Isolation and Characterization of a Novel Lytic Phage, vB_PseuP-SA22, and Its Efficacy against Carbapenem-Resistant Pseudomonas aeruginosa

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a serious public health threat in multiple clinical settings. In this study, we detail the isolation of a lytic bacteriophage, vB_PseuP-SA22, from wastewater using a clinical strain of CRPA. Transmission electron microscopy (TEM) analysis identified that the phage had a podovirus morphology, which agreed with the results of whole genome sequencing. BLASTn search allowed us to classify vB_PseuP-SA22 into the genus Bruynoghevirus. The genome of vB_PseuP-SA22 consisted of 45,458 bp of double-stranded DNA, with a GC content of 52.5%. Of all the open reading frames (ORFs), only 26 (44.8%) were predicted to encode certain functional proteins, whereas the remaining 32 (55.2%) ORFs were annotated as sequences coding functionally uncharacterized hypothetical proteins. The genome lacked genes coding for toxins or markers of lysogenic phages, including integrases, repressors, recombinases, or excisionases. The phage produced round, halo plaques with a diameter of 1.5 ± 2.5 mm on the bacterial lawn. The TEM revealed that vB_PseuP-SA22 has an icosahedral head of 57.5 ± 4.5 nm in length and a short, non-contractile tail (19.5 ± 1.4 nm). The phage showed a latent period of 30 min, a burst size of 300 PFU/infected cells, and a broad host range. vB_PseuP-SA22 was found to be stable between 4–60 °C for 1 h, while the viability of the virus was reduced at temperatures above 60 °C. The phage showed stability at pH levels between 5 and 11. vB_PauP-SA22 reduced the number of live bacteria in P. aeruginosa biofilm by almost five logs. The overall results indicated that the isolated phage could be a candidate to control CRPA infections. However, experimental in vivo studies are essential to ensure the safety and efficacy of vB_PauP-SA22 before its use in humans.


Introduction
Pseudomonas aeruginosa (P. aeruginosa, PA) is a Gram-negative bacterium involved in opportunistic infections in humans. P. aeruginosa is the main cause of nosocomial infections, which can result in the demise of those infected, especially among immunosuppressed patients with severe wounds, chronic obstructive lung disease, cystic fibrosis, ventilatorassociated pneumonia, and catheter-associated chronic urinary tract infections. Some 3 of 24 multidrug-resistant P. aeruginosa infections [23][24][25]. Knowledge about the potential therapeutic phages is vital for the development of phage therapy. Nevertheless, phages are not frequently used therapeutically due to various constraints, such as inadequate clarity of phage formulations, the narrow specific spectrum of activity, poor viability or stability of phage preparations, and a lack of understanding of the mechanism of action of phages and their heterogeneity [26,27]. Despite these constraints, the global increase in MDR pathogenic bacteria has led to the revival of interest in phage-based therapeutic approaches [23].
Phage therapy against P. aeruginosa biofilms has been assessed in both in vivo and in vitro studies. For instance, phage PELP20 showed a 3-log phage reduction of biofilm in a mouse model of cystic fibrosis infection [28]. Similarly, a temperate phage ΦPan70 significantly reduced P. aeruginosa biofilm production in a mouse model [29]. In another study, the combined use of ΦMR299-2 and ΦNH-4 eliminated the biofilm and killed P. aeruginosa growing in the cystic fibrosis bronchial epithelial CFBE41o-cell line. In in vitro experiments, bacteriophages such as M-1, PB1-like, phiKZ-like, and LUZ24-like phages have been shown to be effective in destroying the planktonic cells as well as the biofilm [30,31].
Despite the above-mentioned reports, very limited lytic bacteriophages have been characterized against CRPA infections so far [32]. Hence, this study was conducted to isolate and characterize a lytic bacteriophage targeting CRPA. In addition, the anti-biofilm effect of the isolated phage was investigated.

