1 Introduction

Based on the current literature, it is well-known that more than thirty different Staphylococcus species could be responsible for a great number of infections. Many of these infections are caused by Staphylococcus aureus, and a large number of them colonize the human body [1]. S. aureus, including methicillin-resistant S. aureus (MRSA), are generally transported via contaminated apparatus or through directly contaminated persons more often result in nosocomial infections. These infections usually lead to an additional prolonged hospital stay as well as substantial economic burden [2]. Therefore, alternative approaches are needed to prevent and/or treat these infections. One of such approaches include the use of lytic bacteriophages alone or in combination with conventional antimicrobial agents have been reported [3]. Although phage resistance is less common compared to the antibiotic resistance in bacteria, the narrow host range due to phage receptor variability on the host cell surface limits the use of phages for infections [4]. They can interact with the immune system, potentially leading to harmful immune responses [5,6,7]. So far, little evidence suggests this is a significant concern during phage treatment [8]. However, some phage therapy protocols may require highly purified phage preparations to avoid allergic reactions to bacterial components, like endotoxins found in crude phage lysates. Phages can also release bacterial components when they kill bacteria, similar to how cell-wall-disrupting antibiotics work [6]. In recent years, studies have been conducted regarding the therapeutic efficacy of endolysins for infections that are caused by Gram-positive bacteria including those caused by antibiotic-resistant ones [9,10,11]. Endolysins are enzymes that are encoded and released by phages at the final stage of their life cycle. At this stage, phages degrade host bacterial cell wall to release the newly produced phage particles [12]. Exogenous recombinant endolysin causes bacterial disintegration within a short period of time without the help of holin proteins, which is different from their usual life cycle [13, 14]. Considering the remarkable combined effects of endolysin and antibiotics, their synergistic use demonstrates the potential to enhance the endolysin’s broad-spectrum activity while concurrently preventing the development of resistance in target cells [15,16,17]. Presently, several clinical trials are underway, and some phage endolysin-related preparations have already entered the market, indicating their potentials being better antibacterial agents [18, 19]. The benefits of using phage endolysins over other antimicrobial agents include their specificity, their ability to damage cells without harming the normal microflora, and the rarity of bacteria that develop resistance [20]. Therefore endolysins offer new hope for treating superbugs that regular antibiotics are unable prevent due to their resistance to multiple drugs. These properties of phage endolysins make them alternative antibacterial proteins in medicine, veterinary care, agriculture, food production, and industrial biotechnology [11]. The use of phage endolysins for the treatment of infections and biofilms caused by MRSA [21, 22] as well as free-floating Staphylococcus cells and biofilms, regardless of the surface they were on [23].

In the present study, it was aimed to clone and express the endolysin from S. aureus subsp. aureus ATCC 27,692-B1 bacteriophage 52 and demonstrate its antibacterial potential against Gram-positive bacteria including S. aureus, S. epidermidis, S. haemolyticus, S. pyogenes, S. pneumonia, E. faecalis, E. faecium, and B. atrophaeus.

2 Materials and Methods

2.1 Bacterial Strains, Media, and Growth Conditions

S. aureus, S. epidermidis, and S. haemolyticus previously isolated from various clinical specimens were obtained from the Department of Medical Microbiology Culture Collection, Faculty of Medicine, Karadeniz Technical University, and the bacteriophage host S. aureus subsp. aureus ATCC 27692, and ATCC 27692-B1 bacteriophage 52 was obtained from the American Type Culture Collection (ATCC, USA). Bacterial strains used in this study were presented in Table 1. Chemically competent Escherichia coli One Shot™ Mach1™ cells used for the preparation of recombinant plasmids and the E. coli BL21 (DE3) strain used as a recombinant endolysin expression host were obtained from Thermo Fisher Scientific (USA) and Invitrogen (USA), respectively. All Gram-positive strains except lactobacilli were grown in tryptic soy agar (TSA) and tryptic soy broth (TSB). Lactobacilli were cultured in Demand Rogosa agar (MRSA) and MRSA broth. Gram-negative bacteria were cultured in Luria-Bertani (LB) media. Except for lactobacilli cultures, all microorganisms were grown at 37 °C with shaking aerobically unless otherwise indicated. Lactobacilli cultures were incubated statically in a candle jar at 37 °C.

