

lm, improving survival in MRSA-infected mice

and reducing bacteria in the blood and spleen

[120]. A comparative study also investigated

the effectiveness of chimeric HY-133 endolysin,

daptomycin, and rifampicin against S. aureus

on vascular graft surfaces. Daptomycin demon-

strated rapid bactericidal effects, while HY-133

had moderate activity, especially against mature

biolm. Rifampicin did not achieve bactericidal

effects, even at high concentrations [98].

As previously stated, the ability of CoNS to

create protective biolms, particularly in medi-

cal device-related infections, presents substantial

risks in cases of prolonged infections, restricted

Infect Dis Ther (2025) 14:13–57

treatment choices, and recurrent periods of ill-

ness [121]. Hence, considering the importance

of clinical management for CoNS-induced infec-

tions, evaluation of chimeric P128 endolysin

against these strains demonstrated effective dis-

ruption of biolms and eradication of persister

cells at MIC = 16μg/ml, with synergy observed

when combined with antibiotics against pre-

formed biolms [105]. Although persister cells

in biolms are not inherently resistant to anti-

biotics, they can increase the risk of antibiotic

resistance due to their ability to survive anti-

biotic treatments and lead to chronic disease

[122]. In this regard, the endolysin LysH5 as an

antibiotic adjuvant has displayed potential in

reducing staphylococcal biolms and inhibit-

ing persister cells acquired post-treatment with

rifampicin and ciprooxacin [123].

In summary, endolysins are particularly effec-

tive against S. aureus and S. epidermidis biolms.

The specic mechanisms of action may vary

depending on the type of endolysin and biolm

conditions, but their ability to target and disrupt

the biolm structure makes them instrumental

in combating persistent infections and antibi-

otic resistance.

Synergistic Effects of Endolysins and Other

Antimicrobial Agents

Combining staphylococcal endolysins with

other endolysins or antimicrobial agents, such

as antibiotics, can synergistically enhance their

efcacy against drug-resistant pathogens. These

synergistic combinations provide multiple

advantages, such as decreased dosage, decreased

risk of resistance development, preservation of

benecial microbiota, and enhanced efcacy

against a broader spectrum of pathogens [124,

125]. The synergy among different endolysins

typically arises from their capacity to cleave dis-

tinct peptidoglycan bonds simultaneously, dis-

rupting the overall peptidoglycan structure and

causing rapid substrate lysis. This phenomenon

is commonly seen when endolysins with differ-

ent catalytic properties are combined, result-

ing in a more powerful destructive impact [82].

Indeed, the initial cleavage of peptidoglycan

by one endolysin can create new chain ends or

weaken the peptidoglycan network, facilitating

the access of the second endolysin to the target

site [23]. Additionally, targeting multiple sites

simultaneously makes it more difcult for bac-

teria to develop resistance mechanisms against

all involved lysines [126].

For instance, the combination of LysK,

derived from the staphylococcal bacteriophage

K, and lysostaphin, a metalloendopeptidase

expressed by Staphylococcus simulans, demon-

strated significant synergistic effects against

MRSA strains through checkerboard assays [127].

Another successful synergy was observed with

endolysin SAL200, which decreased the MICs of

standard-of-care (SOC) antibiotics and resulted

in a rapid reduction of over 3-log CFU/ml of S.

aureus counts in time-kill assays. SAL200 also

demonstrated the lowest bacteremia levels when

used alongside standard antibiotics in mouse

models [128]. Similarly, LysP108 endolysin

lysed S. aureus and Pseudomonas aeruginosa with

compromised outer membranes, reducing via-

ble bacterial numbers and disrupting biolms.

Invivo experiments demonstrated a signicant

reduction in subcutaneous abscess size in MRSA-

infected mice following combined treatment

with LysP108 and vancomycin [129].

Another synergistic approach involves the

development of hybrid endolysins capable of

targeting multiple bacterial species simultane-

ously, leading to a wider spectrum of activity

and enhanced efcacy in eradicating bacterial

pathogens [130]. The chimeric endolysin ClyS

effectively lysed MRSA, vancomycin-interme-

diate strains of S. aureus (VISA), and methicil-

lin-sensitive strains of S. aureus (MSSA). This

endolysin displayed synergistic interactions with

vancomycin and oxacillin invitro, and when

combined with oxacillin, it protected against

MRSA-induced septic death in mouse models

[79]. Another chimeric endolysin, P128, created

by merging the enzymatic domain of bacterio-

phage K with the cell wall-binding domain of

lysostaphin, was evaluated both as monotherapy

and in combination with two SoC antibiotics,

vancomycin and daptomycin. P128/SoC anti-

biotic combinations demonstrated synergistic

effects, leading to enhanced survival rates in

mouse models of bacteremia infected with MRSA

and vancomycin-resistant S. aureus (VRSA) [106].

Infect Dis Ther (2025) 14:13–57

In addition to the previously discussed strat-

egies, researchers have considered combining

endolysins with antimicrobial peptides (AMP),

a class of natural or synthetic antibacterials, to

exploit synergistic responses. These synergis-

tic interactions are thought to result from the

membrane-depolarizing action of AMPs, which

can overcome bacterial tolerance to endolysins

and enhance the susceptibility of bacteria to

lysis induced by endolysins. A recent study

revealed that combining antimicrobial peptide

R8K and endolysin Lys11 effectively reduces

bacterial numbers compared to using R8K or

Lys11 alone. This synergy is linked to enhanced

endolysin binding and disruption of the pro-

ton motive force (PMF) in bacterial cells. The

cationic peptide R8K interacts with negatively

charged bacterial envelope components like

teichoic acids, destabilizing the membrane and

increasing endolysin permeability. Alternatively,

R8K binding to the bacterial cell membrane may

form pores for proton leakage, disrupting PMF

and sensitizing cells to Lys11’s action, as the

cells have reduced energy reserves to maintain

their structural integrity and repair post-endoly-

sin damage [131]. Moreover, a study by Olsen

etal. discussed the synergy between endolysin

LysK and poly-N-acetylglucosamine depolymer-

ase DA7 targeting extracellular matrix polysac-

charides as an anti-biolm strategy. The research

showed LysK’s activity against various S. aureus

strains, while the combined use of LysK and DA7

effectively removed biolms from surfaces at low

concentrations in both static and dynamic mod-

els [132].

Hence, the synergistic effects observed when

combined with traditional antimicrobial agents

underscore the potential of endolysins as valu-

able supplements in addressing the growing

threat of staphylococcal infections and pave the

way for more efcient therapeutic interventions

in clinical settings.

Food Biopreservation

Food safety is one of the critical aspects of the

Sustainable Development Goals to protect the

health of humans, animals, and the environ-

ment [133]. Despite signicant advancements

in food hygiene techniques, the global burden

of foodborne illnesses persists, with the World

Health Organization (WHO) reporting approxi-

mately 600 million cases annually, leading to

over 420,000 deaths. Notably, children under

the age of 5 are signicantly affected, account-

ing for approximately 125,000 deaths each year

[134, 135].

