Endolysins are proteins that are expressed late in the bacteriophage virus’s lytic cycle. After the bacteriophage completes its lytic cycle within the host, endolysins break down the host’s cell wall by splitting the host’s peptidoglycan to release offspring virions (Abdelrahman et al., 2021b). Gram-positive bacteriophage endolysins have a modular structure, with EADs at the N-terminus and CBDs attached at the C-terminus via a linker. Endolysins have enzymatic hydrolysis and substrate identification capabilities due to the presence of EADs and CBDs. Typically, modular endolysins have one or two EADs at the N-terminal and CBD at the C-terminal connected by a flexible region known as the linker (Gondil et al., 2021b). The N-terminal EAD of modular endolysins cleaves certain peptidoglycan bonds in the host bacterium’s murein layer, while the C-terminal CBD identifies and connects to numerous epitopes in the cell wall for optimal binding of the EAD’s catalytic actions (Wang et al., 2023). The bacteriophage-derived endolysins of Gram-negative hosts can be structured in different kinds of ways, although most of them have a basic globular EAD domain without a CBD. The latest study also discovered Gram-negative endolysins with globular structures, one or two CBDs at the N-terminus, and the EAD module at the end of the C-terminal region (Briers et al., 2007). Endolysins are categorized based on their cleavage site. These enzymes include lysozymes (N-acetylmuramidases), glycosidases (N-acetyl--d-glucosamidases), N-acetylmuramoyl-l-alanine amidases, and L-alanoyl-d-glutamate endopeptidases (Khan et al., 2023b). Endolysins, which are composed of one of these four N-terminals connected to a distinct cell wall-binding domain, also be known as bacteriophage-encoded cell wall hydrolases (Murray et al., 2021).

Glycosidases: The polymeric structures of N-acetylmuramic acids (MurNAc) and N-acetylglucosamines (GlcNAc) are linked by −1,4 glycosidic linkages that are split by glycosidases. N-acetyl--D-muramidase’s split bonds within GlcNAc and MurNAc residues. N-acetyl--D-glucosidases split bonds within GlcNAc and MurNAc residues (Matamp and Bhat, 2019). Like the other two glycosidases, transglycosylases cleave −1,4 bonds within GlcNAc and MurNAc, but they additionally play a role in an intramolecular mechanism that results in the production of a 1,6-anhydro ring at the MurNAc residue (Rodríguez-Rubio et al., 2016a). Lysozymes, also known as N-acetylmuramidases, eradicate bacteria by specialized hydrolysis. Glycosidases, or N-acetyl-β-d-glucosamidases, control the hydrolysis of the glycosidic link. Glycosidic linkages β-1,4 bind the NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid) monomers of peptidoglycan polymers together. Lysozyme acts to break the structural integrity of the peptidoglycan cell wall by hydrolyzing these connections. This results in an imbalance in the turgor pressure, which kills the bacterial cell (Ragland and Criss, 2017).

Amidases: The mechanism by which N-acetylmuramoyl-L-alanine amidases, also referred to as peptidoglycan amidases act is to break the amide bond that separates the glycan strand from the stem peptide that is located between the L-alanine and N-acetylmuramic acid residues (Abdelrahman et al., 2021b).

Endopeptidases: Enzymes called endopeptidases dissolve the bonds that hold two amino acids together in the stem peptide. Both L-alanoyl-d-glutamate endopeptidases and interpeptide bridge-specific endopeptidases target the peptide that makes up the L-alanoyl-d-glutamate link. Between stem peptides or inside an interpeptide bridge, bonds can break (Rodríguez-Rubio et al., 2016a).