Bacterial Strains and Culture Condition
The clinical strain of CRPA (P. aeruginosa strain B10) was obtained from King Abdulaziz University hospital and used as a host for the isolation of vB_PseuP-SA22 from wastewater. Identification of the organism was carried out using an API 20 E test kit (bioMerieux Industry, Hazelwood, MO, USA), a VITEK identification system (bioMèrieux Inc., Durham, NC, USA) using GN ID REF21341 cards, and 16S rRNA molecular analysis and sequencing. The bacterial strains used to evaluate the host range of vB_PseuP-SA22 are listed in Table 1. Some of these isolates were obtained from the King Fahd Medical Research Center (KFMRC), while the others were purchased from American Type Culture Collection (ATCC). All bacterial isolates used in our study were preserved using 25% glycerol at −20 • C until used.

Antibiotic Sensitivity Test
Antimicrobial susceptibility was assessed using the standard agar disk diffusion method following the CLSI guideline (Supplementary Table S1). P. aeruginosa ATCC 27853 was used as a positive control [33].

Enrichment and Isolation of Bacteriophages
To isolate potential lytic bacteriophages, we collected wastewater samples from Jeddah Wastewater Treatment Plant, Saudi Arabia. The samples were transported to the KFMRC laboratory on ice, stored at 4 • C, and processed within 24 h. The wastewater samples were centrifuged at 10,000× g for 10 min, and the supernatant was passed through 0.22 µm filters (Fischer Scientific, Ottawa, ON, Canada) to remove unnecessary particulates (including bacterial cells) and debris. The filtrate was then directly used for phage isolation [34]. Briefly, 20 mL of the filtrate was added to 20 mL of an overnight culture of CRPA in double-strength nutrient broth supplemented with 2 mM CaCl 2 . The culture was placed in a shaking incubator with gentle shaking (100 rpm) at 37 • C for 48 h. Afterward, it was centrifuged at 6000× g for 12 min at 4 • C, and the supernatant was filtered using 0.22 µm size filters to exclude host bacteria and contaminants. Finally, the filtrates were stored at 4 • C until further use.

Spot Assay
The presence of phage in the lysate was determined by spot assay [35]. In brief, 0.1 mL of exponential phase host culture was mixed with 4 mL of molten soft agar (0.7% w/v agar), then poured on the surface of nutrient agar plates to create a lawn. Thereafter, 5 µL of the crude phage extract was spotted on top of the soft agar and kept in a laminar airflow for 20 minutes to allow for viral adsorption. The inoculated plate was incubated overnight at 37 • C. The sample was considered positive for phage if it produced a clear inhibition (lytic) zone.

Purification
Purification of the phage was carried out through a double-layer agar assay (DLA), as described elsewhere [36]. Briefly, typical phage plaques were picked up from the soft agar layer by touching the top layer using a 1 mL pipette tip and transferred into 500 µL sterile PBS (pH 7.4) suspension. The suspension was then kept at 4 • C overnight for proper diffusion of phages from the soft agar. Thereafter, 10-fold serial dilutions of the purified lysates were conducted, and DLA assay was carried out to quantify the phage titer and Antibiotics 2023, 12, 497 5 of 24 the uniformity of the plaques. The purification process was repeated until similar plaques were obtained. The purified lysate was stored at 4 • C.

Concentration of Phages
A full-plate lysate method was used to increase the titer of bacteriophages, following the technique detailed in [37,38]. Confluent lysis zone was produced in the top agar overlay, and 8 mL of PBS was poured over the top agar. Then, the plate was kept at 4 • C overnight for proper diffusion of the virions from the soft agar into PBS. Thereafter, the suspension was aspirated with a 10 mL sterile syringe and transferred into a test tube. The collected suspension was then centrifuged at 8000× g for 5 min, and the supernatant was passed through 0.22 µm filters to eliminate bacterial debris. The purified phage lysate was then used to determine the titer of phage using the DLA technique.