Table 1 Lytic efficacy of the recombinant LysSA52 against different clinical isolates of Staphylococcus and various Gram-positive and Gram-negative bacteria
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2.2 Phage DNA Isolation, Primer Design, and PCR Amplification of the Endolysin Gene

Phage purification was performed according to Carroll-Portillo et al., 2021 with some modification [24]. DNA isolation from phage was performed by using the phenol-chloroform isoamyl extraction method. Briefly, 450 µL purified phage stock (ca. 1 × 1010 PFU mL− 1) of bacteriophage 52 was treated with lysis buffer (500 mM Tris-HCl; 250 mM EDTA; 2.5% SDS, 20 mg/mL RNase A; and 10 mg/mL DNase I) for about 30 min at 37°C. The lysed phage particles were then centrifuged at 9000 g (Eppendorf Centrifuge 580 R, Germany) for 10 min, and the supernatant was transferred to a new microcentrifuge tube followed by the addition of phenol in a one-to-one ratio before mixing and centrifugation at 9000 g for 10 min. The DNA extraction from supernatant was performed by treatment with a 1:1 volume of chloroform: isoamyl alcohol (24:1) for 10 min at room temperature. After precipitation with two volumes of 96% ethanol, DNA was washed with 70% ethanol and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The purity and concentration of DNA were measured using a NanoDrop 2000/2000c (Thermo Scientific, USA) spectrophotometer. The forward (Lys52F: 5’- ATG CAA GCA AAA CTA ACT AAA AAA G -3’) and reverse (Lys52R: 5’- TTA ACT GAT TTC TCC CCA TAA GTC-3’) primers were designed based on the coding sequence of five staphylococcal phage endolysin sequences (accession numbers; NC_021801.1, NC_010147, NC_011614.1, NC_024391.1, and NC_021863.1) available from the National Center for Biotechnology Information (NCBI) website (www.ncbi.nlm.nih.gov), and were employed to amplify the phage 52 endolysin gene using PCR. The PCR reaction was carried out in 25 µL reaction volume containing 1х PCR buffer (Promega 5х), 2.5 µM of each nucleotide, 10 µM of each primer, 25 mM MgCl2, 0.5 U Taq DNA polymerase (Promega, USA), and 5–10 ng DNA template. A PCR condition was adjusted to an initial denaturation (5 min at 94 °C), 30 cycles of denaturation (1 min at 94 °C), primer annealing (1 min at 52 °C), and extension (1 min at 72 °C), plus a final extension (7 min at 72 °C). The amplified PCR products were analyzed by electrophoresis in 1% agarose gel and visualized under a UV transilluminator.

2.3 Cloning and Sequencing of the Endolysin Gene

The amplified PCR product was purified using the High Pure PCR Product Purification kit (Sigma-Aldrich, USA) and cloned into the pET-SUMO expression vector (Invitrogen, USA) by following the recommendations of the manufacturer. The cloned plasmid was used to transform E. coli One Shot Mach1-T1R competent cells and then transformed cells were plated on LB agar plates containing kanamycin (50 µg/mL), and incubated at 37 °C for overnight. Several colonies were picked and inoculated into 5 mL of LB containing kanamycin (50 µg/mL) for plasmid DNA isolation using a plasmid Miniprep kit (Qiagene, Germany), according to the manufacturer’s protocol. The clones were confirmed by sequencing using an Applied Biosystem ABI 310 DNA Analyzer (Applied Biosystem®, USA) with vector-specific forward and reverse primers. One clone with the correct sequence designated as pET-SUMO-LysSA52 was selected for expression.

