Bacterial characterization
Identification and growth conditions of bacterial isolates
In 2023, twenty clinical isolates of P. aeruginosa-infected UTI patients were gifted from the Rofayda Hospital, Giza, Egypt. The isolates were initially streaked onto selective media (Cetrimide agar, Oxoid, England) and identified based on their characteristic morphology. The identity of the bacterial strains was further verified and confirmed by the Vitek MS automated system (bioMérieux, Marcy l’Étoile, France). The bacterial colonies were then selected and stored in Tryptic Soy Broth (TSB, Oxoid, England) supplemented with 20% (v/v) glycerol at − 80 °C.
Antimicrobial susceptibility testing
The antibiotic profiles of twenty P. aeruginosa isolates were evaluated against ten different antibiotics using the disc diffusion method [22]. The tested antibiotics included piperacillin (PRL; 100 µg), piperacillin-tazobactam (TZP; 110 µg), ticarcillin-clavulanate (TCC; 75/10 µg), ceftazidime (CAZ; 30 µg), cefepime (Feb; 30 µg), imipenem (IPM; 10 µg), gentamicin (CN; 10 µg), tobramycin (NN10; 10 µg), amikacin (AK; 30 µg), and ciprofloxacin (CIP; 5 µg) (Oxoid, UK). The antimicrobial susceptibility of each isolate was assessed by measuring clear zone diameters in triplicate and then calculating the mean values. The results were interpreted according to the clinical and laboratory standards institute (CLSI) 2023 [23]. The reference strain Pseudomonas aeruginosa NCTC 12,903 / ATCC 27,853 was used as a quality control in the susceptibility test. Additionally, the multiple antibiotic resistance (MAR) index for each isolate was determined based on the method described by [24].
Phage isolation, purification, and amplification
Different liquid sewage samples were collected from October Gardens Water Station, Giza, Egypt. The sewage samples were subsequently centrifuged at 8000 rpm for 10 min, then the phage isolation was carried out through enrichment technique. Each collected sewage sample was mixed with an equal volume of a mixed culture of P. aeruginosa isolates in a sterile 50 mL centrifuge tube as previously described [25, 26]. The mixtures were incubated in the shaking incubator for 4 h at 37 °C. After incubation, the samples were centrifuged at 8000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm membrane filter to remove bacterial cells, and 1% chloroform was added.
Following the double agar method described by Clokie & Kropinski, with a slight modifications [27], 80 µL of exponential-phase bacterial host culture was mixed with 5 mL of soft agar (0.5% w/v) maintained at 45–50 °C. The mixture was poured onto a TSA plate, after that 10 µL was spotted onto the bacterial lawns in triplicate, and the plates were incubated at 37 °C for 24 h. Then, single plaques observed on the plates were picked using sterile pipette tips, suspended in 100 µL of sterilized Gelatin-SM buffer [5.8 g NaCl, 2.0 g MgSO₄·7 H₂O, 50 mL 1 M Tris-HCl (pH 7.4), and 1 L dH₂O] and stored at 4 °C for 4 h.
Purification of phages was achieved through a tenfold serial dilution of each single plaque, followed by repeated spot assays (six rounds) to obtain a single phage. To amplify phage concentration, 10 mL of bacterial host culture was infected with 100 µL of a single phage and incubated at 37 °C for 4 h, followed by centrifugation at 8000 rpm for 10 min and transferring the supernatant to a new centrifuge tube. A spot assay was then conducted as previously described to quantify the plaque-forming units (PFU)/mL.
Phage characterization
Pulsed-field gel electrophoresis (PFGE)
PFGE analysis was performed to estimate the purity and the genome size of the isolated phage (vB_Ps_ZCPS13) following the method described by Lingohr et al., with slight modifications [28]. In summary, 100 µL of phage suspension (10⁹ PFU/mL) was added to 100 µL of 1.4% plug agarose in plugs mold. After solidification, the plugs were immersed in lysis buffer containing 1 mg/mL proteinase K (ThermoFisher Scientific, USA), 0.2% w/v SDS (Sigma Aldrich, UK), 100 mM EDTA, and 1% w/v N-Lauryl sarcosine (Sigma Aldrich, UK), and the mixture was incubated at 55 °C for 18 h.