Treatment of A. baumannii infections is complex due to its high capacity for acquiring resistance genes and its natural resistance to a wide range of antibiotics (Poirel et al., 2011). Acinetobacter baumannii infections are frequently treated with colistin as a last-line antibiotic. Still, an increasing number of reports on colistin-resistant strains indicate that the era of antibiotics is challenging (Lim et al., 2019). Carbapenems were once the primary therapy for MDR A. baumannii infections, but their use has resulted in an increase in carbapenem resistance in recent years (Nordmann and Poirel, 2019). Polymyxins are currently routinely used as the antibiotics of choice for treating MDR A. baumannii infections, despite initially being prohibited due to systemic toxicities (nephrotoxicity and neurotoxicity) (Piperaki et al., 2019). Extensive drug-resistant (XDR) A. baumannii is classified as an isolate resistant to three or more of the antibiotic classes (penicillin’s and cephalosporins, which include inhibitor combinations, fluoroquinolones, and aminoglycosides, with carbapenem resistance in the majority of cases). In contrast, pan-drug-resistant (PDR) A. baumannii is an XDR isolate resistant to polymyxins and tigecycline (Mulani et al., 2019). Antibiotic resistance mechanisms fall into three categories. First, resistance can be developed by decreasing membrane permeability or increasing antibiotic efflux, thereby blocking access to the target. Secondly, bacteria can safeguard the antibiotic receptor through genetic mutation or post-translational modification. Finally, antibiotics can be immediately inactivated through hydrolysis or alteration (Blair et al., 2015). One of Acinetobacter’s most powerful weapons is its remarkable genetic adaptation, which allows for rapid genetic changes and rearrangements, as well as the incorporation of foreign determinants carried by mobile genetic components. Insertion sequences are regarded as one of the primary mechanisms shaping bacterial genomes and, ultimately, evolution (Vrancianu et al., 2020). Furthermore, A. baumannii can form biofilms, extending its ability to survive on healthcare equipment, such as ventilators in intensive care units (ICUs) (Pakharukova et al., 2018). However, the link between biofilm development and antibiotic resistance is currently unclear (Yang et al., 2019). Many therapeutic options have been failed by A. baumannii, making treatment of these infections difficult and, in some circumstances, impossible. Given the scarcity of treatment alternatives, there is a need for an innovative strategy and rethinking of remedies for combating this bacterium (Oyejobi et al., 2022b).

The emergence of antibiotic-resistant A. baumannii necessitates the development of alternative treatment options, with many global pharmaceutical industry players strategically choosing to discontinue or outsource novel antibiotic discovery programs. One of the most promising methods is endolysin therapy, which uses endolysin to combat bacterial pathogens (Khan et al., 2021). Endolysins are peptidoglycan hydrolytic enzymes encoded by bacteriophages that have great potential as a new class of antimicrobial therapeutic agents for multidrug-resistant bacteria (Gondil et al., 2021a). Due to the widespread emergence of antibiotic resistance, endolysins have drawn more attention as an alternative therapy. There is strong evidence that endolysins can externally degrade peptidoglycan without the aid of a bacteriophage. Their incorporation into therapeutic approaches has consequently created new opportunities for treating bacterial infections in the human, veterinary, agricultural, and biotechnology fields (Abdelrahman et al., 2021a). Endolysins are a desirable alternative due to their lytic potential against various bacterial species that cause human infections. Endolysins are the most successful alternative therapy for treating and curing MDR bacteria. According to reports numerous endolysins have been used successfully to treat bacteria resistant to antibiotics. The antibacterial treatment through bacteriophage-derived endolysin is tested in animal experiment models. Due to their extraordinary therapeutic potential against multidrug-resistant bacteria, endolysin has attracted much attention in recent years (Mehmood Khan et al., 2023). Endolysin are bacteriophage-derived proteins which have high specificity for bacteria, they have no detrimental effects on human or animal cells. Endolysin lacks many features that make antibiotics superior to them, such as no resistance and high specificity (Abdelrahman et al., 2021a; Gondil et al., 2021b). Most bacteriophage endolysins indicate near-species specificity, which is thought to be one of their most beneficial features in this age of broad-range resistance to antibiotics as it avoids selective pressure on naturally occurring beneficial microbiota. Furthermore, resistance to endolysins is improbable for a variety of reasons, Endolysins have evolved to attach to and break highly conserved components in the cell wall of the pathogenic bacteria, secondly, as the bacteriophage and host bacteria coevolution occurs so there are fewer chances of resistance development to endolysin. Furthermore, because endolysins are applied externally and act on the cell wall rather than entering the bacterial cell, they avoid the majority of possible resistance mechanisms (e.g., active efflux from the cell or decreased membrane permeability) that contribute to resistance to most conventional antibiotics. Several endolysins include two catalytic domains that hydrolyze distinct bonds in the peptidoglycan, which is thought to limit the likelihood of resistance formation (Haddad Kashani et al., 2018).

Endolysin LysAB1245 showed broad lytic range efficacy against A. baumannii isolates from various capsular variants. Furthermore, LysAB1245 exhibited quick antibacterial activity and stability under different ranges of pH and temperature conditions. This study tested the possibility of LysAB1245 as a novel therapeutic agent for the treatment of MDR A. baumannii bacterial infections (Soontarach et al., 2024). Table 1 shows the summary of the therapeutic potentials of bacteriophage derived endolysins against A. baumannii.