Bacteriophage Titer Determination
The purified phage lysate was diluted 10-fold in PBS solution and plated using the DLA technique. The plaques on each plate were individually counted. To calculate the phage titer, we used the last plate, which had plaque counts ranging from 30-300. The titer was calculated, and the results were recorded as PFU mL − 1 [39]. The size of the plaque was measured using a ruler, and photographs were captured using a digital camera.  [40]. Briefly, micro-centrifuge tubes containing a mixture of 100 µL of the purified phage lysate (1 × 10 8 FU/mL) and 900 µL PBS (pH 7.4) were incubated in a water bath at different temperatures (4 • C, 40 • C, 50 • C, 60 • C, 70 • C, 80 • C, or 90 • C) for 1 h. Phage suspended in an equal volume of PBS (pH 7.5) kept at 4 • C was used as a control. Then, the phage titer after treatment was determined by the DLA method. This experiment was carried out in triplicate.

Phage pH Stability Test
The pH stability of the vB_PseuP-SA22 was tested as described elsewhere, with minor adjustments [41]. The pH of fresh/sterile nutrient broth (pH 2-14) was adjusted, using a pH meter, by adding 1 M HCl and NaOH drop by drop. Nine milliliters of pH-adjusted medium was mixed with 1 mL of phage suspension and incubated at 37 • C for 1 h. Then, the titers of vB_PseuP-SA22 in each suspension were determined using the DLA method. This experiment was carried out in triplicate.

Bacterial Reduction Assay
The in vitro host reduction ability of vB_PseuP-SA22 was assessed as described elsewhere, with minor modifications [42]. Briefly, 100 µL of P. aeruginosa culture (OD 0.4, 10 7 CFU/mL) was added to 100 µL of diluted isolated phage (10 4 -10 9 PFU/mL) in microtiter plate wells. Thus, 0.001, 0.01, 0.1, 1, 10, and 100 multiplicities of infection (MOI) were generated. The bacterial reduction potential was measured by DLA at different time intervals over 48 h. Bacterial culture without phage (OD 0.4) was used as a control (100 µL of bacterial culture mixed with 100 µL of fresh nutrient broth). The results are presented as mean values ± SD from three independent experiments, and the bacterial reduction curve (CFU/mL vs. time) was also plotted.

Determination of Optimal Multiplicity of Infection (MOI)
The bacterial cultures in the exponential phase were washed away with PBS three times and adjusted to analogous concentrations of 10 6 , 10 7 , 10 8 , 10 8 , 10 8 , and 10 8 CFU/mL, then the phages and bacteria were mixed with MOIs of 100, 10, 1, 0.1, 0.01, and 0.001, respectively. Thereafter, the suspension was incubated for 2 h at 37 • C, and the titer of phages was carried out by the DLA method [43]. The optimal MOI was determined according to the ratio of the specified titer (PFU/mL) of phages to the specified concentration of host bacteria (CFU/mL); in particular, the MOI value attaining the highest phage titer was considered the optimal MOI.

Phage Adsorption Assay
The adsorption rate of phages onto the surface of host cell was assessed based on a formerly used protocol [44]. The exponential phase bacterial culture was collected and re-suspended in sterile nutrient broth to reach an OD 600 of 0.4. Then, the phage was added at an MOI of 0.1, and the adsorption was monitored at 37 • C for 90 min. One milliliter aliquot was collected at regular time intervals and centrifuged (10,000× g, 10 min). The supernatant was filtrated using a 0.22 µm pore-size filter. The titers of the phage in the original phage stock (t0) and supernatant (t1) were determined by the DLA technique. The adsorption rate was estimated as (t0 − t1)/t0.

One-Step Growth Curve
A cycle growth pattern test was performed using vB_PseuP-SA22 and its host bacteria in order to investigate the latent period and the burst size, as described by others, with minor modifications [35]. Briefly, the experiment was initiated at an MOI of 0.1 in a sterile centrifuge tube containing phage (1 × 10 7 PFU/mL) and its host strains (1 × 10 8 CFU/mL) in 10 mL nutrient broth. The mixture was placed in a shaker incubator and incubated at 37 • C for 10 min for proper adsorption of phage onto the host cell surface. Afterward, the suspension was centrifuged at 12,000× g for 10 min. After removing the supernatant, the pellet of infected cells was mixed with 10 mL of pre-warmed nutrient broth and incubated in a shaker incubator (120 rpm) at 37 • C. Thereafter, 100 µL of sample was collected at 10-min intervals over a period of 60 min. To determine the phage titers, we diluted aliquots with PBS (pH 7.4), and the soft agar overlying technique was used. The time difference between adsorption (excluding the first 20 min of pre-incubation) and the initial point of the first burst was considered as the latent period, and the ratio of the end count of progeny virions to the number of infected bacterial cells found at the initial point during the latent period was considered as the burst size.