S. aureus is one of the most common food

pathogens that cause staphylococcal food poi-

soning (SFP) due to the production of heat-

stable enterotoxins [136]. Food contamination

can occur at any point in the food supply chain,

including production, distribution, processing,

or storage [137]. Also, humans carrying entero-

toxin-producing S. aureus are a signicant source

of food contamination through hand contact or

respiratory secretions [138].

Endolysins derived from bacteriophages can

effectively decrease the levels of S. aureus and

other foodborne pathogens in food products.

These endolysins have unique properties, such

as high specicity, lack of resistance, preserva-

tion of benecial bacteria in food, and non-dis-

ruption of the body’s microbiota, which makes

them potential substitutes for traditional anti-

biotics and disinfectants [61]. They can also

destroy the biolms produced by staphylococci

that allow them to survive in food processing

environments [139]. For instance, the LysCSA13

endolysin effectively removed staphylococcal

strains and their biolms from different food

contact surfaces including polystyrene, glass,

and stainless steel, resulting in an 80–90%

reduction in biolm mass [140]. Similarly, the

LysSA11 endolysin reduced MRSA contamina-

tion in food products such as pasteurized milk

and ham, as well as utensils such as polypropyl-

ene plastic and stainless steel (4-log CFU/cm2)

[141].

Nevertheless, the use of endolysins in food

safety also brings challenges. Some of the

main obstacles include the necessity to assess

the performance of endolysins in food set-

tings, considering factors such as temperature

stability, pH, and ionic composition [142]. In

this regard, the antibacterial effectiveness of

LysH5 endolysin was assessed against bovine

and human S. aureus strains, as well as human

S. epidermidis in pasteurized milk. The results

Infect Dis Ther (2025) 14:13–57

revealed signicant lytic activity and optimal

performance at pH 7.0 and 37°C, while sensi-

tivity to high temperatures was observed [143].

Building upon the previous ndings, a study

delved into the optimal lytic activity of LysH5

under specic ionic conditions. The research

highlighted that Mn2+ and Zn2+ hindered its

activity, whereas Ca2+, Mg2+, and NaCl bol-

stered its effectiveness. Furthermore, combin-

ing LysH5 with nisin resulted in a strong syn-

ergistic effect and effectively cleared S. aureus

in pasteurized milk [144]. In another study

conducted in 2011, the HydH5 endolysin dem-

onstrated considerable lytic activity against S.

aureus at 45°C without needing divalent cati-

ons. Moreover, it preserved 72% of its activity

after 5 min at 100°C. These attributes position

HydH5 as an appealing antimicrobial agent

with noteworthy thermal stability, making it

suitable for incorporation into high-tempera-

ture hygiene practices like food preservation

[145]. LysSAP27 is another endolysin that dem-

onstrated high antimicrobial efficacy when

employed in milk contaminated with S. aureus

at a neutral pH, a temperature of 30°C, and in

the presence of calcium ions [146].

In addition to the aforementioned instances,

the development of endolysins presents disad-

vantages that restrict their application in the

food industry. These include decreased activity

over time and bacterial regrowth, highlight-

ing the necessity for combined use with other

antimicrobials [147]. Moreover, the expense of

endolysins and large-scale production necessi-

tates strategies to minimize the amount of lytic

protein required [61]. The recent study of Yusuf

etal. (2023), explored the combined utilization

of bacteriophage phiIPLA-RODI, engineered

lytic protein LysRODIΔAmi, and nisin to com-

bat S. aureus contamination in dairy products.

This combination led to a signicant reduction

in the pathogen population compared to indi-

vidual treatments. Interestingly, elevated cal-

cium concentrations enhanced endolysin activ-

ity, allowing for a roughly tenfold reduction in

protein usage while still effectively controlling

contamination [148]. Also, the study conducted

in 2022 conrmed that the combined treatment

involving LysSAP8 and nisin reduces the pro-

liferation of S. aureus, including MRSA strains

[149]. Combining nisin, an approved food

preservative, with endolysin could reduce the

required lytic protein amount and inhibit bacte-

rial regrowth [150]. On the other hand, increas-

ing calcium concentration during endolysin use

can enhance the cost-effectiveness of biopreser-

vation [151]. Carvacrol, extracted from essen-

tial oil, was also researched for its antibacterial

activity in combination with LysSA97 endoly-

sin in food products such as milk and beef. This

combination demonstrated antibacterial effects

against various strains of S. aureus. Furthermore,

it was observed that the fat content in the foods

impacted the extent of bacteria inactivation,

resulting in greater clearance in skim milk com-

pared to whole milk [152].

Therefore, according to the studies conducted

so far, endolysins, despite facing various chal-

lenges, are innovative biocontrol strategies for

mitigating the risk of foodborne diseases associ-

ated with bacterial contamination and mainte-

nance of public health.

Veterinary Medicine

Bovine mastitis is a chronic inammatory con-

dition affecting udder tissue in cows, primar-

ily transmitted during milking through milk-

ing equipment or the hands of milkers acting

as fomites [153]. S. aureus and CoNS are com-

mon pathogens in subclinical bovine mastitis,

causing damage to udder tissue by the secretion

of enzymes and toxins [154, 155]. The global

dairy industry faces annual costs ranging from

US $19.7 to US $32 billion due to mastitis, with

the U.S. alone experiencing an estimated loss of

US $2 billion annually [156, 157]. The cost of a

single case of clinical mastitis can range from US

$128 to US $444. This cost breakdown includes

decreased milk production and quality, veteri-

nary and drug expenses, excess labor require-

ments, and the risk of culling infected cows

[158]. These gures demonstrate the substantial

nancial burden that bovine mastitis imposes

on dairy producers globally and underscore the

importance of effective management strategies

to mitigate these costs.

The common antibiotic treatment for

bovine mastitis includes penicillin, ampicillin,

Infect Dis Ther (2025) 14:13–57

tetracycline, streptomycin, and other related

drugs, typically administered via intramam-

mary infusion [159]. However, the overuse and

misuse of antibiotics can lead to the emergence

of antibiotic-resistant bacterial strains and the

spread of resistance within the population [160].

Additionally, antibiotic residues in milk can

result in sales restrictions and consumer safety

worries [161]. To address these risks, alternative

treatments are being explored, such as natural

compounds, endolysins, nanotechnology, and

vaccinations [162].

Endolysins offer several advantages over anti-

biotics in the treatment of bovine mastitis. They

are effective against biolms, a common cause

of persistent infections, do not remain in milk,

and contribute to safeguarding the well-being

of both humans and animals by reducing the

development of antibiotic resistance [163, 164].

Moreover, replacing antibiotics with endolysins

may offer cost savings due to their lower produc-

tion costs, shorter treatment periods, and faster

recovery [165].

