Isolation and phenotypic characterization of bacteriophage SA14 with lytic- and anti-biofilm activity against multidrug-resistant Enterococcus faecalis

Abstract

Background

Antimicrobial resistance is a growing global health concern demanding more attention and action at the international-, national- and regional levels. In the present study, bacteriophage was sought as a potential alternative to traditional antibiotics.

Results

Vancomycin-resistant Enterococcus faecalis was isolated from a urine sample. Partial 16S rRNA-gene sequencing and VITEK®2 system were employed for its identification, biochemical characterization, and antibiotic susceptibility testing. The isolate was resistant to eight antibiotics (out of 11): vancomycin, gentamicin (high-level synergy), streptomycin (high-level synergy), ciprofloxacin, levofloxacin, erythromycin, quinupristin/dalfopristin, and tetracycline. Bacteriophage SA14 was isolated from sewage water using the multidrug-resistant isolate as a host. Transmission electron micrographs revealed that phage SA14 is a member of the Siphoviridae family displaying the typical circular head and long non-contractile tail. The phage showed characteristic stability to a wide range of solution pH and temperatures, with optimal stability at pH 7.4 and 4 °C, while showing high specificity toward their host. Based on the one-step growth curve, the phage's latent period was 25 min, and the burst size was 20 PFU/ml. The lytic activity of phage SA14 was evaluated at various multiplicities of infection (MOI), all considerably suppressed the growth of the host organism. Moreover, phage SA14 displayed a characteristic anti-biofilm activity as observed by the reduction in adhered biomass and -viable cells in the pre-formed biofilm by 19.1-fold and 2.5-fold, respectively.

Conclusion

Phage therapy can be a valuable alternative to antibiotics against multi-drug resistant microorganisms.

1 Background

The current status of limited antibiotic discovery besides the inappropriate use (misuse and overuse) of available antibiotics are major contributors to the rise of antimicrobial resistance (AMR) [1]. Consequently, World Health Organization (WHO) has announced the emergence of the post-antibiotic era where antibiotics will no longer be effective, leaving microbial infections with limited treatment options [2].

Enterococci are Gram-positive lactic acid bacteria that naturally inhabit the human gut [3]. Some strains are used as probiotics, while others are incorporated in the manufacture of fermented foods [4]. Yet, in the last few decades, enterococci have become a major public health concern causing a variety of hospital- and community-acquired infections [5,6,7]. For instance, Enterococcus faecalis accounts for the majority of enterococcal infections overall, and is the second most detected vancomycin-resistant enterococci (VRE). The largest fraction of hospital-acquired enterococcal infections, however, is attributed to Enterococcus faecium [8].

Most enterococci are intrinsically resistant to clindamycin, fluoroquinolones, trimethoprim/sulfamethoxazole, and low concentrations of β-lactams and aminoglycosides [9]. Moreover, they can acquire resistance to glycopeptides, tetracycline, erythromycin, fluoroquinolones, rifampin, chloramphenicol, fusidic acid, nitrofurantoin, and high concentrations of β-lactams and aminoglycosides [9]. This exceptional AMR pattern complicates their management [10,11,12,13]. So, the protocol for managing enterococcal infections is typically a combination of antibiotics [5].

The ability of enterococci to build up and grow within biofilms is an extra burden [14]. During biofilm formation, the microorganism adheres to and multiplies on a variety of surfaces, creating extracellular polymeric substances (EPS) as a matrix [15]. The EPS encourages bacterial adherence and protects against surrounding threats like the immune system and antimicrobials [16]. Therefore, biofilms are more often associated with a lower sensitivity of bacterial cells to antibiotics, resulting in prolonged infections and the development of AMR [17].

Bacteriophages, nanobiotics, enzybiotics, and vaccines, among others, have been suggested as potential alternatives to traditional antibiotics [18,19,20]. However, since bacteriophages are the natural predators of bacteria in the environment, they received more attention as a tool for limiting the expansion of bacterial populations [19]. Phage therapy is a term used to describe the removal of harmful germs through the application of bacteriophages [21]. In 1919, bacteriophages were utilized for the first time as medicinal agents [22] and were proven to be more effective than their chemical counterparts on several occasions [19, 23,24,25]. Moreover, they are non-toxic to humans, animals, and plants.