Host Spectrum
The host range of vB_PseuP-SA22 was determined by a spot assay, as described elsewhere [45], using a panel of 23 species of bacteria with different antimicrobial sensitivity profiles (Table 1). Briefly, 5 µL of the isolated phage (10 9 PFU/mL) was dropped onto the lawn of the tested species. Then, after overnight incubation at 37 • C, the presence of plaques (lytic zones) on the spotted area was determined to be positive, whereas the absence of any lytic zone was considered negative for the test. Positive results were further confirmed by the DLA method.

Transmission Electron Microscopy (TEM)
The morphology of vB_PseuP-SA22 was determined by TEM. The purified phage lysate (1 × 10 9 PFU/mL) was placed onto thin carbon films, and the adsorbed phages were negatively stained with 2% (w/v; pH 5.0) uranyl acetate [46]. The virions were observed using a TEM 910 (Carl Zeiss, Oberkochen, Germany) at 80 kV. The morphology of the virions was captured using a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany). Certain parameters, such as the head and tail length of the virion, were measured twice using the ImageJ software. The identification guidelines indicated by the International Committee of Taxonomy of Viruses were implemented to assess the phenotypic diversity of the virion [47].

Test Tube Biofilm Production Assay
The biofilm production capacity of CRPA was determined according to the tube biofilm production method (TM) described by Di Domenico et al. (2016) [48]. The test organism was first inoculated in a polystyrene test tube which contained nutrient broth, then incubated at 37 • C for 24 h. Next, the planktonic cells found in all tubes were discarded and rinsed twice with PBS. Thereafter, the emptied polystyrene tubes were stained with 0.1% crystal violet (CV; HiMedia Labs Pvt. Ltd., Dindori, Nashik, India) for 1 h. Then, after air-drying, if stained visible film lined the inside walls, the bottom of the tube was considered to be positive for biofilm production. A biofilm-negative culture of Escherichia coli (E. coli) was used as a negative control.

Microtiter Plate Biofilm Production and Reduction Assay
The anti-biofilm activity of vB_PseuP-SA22 was assessed in optically clear, flat-bottom 96-well plates (SPL, Pocheon, Korea), in accordance with the methods stated by Fong et al. [49], with slight modifications. Briefly, the overnight grown culture (1 × 10 7 cells/mL) was inoculated into the sterile nutrient broth, following which aliquots (200 µL) were distributed to individual wells. Two different microplates, one for staining and another for enumeration of bacteria, were then incubated at 37 • C for 12 h with slight shaking (120 rpm). The supernatant of each individual well was discarded and rinsed twice with PBS (pH 7.4) to eliminate all planktonic cells, followed by treatment with 200 µL of phage suspension (1 × 10 6 PFU/mL) for 4, 8, and 12 h. The plates were then washed twice with PBS and air-dried. The number of bacterial cells in the biofilm was enumerated by re-suspending the biofilm in PBS after scraping the well with a sterile pipette tip, following which the suspension was diluted and plated. The total biomass of the biofilm was quantified by staining with CV (0.1%, w/v) for 15 min, and the wells were then rinsed with PBS. CV was dissolved in an acetone-ethanol solution (20:80, v/v), and the intensity of CV staining (OD value) was quantified at 595 nm using a Plate Reader Infinite 200 pro (MTX Lab Systems, Austria) [49]. This assay was repeated three times. The OD values higher than the control or blank well were considered to indicate biofilm producers.