Establishing the in vivo safety and efficacy of phage-encoded endolysin as medicinal products is an essential step in their development (Lai et al., 2020a). Endolysin can be administered by various methods, such as intravenously and intraperitoneal injections, topical treatments (creams, ointments, and gels), and trans nasal, vaginal, and oral delivery systems (Abdelrahman et al., 2021b). Endolysins, which target pathogens in various tissues and organs, are currently in clinical trials. Schmelcher et al. have briefly addressed the systemic use of endolysins to treat bacteria-related infections of the bloodstream, organs, and tissues (Schmelcher M. and Loessner M. J. J. C. O. I. B., 2021). In an A. baumannii mouse model of burn wound infection, a single dosage of 14 µg/mouse of endolysin LysP53 resulted in a 3-log reduction in bacterial load. The findings suggested that LysP53 can be a promising choice for the treatment of topical infections caused by A. baumannii and other Gram-negative bacteria (Li et al., 2021). ElyA1 was used to treat skin infections of A. baumannii in Galleria mellonella. ElyA1 showed a confirmed significant reduction in the number of bacteria (Blasco et al., 2020a). The P307_SQ-8C reduced the bacterial burden by two logs in a mice model of A. baumannii cutaneous infection in 2 h (Thandar et al., 2016). LysAB2 P3 reduced bacterial burden in ascites and blood by 13-fold and 27-fold, respectively, in a mouse intraperitoneal infection model 4 h after bacterial injection. Furthermore, LysAB2 P3 saved 60% of mice infected with A. baumannii from deadly bacteremia (Peng et al., 2017). Repeated exposure to endolysins LysAm24, LysAp22, LysECD7, and LysSi3 that target Gram-negative bacteria are tested to determine the development of resistance. These endolysins function well in animal models of wound and burn skin infections, have a broad spectrum of activity, and are active against both planktonic bacteria and bacterial biofilms. Regarding safety, these enzymes do not cause cytotoxicity, do not contribute to the development of resistance, and do not disrupt the natural intestinal microbiota in vivo (Vasina et al., 2021). Endolysins specifically prevent specific species or subspecies of pathogenic bacteria, without disrupting the surrounding normal microbiota (Yoong et al., 2004). The endolysin PlyV12 causes less disruption of the normal microbiota when used to treat antibiotic-resistant bacterial strains and they do not transfer resistance genes or bacterial toxins and do not eliminate the colonized beneficial normal flora of mucous membranes (Rahimzadeh et al., 2018).

Due to several resistance mechanisms, including biofilm formation, A. baumannii can survive and spread in the hospital environment (Salmani et al., 2020). Endolysin chemotherapy has a significant antibacterial effect on biofilm-associated bacteria, which are difficult to eliminate due to their minimal metabolic activity (Chang et al., 2022). Endolysin Abtn-4 treatment inhibited A. baumannii biofilm growth by more than 30%, suggesting that endolysin is a possibly effective antibacterial agent in regulating biofilm development (Yuan et al., 2021). The pre-treatment of catheters with endolysin PlyF307 decreased A. baumannii biofilm on the surface by 1.6 log10, resulting in a substantial decrease in total biofilm growth (Lood et al., 2015a). In an additional in vivo study, Lood et al. investigated the efficiency of PlyF307 in eliminating biofilm from subcutaneously implanted catheters colonized with 2-day-old A. baumannii biofilms in the intestines of mice. Their findings revealed that mice with fatal A. baumannii bacteremia were saved by PlyF307 (Lood et al., 2015a). Endolysin anti-biofilm activity should be evaluated in future studies implementing more clinically relevant models such as multispecies biofilm matrices and flow cell-based biofilm models, as well as pre-treatment processes on diverse substrates and surfaces in hospitals (Chang et al., 2022). The endolysin LysAB1245 showed broad lytic spectrum efficacy against MDR A. baumannii isolates from various main capsular types. Furthermore, LysAB1245 has the potential for use with nosocomial MDR A. baumannii infections and can control the biofilms in the clinical healthcare environment (Soontarach et al., 2024).