To date, extensive research has been done on

the potential application of endolysins in vet-

erinary medicine, particularly in bovine mas-

titis management. A recent study explored the

potential of endolysin LysRODI as a prophylac-

tic treatment for mastitis. This endolysin dem-

onstrated a high killing rate against S. aureus

and S. epidermidis without inducing resistant

mutations. In zebrash and mouse models, Lys-

RODI was non-toxic at concentrations close to

the MIC, providing complete protection against

these pathogens when administered intramam-

mary [166]. In parallel research efforts, chi-

meric endolysin HY-133 displayed high activity

against Livestock-associated methicillin-resistant

S. aureus (LA-MRSA) isolates, offering a hopeful

solution for eliminating MRSA strains common

in animal healthcare settings [99]. Fusion pro-

teins have also provided an alternative approach

to combat bovine mastitis caused by S. aureus.

For instance, the proteins λSA2-E-Lyso-SH3b and

λSA2-E-LysK-SH3b, which combine streptococ-

cal and staphylococcal domains, were assessed

for their efcacy against penicillin-resistant S.

aureus strains associated with mastitis. They

displayed potent activity against the targeted

organisms, reducing the bacterial load by up to

3 logs within 3 h at 100 µg/ml in processed cow

milk. These fusion proteins also demonstrated

synergy when combined with lysostaphin [72].

Furthermore, a study by Fan etal. in 2016 aimed

at developing a new antibacterial agent to com-

bat MRSA in bovine mastitis demonstrated that

the recombinant endolysin Trx-SA1 from bacte-

riophage IME-SA1 can effectively reduce bacte-

rial levels in the mammary gland and manage

mild clinical mastitis induced by S. aureus [167].

Similarly, the recombinant endolysin LysSA4,

expressed in E. coli, was demonstrated as a

proper therapeutic choice due to its lytic activ-

ity against mastitogenic Staphylococcus isolates

of bovine origin [168]. Further instances pertain

to S. aureus bacteriophage phi11 endolysin and

endolysin ΦSH2 from Staphylococcus haemolyti-

cus, which effectively lysed various staphylo-

cocci strains, including MRSA, mastitis isolates,

and CoNS. These endolysins were studied in var-

ying conditions to optimize their effectiveness.

The lytic activity of endolysin phi11 remained

stable under the physiological pH and calcium

concentrations found in milk, and even short

proteins derived from it displayed antimicro-

bial activity against staphylococci. Conversely,

the CHAP domain of endolysin ΦSH2 exhibited

notable lytic activity, especially in physiologi-

cal saline conditions, with increased activity in

the presence of Ca2+. The SH3b domain played a

crucial role in the complete functionality of this

endolysin [169, 170].

It is important to note that despite all the

advantages that endolysins offer compared

to antibiotics, their utilization is not without

constraints. Endolysins target specific bacte-

rial strains, which can limit treating infections

caused by multiple strains or mixed infections.

In contrast, antibiotics may have a broader spec-

trum of activity [124]. On the other hand, these

compounds are not yet widely available for com-

mercial use in veterinary medicine, which can

hinder their widespread adoption as a treatment

option for bovine mastitis [163]. The effective-

ness of endolysins in real-world settings may

differ from controlled laboratory environments,

necessitating validation through eld trials and

practical applications [171]. In addition, regu-

latory approval processes for treatments of an

innovative nature such as endolysins can be

Infect Dis Ther (2025) 14:13–57

intricate and time-consuming, potentially delay-

ing their introduction into routine veterinary

practice [172]. Hence, overcoming these limita-

tions through continued research, development,

and regulatory efforts will be crucial to fully har-

ness the potential of endolysins as an alternative

treatment for bovine mastitis.

Detection of Pathogens

The wide range of diseases caused by staphylo-

cocci, emphasizes the necessity of developing

fast and accurate diagnostic methods in research

and clinical settings. Delayed recognition of

pathogens, especially drug-resistant strains like

MRSA, can lead to increased mortality from

acute bacterial infections, inappropriate treat-

ment, rising healthcare costs, spread of infection

to other patients, and development of antibiotic

resistance [173].

Traditional culture-based detection meth-

ods may be less expensive in terms of instru-

ments and consumables but require signicant

resources like media and reagents, time-con-

suming processes, and specic data collection

techniques [174]. Rapid detection methods, such

as molecular methods (PCR, NASBA, LAMP, and

microarray), offer advantages including high

accuracy, sensitivity, and specicity, and the

ability to detect several pathogens simultane-

ously. However, they require intensive nucleic

acid extraction techniques and can be expensive

due to the need for specialized equipment and

trained personnel. Also, the inability to distin-

guish between the target and other non-target

endogenous microorganisms of the same species

or genus, the lack of differentiation between liv-

ing and non-living cells, and sensitivity to the

presence of inhibitors in the sample, are limita-

tions that should be considered. Immunologi-

cal assays are less expensive and can identify

bacterial toxins, but compared to molecular

methods, they require initial enrichment and

are less sensitive [175, 176]. In recent years, bio-

sensors based on endolysin have been studied as

novel diagnostic tools to overcome the limita-

tions of conventional bacterial detection meth-

ods. These bio-probes offer rapid and real-time

pathogen detection with notable sensitivity and

specicity. They also provide advantages such

as automated detection without pre-enrichment,

miniaturization and portability for on-site test-

ing, discrimination between live and dead cells,

and the potential for targeted engineering to

enhance efcacy and stability [174, 177].

Research in developing innovative diagnos-

tic platforms utilizing staphylococcal endolysins

has demonstrated the capability of endolysins

LysK and Φ11 to efciently bind to staphylo-

cocci when immobilized on silicon wafers [178].

In addition, a new ow cytometry method has

been designed to identify various staphylococcal

strains. This technique, by exploiting different

domains of endolysin E-LM12 in combination

with green uorescent protein, enabled rapid

and specific detection of Staphylococcus (1–5

CFU/ml after 16 h of enrichment step) in blood

culture samples [179]. These advances highlight

the crucial role of precise diagnosis in the ef-

cient management of staphylococcal infections.

The characteristics of the phage-encoded

endolysins with the outcomes following the

experiments or trials are shown in Table2.