After the plugs were washed and loaded onto a PFGE gel composed of 1.5% agarose along with a Lambda PFG Ladder (Biolabs, UK) as a size standard, electrophoresis was carried out using a Bio-Rad CHEF DRII system (Bio-Rad, USA) for 18 h at 200 V (6 V/cm), with a switch time ranging from 30 to 60 s. The gel was then stained with 5 µL of ethidium bromide (Carl Roth, Germany) for visualization, and washed in distilled water, then analyzed under UV light using the ChemiDoc imaging system (Bio-Rad, USA).
Morphological characterization by transmission electron microscopy (TEM)
Phage vB_Ps_ZCPS13 was visualized using a JEOL 1230 transmission electron microscope (TEM) at the Faculty of Science, Alexandria University, Egypt. The phage suspension (10⁹ PFU/mL) was prepared in SM buffer, filtered, and applied to Formvar carbon-coated copper grids (Pelco International). The grids were stained with 2% phosphotungstic acid (pH 7.0) and dried before TEM examination.
Host range analysis
The lytic activity of the isolated phage was evaluated against a total of 30 clinical isolates of P. aeruginosa using the spot assay, which was performed in triplicate as previously described [29]. This included 20 primary isolates obtained from Rofayda Hospital, Giza, Egypt, which were fully characterized in this study, and an additional 10 clinical isolates to extend the host range assessment.
Briefly, to evaluate each bacterial isolate, 80 µL of fresh culture was added to 5 mL of soft agar (0.5% w/v) and poured onto a TSA plate. Afterwards,10 µL of phage lysate was spotted onto the bacterial lawns, and the plates were incubated at 37 °C for 24 h.
Relative efficiency of plating (EOP)
The effectiveness of the phage against P. aeruginosa host strains was further evaluated using the efficiency of plating (EOP) method as previously described [29]. In this method, the phage stock was serially diluted 10-fold, ranging from 10¹ to 10⁸, and 10 µL of each dilution was spotted in triplicate onto a fresh layer of each susceptible bacterial isolate identified through the host range assay. Plaques with the highest phage titer were counted after overnight incubation, and the average plaque-forming unit (PFU) count for each spot was calculated in triplicate for each bacterial isolate. The EOP was calculated by dividing the average PFU count of the target bacteria by the average PFU count of the host bacteria.
Determination of the frequency of bacteriophage insensitive mutants (BIMs)
To determine the BIM, the susceptible bacterial host Ps13 was treated with the phage at a multiplicity of infection (MOI) of 100. After 10 min of incubation at 37 °C, the suspension was serially diluted and spotted onto plates using the double agar overlay plaque assay in triplicate, then the plates were incubated overnight. BIM was calculated by dividing the number of viable bacteria remaining after phage infection by the initial viable bacterial count [30].
Physical stability of phage vB_Ps_ZCPS13
The stability of phage vB_Ps_ZCPS13 was evaluated under various conditions, including temperature, pH, and UV exposure. The thermal stability was evaluated by incubating the phage suspended in SM buffer at temperatures of -20 °C, 4 °C, 37 °C, 50 °C, 60 °C, 70 °C, 75 °C, and 80 °C for 1 h. Following incubation, the phage was quantified via tenfold serial dilution and assessed in triplicate via the spot test assay as outlined in previous study [31].
Furthermore, the pH stability was assessed by incubating the phage suspended in deionized water, adjusted to pH values of 2, 3, 5, 7, 9, 11, and 12 with HCl and NaOH for 1 h. The phage was subsequently quantified using the spot test assay.
Additionally, the susceptibility of the phage to UV inactivation was assessed by directly exposing the phage suspended in SM buffer to UV radiation (λ = 253 nm). Samples were collected at 15-minute intervals over the course of 1 h, and phage titers were determined using a spot test assay performed in triplicate.
In vitro experiments to study phage replication dynamics at different MOIs
The bacteriolytic activity of the phage was evaluated in vitro against the bacterial host (Ps13) at multiple multiplicities of infection (MOIs) [31]. Phage lysates with concentrations of 105, 106, and 107 PFU/mL were prepared to achieve MOIs of 0.1, 1, and 10, respectively. The bacterial host was cultured in TSB to reach the mid-log phase, with a concentration of 106 colony-forming units (CFU)/mL, confirmed by serial dilution and spot assay on agar plates. For each MOI, 20 mL of bacterial suspension was divided into two 10 mL aliquots: one for testing and the other for the control. The volume of the phage lysate was calculated based on each MOI. The test and control tubes were incubated in a shaker incubator at 37 °C and 100 rpm/min.