Phage therapy has been used for enteric and systemic therapies in nations like Russia, Poland, and Eastern Europe [26]. Georgia's Institute of Bacteriophage, Microbiology, and Virology is an institution for phage therapy [27]. Lately, phage management has gained more attention as a consequence of the increase in the frequency of bacterial multi-, extensively, and pandrug resistance [24]. There is, however, no single variety of phages that can kill all the harmful bacteria [19].

To date, only a few bacteriophages have been isolated and characterized against E. faecalis. The current investigation aims to isolate, identify, and phenotypically characterize a bacteriophage against a multi-drug-resistant clinical Enterococcus isolate and to assess its potential lytic and anti-biofilm activity.

2 Methods

2.1 Culture media and chemicals

Bile esculin agar (Cat. No. M9721), Muller Hinton agar (Cat. No. M173), MRS broth (Cat. No. M369), Luria Bertani broth (Cat. No. M1245), tryptone soy broth (TSB) (Cat. No. LQ508), and bacteriological agar were products of HiMedia (HiMedia Laboratories Pvt. Ltd., Mumbai, India). Bile Esculin Agar contains per liter: 5-g peptic digest of animal tissue, 3-g beef extract, 1-g esculin, 40-g bile salts, 0.5-g ferric citrate, and 15-g agar (pH 6.6), while Muller Hinton Agar contains per liter: 300-g beef infusion, 17.5-g casein acid hydrolysate, 1.5-g starch, and 17-g agar (pH 7.3). Sodium chloride, MgSO₄.7H₂O, Tris HCl, gelatin, glycerol, and sodium hydroxide were products of Merck (Merck, Germany).

2.2 Isolation of VRE from a clinical sample

Urine samples were obtained from biochemical analysis laboratories (private sector) and used as a source for the isolation of VRE. A urine sample (10 ml) was collected in a 15-ml sterile Falcon tube and then centrifuged at 3,000 g for 15 min. The supernatant was discarded, and the pellet was resuspended in 5 ml of sterile saline solution and streaked on bile esculin agar. The plates were cultured at 37 °C for 24 h. The colonies showing black precipitate were further examined by Gram-staining. Gram-positive isolates with cocci morphology were diluted serially in sterile saline solution and then cultured on Muller Hinton agar plates containing 16 μg/μl vancomycin as a selective medium for isolation of VRE. The plates were then incubated at 37 °C for 24 h. The resulting colonies were transferred to 2 ml of MRS broth supplemented with 30% glycerol for extended storage at – 20 °C. Only one isolate was chosen for further study and as a host for bacteriophage isolation.

2.3 Identification of the selected isolate

The identity of the selected isolate was determined using two different techniques; partial 16S rRNA gene sequencing and the VITEK®2 compact system (bioMérieux, Marcy l’Etoile, France).

2.3.1 Colorimetric identification and biochemical tests using the VITEK technique

A variety of biochemical experiments were performed on the selected isolate, including gelatin liquefaction and sugar fermentation using sorbitol, arabinose, arginine, and mannitol as carbon sources in the latter. The complete biochemical analysis and identification of the isolate were further done using the VITEK®2 compact system following the manufacturer's guidelines for sample preparation and operation.

2.3.2 Confirming the identity of the selected isolate by partial 16S rRNA gene sequencing

The bacterial genomic DNA was extracted as described earlier [28]. The following specific primers (Forward: F-1,5'-AAACTCAAATGAATTGACGG-3'; and Reverse: R-1, 5'-ACGGGCGGTGTGTAC-3') were used to amplify a portion of the 16S rRNA encoding gene using the polymerase chain reaction (PCR) thermocycler (Biometra thermocycler TAdvanced, Goettingen, Germany) [29]. The reaction mixture (25 μl) contained 12.5 μl 2X master mix, 1 μl forward primer (1.5 μM), 1 μl reverse primer (1.5 μM), 2 μl of template DNA, and up to 25 μl of water. The following conditions were used for the PCR reaction: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, and finally a single elongation step at 72 °C for 5 min. The amplicon size was determined using 1.5% w/v agarose gel electrophoresis. The gel was stained using ethidium bromide and visualized using a UV trans-illuminator (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany) using a 1 kb HyperLadder (Bioline reagents Ltd, London, UK).