The outer membrane of Gram-negative bacteria acts as a barrier for many endolysins, and very few endolysins with exogenous activity against Gram-negative bacteria have been observed. Endolysins can target Gram-negative bacteria if the outer membrane is first permeated with chemicals such as Ethylenediaminetetraacetic acid (EDTA), which destabilizes the lipopolysaccharides of the outer membrane; but the combined use of endolysin and EDTA is restricted to superficial treatment of localized infection (Briers et al., 2011; Thummeepak et al., 2016b). Several studies have tried enhancing the muralytic effect of endolysins by using them with different antibiotics to take on their combined synergistic effects (Thummeepak et al., 2016b; Letrado et al., 2018). Using endolysin and antibiotics together to target Gram-negative bacteria has shown some promising outcomes (Lai et al., 2020a). The synergistic effect of LysABP-01 endolysin with seven commonly prescribed antibiotics against the MDR strain of A. baumannii was assessed. LysABP-01 and colistin together had a synergistic effect in vitro, with a fractional inhibitory concentration index ranging from 0.156 to 0.188. The encouraging findings demonstrated that colistin and LysABP-01’s minimum inhibitory concentrations (MICs) were lowered by up to 8 and 32 fold, respectively (Thummeepak et al., 2016a). Colistin, a ‘last-resort’ antibiotic, is commonly used to treat infections caused by MDR Gram-negative bacteria. However, due to its nephrotoxicity and neurotoxicity, the dose of colistin is extremely restrictive (Nation and Li, 2009). As a result, combining colistin with lysins may help optimize the clinical use of colistin at a lower dose. Blasco et al. recently demonstrated the synergistic effects of the endolysin ElyA1 and colistin in targeting various MDR Gram-negative bacteria including A. baumannii, in both in vitro and in vivo conditions. When colistin was combined with ELyA1, the MIC of colistin was lowered by at least 4-fold for all tested A. baumannii strains (Blasco et al., 2020a). In both skin and lung infection models, the in vivo survival rate for A. baumannii infected G. mellonella and bacterial reduction of infected mice treated with ELyA1 and colistin combination was considerably greater than that of the colistin-alone. Colistin can operate as an OMPto break the integrity of the OM, allowing lysin access to the PG substrates. Simultaneously, peptidoglycan cleavage by endolysin may increase antibiotic absorption and hence promote antibiotic action (Gerstmans et al., 2018a). These positive results show that combining endolysin and antibiotics has the potential to resensitize bacterial pathogens with drug resistance to antibiotics. And to slow the spread of antibiotic resistance by using fewer antibiotic treatments of low dosages (Lai et al., 2020a). Combined use of endolysin LysAB-vT2-fusion with colistin, polymyxin B, or copper showed synergistic against A. baumannii, which may be used for the control of A. baumannii infection (Sitthisak et al., 2023b).

In Gram-negative bacteria, the outer membrane functions as a barrier to several endolysins.

Few endolysins with exogenous activity against Gram-negative bacteria have been reported and the majority are synthetically engineered endolysins (Lai et al., 2011b; Briers et al., 2014a; Briers et al., 2014b; Lim et al., 2014; Lood et al., 2015; Soontarach et al., 2024).

OMPs have the potential to significantly increase the antibacterial activity of endolysin while also broadening their host spectrum. However, the in-vivo study of the efficacy of endolysin and OMPs in combination is rarely described in the literature. The harmful effects of the most often used OMPs such as EDTA through various delivery routes, such as oral, intravenous, intraperitoneal, topical, and inhalation, have been investigated (Lanigan and Yamarik, 2002). EDTA is cytotoxic, slightly genotoxic, and has an anticoagulant effect. It has been observed that the low EDTA dose required to cause toxicity in rats was 750 mg/kg/day. As a result, the use of EDTA with endolysin should be restricted to topical applications because it cannot affect the skin and does not produce skin hypersensitivity (Lanigan and Yamarik, 2002). Citric acid is also used as an OMP, but despite having a much better safety profile than EDTA, more testing is required because citric acid also has hazardous consequences, When used in combination with endolysin, particularly for the inhalation route, as it has been linked to animal obstruction of the airway and cause reactions in humans coughing (Allott et al., 1980). With further work being done on combining endolysin with polycationic peptides to enable them to target Gram-negative bacteria, their therapeutic use should be carefully evaluated. This is because endolysin with OM-disrupting characteristics or those coupled with peptide OMPs may also interact with eukaryotic cell membranes, raising safety issues (Schmelcher et al., 2012).