Scanning Electron Microscopy
Scanning electron microscopy (SEM) was conducted at KFMRC in order to assess the biofilm reduction potential of vB_PseuP-SA22, following a previously described method [50,51]. Biofilms were grown on a glass coverslip initially placed into a 96-well microtiter plate, then rinsed twice with PBS and dried in a bacteriological incubator at 37 • C for 20 h. The mounted biofilms were fixed with glutaraldehyde (2.5%) and dehydrated continuously with different concentrations of ethanol (30-100%) for 5 min in each. Thereafter, they were sputtered with gold, and the status of the biofilms was examined using SEM (SEM; Norcada Inc., Edmonton, AB, Canada).

Extraction of Bacteriophage DNA
The DNA of the concentrated phage (1 × 10 12 PFU/mL) was extracted using a phage DNA extraction kit (QIAGEN, Germany) based on the instructions provided by the manufacturers. The concentration and purity of the extracted DNA were assessed using a spectrophotometer (Invitrogen Qubit) and examined by agarose gel electrophoresis [52].

Whole-Genome Sequence and Bioinformatic Analysis
Sequencing of the vB_PseuP-SA22 genomic DNA was performed at the Pittsburgh genome sequencing center (USA) using an Illumina HiSeq platform. The sequence output was assembled using ABySS v2.0.2, and the resulting contigs were assembled using the assembly algorithm Newbler version 3.0, with default parameters. RAST and GeneMark were used to predict and annotate potential open reading frames. The circular map was built using the CGView online tool, and the GC content and GC skew of the genome were assessed accordingly [53]. We used the PHIRE platform to generate promoters specific to the vB_PseuP-SA22 DNA sequence [54]. The ARNOLD online tool was used to identify the number and types of Rho-factor independent terminators [55]. GeneMarks [56] and PHAST were employed to locate the open reading frames (ORFs) [57]. The function of each coding sequence was predicted by the protein basic local alignment search tool (Blastp) of the NCBI server [58]. GtRNAdb and tRNA Scan-SE tools were used to predict putative tRNAs [59,60]. The existence of allergic proteins in the predicted coding sequences was also inspected using the food and allergy search tool. Finally, the existence of virulence factors was assessed by ResFinder and the virulence factor database (VFDB) [61,62].

Phylogenetic Analysis
Nucleotide sequence alignment was performed using ClustalW, and the resulting aligned sequence was used to construct the phylogenetic tree using the MAFFT v.7 software. During the construction of the tree, the neighbor-joining method and 1000 bootstrap replicates were applied [63,64].

Comparative Genomic Analysis
The comparative genomic sequence was conducted using the circoletto program, and a circular ideogram was built according to a method described elsewhere [65].

Statistical Analysis
The data were recorded as means ± SE of triplicate experiments. The statistical analysis was conducted using two-way analysis of variance (ANOVA). We used version 6 of the GraphPad Prism software to perform the analyses. The significance level was set at p < 0.05.

Nucleotide Sequence Accession Number
The sequence data for the P. aeruginosa phage vB_PseuP-SA22 was deposited in the GenBank under accession number OP793496.

Identification of the Host Bacteria and Its Antibiotic Sensitivity Profile
The host bacteria were identified phenotypically by API 20 (Supplementary Figure S3) and Vitek (Supplementary Table S3) and further characterized at the molecular level, with 16s rRNA sequence analysis carried out for confirmation. The BLAST nucleotide similarity search revealed that the used CRPA showed 99% sequence similarity to P. aeruginosa deposited in the national database (Accession: JQ900545.1).

Phage Isolation and Morphology
We isolated a novel wastewater phage, vB_PseuP-SA22, using CRPA P. aeruginosa strain B10 as a host. The isolated phage produced round, halo plaques of 1.5 ± 2.5 mm in diameter on a lawn of the host cells ( Figure 2B). The TEM results revealed that vB_PseuP-SA22 had an icosahedral head of 57.5 ± 4.5 nm in length and had a short, non-contractile tail (19.5 ± 1.4 nm). It was found to have a podophage morphology ( Figure 2C).

Phage Isolation and Morphology
We isolated a novel wastewater phage, vB_PseuP-SA22, using CRPA P. aeruginosa strain B10 as a host. The isolated phage produced round, halo plaques of 1.5 ± 2.5 mm in diameter on a lawn of the host cells ( Figure 2B). The TEM results revealed that vB_PseuP-SA22 had an icosahedral head of 57.5 ± 4.5 nm in length and had a short, non-contractile tail (19.5 ± 1.4 nm). It was found to have a podophage morphology ( Figure 2C).