DNA Clean & ConcentratorTM-5 (Zymo Research, Orange, CA, USA) was employed to separate the amplicons from the reaction mixture, which were sequenced in both the forward and the reverse directions (Macrogen Inc., Seoul, Republic of Korea). BioEdit software (version 7.2.5) was utilized for sequence editing and building up of the contigs. Finally, the resulting sequence was used to query the GenBank database to retrieve the sequences of the closest matching-type strains using the mega BLASt tool (https://www.ncbi.nlm.nih.gov/BLAST).

2.3.3 Antibiotic susceptibility testing (AST)

The isolate's resistance to 11 different antibiotics and antibiotic combinations including gentamicin/ampicillin, streptomycin/ampicillin, ciprofloxacin, levofloxacin, erythromycin, tetracycline, quinupristin/dalfopristin, vancomycin, linezolid, nitrofurantoin, and tigecycline was examined. Antibiotic Susceptibility Test (AST) cards that had been inoculated following the manufacturer's instructions were used in the analyses using the VITEK®2 system.

2.4 Isolation, propagation, purification and storage of Enterococcus phage

Sewage water obtained from a wastewater treatment facility in Beni-Suef, Egypt, was used as a source for Enterococcus bacteriophage. The sewage water was centrifuged at 10,000 g and 4 °C for 10 min to remove particulate matter. The supernatant was filtered through a 0.22-μm syringe filter to remove residual impurities and bacterial cells. The filtrate was combined with an equal volume of a double-strength LB broth, and then 200 μl of the overnight culture of the host organism (E. faecalis clinical isolate) was added to the mixture which was incubated in a shaker incubator at 37 °C and 120 rpm for 18 h. The culture was then centrifuged and filtered through a 0.22-μm syringe filter to remove cell debris, and the filtrate was used to measure the bacteriophage's ability to lyse bacteria using spot and plaque assays [30].

For the spot assay, 4 ml of a sterile LB soft agar (LB broth with 7 g/l bacteriological agar) was mixed with 1 ml of an overnight culture of the bacterial isolate. The mixture was then spread onto a sterile LB agar plate (LB broth containing 15 g/l bacteriological agar). The soft agar was then spotted with the clarified solution of the bacteriophage, and the plates were incubated at 37 °C for 18 h [31]. The resulting transparent spots are expected to be bacteriophages. The spots were then removed, and placed in a 2-ml Eppendorf tube with 1.5 ml of the SM buffer (5.8 g/l NaCl, 2 g/l MgSO₄.7H₂O, 50 ml/l 1 M Tris HCl at pH 7.5, and 5 ml/l 2% gelatin; pH 7.4), incubated at 30 °C for 2 h, and then stored as isolated bacteriophage stock.

For purification of the bacteriophage, a tenfold serial dilution of the isolated bacteriophage stock solution was generated, and 100 µl of each dilution was added to 100 µl of an overnight culture of the isolate, which was then poured into a soft agar tube to be overlaid on LB agar plate. The plates were incubated at 37 °C overnight until plaques were detectable. A small number of plaques were removed, placed in a 2-ml Eppendorf tube with 1.5 ml of SM buffer, and then incubated at 30 °C for 2 h. The tube was then preserved as an isolated bacteriophage stock. The steps of phage enrichment and purification were repeated several times until plaques having uniform sizes were obtained [32]. The bacteriophage solution in SM buffer was stored at 4 °C and used as a stock for further experiments.

2.5 Characterization of the bacteriophage against the clinical isolate

2.5.1 Determination of the Enterococcus phage morphology by TEM

The morphology of the isolated bacteriophage was determined using the procedures described elsewhere [33]. The solution of the phage (10⁶ PFU/ml) was purified by centrifugation at 14,000 g, at 4 °C for 1 h in 0.1 M ammonium acetate. The supernatant was discarded, and this step was repeated three times, respectively. The supernatant was then discarded, and the phage pellet was resuspended in SM buffer (pH 7.4). For negative staining of the phage sample, 2% uranyl acetate was added to the phage dispersion on copper grids. The grid was let to dry completely in the air before examination with an electron microscope using a magnification power of 371,000 × at 80 kV.