100 µL were taken from each tube at 15 distinct time points: 0, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, and 300 min. The collected samples were serially diluted tenfold in TSB, and each dilution was spotted onto agar plates. The bacterial counts in the control tube were calculated as CFU/mL. The surviving bacterial counts and free phage titers were calculated as CFU/mL and PFU/mL, respectively. The bacterial survival rates at the different MOIs were analyzed to identify the MOI with the most efficient bacteriolytic activity. These data provide insight into the optimal phage-to-bacterium ratio for effective bacterial eradication [32].
Genomic DNA extraction and sequencing
The genomic DNA of the isolated phage was extracted from 10 mL of purified high-titer phage lysate (10¹⁰ PFU/mL) using the phenol–chloroform–isoamyl alcohol method as previously described [33]. The DNA concentration and quality were then measured using the FLUOstar Omega Microplate Reader (BMG LABTECH, Germany). Nucleotide sequencing was subsequently performed on the Illumina MiSeq platform. The sequence reads were de novo assembled using Unicycler (v0.4.8) via the BV-BRC portal. The accuracy of the paired-end DNA reads was assessed through FASTQC [34].
Bioinformatics analysis of phage vB_Ps_ZCPS13
Genome visualization, comparison, and orientation were carried out using ProgressiveMauve and Ugene, with the Pseudomonas phage PAK_P4 (accession number NC_022986) as a reference [35, 36]. The assembled genome was annotated using the Rapid Annotation using the Subsystem Technology Toolkit (RASTtk) [37]. Another round of annotation was conducted after RASTtk annotation to assign functions to proteins with unassigned functions. For this purpose, tools such as NCBI BLASTp, UniProt Blast, PhageScope, HHPred, and InterProScan were used. The circular genomic map was generated utilizing CGView on the PROKSEE server [38].
The identification of temperate genetic markers, bacterial virulence factors, and antimicrobial resistance genes was conducted using BACterioPHage LIfestyle Predictor (BACPHLIP) and PhageLeads [39, 40]. The topology of the phage-predicted proteome was analyzed for transmembrane domains (TMDs) using DeepTMHMM [41]. Furthermore, genes with putative depolymerase function were also detected by Phage Depolymerase Finder (PhageDPO) [42].
Phylogenetic analysis
Circular and rectangular proteomic trees were constructed using the ViPTree server [43]. The results from ViPTree were used to identify phages exhibiting the highest tBLASTx scores (SG) and outgroup phages with the lowest SG scores. These, along with the top BLASTn hits from NCBI were used as inputs for the virus intergenomic distance calculator (VIRIDIC). The VIRIDIC tool is used to calculate pairwise intergenomic similarities between phage vB_Ps_ZCPS13 and the selected phages [44]. To address the taxonomic status of vB_Ps_ZCPS13, all established species of Pakpunavirus listed in the ICTV database were included in the analysis [45]. VIRIDIC was re-run accordingly to clarify whether vB_Ps_ZCPS13 represents a novel species or a new strain within the genus Pakpunavirus.
Further analysis of the closest-related phages was conducted using CoreGenes 5.0 to identify genes conserved across the genus and family levels [46]. The terminase large subunit (TerL), a signature gene, was used to construct a protein-based phylogenetic tree using MEGA11 [47], employing the CLUSTAL-W aligner and the best maximum likelihood fit model.
Screening for bacteriophage potency against bacterial biofilm
Phage antibiofilm activity was assessed at various MOIs (100, 10, 1, 0.1, 0.01, 0.001, and 0.0001) for two phenotypes: inhibition of biofilm formation and clearance of preformed biofilm. The biofilm formation, staining, and measurement were performed using the microtiter plate biofilm assay as previously described with slight modifications [48].
A fresh bacterial culture in TSB was adjusted to an exponential phase concentration of 10⁵ CFU/mL, confirmed by serial dilution and spot assay on agar plates, and 180 µl of this culture was added into the wells of polystyrene microtiter plates (Greiner Bio-One, Portugal). For controls, a row of six wells on each microtiter plate contained 200 µl of untreated culture, consisting of 180 µl of bacterial suspension and 20 µl of TSB (the same diluent used in phage MOI preparations).