Phage Adsorption Assay and One-Step Growth Curve
The rate of adsorption of the vB_PseuP-SA22 phage on the host cell surface is presented in Figure 3A. The virions attached to the host bacterial cell surface quickly, and 60% had adsorbed at 20 min post-infection. Nearly all virions were attached to the surface of the host cells at 23 min post-infection ( Figure 3A).
A one-step growth experiment was carried out to assess the burst size and the latent

Phage Adsorption Assay and One-Step Growth Curve
The rate of adsorption of the vB_PseuP-SA22 phage on the host cell surface is presented in Figure 3A. The virions attached to the host bacterial cell surface quickly, and 60% had adsorbed at 20 min post-infection. Nearly all virions were attached to the surface of the host cells at 23 min post-infection ( Figure 3A).

Stability Test and Bacterial Challenge Test
The temperature stability assessment indicated that vB_PseuP-SA22 was stable tween 4-60 °C for 1 h, while the viability of the virus was reduced at temperatures o 60 °C. The approximate titer was 8 log10 PFU/mL upon incubation at 4 °C, 37 °C, 40 °C °C, or 60 °C for 1 h, and no significant differences (p > 0.05) were noticed in this temp ture range. Nevertheless, we noticed that the thermal resistance rate declined to roug A one-step growth experiment was carried out to assess the burst size and the latent period of vB_PseuP-SA22. The growth pattern indicated that the latent period of vB_PseuP-SA22 was roughly 30 min, and the mean burst size was found to be 300 PFU/infected cell ( Figure 3B).

Stability Test and Bacterial Challenge Test
The temperature stability assessment indicated that vB_PseuP-SA22 was stable between 4-60 • C for 1 h, while the viability of the virus was reduced at temperatures over 60 • C. The approximate titer was 8 log 10 PFU/mL upon incubation at 4 • C, 37 • C, 40 • C, 50 • C, or 60 • C for 1 h, and no significant differences (p > 0.05) were noticed in this temperature range. Nevertheless, we noticed that the thermal resistance rate declined to roughly 5.35 log 10 PFU/ml (p < 0.05) at 70 • C, and the phage fully lost viability at 80 • C, as shown in Figure 4A. The lysis kinetics of vB_PseuP-SA22 were determined at different MOIs (100, 10 0.1, 0.01, and 0.001). Based on two-way ANOVA tests, significant differences were served for all MOIs in comparison to the control (0 MOI; p < 0.05). As shown in Figure 4 the density of bacteria in the untreated control increased by ∼3.05 log CFU/mL at 12 h incubation. The highest reduction in the concentration of live bacteria, by 4.67 lo CFU/mL at 12 h, was recorded at an MOI of 0.1 compared to the original concentration < 0.05). Similar reductions (by approximately 2.85 log10 CFU/mL) were observed at MO of 1, 10, and 100.

Host Range
The spectrum of lytic activity of vB_PseuP-SA22 was assessed by spot assay and co firmed by DLA. To do this, we used a phage stock with a titer of 1 × 10 8 PFU/mL agai a panel of 23 bacterial strains ( Table 1). The results revealed that vB_PseuP-SA22 show lytic activity against 47.8% (n = 11) of tested bacterial strains, including 8/11 (72.7%) of aeruginosa strains (Table 1). These results, therefore, indicated that vB_PseuP-SA22 ha broad host range.