2.5.2 Host range determination of the Enterococcus phage

Bacteriophage activity was examined against a wide range of bacteria including four different clinical E. faecium strains [34], nine different Enterococcus spp. (pre-isolated from urine and stool samples—provided by Dr. Ahmed F. Azmy), one vancomycin-resistant E. faecalis ATCC583, one vancomycin-resistant S. aureus ATCC43300, one E. coli O157 ATCC6933, and one P. aeruginosa ATCC9027. The phage's specificity to the different strains was determined using the spot test.

2.5.3 One-step growth curve (OSG) of the Enterococcus phage

Ten milliliters of the mid-exponential phase culture (OD_620nm = 0.4) were centrifuged at 7,000 g and 4 °C for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 5 ml of fresh LB media to reach a final OD_620nm of 1. This suspension was mixed with 5 ml of 10⁵ PFU/ml phage solution (multiplicity of infection (MOI) of 0.1) and then incubated for 10 min at 37 °C to allow the adsorption to take place. The mixture was centrifuged at 7,000 g and 4 °C for 5 min, and the pellet was re-suspended in 5 ml of fresh LB broth and then incubated at 37 °C. Over the course of 30 min, samples were collected every 5 min; after that, they were taken every 10 min for the next hour. For analysis, 100 µl of tenfold dilutions of each sample were mixed with an equal quantity of overnight culture of host strain and 5 ml of fresh LB soft agar and was poured as an overlay. After 24 h of incubation at 37 °C, the plates were examined.

2.5.4 Thermal and pH stability of the Enterococcus phage

The thermal stability of phage (10⁶ PFU/ml) was tested over a period of 1 h at 37 °C, 44 °C, 45 °C, 50 °C, 65 °C, and 75 °C [35]. At 10-min intervals, samples were collected and serially diluted in 900 µl of SM buffer. The phage titer was calculated using the plaque assay.

For determination of the pH stability, the phage was incubated at 37 °C for 1 h in SM buffers with different pH values (2.5, 5, 7, 9, and 11) [36]. Subsequently, the number of plaque-forming units was counted using plaque assay.

2.5.5 Effect of divalent ions on the Enterococcus phage adsorption

The effect of Ca⁺² or Mg⁺² ions on the phage adsorption was investigated by combining bacterial cells (25 ml in 100-ml flask) with 250 µl of the phage solution (at MOI = 0.5) in one flask (control), and with 250 µl of the phage solution and 250 µl of 10 mM CaCl₂ or 0.1 mM MgCl₂ in the other flasks. The mixtures were incubated at 37 °C for 20 min and then centrifuged at 10,000 g, for 5 min at room temperature. The supernatant was collected for counting the un-adsorbed phage. The influence of divalent ions on phage adsorption to the host bacterium was evaluated.

2.5.6 The anti-biofilm activity of the Enterococcus phage against the host bacterial strain

The effect of phage SA14 on the generated biofilm was assessed using previously developed methods [37]. Two different techniques were used for determining cell adherence; the overall biomass loss and the viable count (colony forming unit—CFU) techniques.

To measure the overall biomass loss, the host bacteria were grown overnight at 37 °C in TSB containing 1% glucose. One hundred microliters of the resulting culture were transferred to a 96-well microtiter plate, supplemented with 100-µl TSB containing 1% glucose and incubated at 37 °C for 48 h. The plate was then emptied and cleaned three times with a sterilized phosphate-buffered saline (pH 7.4). Subsequently, each well was filled with 100 µl of the phage solution in SM buffer (pH 7.4) at various MOIs (1, 10, and 100), and the plate was incubated for 2 h at 37 °C. The wells were emptied again, and the plate was placed in an oven at 60 °C for 15 min to fix the developed biofilm, after which 150 μl of 1% crystal violet was added to each well, and the place was kept standing for 15 min at room temperature. The wells were then cleaned using sterile water. The residual bound stain was solubilized in 150 μl of 95% ethanol for 30 min. The reduction in the biomass was calculated from the absorbance difference at 570 nm between the control and phage-treated wells.

For counting the residual viable bacterial cells, the biofilm was initially generated in a six-well plate. Subsequently, all the wells were immersed in the phage solution for 2 h. The solution was discarded, and the residual biofilm was immersed in sterile saline solution and subjected to sonication for 1 min before performing viable counting. Both the test and the control were carried out in three independent replicates.