TARGETED DELIVERY SYSTEMS

Although numerous studies have demonstrated

the effectiveness of endolysins against bacte-

rial pathogens, selecting appropriate delivery

mechanisms, particularly in clinical cases, can

impact their optimal efciency. Developing a

proper delivery system requires assessing mul-

tiple factors to ensure controlled release, tar-

geted delivery, patient safety, and enhanced

bioavailability [195]. Various drug delivery

methods, including injections, oral, inhala-

tion, transnasal, vaginal, and topical routes,

each offer unique advantages and challenges

in the eld of endolysin therapy [19]. Injec-

tions are a common method for administering

endolysins via intravenous and intraperito-

neal routes [30]. These methods provide direct

access to the target site but face disadvantages

like rapid clearance, immunogenicity, and sec-

ondary bacterial infections [196]. Oral deliv-

ery of endolysins offers unique advantages in

terms of ease of administration and dosage

Infect Dis Ther (2025) 14:13–57

exibility, but it brings problems such as sen-

sitivity to acidic pH in the gastrointestinal

tract, enzymatic degradation, and short serum

half-life, resulting in low bioavailability and

protein structure damage [197]. The topical

application of endolysins involves using oint-

ments, creams, and gels for direct delivery to

infection sites. However, the stratum corneum

can hinder penetration into deeper skin lay-

ers, affecting the target site reach. Maintain-

ing stability in formulations and preventing

enzymatic degradation may also limit thera-

peutic efcacy [198]. Inhalations, trans-nasal,

and vaginal administration of endolysins also

involve delivering the enzymes through the

respiratory tract, nasal cavity, or vaginal tract,

respectively. These routes bypass the gastroin-

testinal tract and hepatic metabolism, lowering

the risk of enzymatic degradation and acidic

pH exposure. Additionally, the high perme-

ability and abundant vasculature in the nasal

cavity and vaginal route facilitate maximum

drug absorption for rapid systemic distribution,

although challenges like mucociliary clearance

and short serum half-life may still arise [196].

In recent years, the development of novel

delivery methods for endolysins has become a

key strategy in combating bacterial infections,

especially in the face of growing antibiotic resist-

ance. Nanoencapsulation is one of these innova-

tive approaches that has emerged to enhance the

stability, shelf life, and therapeutic efcacy of

endolysins [199]. An interesting trial in this eld

utilized the increase in skin temperature during

infection to trigger the controlled release of an

enzybiotic cocktail containing truncated endoly-

sin CHAPK and lysostaphin encapsulated in poly

(N-isopropylacrylamide) nanoparticles. These

particles released endolysins at the infected site

in response to heat (37°C), allowing targeted

delivery and bacterial lysis while maintaining

microora growth at a healthy skin temperature

(32°C) [184]. Similarly, temperature-sensitive

poly (N-isopropylacrylamide) hydrogels as a

delivery system for the phage endolysin Lys84

demonstrated uniform pores, efcient release

of endolysin, and over 99.9% bactericidal activ-

ity against multidrug-resistant S. aureus [185].

In a study conducted in 2020, encapsulation

of endolysin LysMR-5 in alginate-chitosan

nanoparticles (Alg-Chi NPs) demonstrated struc-

tural integrity, consistent bioactivity, and a sus-

tained release pattern, enhancing antibacterial

efcacy against S. aureus [180]. Besides, another

study in the same year exhibited that pH-sensi-

tive liposomes encapsulating endolysin LysRODI

enabled targeted delivery against S. aureus under

mildly acidic conditions, effectively reducing

cell counts in both planktonic and biolm forms

[183]. Further research on the development of

new delivery routes tailored to the site of infec-

tion and the intended therapeutic outcome will

increase the effectiveness of endolysins, particu-

larly against resistant strains like MRSA.

PRECLINICAL EXPERIMENTS AND

CLINICAL TRIALS

Despite extensive efforts to combat staphylococ-

cal infections, the increasing complexity and

prevalence of these infections, the opportunis-

tic nature of staphylococci, and their increasing

antibiotic resistance have challenged current

treatment strategies and necessitated the search

for effective treatments [200]. To date, multiple

trials have been undertaken to assess the effec-

tiveness of anti-staphylococcal treatments utiliz-

ing endolysins.

Between 2013 and 2016, a research team in

India assessed the effectiveness of combining the

staphylococcal endolysin MR-10 with common

antibiotics for treating MRSA-induced diabetic

wound and burn wound infections in mouse

models. The results indicated that monotherapy

with MR-10 had comparable outcomes to lin-

ezolid in treating local foot infections in diabetic

mice. The synergy between the two therapeutic

agents was signicantly more effective in con-

trolling infection and accelerating tissue heal-

ing. Additionally, the combination of MR-10 and

minocycline led to a 100% survival rate among

mice with systemic burn wound infections. The

group that underwent the combined treatment

exhibited the most signicant decrease in bac-

terial load in various organs, with no signs of

inammation noted histologically [193, 194].

Likewise, in a 2011 study by Gu etal., the

endolysin LysGH15, derived from phage

Infect Dis Ther (2025) 14:13–57

Table 2 Characteristics of the phage-encoded endolysins with antibacterial activity

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

LysMR-5 S. aureus Phage MR-5 S. aureus

S. epidermidis

e functionality of

alginate-chitosan

nanoparticles as a car-

rier to protect endoly-

sin from degradation

and enhance bacterial

interaction

[180]

Full ecacy against

MRSA and 95% of S.

epidermidis strains

[37]

LysK S. aureus phageK S. aureus Synergistic eects with

poly-N-acetylglucosa-

mine depolymerase

DA7 against staphylo-

coccal biolms

[132]

S. aureus

S. epidermidis

Utilization as an e-

cient and easy-to-use

biosensor for detecting

staphylococci

[178]

S. aureus Eective synergy

between endolysin and

lysostaphin as antimi-

crobial agents

[127]

S. aureus

S. epidermidis

S. haemolyticus

S. hominis

S. saprophyticus

S. capitis

S.chromogenes

S.caprae

S.hyicus

Cloning and expres-

sion of endolysin in L.

lactis and lytic activity

against bovine and

human staphylococci

[48]

LysKΔamidase S. aureus LysK amidase domain

deletion

S. aureus

S. epidermidis

S. haemolyticus

S. hominis

Extensive lytic activity

against MRSA and

MSSA strains isolated

from bovine milk and

humans

[92]

Infect Dis Ther (2025) 14:13–57

Table 2 continu ed

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

LysGH15 S. aureus PHAGE GH15 S. aureus Eective treatment of

MRSA-induced pneu-

monia, in combination

with apigenin com-

pared to monotherapy

[181]

S. aureus Lytic activity against

various strains of S.

aureus and protection

of mice from MRSA

bacteremia

[182]

S. aureus Reduced bacterial levels

and inammation-

related cytokine

expressions in mice

infected with MRSA

[55]

S. aureus Reduction in pro-

inammatory

cytokines; no impact

of humoral immunity

on its eectiveness

against MRSA infec-

tion in mice

[56]

S. aureus

S. epidermidis

S. haemolyticus

S. hominis

Antimicrobial eects

against planktonic cells

and biolms of various

staphylococcal species

[119]

CHAPLysGH15 S. aureus Catalytic domain of

LysGH15

S. aureus Signicant lytic activity

against S. aureus in

whole and skim milk

[87]

LysGH15B S. aureus SH3b Domain of

LysGH15

S. aureus

S. epidermidis

Specic binding anity

to staphylococcal iso-

lates, especially MRSA

[88]