Genomic Analysis of the vB_PseuP-SA22 Phage Genome
Whole-genome sequence analysis revealed that the genome of vB_PseuP-SA22 w 45,458 bp (45.5 Kb) in length, with a GC content of 52.5% ( Figure 5). Using Artemis a BLAST analysis, 58 ORFs were predicted as protein-coding sequences (CDSs) transcrib in both directions. Of the 58 putative ORFs, 32 ORFs were on the negative strand, wh The pH stability test results indicated that the activity of vB_PseuP-SA22 was relatively stable at pH values between 5 and 11; in particular, vB_PseuP-SA22 retained approximately 8 log 10 PFU/mL from pH 5-11 for 1 h. Nevertheless, it displayed a significant reduction (p < 0.05) in titer at pH 4 and 12, with titers of 3.5 and 3.15 log 10 PFU/mL, respectively. Furthermore, vB_PseuP-SA22 was inactivated at pH ≤ 2 and pH ≥ 12. pH 7 and 8 were found to be the optimal pH values for vB_PseuP-SA22 ( Figure 4B).
The lysis kinetics of vB_PseuP-SA22 were determined at different MOIs (100, 10, 1, 0.1, 0.01, and 0.001). Based on two-way ANOVA tests, significant differences were observed for all MOIs in comparison to the control (0 MOI; p < 0.05). As shown in Figure 4D, the density of bacteria in the untreated control increased by ∼3.05 log CFU/mL at 12 h of incubation. The highest reduction in the concentration of live bacteria, by 4.67 log 10 CFU/mL at 12 h, Antibiotics 2023, 12, 497 13 of 24 was recorded at an MOI of 0.1 compared to the original concentration (p < 0.05). Similar reductions (by approximately 2.85 log 10 CFU/mL) were observed at MOIs of 1, 10, and 100.

Host Range
The spectrum of lytic activity of vB_PseuP-SA22 was assessed by spot assay and confirmed by DLA. To do this, we used a phage stock with a titer of 1 × 10 8 PFU/mL against a panel of 23 bacterial strains ( Table 1). The results revealed that vB_PseuP-SA22 showed lytic activity against 47.8% (n = 11) of tested bacterial strains, including 8/11 (72.7%) of P. aeruginosa strains (Table 1). These results, therefore, indicated that vB_PseuP-SA22 has a broad host range.
The genome of vB_PseuP-SA22 was identified to be linear, as it was cleaved by the exonuclease Bal31, which degrades only double-stranded linear DNA from both ends simultaneously. It does not comprise genes coding for recombinases, integrases, excisionases, or repressors, which are the chief markers of lysogenic bacteriophages. Hence, we confirmed that our isolate was likely an obligate lytic phage following only a lytic cycle. Additionally, no genes were found that encode virulence factors, as assessed by testing against the VFDB. Thus, the results suggest that vB_PseuP-SA22 meets multiple requirements of a safe and virulent phage and, therefore, is a candidate for use in treating P. aeruginosa-associated infections.    The genome of vB_PseuP-SA22 was identified to be linear, as it was cleaved by the exonuclease Bal31, which degrades only double-stranded linear DNA from both ends simultaneously. It does not comprise genes coding for recombinases, integrases, excisionases, or repressors, which are the chief markers of lysogenic bacteriophages. Hence, we confirmed that our isolate was likely an obligate lytic phage following only a lytic cycle. Additionally, no genes were found that encode virulence factors, as assessed by testing against the VFDB. Thus, the results suggest that vB_PseuP-SA22 meets multiple requirements of a safe and virulent phage and, therefore, is a candidate for use in treating P. aeruginosaassociated infections.

Predicted tRNA and Rho-Independent Transcription Terminators
A total of three tRNAs were predicted using the tRNAscan-SE de facto tool. They were asparagine (Asn), aspartic acid (Asp), and proline (Pro) tRNAs with GTT, GTC, and TGG anti-codons, respectively (Table 3). The predicted tRNAs were situated at different locations in the whole genomic sequence with the specified isotype score (Supplementary Figure S2). A total of eight rho-independent terminators were predicted by ARNold (RNAmotif and/or Erpin tool). The terminators were composed of loops and stems located at different regions of the genomic sequence (Supplementary Figure S1).