2.4 Over-expression and Purification of the Endolysin

The pET-SUMO-LysSA52 plasmid with correct orientation was transformed into competent E. coli BL21 (DE3) cells for endolysin protein expression using a standard calcium chloride transformation procedure. A single colony from the transformed cells was grown in 10 mL LB medium with appropriate antibiotic (50 µg/mL of kanamycin) and incubated overnight at 37 °C with shaking. Then, 100 mL of LB media containing kanamycin (50 µg/mL) and 1% glucose was inoculated with 1 mL of the overnight culture and grown at 37 °C with shaking until it reached an optical density at 600 nm (OD600 nm) of 0.5. Then, culture was induced for 18 h with 1 mM IPTG at 20 °C with shaking. The induced cells were collected by centrifugation at 5000 xg at 4 °C for 30 min, weighed, and resuspended as a 10 mL/g wet pellet in lysis buffer (400 mM NaCl; 50 mM NaH2PO4 buffer pH 7,8; 0.5% Triton X-100; 10% glycerol; and 10 mM imidazole) and performed three freeze-thaw cycles at -80 °C and 37 °C. The cells were sonicated (Vibra Cell™ Sonics, USA) at 50% amplitude via 18 × 10 s bursts with 10 s rest time. Lastly, the homogenized suspension was centrifuged at 9000 xg for 30 min and the clear lysate was filtered through Millex® Syringe Filters (Merck KGaA, Germany) with a 0.2 μm pore size, and polyhistidine-containing recombinant fusion protein (SUMO-LysSA52) was purified using a ProBond™ Purification System kit (Novex, USA), in accordance with the manufacturer’s recommendations. All extracted fractions including fusion protein were collected and analyzed using 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing the fusion protein were combined and dialyzed (SnakeSkin™ Dialysis Tubing, Thermo Scientific, USA) at 4 °C against 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. The protein concentration was measured using Bradford Assay and stored in a -20 °C freezer until use.

2.5 Lytic Activity and Lytic Spectrum of Endolysin

The expressed endolysin (LysSA52) activity was determined via agar spot-on lawn antimicrobial assay. In brief, 10 µL of purified endolysin (0.5-1 mg/mL) was spotted onto agar plates pre-seeded with mid-log phase bacterial culture and grown at 37 °C for 24 h. Lytic activity was determined as the presence of bacterial lysis with clear spots on the agar cultures was considered to indicate positive endolysin activity.

The host spectrum of LysSA52 endolysin was tested against 90 strains of 10 Gram-positive species including 49 S. aureus strains, 18 of which were MRSA, 13 S. epidermidis, 3 S. haemolyticus, and S. pneumonia ATCC 49,619, S. pyogenes ATCC 19,615, E. faecium ATCC 6057, E. faecalis ATCC 29,212, B. atrophaeus ATCC 9372, Listeria monocytogenes ATCC 43,251, Lactobacillus plantarum ATCC 14,917, and two Gram-negative bacterial strains including E. coli ATCC 35,218, and Salmonella typhimurium ATCC 14,028 (Table 1).

2.6 Effects of pH and Heat on the Activity of LysSA52 Endolysin

The effects of pH and heat sensitivity (thermostability) on LysSA52 activity were analyzed by using the S. aureus TRSA 2 strain cultured to the logarithmic growth phase. To evaluate the effect of pH on LysSA52, bacterial host cells were suspended in three different pH buffers (sodium acetate (pH 4.0–6.0), HEPES (pH 6.8–8.2), and Tris (pH 8.0–9.0), mixed with an equal volume of endolysin solution (1 mg/mL) in a 96-well plate with a 200 µL total volume, and incubated plates at 37 °C for 12 h. The buffer concentration was 20 mM for all experiments. Surviving cells were determined by colony count and the cell killing rate of the enzymes was expressed as a percentage.

The heat sensitivity of endolysin was reported previously [25]. Briefly, LysSA52 was incubated at various temperatures between 30 and 90 °C at 10 °C intervals for 30 min followed by a 20 min cooling step on ice, and was spotted on an agar culture of the S. aureus TRSA 2 strain. Endolysin solution without heat treatment was used as a positive control. The inhibition zone diameters of heat-treated endolysins were measured and compared to that of positive control which was kept on ice.

2.7 Storage Stability

Storage stability of the LysSA52 endolysin was examined by exposing it to various temperatures (room temperature, 4 °C, and − 20 °C) for nine weeks. The enzyme samples were stored in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.1 mM EDTA, and 50% (v/v) glycerol. Endolysin samples were assessed for their antimicrobial activity using the agar spot-on lawn antimicrobial assay following nine weeks of storage.