The log-growth phase bacteria are more susceptible to endolysin treatments than the stationary phase bacteria. Oliveira et al. presented that the lower bactericidal efficiency against the bacterial stationary cells is due to modifications to the structure in the outer membrane, such as LPS biosynthesis, which affects endolysin permeability that even in the presence of OMPs, or chemical modifications of the peptidoglycan compositions (Oliveira et al., 2014). These alterations could aid bacterial cells in coping with endolysins via acetylation, N-deacetylation, and amidation (Typas et al., 2012). A significant number of findings show anti-bacterial efficacy against the log-phase bacteria. To overcome this research gap, and to completely investigate the potency of endolysin against Gram-negative bacteria, further research is required to evaluate the antibacterial efficiency of endolysins against the stationary phase bacteria. Regarding the concern of the resistance of bacteria to endolysin; The resistance development of endolysin was evaluated in which bacterial strains were repeatedly challenged with subinhibitory doses of endolysins or artilysins failed to give rise to resistant mutants, although bacterial strains exposed to control antibiotics got resistance. The probability of developing resistance against endolysins and artilysins is thus regarded as minimal, especially when compared to standard antibiotics (Fischetti, 2005; Briers et al., 2014a).

The stability of the endolysin during production, storage, and administration is essential for its prospective use as an antibacterial agent. Several stability concerns of Gram-negative endolysins have been observed in preclinical studies and must be solved for further research and development (Lai et al., 2020a). The oral delivery route is the most widely used for gut-targeted medicines and is also the most well-liked by patients. Most medications available on the market are used orally and come in tablet or liquid form. This is not without its challenges, though. Because medications of this kind have to pass through the stomach and digestive tract, they are instantly exposed to a variety of enzymes, varying pH levels, and mechanical digestion. All of these may have an impact on the drug’s molecular integrity and systemic bioavailability (Murray et al., 2021). Endolysins are proteinaceous, therefore they can be swiftly damaged by these processes, rendering them ineffective. Stomach acid has the potential to damage the structure of several endolysins. Proteases such as trypsin, chymotrypsin, pepsin, and peptidase degrade proteins. Many endolysins contain cleavage sites that these enzymes can target (Bruno et al., 2013).

One of the limitations of endolysin is its short in vivo half-life, caused by the inflammatory response of cytokines and neutralizing antibodies. Endolysin elicits an immune system response when used repeatedly, and as a result of the immune response, it loses its enzyme-mediated lytic activity in vivo (Nau and Eiffert, 2002; Jado et al., 2003; Abdelrahman et al., 2021a). Raz et al. described a novel approach to discovering immune-derived engineered endolysins. The engineering of CBDs of different endolysins fusions to the Fc receptor of human IgG antibodies. The engineered lysin called Lysibodies binds to S. aureus cells and opsonizes them, causing immune complement system activation, which can cause phagocytosis and clearance of the bacteria. Through this approach, endolysin can be used to target an immune response toward pathogenic bacteria (Raz et al., 2018). A similar approach without using antibody fragments was described for a protein derived from the CBD of the endolysin PlyV12 (Yang et al., 2018). As some of the Gram-negative endolysin enter preclinical development, concerns about immunogenicity, toxicity from lipopolysaccharide release during the bactericidal process, and pharmacokinetic issues due to the complexity of the Gram-negative cell wall must be addressed (Ghose and Euler, 2020). We suggested a strategy to minimize the body’s immune responses to endolysin. Engineered endolysins should be designed with human immune cells. When such proteins are expressed, the immune cells will recognize them as their body proteins, so there will be fewer immune reactions to these foreign proteins and less chance of the clearance of such foreign proteins in the body.

In February 2020, the FDA recognized endolysin innovations as antibacterial biological drugs in the phase iii study as “Breakthrough Therapy” (Harhala et al., 2022). However, the fundamental obstacle to the use of endolysins as a therapeutic agent is the lack of proper clinical research to provide an excellent scientific fundamental basis for approval. We suggested that additional research on endolysins affecting their safety in humans will be beneficial to get past the reservations point of view on endolysins’ usage and create the legal framework for the approval of endolysins. Preclinical trials of more endolysins are needed to bring endolysin into the medical market for treatment purposes.

Numerous potential endolysin applications are currently being reported in medicine, but there are no published guidelines and regulations for endolysins. Early and effective communication with the relevant authorities is essential to establish endolysin regulatory pathways in their use as a medicine in the health sector and medicine industry. However, there are currently no clear legal principles or laws governing the use of endolysins.