2.6 Statistical analysis

The present data are the mean ± standard deviation over three replicates. The Student's t test was employed to look for significant differences between treatment means at a level of significance of 0.05, unless otherwise stated.

3 Results

3.1 Isolation, characterization, and identification of VRE

The selected bacterial isolate was a Gram-positive, round-shaped, which produced a black precipitate upon cultivation on the bile esculin agar. Enterococci can grow in the presence of 40% bile salts and hydrolyze the esculin with the formation of a black precipitate. Biochemical characterization revealed a positive result for sugar fermentation utilizing sorbitol, mannitol, and arginine, but a negative result for arabinose and gelatin liquefaction. A complete biochemical characterization was obtained using the VITEK®2 system, upon which the isolate was identified as E. faecalis (Additional file 1: Table S1). Subsequently, a fragment (1073 bp) of the 16S rRNA gene was amplified and used to confirm the isolate's identity. The sequence was deposited in the GenBank (Accession No.: ON999041) which showed high similarity with various sequences of E. faecalis type strains as revealed by BLAST analysis.

The antimicrobial susceptibility of the isolate was determined using the VITEK®2 system (Table 1). The bacterial isolate showed a multidrug-resistant pattern against high-level gentamicin (synergy with ampicillin), high-level streptomycin (synergy with ampicillin), ciprofloxacin, levofloxacin, erythromycin, vancomycin, tetracycline, and quinupristin/dalfopristin, while was sensitive to linezolid, tigecycline, and nitrofurantoin.

Table 1 Antibiotics susceptibility testing for the clinical E. faecalis isolate using the VITEK®2 system

3.2 Isolation, enrichment, and purification of Enterococcus phage

Sewage water from a wastewater plant in Beni-Suef was used as a source for Enterococcus bacteriophage. A clear zone of a bacteriophage was obtained against the selected isolate, which was further verified using the soft agar technique [38]. Moreover, the presence of distinct plaques on bacterial lawns (1.5 – 2.5 mm, with well-defined boundaries) revealed the lytic nature of this phage. Further purification of the resulting phage resulted in a homogeneous plaque size that was obtained after 13 purification rounds (Additional file 1: Fig. S1).

3.3 Morphology of isolated phage by TEM

According to TEM pictures, the isolated phage has a circular core and a lengthy, non-contractile tail (Fig. 1). Therefore, it was allocated under the Siphoviridae family and was given the designation vb_EFS_SA14 (abbreviated: SA14).

Fig. 1

Morphology of Enterococcus phage SA14 as seen by transmission electron microscopy (TEM). Phage SA14 was treated with 2% phosphotungstic acid negatively implanted and visualized using TEM at an accelerating voltage of 80 kV. The bars have a length of 100 nm. Two arrows point at the phage particles, showing a circular head and a long tail, a typical morphology of the Siphoviridae family. (A) and (B) show different phage particles at magnification powers of 247000 X and 371000 X, respectively

3.4 One-step growth curve of phage SA14

The OSG experiment was performed to assess the phage's latent period (the time it takes for newly released phages) and average burst size (the number of released phages per cell). Phage SA14 had a latent period of 25 min and the average number of new phage particles that were released from one infected cell (burst size) was estimated to be 20 PFU/ml (Fig. 2).

Fig. 2

One-step growth curve of phage SA14 showing a typical tri-phasic pattern. The latent period was 25 min, and the burst size is explained on the curve as about 20 PFU/ml. Experiments were run in three replicates; the presented data are the average of these replicates ± standard deviation

3.5 Host range determination of phage SA14

The host spectrum of phage SA14 was determined using seventeen different bacterial strains representing four different genera; Enterococcus, Escherichia, Staphylococcus, and Pseudomonas. All the test strains were insensitive to phage SA14 except E. faecium strain FM65 which showed minor sensitivity. This indicates that phage SA14 is highly specific to its host E. faecalis (clinical isolate).

3.6 Thermal and pH stability of phage SA14

Investigations were conducted to determine the temperature tolerance of the phage SA14 at pH 7. Initially, at 4 °C, the phage titer remained constant around 100% of its initial over a period of 1 h (Fig. 3A, Additional file 1: Fig. S2), while at 37 °C and 50 °C, the final titer was reduced to around 60% of the initial over the same period. For temperatures above 50 °C, only 34% and 20% of the phages’ titer was retained after incubation for 1 h at 65 °C and 75 °C, respectively (Fig. 3A, Additional file 1: Fig. S2).