2.8 Anti-biofilm Properties of Endolysin

The biofilm removal performance of the LysSA52 endolysin was performed by using a microtiter plate assay as described previously with slight modifications [26]. Briefly, S. aureus and S. epidermidis strains were diluted with 0.25% wv D-(+)-glucose to attain an initial inoculum level of 106 CFU/mL. From each strain, 200 µL of prepared inoculums was added into 96 wells of a flat-bottomed microtiter plate and 200 µL TSB was added in separate wells as a negative control. Biofilms were established by incubating the plate at 37 °C without agitation for 48 h. Then, the content of the wells was discarded, and biofilms were washed two times with sterile phosphate-buffered saline (PBS). Following this, biofilms were treated with LysSA52 endolysin for 12 h at 37 °C. The content of the wells was discarded and washed two times with PBS. Wells were stained for 30 min by adding 200 µL of 1% (/v) crystal violet solution. The unbound stain was removed, and stained biofilms were washed with PBS, and then solubilized by the addition of 200 µL of ethanol-acetone (80:20) solution to each well. The remaining biofilms after endolysin treatment were quantified using a plate reader at OD595 nm and the percentage reduction in biofilms was compared with untreated control. The mean of four wells was used and the experiments were repeated three times.

3 Results

3.1 PCR Amplification, Cloning, and Sequence Analysis of LysSA52 Genes

The DNA from S. aureus subsp. aureus bacteriophage 52 (ATCC®27692-B1™) obtained from ATCC was used to amplify the endolysin gene of 1446 bp (Fig. 1). The amplified product was cleaned and cloned directly into the pET-SUMO vector (Invitrogen, USA) and the pET-SUMO-LySA52 recombinant clone was sequenced to confirmed for the presence of the 1446 bp insert with correct orientation encoding a single open reading frame beginning with a start codon (ATG) and ending with a stop codon (TAA). The nucleotide sequence data were submitted to the GenBank database (NCB, USA) and assigned accession number: (MH104949). Neighbor-joining phylogenetic tree analysis showed that the LysSA52 endolysin gene was closely related to the staphylococcal phages DW2, SA12, and SA13 (Fig. 2).

Fig. 1
figure 1

PCR amplification of LysSA52 gene from S. aureus subsp. aureus bacteriophage 52 DNA. M; molecular weight marker, 1; endolysin gene product

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Fig. 2
figure 2

Phylogenetic position of LysSA52 endolysin gene derived from S. aureus subsp. aureus bacteriophage 52

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The amino acid sequence (481 amino acids) analysis using Simple Modular Architecture Research Tool (SMART) available at http://smart.embl-heidelberg.de/ revealed that LysSA52 has three distinct domains; CHAP (cysteine, histidine-dependent) domain, amidase (Ami-2) domain, and SH3b domain (Fig. 3).

Fig. 3
figure 3

Domain organization of the endolysins LysSA52. Two catalytic domains CHAP and Ami-2; catalytic domains, SH3b; cell wall binding domain Numbers indicate amino acid positions

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3.2 Expression of LysSA52 Endolysin

The culture of E. coli BL21 (DE3) carrying the pET-SUMO-LysSA52 plasmid was induced with 1 mM IPTG. The LysSA52 endolysin with a molecular mass of approximately 53 kDa was successfully co-expressed with 13 kDa SUMO protein and polyhistidine [6-His]-tag at the N terminus resulting in a 66 kDa fusion protein (Fig. 4A). The expressed his-tagged protein was purified from crude cell lysate by nickel chelating affinity chromatography. The eluted polyhistidine-containing recombinant SUMO-fused endolysin LysSA52 was demonstrated in SDS-PAGE analysis (Fig. 4B). The LysSA52-SUMO fusion protein was used for the lytic activity assays without further purification since the SUMO protease cleavage was ineffective despite the manufacturer’s recommendations.

Fig. 4
figure 4

SDS-PAGE analysis of expressed His-SUMO-LysSA52 endolysin. A; total cell proteins of E. coli BL 21 carrying pET-SUMO. LysSA52 plasmid, M; protein molecular weight marker, 1; uninduced cell proteins, 2 and 3; IPTG induced LysSA52 for 18 and 20 h, respectively. B; SDS-PAGE analysis of His-SUMO-LysSA52 fusion protein before and after purifications. M; protein molecular weight marker, 1; total cell proteins before purification, 2 to 6; eluted fractions

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3.3 Lytic Activity and Host Range of LysSA52 Endolysin

The LysSA52 endolysin lytic activity and the lytic spectrum were demonstrated by the agar spot-on lawn antimicrobial assay and indicated by a clear lysis zone on the endolysin spotted area compared to the control, containing total cell proteins of uninduced E. coli BL 21 cells carrying the pET-SUMO-LysSA52 plasmid (Fig. 5). With respect to the lytic spectrum, the recombinant endolysin LysSA52 showed lytic activity against 74 (89%) out of 83 staphylococcal strains, including S. aureus, MRSA, S. epidermidis, and S. haemolyticus, irrespective of their origin (Table 1). In addition, LysSA52 lysed E. faecium ATCC 6057, E. faecalis ATCC 29,212, S. pneumonia ATCC 49,619, S. pyogenes ATCC 19,615, and B. atrophaeus ATCC 9372 but did not show any lytic activity against L. plantarum ATCC, 14917, L. monocytogenes ATCC 43251, and S. Typhimurium ATCC 14,028 (Table 1).