Infect Dis Ther (2025) 14:13–57

Table 2 continu ed

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

LysRODI S. aureus Myophage phiIPLA-

RODI

S. aureus Encapsulation of endoly-

sin in pH-responsive

liposomes for con-

trolled release against

plankton and S. aureus

biolm in mild acidity

[183]

S. aureus

S. epidermidis

S. sciuri

S. hominis

S. pasteuri

S. xylosus

S. saprophyticus

S. arlattae

S. haemolyticus

S. gallinarum

S. kloosii

Lysis of staphylococci

with low MIC values,

antibiolm activity

and prevention of

mammary infections

in mice

[166]

S. aureus Combined use of

bacteriocins and bac-

teriophages in order

to control S. aureus

contamination in dairy

production

[148]

LysRODIΔAmi S. aureus LysRODI amidase

domain deletion

S. aureus Reduction of staphylo-

coccal contamination

to undetectable levels

during fresh cheese

production

[95]

S. aureus

S. epidermidis

S. saprophyticus

S. haemolyticus

S. hominis

S. sciuri

S. pasteuri

S. xylosus

S. arlattae

S. gallinarum

S. kloosii

Broad lytic spectrum,

high stability, strong

anti-biolm activity,

non-toxicity to human

keratinocytes, and

complete eradication

of S. aureus in a pig

skin model

[94]

Infect Dis Ther (2025) 14:13–57

Table 2 continu ed

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

CHAPKS. aureus LysK CHAP domain S. aureus Targeted release

of endolysin and

lysostaphin enclosed

within thermally trig-

gered nanoparticles at

the sites of S. aureus

infection

[184]

Staphylococcus Superior lytic activity

against viable staphy-

lococcal strains invitro

compared to native

form

[84]

S. aureus Biocidal agent for

preventing and treating

biolm-associated

staphylococcal infec-

tions

[85]

S. aureus High specicity and

solubility and eective

removal of S. aureus

from the mouse nares

[86]

LysSAP26 S. aureus Phage SAP-26 A. baumannii

E. coli

K. pneumoniae

P. aeruginosa

S. aureus

E. faecium

Inhibitory eects

against various types

of multidrug-resistant

bacteria and protection

of mice infected with

A. baumannii

[40]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

Lys84 S. aureus Phage qdsa002 S. aureus Broader bactericidal

range and greater

anti-biolm activity

of endolysin and its

domains compared to

the parent phage

[117]

S. aureus Notable antibacterial

properties of Lys8-

containing hydrogels

as an ecient deliv-

ery system against

multidrug-resistant S.

aureus

[185]

LysSAP33 S. aureus Phage SAP33 S. aureus Broader lytic activity

and higher ecacy

against biolms in

comparison to LysK-

like endolysin

[118]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

SAL200 S. aureus Recombinant form of

endolysin SAL-1

S. aureus Higher survival rate,

reduced bacterial load

in lung and blood,

and increased lung

histopathology follow-

ing intranasal admin-

istration of endolysin

in mouse pneumonia

model

[186]

S. aureus Synergistic eects with

standard-of-care

(SOC) antibiotics

against S. aureus in

both vitro and invivo

[128]

S. aureus Eective removal of

plankton cells and S.

aureus biolm and

increased survival of

MRSA-infected mice

[120]

Lack of adverse eects

in safety and pharma-

cokinetic assessments

conducted in animal

models

[187]

Acceptable safety and

tolerability of intra-

venous endolysin in a

phase I human trial

[188]

Tolerance of intravenous

endolysin injection in

monkeys without any

adverse eects

[189]

LysCSA13 S. aureus Phage CSA13 S. aureus Elimination of staphy-

lococcal cells and

biolms from food

processing surfaces

[140]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

LysP108 S. aureus Phage P108 S. aureus

P. aeruginosa

Potent antibacterial and

antibiolm activity,

alongside synergistic

eects with vancomy-

cin invivo

[129]

LysSA11 S. aureus Phage SA11 S. aureus Potential of a yeast

surface display system

for expressing endoly-

sin with improved

bactericidal activity

and stability

[45]

S. aureus Robust anti-staphylo-

coccal activity in food

products and utensils

[141]

LysSA52 S. aureus Phage 52 S. aureus

S. epidermidis,

S. haemolyticus

S. pneumonia

S. pyogenes

E. faecium

E. faecalis

B. atrophaeus

Anti-biolm activity and

extensive lytic eects

against antibiotic-

resistant Gram-positive

bacteria

[41]

LysSAP8 S. aureus Phage SAP8 S. aureus Signicant decrease in

the proliferation of

MRSA strains with

combined treatment of

endolysin and nisin

[149]

S. aureus

S. epidermidis

S. hominis

S. sciuri

S. warneri

S. xylosus

Lytic activity against

a broad spectrum of

staphylococcal strains

[38]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

PlyGRCS S. aureus Phage GRCS S. aureus

S. epidermidis

Dose-dependent antimi-

crobial activity against

both planktonic and

biolm forms of S.

aureus and lytic activ-

ity on all tested strains

of S. epidermidis and S.

aureus

[33]

S. aureus Strong lytic activity

against MRSA, reduc-

tion of CspC–nucleic

acid binding by com-

plex with CspC and

possibly downregulat-

ing of its function in

infection

[25]

LysSA97 S. aureus Phage SA97 S. aureus Synergistic eect of

endolysin and carvac-

rol in inactivation of S.

aureus in milk and beef

[152]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

LysH5 S. aureus Phage vB_SauS-phi-

IPLA88

S. aureus Eradication of S. aureus

in milk by combined

use of endolysin and

nisin

[144]

S. aureus

S. epidermidis

Antibacterial activity

against bovine and

human S. aureus,

as well as human S.

epidermidis strains,

and the inhibition of

S. aureus growth in

pasteurized milk

[143]

Cloning and express-

ing of endolysin in

the L. lactis as a food

preservative additive

[49]

S. aureus

S. epidermidis

Eective activity against

S. aureus and S. epi-

dermidis biolms and

inhibition of persister

cells

[123]

Trx-sa1 S. aureus Phage IME-SA1 S. aureus Treatment of mild

bovine mastitis caused

by S. aureus

[167]

SAL-2 S. aureus Phage SAP-2 S. aureus

S. epidermidis

S. haemolyticus

Greater anti-biolm

activity against S.

aureus compared to

SAP-2

[115]

P-27/HP S. aureus Phage P-27/HP S. aureus Extensive lytic activity

against antibiotic-

resistant S. aureus

strains and protection

of mice from bactere-

mia without harmful

physiological eects

[190]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

LysSA4 S. aureus Phage SA4 MastitogenicStaphy-

lococcusisolates of

bovine origin

Mild lytic eect against

Staphylococcus isolates

responsible for bovine

mastitis

[168]