Fig. 3

A Stability of phage SA14 under various temperature conditions. Infectivity of phage SA14 after exposure to a temperature ranging from 4 to 75 °C for 1 h. B Stability of phage SA14 in various solution pHs. Infectivity of phage SA14 after exposure to pH values ranging from (2.5 to 11) at 37 °C for 1 h. Asterisks indicate a significant difference from the control (**P < 0.01). Experiments were run in three replicates; the presented data are the average of these replicates ± standard deviation

With regard to pH stability, phage SA14 titer was very steady at pH 7.4 at 37 °C, slightly stable at high pH of 9 and 11, and at low pH of 5 (Fig. 3B), while a sharp reduction in phage titer was observed upon incubation under extremely acidic conditions (pH 2.5). The optimal pH for phage stability was 7.4, where the plaque forming units were significantly higher than that obtained at pH values of 2.5, 5, 8 (P < 0.01), and 11 (P < 0.05).

3.7 Effect of divalent ions on the adsorption of phage SA14

To investigate the effect of calcium and magnesium ions on phage adsorption, CaCl₂ and MgCl₂ (10 mM) were added, respectively, to a mixture of the phage solution and the test strain. The plaque assay was used to determine the number of free-floating phages after 20 min. As shown in Fig. 4, there was a significant reduction in the number of free bacteriophages in the solution in the presence of CaCl₂ compared to the control and MgCl₂ (P < 0.01). On the other hand, this difference was not significant in the case of MgCl₂ compared to the control.

Fig. 4

Effect of Ca⁺² and Mg⁺² ions on the number of free-floating phage SA14 particles after 20 min of incubation with the host microorganism. This experiment is a measure of the phage’s rate of adsorption. Asterisks indicate a significant difference from the control (**P  < 0.01). Experiments were run in three replicates; the presented data are the average of these replicates ± standard deviation

3.8 Anti-biofilm activity of phage SA14

In contrast to the untreated control, phage SA14 decreased the number of viable bacterial cells (P < 0.01) within the pre-formed biofilm (Fig. 5A). The viable count of the control was 8.2 × 10⁸ CFU/ml, while the count at MOI = 100, 10, and 1 reached 4.3 × 10⁷, 6.6 × 10⁷, and 2.8 × 10⁸ CFU/ml, respectively. Moreover, applying the crystal violet to determine the overall biomass loss showed that the stain intensity in the case of the phage-treated biofilm was substantially lower than that of the untreated control (P < 0.01) (Fig. 5B).

Fig. 5

Anti-biofilm activity of Enterococcus phage SA14 at different multiplicities of infection (MOI), A through analysis of viable bacterial count, and B through analysis of total biomass loss using crystal violet as a bacterial stain. Asterisks indicate a significant difference from the control (**P < 0.01). Stars indicate a significant difference between variables where: (^♦P < 0.05) and (^♦♦P < 0.01). Experiments were run in three replicates; the presented data are the average of these replicates ± standard deviation

3.9 Lytic activity of phage SA14

Phage SA14 exerts lytic activity against E. faecalis clinical isolate. When the phage solution was administered to the bacterial suspension at different MOIs (1000, 100, 10, 1), the bacterial density was considerably reduced as compared to the control (OD_{570 nm} of 0.35) (Fig. 6). The OD_{570 nm} of a bacterial suspension at MOI = 1000 scored less than 0.1, at MOI = 100 was equal to 0.1, and at both MOI 10 or 1 was between 0.1 and 0.15.

Fig. 6

Lytic activity of phage SA14 against host bacterial culture at different multiplicities of infection (MOI) followed by measuring the bacterial OD_570nm of overnight culture. [(■) Control, (◆) MOI:1, (●) MOI: 10, (▲) MOI:100, (*) MOI:1000]

4 Discussion

The extensive use of broad-spectrum antibiotics in the intensive care units exerts a selective pressure that encourages the proliferation of intrinsically resistant commensal enterococci. According to a recent surveillance of AMR in Europe covering eight different bacterial species, resistant E. faecalis and E. faecium were ranked fourth (8.4%) and sixth (5.5%), respectively, among the bacterial species under investigation [39]. Septicemia, endocarditis, urinary tract infections (UTIs), and wound infections were the most typical clinical enterococcal infections [29]. With regard to UTIs, a recent study by Kraszewska et al. [40] performed over a period of 5 years reported that enterococci were responsible for about 10% of UTIs.