Endolysins have made great progress towards clinical application as novel antibacterial treatments. Another major problem that must be addressed is the large-scale industrial manufacture of endolysin. The present cost of endolysin production is projected to be expensive, which may be a major barrier to the use of endolysins as antibacterial replacements (Oliveira et al., 2012). The high cost arises mostly from the necessity for the development of recombination engineering, effective, and safe expression platforms for endolysin expression and purification. As a result, technical techniques to increase the solubility and expression levels of target endolysins, as well as cost-effective expression systems, are required to make endolysins commercially acceptable antibacterial agents worth investing in (Lee et al., 2023). When transferring the therapeutic endolysin expression and purification from the lab to the industrial scale, manufacturing costs and safety concerns must be addressed. This applies to the entire manufacturing process, from the selection of the expression host. From the beginning of protein expression until the last stages of processing, there are numerous chances to lower production costs and improve safety. Cell harvesting, cell lysis, and purification are examples of downstream processing procedures that are carried out once endolysins have been engineered and reassembled in a particular expression system. Toxic effects must be avoided, especially when it comes to protein expression in bacteria where endotoxin must be eliminated. Furthermore, early in the production process, choosing the best expression hosts—such as bacteria, yeast, or plants—is a crucial step toward achieving overexpression and cheap expression costs. Because they are inexpensive to produce, plants have been suggested as potential hosts for recombinant protein production. Furthermore, plant-produced proteins are likely safer than those made from bacteria or animal cells (Magnusdottir et al., 2013).

So far, the effectiveness of Gram-negative endolysin in vivo has only been assessed using acute infection models. There are fewer documented chronic in vivo models for endolysin assessment, except for topical infection research investigations (Lai et al., 2020b). The current in vivo research on Gram-negative endolysin has been focused on assessing the rates of survival and/or reduction of bacterial burden to validate their effectiveness. Before continued research, issues with immunogenicity, pharmacokinetics, and the production of proinflammatory components during bacteriolysis must be resolved for the systemic use of Gram-negative endolysin. Many Gram-positive endolysins had problems being cleared by neutralizing antibodies because they were proteinaceous, which resulted in a relatively short half-life in vivo—between 4 and 40 min, depending on the type of endolysin (Gerstmans et al., 2018b).

Formulation techniques include the use of different carrier systems. The use of OMP (chelators such as citric acid, lactic acid, malic acid, acetic acid, benzoic acid and EDTA in the formulation is useful in permeabilizing the outer membrane of Gram-negative bacteria to endolysins (Oliveira et al., 2016a). Using OMP is the simplest way to get over the outer membrane barrier. The most often utilized permeabilizer in this scenario is EDTA, which works by chelating the outer membrane stabilizing divalent cations (Kocot et al., 2023).

Despite their therapeutic effects, bacteriophages and endolysins face some practical challenges posed by the host system, such as low bioavailability, losing activity, non-targeted delivery, rapid elimination by the retinal endothelial system, and antibody-mediated deactivation. Against this context, there has been an increase of interest among scientists to investigate the possibilities of delivery systems for encapsulating bacteriophages and endolysins. In recent years, a multitude of bacteriophage and endolysin encapsulation strategies have been reported (Loh et al., 2021). Similarly, nanoencapsulation can help improve the therapeutic potential by not only shielding endolysin from degradation but also allowing for continuous release, potentially boosting stability, shelf life, and therapeutic efficacy (Gondil and Chhibber, 2021). Liposomes’ ability to penetrate bacterial outer membranes through membrane fusion is also a viable way for delivering endolysin to Gram-negative bacteria. The authors achieved successful lysis of both Salmonella typhimurium and E. coli with endolysin BSP16Lys-containing liposomes without the need for a membrane permeabilizer (Bai et al., 2019b). These examples highlight the need to utilize these existing strategies to improve the potential of endolysin as an effective and sustainable therapy.

It has been observed that endolysin encapsulated in liposomes enhances the efficacy of endolysins against Gram-negative bacteria (Bai et al., 2019a). The method of packing lytic proteins derived from bacteriophage in the structure of liposomes appears to be a promising weapon against Gram-negative bacteria. Like dendrimers, liposomes can also have a protective role that maintains the endolysin activity (Kocot et al., 2023).