Fig. 5
figure 5

Lytic activity of the LysSA52 endolysin against S. aureus, MRSA, S. epidermidis, and S. haemolyticus by the agar spot-on lawn antimicrobial assay. 1; LysSA52; C: control

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3.4 Enzymatic Properties of LysSA52

Using the agar spot-on lawn antimicrobial assay and colony count, the activity of LysSA52 was evaluated over a range of pH and temperature, (Fig. 6A-B). The LysSA52 endolysin works best in the alkaline pH range with maximum or optimum activity at pH 8.0, and its activity decreased significantly below pH 7.0 (Fig. 6A). The LysSA52 endolysin retained more than 85% of its antibacterial activity at 30-50 °C for 30 min (Fig. 6B). However, its activity sharply decreased at or above 60 °C. The storage stability of LysSA52 endolysin was examined by exposing the enzyme at room temperature, 4 °C, and − 20 °C and assessing its activity after nine weeks. The result showed that LysSA52 retained its activity for 9 weeks of storage at 4 °C (Fig. 7).

Fig. 6
figure 6

Biochemical properties of LysSA52. A; Influence of pH on the bacterial activity of LysSA52. The activity was determined by viable cell count and expressed in percentage. B; Temperature stability of LysSA52 endolysin. The activity was determined by measuring inhibition zones and expressing the results in percentage relative to control (non-heat treatment endolysin kept at room temperature). Each column represents the mean of triplicate assays, and error bars indicate the standard deviation

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Fig. 7
figure 7

Storage stability of LysSA52 endolysin. A; LysSA52 test at the beginning of the experiment on S. aureus TRSA 8, B; LysSA52 test result after nine weeks on the same strain. In each case 5 µL of LysSA52 was dropped on agar plate containing bacterial lawn (endolysin stored at room temperature started to loosing activity only after nine weeks)

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3.5 Biofilm Removal Assay

In order to assay the biofilm removal capacity of LysSA52 endolysin using crystal violet assay, 48 h mature biofilms of S. aureus TRSA 8 and S. epidermidis TRSA 31 established in 96 well polystyrene microtiter plates were treated with LysSA52 endolysin at three different concentrations (0.20 mg/mL, 0.30 mg/mL, and 0.50 mg/mL) for 12 h. The remaining biofilm mass was stained with crystal violet and the remaining biofilm was estimated by spectrophotometric measurement of the solubilized biofilm. The data demonstrated that the LysSA52 treatment reduced biofilms of both S. aureus and S. epidermidis relative to the control group at all concentrations tested as shown in Fig. 8. The reduction was markedly sensitive to 0.50 mg/mL, which removed about 60% of established S. aureus and S. epidermidis biofilms in 12 h.

Fig. 8
figure 8

Biofilm removal capacity of LysSA52 endolysins at different concentrations as determined by 96 well microtiter plate assay. Error bars represent the SD of three independent experiments

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4 Discussion

S. aureus is one of the main human pathogens that cause local, systemic, and nosocomial infections amongst coagulase-positive staphylococci [27]. In previous studies, it has been reported that treatment of these infections has challenges because of the high rate of MRSA infection [28]. Lytic phages are used individually or in combination with conventional antimicrobial agents to treat bacterial infections [3]. However, the narrow host range, which is largely due to the variability in phage receptor molecules on the host cell surface, limits the application of bacteriophage therapy [4]. Recently, the use of phage endolysin has attracted considerable interest in combating pathogenic bacteria as an alternative to conventional antibiotics [9]. Phage endolysins can kill target bacteria upon exposure and do not harm the normal microflora, and it is very rare for bacteria to develop resistance to them [20]. The demand for recombinant antimicrobial proteins, including phage endolysins as enzybiotics, which can be produced with high quality in large amounts under lower production costs, has increased due to their utilization in various sectors, such as healthcare, agriculture, and the food industry [29].