Several studies have reported the isolation of phages against VRE from sewage, compost, and water channels [41,42,43]. In the present study, phage SA14 was isolated from sewage water against the MDR clinical E. faecalis. The temperature, the pH, the presence of divalent ions, and the host’s physiological stage are all factors that influence the activity of the phage [44, 45].

Phage SA14 demonstrated great neutral to alkaline pH stability while gradually losing infectivity at extreme acidic pH. This was in agreement with a previous study that showed an increased phage activity in the range from 5 to 11 [42]. The neutral to alkaline nature of the sewage water which is the biological environment surrounding the isolated phage could be the reason for this behavior. The observed pH stability of phage SA14 is highly desirable, since it is compatible with the physiological and intestinal pH (7 – 9) as well as that of the urine (pH 4.8 – 8).

Temperature is another crucial factor to be taken into consideration for phage survival and growth. It may have an impact on phage adhesion, entry, and propagation as well as the creation of therapeutic phage storage systems. Additionally, phages that can endure a variety of temperatures may result in lower maintenance expenses. Phage SA14 showed great stability at pH 4 °C, which indicates its high storage stability. At the operating temperature of 37 °C, the phage starts gradually losing its infectivity after 30 min reaching 70% of the initial count within 1 h. A somewhat similar behavior was observed at 50 °C. However, at higher temperatures, a sharp drop in phage SA14 infectivity was observed within 10 min highlighting very low stability at these temperatures. This behavior will have a negative impact on the phage stability during manufacturing, formulation and storage.

Divalent ions, mainly calcium, were reported to stimulate an alteration in cell surface receptors leading to increased virion concentration on the cell surface. They also stabilize the weak phage-receptor interaction and accelerate phage nucleic acid translocation resulting in enhanced host-killing ability [44, 46, 47].

The one-step growth curve of phage SA14 displayed a typical tri-phasic pattern. The phage's latent period was calculated from the OSG graph and is considered somewhat short. Previous studies have linked optimal latent time qualities to good viral fitness [48]. The latent duration of phage SA14 after infection was 25 min, which is less than that of other E. faecalis phages reported earlier (30–50 min) [49].

Several pathogenic bacterial strains were used to assess the host range of phage SA14, and the results showed that the phage was highly specific to its host, as is typical for other Enterococcus phages. Furthermore, phage SA14 showed strong lytic activity against its host at various MOIs between 1 and 1000.

The ability of phage SA14 to destroy the enterococcal biofilm, as demonstrated by crystal violet and viable count techniques, is a looked-for feature. Phage SA14 greatly reduced E. faecalis cell counts (for roughly 1.3 log cycles after 2 h at MOI of 100). This reduction is close to that reported earlier in another study showing that the lytic Enterococcus phages; Max and Zip, induced a reduction by 1.5–2 log cycles of E. faecalis and E. faecium, respectively, in the biofilm after 3–6 h of treatment [42]. Phage SA14 was also found to be capable of reducing the biofilm biomass by 2.5-folds when compared to the biomass content of the untreated biofilm. The age of the biofilm could have impacted the treatment process. In younger biofilms, exopolymeric components are more easily degraded by phages due to the high bacterial cell density and the rapid phage dispersion in the biofilm structure [50]; however, older biofilms prevent the phage from entering the core layers due to the formed thick matrix [51].

5 Conclusion

Finding an alternative approach for combating multidrug-resistant enterococci is crucial. Anti-E. faecalis phages have the potential to replace conventional antibiotics. The phage's stability throughout a wide range of temperatures and pH values, as well as, its brief latent period, are further advantages and lend credence to the phage's therapeutic utility in reducing human infections. The phage's potency against biofilms suggests that it could be used as a therapeutic agent either alone or in combination with antibiotics; nevertheless, more research is needed. Moreover, investigation of the phage-derived lytic enzymes (endolysins) and the mechanism of biofilm degradation is highly recommended.