HydH5 S. aureus Phage vB_SauS-phiI-

PLA88

S. aureus ermal stability and

potent lytic activity

against S. aureus, all

without the need for

divalent cations

[145]

Bovine and human

S. aureusstrains

S. epidermidis

Staphylolytic activity

of fusion proteins and

truncated derivatives of

endolysin

[191]

Endo88 S. aureus Phage 88 S. aureus

S. epidermidis

Lytic eect of recom-

binant endolysin

expressed and secreted

in L. lactis against

multidrug-resistant S.

aureus

[50]

Lys-phiSA012 S. aureus Phage phiSA012 S. aureus

S. pseudintermedius

S. haemolyticus

Improving the lytic

capacity of endolysin

by adding Ca2 + and

Zn2 + while reducing

the amount of oxacillin

required to inhibit bac-

terial growth

[90]

S. pseudintermedius

S. schleiferi

S. intermedius

Greater lytic activ-

ity than the parent

bacteriophage against

staphylococcal isolates

obtained from dog skin

[192]

Infect Dis Ther (2025) 14:13–57

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

MR-10 S. aureus Phage MR-10 S. aureus Full protection of mice

from MRSA burn

infection through the

combined action of

endolysin and mino-

cycline

[193]

S. aureus e combined eect of

endolysin and linezolid

to accelerate tissue

healing and treat S.

aureus diabetic foot

infection in mice

[194]

MV-L S. aureus Phage phi MR11 S. aureus

S. simulans

Elimination of MRSA

from the nares of mice

and protection against

septic death without

adverse eects

[53]

phi11

(Lys11)

S. aureus Phage phi11 S. aureus

S. chromogenes

S. epidermidis

S. hyicus

S. simulans

S. warneri

S. xylosus

Eective lysis of

staphylococcal mastitis

pathogens

[169]

S. aureus

S. epidermidis

S. simulans

Anti-biolm eect

against staphylococcal

isolates

[93]

S. aureus Synergy of antimicro-

bial peptide R8K and

endolysin to enhance

bacteriolysis (1–3 log

reduction in CFU/ml)

[131]

Infect Dis Ther (2025) 14:13–57

GH15, effectively killed various strains of S.

aureus, including MRSA, invitro. Animal stud-

ies revealed that intraperitoneal injection of

LysGH15 protected mice from MRSA bactere-

mia [182]. Afterward, a study in 2016 exam-

ined the potential of a combination therapy

involving LysGH15 and apigenin, a natural

plant avonoid, against S. aureus pneumonia in

mice. This combination approach demonstrated

greater efcacy than monotherapy, signicantly

reducing lung bacterial load, inflammatory

responses, and lung tissue damage [181]. Moreo-

ver, endophthalmitis induced by ocular patho-

gens like S. aureus was successfully treated for the

Table 2 continued

Endolysin/

endolysin

derivative

Origin of phage Phage Target Outcome Refs.

E-LM12 S. aureus Phage vB_SauM-LM12

(LM12)

S. aureus Strong antibiolm

eect and wide host

range against dierent

isolates of S. aureus

[116]

S. aureus

S. epidermidis

S. equorum

S. haemolyticus

S. hominis

S. warneri

S. capitis

Sensitive and specic

detection of Staphy-

lococcus in blood by

exploiting dierent

domains of endolysin

through ow cytom-

etry

[179]

LysSAP27 S. aureus Phage vB_SauS-

SAP27(ϕSAP27)

S. aureus

S. condimenti

S. hominis

S. sciuri

S. warneri

Strong lytic activity

against S. aureus in

contaminated milk

[146]

SAL-1 S. aureus Phage SAP-1 S. aureus

S. saprophyticus

S. epidermidis

Greater bacterial cell

wall hydrolyzing activ-

ity than LysK, due to

a specic amino acid

residue change

[24]

Ph28 S. epidermidis Phage PH15 S. epidermidis More eective lysis of

S. epidermidis cells

compared to other

staphylococcal strains

[35]

ΦSH2 S. haemolyticus Prophage ΦSH2 S. aureus

S. chromogenes

S. hyicus

S. simulans

S. warneri

S. xylocus

Eective removal of

MRSA, mastitis

isolates, and CoNs by

complete lysine and its

CHAP domain

[170]

Infect Dis Ther (2025) 14:13–57

rst time utilizing Ply187 chimeric endolysin in

an experimental model. The laboratory experi-

ments illustrated reduced bacterial turbidity,

biolm disruption, and sustained antimicrobial

efficacy across multiple generations without

resistance. Intravitreal administration of endoly-

sin in infected mice maintained retinal integrity

and visual function while decreasing bacterial

load and inammatory cytokine levels [101].

Recombinant endolysin SAL200 (N-Rephasin®

SAL200) was another therapeutic candidate that

was subjected to laboratory and clinical studies

by Jun etal. It should be noted that the strong

bactericidal activity, synergistic potential, and

anti-biolm effects of this endolysin against

MRSA were previously documented by the same

researchers in both invitro and invivo stud-

ies [24, 120, 128]. Safety and pharmacokinetic

assessments of intravenous SAL200 were then

conducted in animal models, such as mice, dogs,

and monkeys, reporting no adverse effects or

abnormal ndings [120, 187, 189]. In the fol-

lowing, based on the acceptable outcomes of

preclinical studies, a phase I human trial was

designed to evaluate the tolerance, pharma-

cokinetics, and pharmacodynamics of a single

intravenous dose of SAL200 at increasing con-

centrations (0.1 to 10 mg/kg) in 34 healthy male

participants. The results of this trial exhibited

acceptable safety and tolerability in humans,

without serious adverse events or clinically sig-

nicant changes [188]. Finally, in 2017 (Korea),

a phase IIa clinical trial was initiated in patients

with persistent bacteremia caused by S. aureus,

which was terminated before completion. To

complement prior studies that focused on intra-

venous injection, Bae etal. in 2019, explored

the effectiveness of intranasal delivery of this

endolysin in treating lethal murine pneumo-

nia caused by MSSA. The positive results of

this study included increased survival rates,

improved lung histopathology, and no change

in serum cytokine levels after treatment [186].

Besides, two other recombinant endolysins,

Staphefekt SA.100 and XZ.700, are in clinical

development for acute and chronic staphy-

lococcal skin infections. In a 2022 study by

Eichenseher etal., XZ.700 demonstrated supe-

rior activity compared to Staphefekt SA.100 in

lab tests, selectively killing S. aureus without

inducing resistance. In practical applications,

XZ.700 formulated as cream and gel eliminated

S. aureus on human skin models and notably

decreased bacterial counts in a mouse model

of skin infection [64]. In the wake of that, an

exvivo study at the University of Copenhagen

in 2023 revealed that XZ.700 hindered the colo-

nization and growth of pathogenic S. aureus on

the skin, reduced inducible cytokine produc-

tion, and prevented the bacterium’s potential

to stimulate tumor development in malignant T

cells within cutaneous T-cell lymphoma (CTCL)

[67]. Moreover, considering that an imbalance

in the skin’s microbial community can elevate

the susceptibility to infections by resistant bac-

teria, especially S. aureus [201], a recent interven-

tion study illustrated the efcacy of XZ.700 in

selectively inhibiting S. aureus on porcine skin

without impacting the resident microbiota.