Biofilm inhibition assay
The bacterial cultures were subjected to phage treatment from the start of the experiment and incubated at 37 °C for 48 h. Each MOI was tested in six replicate wells, with MOIs ranging from 0.0001 to 100, corresponding to phage concentrations of 10² PFU/mL to 10⁸ PFU/mL. A 20 µL volume of phage, adjusted to the required MOI, was added to each well.
Biofilm clearance assay
The biofilm clearance assay was conducted in the same approach as the inhibition assay, with the exception that bacterial cultures were first incubated in the wells for 48 h without phage treatment to allow for mature biofilm formation. Following incubation, phage preparations were introduced at various MOIs to the untreated wells and incubated at 37 °C for 24 h.
Planktonic cells and media were discarded in both assays, and the wells were washed by PBS to remove unattached cells. The plates were then inverted to remove residual liquid and air-dried. The adhered biofilm was stained with 200 µl of freshly prepared 0.1% (w/v) crystal violet. The excess stain was discarded, and the plates were rewashed and dried at room temperature. The stain was solubilized in 200 µl of 30% (v/v) acetic acid to quantify the biofilm, and the optical density of the solubilized biofilm was measured at 590 nm using the FLUOstar® Omega Microplate reader (BMG LABTECH, Germany).
Bacteriophage potency against bacterial biofilm on urinary catheter surfaces
The antibiofilm activity of the phage on urinary catheter surfaces was evaluated at the chosen MOI 1 for the two phenotypes: biofilm inhibition and biofilm clearance. Biofilm experiments were conducted in Wasserman tubes, containing silicone urinary catheters (size 14) cut into 1 cm segments, each placed in individual tubes. Fresh TSB-diluted bacterial culture was subsequently grown to a concentration of 105 CFU/mL, confirmed by serial dilution and spot assay on agar plates, and phage lysate with a concentration of 106 PFU/mL. For each sample, the catheter segment with untreated culture was used as a negative control. Biofilm inhibition and clearance followed the same methodology as described for the biofilm assay performed in the microtiter plate, except that the total volume used was 4 mL (3.6 mL bacterial culture and 0.4 mL phage lysate).
After incubation at 37 °C, each catheter segment was washed three times with PBS to remove planktonic cells. The catheter segments were transferred to new Wasserman tubes containing 1mL saline and followed the vortexing–sonication–vortexing (V-S-V) method to dislodge adherent cells from the catheter surface [49]. The number of viable bacterial cells was subsequently quantified by performing a spot test assay on serially diluted samples. For further qualitative analysis using a scanning electron microscopy (SEM), catheter samples were fixed with 2.5% glutaraldehyde, followed by gold sputter coating.
Phage cytotoxicity effect on normal human cell lines
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used to determine the effect of the phage on the viability of normal human skin fibroblast (HSF) cells. The HSF cells were grown in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Biowest, France) which contained 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. The cells were seeded at a density of 8 × 10³ cells per well in 96-well plates (CELLSTAR, Greiner Bio-One, Portugal) and incubated at 37 °C in a carbon dioxide incubator.
A purified lysate of the phage at a concentration of 10¹⁰ PFU/mL was applied to a proliferative monolayer of fibroblasts in wells containing a total volume of 200 µL. The effect of the phage on cell growth was examined after 24 h. Untreated cells in DMEM served as a negative control, while SM buffer diluted in DMEM with untreated cells served as the vehicle control.
Following incubation, the DMEM media was replaced with 100 µL of fresh DMEM containing 10% MTT labeling reagent (final concentration of 0.5 mg/mL) and incubated for an additional 4 h. The media was then carefully removed, and 100 µL of DMSO was added to each well to dissolve the formazan crystals formed. The absorbance of the resulting solution was measured at 570 nm using a FLUOstar Omega Microplate Reader. The optical density (OD) values were used to calculate cell viability as a percentage of the untreated cells (negative control) using the following formula [50, 51].
$$begin{aligned}&Cell:viability\& quad =100-left(left(:1-frac{{OD}_{phage-treated:cells}-{OD}_{blank}}{{OD}_{untreated:cells}-{OD}_{blank}}:right) times:100right)end{aligned}$$
Statistical analysis
All experiments were performed in triplicate, and the results were presented as the mean ± standard deviation (SD). Statistical analyses and graph generation were carried out using GraphPad Prism software. ANOVA and t-tests were used throughout the study to determine the significance values.