Encapsulation in alginate-chitosan nanoparticles is another strategy for increasing the efficacy of endolysin, particularly for use in therapy (Gondil and Chhibber, 2021). Chitosan-based formulations provide benefits over alternative delivery systems in that they boost therapeutic agent bioavailability while also properly removing from the host after the release of the agent (Gondil and Chhibber, 2021).

Because the outer membrane (OM) of Gram-negative bacteria prevents externally applied endolysins from accessing peptidoglycan, controlling the growth of these Gram-negative pathogenic bacteria is difficult (Sisson et al., 2024b).

Some endolysins contain peptides, typically at their C-terminus, that have a strong antibacterial activity that is not dependent on peptidoglycan breakdown. These antimicrobial peptides (AMPs) are responsible for some endolysins natural capacity to penetrate the outer membrane of Gram-negative bacteria (Szadkowska et al., 2022; Kocot et al., 2023). Predicting AMP-like regions is a good technique for designing endolysin with an intrinsic antibacterial activity that can serve as a framework for further engineering (Kocot et al., 2023).

However, Gram-negative bacteria are difficult to regulate due to the presence of an outer membrane that protects the peptidoglycan layer from enzymatic breakdown. To surpass this threshold, the fusion of endolysin with a sensitizer peptide is known to extend efficacy by disrupting the outer membrane of Gram-negative bacteria (Son et al., 2023). The sensitizer peptide-fused endolysin Lys1S-L9P has highly effective bactericidal activity against various MDR Gram-negative bacteria. Lys1S-L9P displayed strong antibacterial action against MDR Gram-negative bacteria in the G. mellonella in vivo model, with no harmful effect. Sensitizer peptide-fused endolysin could be a useful biocontrol agent against MDR Gram-negative bacteria (Son et al., 2023).

A. baumannii is a leading contributor to nosocomial infections; it is becoming more and more linked to different epidemics and is a serious worry in intensive care units of hospitals across the globe (Almasaudi, 2018). Nosocomial infections resulting from multidrug-resistant bacteria are life-threatening (Choi et al., 2022). Secondly, the formation of bacterial biofilms on medical equipment is one of the biggest problems in the healthcare system (Gutiérrez et al., 2012). Bacteria trapped in biofilms are less sensitive because they grow more slowly and have restricted access to antibiotics and disinfectants (Davies, 2003). The bacteria-reduced susceptibility to antimicrobial agents within the biofilm that exhibited a more resistant phenotypic condition has also been linked to recurring infections (Lewis, 2007). Endolysins break down the bacterial cell wall peptidoglycan and could potentially help in controlling the spread of infections of MDR bacteria seen in hospitals (Choi et al., 2022). This revealed the endolysin potential as an environmentally friendly replacement for hospital-use harmful chemical disinfectants (Choi et al., 2022).

Vaccination, which stimulates the immune system to enhance adaptive immunity through the injection of antigenic material, is one of the greatest public health achievements. Viral nanoparticles (VNPs) have been a focus of recent research as delivery vehicles for protein and peptide-based vaccinations (Rodríguez-Rubio et al., 2016b). Bacteriophage-derived nanoparticles have drawn the attention of researchers since foreign peptides or proteins can be expressed on the surface of phages as fusion proteins. As a result, phage-displayed peptides or proteins have been proven to be functionally and immunologically active, making them appropriate for vaccine development (Hamzeh-Mivehroud et al., 2013). Endolysin can also be employed as a binding agent for displaying heterologous proteins on the surfaces of bacteria in the hunt for live vaccination delivery systems. The endolysin CBDs, can be fused with various other proteins such as antigens to show them at the bacterial surface while retaining their structure and activity (Loessner et al., 2002). Surface display technology based on bacteriophage-derived endolysins has been developed using lactic acid bacteria (Hu et al., 2010; Ribelles et al., 2013). It has been shown that the external addition of the E7 antigen from human papillomavirus type-16 (HPV-16) when fixed on the surface of lactic acid bacteria by the CBD of Lactobacillus casei A2 phage endolysin, secure mice from an HPV-16 attack and its associated tumors. After being immunized via intranasal vaccination with lactic acid bacteria expressing E7 antigen, more than 60% of the mice remained tumor-free. Thus, the surface display of endolysins CBDs is a fascinating method for effectively and safely delivering therapeutic proteins (Ribelles et al., 2013). Figure 2, illustrates the applications of bacteriophage-derived endolysin.