This inhibition led to faster wound healing and

the restoration of microbial diversity [68]. The

commercialization of this endolysin for treating

patients with atopic dermatitis is advancing fol-

lowing the beginning of the phase I/IIa clinical

trial in 2020 by the Micreos Biotech Company

in the Netherlands. In this study, the safety and

efcacy of endolysin will be evaluated in 48

patients by applying a topical cream containing

XZ.700 at three varying concentrations over 14

days (www. trial regis ter. nl/ trial/ 8876).

Staphefekt SA.100, marketed as Gladskin, is

another Micreos company product approved

by the European Medical Agency [124]. It is

available over-the-counter in cream and gel

forms at pharmacies and used to treat chronic

skin conditions like eczema and acne caused

by S. aureus [70]. This endolysin during the

invitro assessments in 2014 demonstrated a

dose-dependent and effective response against

MSSA and MRSA strains in a lysis assay with

a greater reduction in OD for MSSA, while

other coagulase-negative staphylococci strains

were minimally affected. Both MRSA and

MSSA strains displayed similar sensitivity to

the endolysin, and resistance induction was

not observed in lab tests [69]. Furthermore, in

2016, Totté etal. initiated a phase I/II clini-

cal trial with 100 participants diagnosed with

moderate to severe atopic dermatitis and recent

corticosteroid utilization. This study aimed to

Infect Dis Ther (2025) 14:13–57

compare the need for corticosteroid adjuvant

therapy, clinical efcacy, quality of life, and

microbial composition between the Staphefekt

and placebo groups over a 3-month treatment

regimen using a cetomacrogel-based cream.

Initial results indicated good drug tolerance,

reduced bacterial load, and no adverse effects

[202]. Subsequently, in 2017, this research

group published a case series highlighting the

potential of Staphefekt in improving chronic

skin dermatosis associated with S. aureus. The

study included three patients with skin mani-

festations such as nummular eczema with

impetigo, and bacterial folliculitis on their

trunk and legs. Previous treatments with stand-

ard steroids and antibiotics had led to infection

recurrence and treatment resistance. Treatment

with Gladskin resulted in a signicant reduc-

tion in clinical symptoms without side effects

or drug resistance during the treatment period.

Nevertheless, discontinuing the treatment led

to the recolonization of S. aureus and a recur-

rence of symptoms [70].

In addition to the endolysin discussed ear-

lier, chimeric endolysin P128 has progressed

to clinical phase II in Singapore. Extensive

laboratory research has reported signicant

benets of P128, including its specicity in

targeting the Staphylococcus genus, anti-bio-

lm properties, a low probability of resistance

development, lack of cytotoxicity, and syn-

ergistic potential with other antibiotics [74,

105, 203, 204]. Moreover, preclinical studies

using animal models, like mice and dogs, have

demonstrated promising results in nasal decol-

onization, improving symptoms of MRSA bac-

teremia, and treating staphylococcal pyoderma

[102, 106, 205–207]. The preliminary ndings

of the clinical trial of P128 in both healthy

individuals and patients with MRSA nasal car-

riage have indicated that intranasal adminis-

tration of this endolysin is well tolerated and

effective in reducing the bacterial load in the

nasal cavity (ClinicalTrials.gov NCT01746654).

The scientic community’s growing interest

in endolysins and the successful outcomes of

preclinical and clinical studies have propelled

several endolysins beyond the research labo-

ratory to be considered for commercial and

medical applications. However, numerous

challenges in the path to the clinical develop-

ment of endolysins, such as potential stimula-

tion of immune responses, limited cell penetra-

tion, the need for multiple doses due to short

half-life, high production costs, limitations in

drug delivery methods, and the intricate regu-

latory approval processes, have led to only one

endolysin-based treatment (Staphefekt SA.100)

being accepted as a medical product for human

use [51, 124, 208]. Hence, ongoing efforts to

address these challenges through innovative

research, biotechnological advancements, and

strategic collaborations are imperative to fully

harness the potential of endolysins as effective

antimicrobial treatments.

Author Contributions. Mina Golban and

Hamid Heidari contributed to the conceptu-

alization, design, search, and writing of the

manuscript. Hamid Heidari, Javad Charostad,

and Hossein Kazemian reviewed and edited the

manuscript. All authors approved the nal ver-

sion for submission.

Funding. No funding or sponsorship was

received for this study or publication of this

article.

Declarations

Conict of Interest. Mina Golban, Javad

Charostad, Hossein Kazemian, and Hamid Hei-

dari declare no conicts of interest.

Ethical Approval. This article is based on

previously conducted studies and does not con-

tain any new studies with human participants or

animals performed by any of the authors.

Open Access. This article is licensed under a

Creative Commons Attribution-NonCommercial

4.0 International License, which permits any

non-commercial use, sharing, adaptation, distri-

bution and reproduction in any medium or for-

mat, as long as you give appropriate credit to the

original author(s) and the source, provide a link

to the Creative Commons licence, and indicate

if changes were made. The images or other third

party material in this article are included in the

Infect Dis Ther (2025) 14:13–57

article’s Creative Commons licence, unless indi-

cated otherwise in a credit line to the material.

If material is not included in the article’s Crea-

tive Commons licence and your intended use is

not permitted by statutory regulation or exceeds

the permitted use, you will need to obtain per-

mission directly from the copyright holder. To

view a copy of this licence, visit http:// creat iveco

mmons. org/ licen ses/ by- nc/4. 0/.

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The Unique Capability of Endolysin to Tackle Antibiotic Resistance: Cracking the Barrier

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The lack of new antibacterial medicines and the rapid rise in bacterial resistance to antibiotics pose a major threat to individuals and healthcare systems. Despite the availability of various antibiotics, bacterial resistance has emerged for almost every antibiotic discovered to date. The increasing prevalence of multidrug-resistant bacterial strains has rendered some infections nearly untreatable, posing severe challenges to health care. Thus, the development of alternatives to conventional antibiotics is critical for the treatment of both humans and food-producing animals. Endolysins, which are peptidoglycan hydrolases encoded by bacteriophages, represent a promising new class of antimicrobials. Preliminary research suggests that endolysins are more effective against Gram-positive bacteria than Gram-negative bacteria when administered exogenously, although they can still damage the cell wall of Gram-negative bacteria. Numerous endolysins have a modular domain structure that divides their binding and catalytic activity into distinct subunits, which helps maximize their bioengineering and potential drug development. Endolysins and endolysin-derived antimicrobials offer several advantages as antibiotic substitutes. They have a unique mechanism of action and efficacy against bacterial persisters (without requiring an active host metabolism); subsequently, they target both Gram-positive and Gram-negative bacteria (including antibiotic-resistant strains), and mycobacteria. Furthermore, there has been limited evidence of endolysin being resistant. Because these enzymes target highly conserved links, resistance may develop more slowly compared to traditional antibiotics. This review provides an overview and insight of the potential applications of endolysins as novel antimicrobials.