In this study, we aimed to clone and express the endolysin gene from S. aureus subsp. aureus ATCC 27,692-B1 bacteriophage 52 and assess its antibacterial activity against staphylococci and other various Gram-positive pathogens. The lytic spectrum of LysSA52 was evaluated via the agar spot-on lawn antimicrobial assay method. LysSA52 displayed strong lytic activity against a wide range of Staphylococcus isolates, including methicillin-sensitive S. aureus (MSSA), MRSA, S. epidermidis, and S. haemolyticus of clinical origin. LysSA52 also showed cross-species lytic efficacy for several Gram-positive bacteria, including E. faecium, E. faecalis, S. pneumoniae, S. pyogenes, and B. atrophaeus. These results demonstrated the therapeutic potential of this recombinant endolysin. On the other hand, it was ineffective against L. monocytogenes ATCC 43,251, L. plantarum ATCC 14,917, and it did not exhibit lytic activity against Gram-negative bacteria such as E. coli and S. Typhimurium.

Numerous studies have been conducted regarding bacteriophage endolysins and their structures and applications as antibacterials in controlling and treating different bacterial infections [4, 25, 29,30,31]. Although many phage endolysins studied so far have demonstrated lytic activity against only their respective phage host strains, a number of endolysins from phages of Gram-negative and -positive bacteria exhibit cross-species lytic activity. The phage endolysin LysAB2 from Acinetobacter baumanii phage AB2 exhibits bacteriolytic activity against S. aureus in addition to (A) baumannii [32]. LysBPS13, an endolysin derived from Bacillus cereus bacteriophage was found to lyse (B) cereus and Bacillus thuringiensis and it was also active against EDTA-treated E. coli, Shigella, Salmonella, and Coronobacter sakazakii [33]. The Streptococcus suis phage endolysin, PlySs2, showed a broad lytic spectrum against a number of Gram-positive bacteria including vancomycin-intermediate S. aureus, MRSA, Staphylococcus simulans, S. epidermidis, Listeria, and enterococci as well as a number of streptococci [34]. An endolysin originated from a Stenotrophomonas maltophilia bacteriophage, P28, demonstrated high lytic activity against three Gram-negative bacteria; Xanthomonas citri Malvacearum sp. Shigella flexneri, and Klebsiella pneumonia ssp.pneumoniae, in addition to B. cereus, Bacillus subtilis, L. monocytogenes, and S. aureus [35]. Similarly, the TSPphg lysin from Thermus sp. TC4 phage-displayed lytic activity against both Gram-positive (Bacillus subtilis and S. aureus) and Gram-negative bacteria (K. pneumonia, E. coli O157, and Salmonella paratyphi B) [36]. In the present study, we cloned, expressed, and assessed the lytic activity of the S. aureus phage 52 endolysin, LysSA52.We demonstrated the ability of LysSA52 to inactivate not only approximately 90% of clinical strains of staphylococci, such as MRSA, S. epidermidis, and S. haemolyticus, but also a number of Gram-positive bacteria, including E. faecium, E. faecalis, S. pneumonia, S. pyogenes, and B. atrophaeus. However, LysSA52 was ineffective against commensal probiotic bacteria, such as L. plantarum, L. monocytogenes, E. coli, and S. Typhimurium. The LysSA52 endolysin proved to be quite stable; retaining activity for at least 9 weeks of storage at 4oC. Stability was also found to be a property of several other characterized bacteriophage endolysins. The lysK endolysin derived from S. aureus phage K remained active for at least 60 days of storage at 4 °C [37]. The Cpl-1 lysin of S. pneumoniae phage Cp -1 was stable for more than 6 months at 4 °C [38].

The potential of LysSA52 as a therapeutic agent for biofilm-associated S. aureus and S. epidermids was evaluated in vitro setup and showed about a 60% reduction of the already established biofilms. We considered this; it is one of the valuable characteristics of LysSA52 because those bacteria involved in biofilms are far more difficult to eradicate than their free-living counterparts [39].

In conclusion, the LysSA52 endolysin is a promising candidate for application, either individually or in combination with other antibacterial agents or each other with different cleavage specificities, as a disinfectant/therapeutic enzybiotic to control nosocomial infections caused by multiple drug-resistant bacteria. Further investigation is required to increase the lytic activity of this endolysin by genetic modifications of its relevant domains.