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Food contamination by pathogenic and spoilage bacteria causes approximately 47 million cases of foodborne diseases in the United States and leads to tons of food spoilage, worsening the food loss situation worldwide. In addition, conventional preservation treatments implemented in the food industry decrease food’s nutritional and organoleptic quality. Therefore, there is a need for new alternatives to counteract food contamination without altering its characteristics. Endolysins are a promising strategy due to their unique properties, such as host specificity, synergism with other antibacterial agents, mode of action, and low probability of resistance development. These characteristics differentiate them from other antibacterial agents used in the food industry. Endolysins are enzymes produced by bacteriophages during the process of bacterial infection and lysis. This review describes the advances related to endolysin application systems in food, considering their potential for food safety and an overview of the application conditions according to the type of food and bacteria to be controlled. We also highlight the need for new studies on endolysin encapsulation and prolongation of the action time in cases of outbreaks that allow obtaining key information to improve the application of endolysins in different food matrices during food processing and storage

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The rise of Multi-Drug Resistant (MDR) bacterial pathogens to most, if not all, currently available antibacterial agents has become a global threat. As a consequence of the antibiotic resistance epidemic, phage therapy has emerged as a potential alternative to conventional antibiotics. Despite the high therapeutic advantages of phage therapy, they have not yet been successfully used in the clinic due to various limitations of narrow host specificity compared to antibiotics, poor adhesion on biofilm surface, and susceptibility to both human and bacterial defences. This review focuses on the antibacterial effect of bacteriophage and their recent clinical trials with a special emphasis on the underlying mechanism of lytic phage action with the help of endolysin and holin. Furthermore, recent clinical trials of natural and modified endolysins and some marketed products have also been emphasized with future prospective.

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Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of A. baumannii and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic in vitro characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins.

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Methicillin Resistant Staphylococcus aureus: Molecular Mechanisms Underlying Drug Resistance Development and Novel Strategies to Combat

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Antimicrobial resistance (AMR) represents a major threat to global health. Infection caused by Methicillin-resistant Staphylococcus aureus (MRSA) is one of the well-recognized global public health problem globally. In some regions, as many as 90% of S. aureus infections are reported to be MRSA, which cannot be treated with standard antibiotics. WHO reports indicated that MRSA is circulating in every province worldwide, significantly increasing the risk of death by 64% compared to drug-sensitive forms of the infection which is attributed to its antibiotic resistance. The emergence and spread of antibiotic-resistant MRSA strains have contributed to its increased prevalence in both healthcare and community settings. The resistance of S. aureus to methicillin is due to expression of penicillin-binding protein 2a (PBP2a), which renders it impervious to the action of β-lactam antibiotics including methicillin. The other is through the production of beta-lactamases. Although the treatment options for MRSA are limited, there are promising alternatives to antibiotics to combat the infections. Innovative therapeutic strategies with wide range of activity and modes of action are yet to be explored. The review highlights the global challenges posed by MRSA, elucidates the mechanisms underlying its resistance development, and explores mitigation strategies. Furthermore, it focuses on alternative therapies such as bacteriophages, immunotherapy, nanobiotics, and antimicrobial peptides, emphasizing their synergistic effects and efficacy against MRSA. By examining these alternative approaches, this review provides insights into the potential strategies for tackling MRSA infections and combatting the escalating threat of AMR. Ultimately, a multifaceted approach encompassing both conventional and novel interventions is imperative to mitigate the impact of MRSA and ensure a sustainable future for global healthcare.

Antimicrobial Resistance of Cattle Mastitis-Causing Bacteria: How to Treat?

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Cattle mastitis continues to be a global burden for the dairy industry, and its control depends on preventive measures, rapid detection and identification of involved pathogens and accurate antimicrobial treatment. The (mis)use of antimicrobials initiated a rapid evolutionary process of bacterial resistance by natural selection and led to the increased frequency and spread of bacterial antimicrobial resistance (AMR). The global AMR emergency and the prudent use of antimicrobials in cows have raised questions about alternative treatment approaches; however, the use of antimicrobials remains the principal method for mastitis therapy. This chapter summarise the current knowledge on AMR in cattle mastitis as a multifactorial global problem, the trends in AMR patterns in the most common mastitis-causing bacterial pathogens and altering factors, the policies and actions restricting the use of antimicrobials in cows and related challenges in the treatment. The reasons influencing the outcome of treating an intramammary infection, such as the selection of appropriate antimicrobial agents, optimal drug regimens, the gaps in antimicrobial susceptibility testing (AST) of mas-titis pathogens and interpretation criteria, and the paradoxical relationship between antimicrobial in vitro activity and in vivo efficacy are discussed. The importance of effective mastitis control programmes is emphasised by an overview of (accurate) diagnosis, the evaluation of the therapy, cow health control and farm management practices.

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Preparation and properties of hydrogels loaded with phage endolysin Lys84

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Sep 2023
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This study investigated the application of poly(N-isopropylacrylamide)-based interpenetrating network temperature-sensitive hydrogels (notation: IPNT) as the delivery vehicle for phage endolysin Lys84 and the potential of drug-loaded hydrogels as antimicrobial materials. Interpenetrating network temperature-sensitive hydrogels were prepared by free radical polymerization of sodium alginate and N-isopropylacrylamide. Drug-loaded hydrogels (IPNT-Lys84) were obtained by dry soaking method with the endolysin Lys84 of Staphylococcus aureus phage. The physical properties of the hydrogels with and without drug loading were characterized by infrared spectroscopy, scanning electron microscopy, and differential scanning calorimetry. The swelling and deswelling of the hydrogels as well as the release of endolysin Lys84 were investigated. Moreover, the antibacterial properties of IPNT-Lys84 hydrogels at different temperatures and concentrations of the drug solution were studied. The results showed that IPNT-Lys84 hydrogel had uniform pores and a low critical solubility temperature (LCST) of 32 ℃. The equilibrium swelling of the hydrogel was 30 g/g, and the water loss rate was 88% upon deswelling. The release rate of endolysin reached more than 70% within 6 h at 37 ℃. The bactericidal rate of IPNT-Lys84 hydrogel was over 99.9%. The research results showed the feasibility of using IPNT to deliver the endolysin Lys84, and IPNT-Lys84 hydrogel might be an effective antimicrobial material against multi-drug resistant Staphylococcus aureus.

Phage-Derived Endolysins Against Resistant Staphylococcus spp.: A Review of Features, Antibacterial Activities, and Recent Applications

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