Phylogenetic Analysis
Phylogenetic analyses of vB_PseuP-SA22 were conducted using BLASTn in comparison to the reference phage sequences deposited in a public database (NR, NCBI), and the tree was constructed using MEGA software. The whole-genome phylogenetic analysis indicated that the isolated phage showed high homology with Pseudomonas phages that belong to the family Podoviridae, genus Bruynoghevirus, as shown in Figure 6A. It had 96%, 94%, and 90% similarity with 95%, 98%, and 74% query coverage with Pseudomonas phages PSA16 (MZ089733.1), SaPL (MH973725.1), and Epa1 (MT108723.1), respectively. This suggests that vB_PseuP-SA22 also belongs to the family Podoviridae, genus Bruynoghevirus. Similarly, the constructed phylogenetic analysis relying on the terminase large sub-units indicated that the upper five phages classified under the Podoviridae family-namely, Pseudomonas phage phiPA01_302, Pseudomonas phage oldone, Pseudomonas phage U47, Pseudomonas virus Pa22, and Pseudomonas phage vB_PaeP_C2-10_Ab22-showed higher sequence similarity than the other phages classified under the same family ( Figure 6B).

Comparative Genomic Analysis
Comparative genomic analysis was performed using the circoletto program in order to determine the sequence similarity between the selected four Pseudomonas phage genomes and vB_PseuP-SA22. Each quadrant represents an individual genome, and the ribbons connecting genomes represent local alignments produced by BLAST (Figure 7). The high sequence similarity is represented by red, followed by orange and blue colors. The results revealed that all four BLAST sequences displayed high-level sequence similarity to the query phage sequence (vB_PseuP-SA22).

Biofilm Production
In the first experiment, the test tube ring assay technique was used to assess the biofilm production capacity of P. aeruginosa strain B10. In this assay, the overnight culture of planktonic bacteria was gently removed, and the remaining biofilm (Supplementary Figure S4A,B) was stained using crystal violet and thus visualized, as shown in Supplementary Figure S4C. The three polystyrene test tubes inoculated with P. aeruginosa strain B10 produced a white clump on the inner wall of the test tube, which was stained violet. These results indicated that the organism was positive for biofilm production. Similarly, in the second experiment, biofilm production was assessed and quantified using a microtiter plate assay. The round violet rings on the inner wall and the bottom of the microtiter plate were indicative of biofilm production. PSA16 (MZ089733.1), SaPL (MH973725.1), and Epa1 (MT108723.1), respectively. This suggests that vB_PseuP-SA22 also belongs to the family Podoviridae, genus Bruynoghevirus. Similarly, the constructed phylogenetic analysis relying on the terminase large sub-units indicated that the upper five phages classified under the Podoviridae family-namely, Pseudomonas phage phiPA01_302, Pseudomonas phage oldone, Pseudomonas phage U47, Pseudomonas virus Pa22, and Pseudomonas phage vB_PaeP_C2-10_Ab22-showed higher sequence similarity than the other phages classified under the same family ( Figure 6B). nomes and vB_PseuP-SA22. Each quadrant represents an individual genome, and th bons connecting genomes represent local alignments produced by BLAST (Figure 7) high sequence similarity is represented by red, followed by orange and blue colors results revealed that all four BLAST sequences displayed high-level sequence simil to the query phage sequence (vB_PseuP-SA22).

Biofilm Production
In the first experiment, the test tube ring assay technique was used to assess the film production capacity of P. aeruginosa strain B10. In this assay, the overnight cultu planktonic bacteria was gently removed, and the remaining biofilm (Supplementary ure S4A,B) was stained using crystal violet and thus visualized, as shown in Supple tary Figure S4C. The three polystyrene test tubes inoculated with P. aeruginosa strain produced a white clump on the inner wall of the test tube, which was stained violet. T results indicated that the organism was positive for biofilm production. Similarly, i second experiment, biofilm production was assessed and quantified using a micro

Biofilm Reduction Assay
The anti-biofilm activity of vB_PseuP-SA22 was assessed in 96-well plates, as described in Materials and Methods ( Figure 7A). The results revealed that the anti-biofilm activity of vB_PseuP-SA22 was noticeable at an MOI of 0.1. The total biomass of biofilm showed a significant reduction (p < 0.001) at 4, 8, and 12 h. The viable bacterial cell count inside the biofilm was also significantly reduced (p < 0.001) throughout the incubation period, resulting in 2.25, 1.25, 0.34, and 0 log 10 CFU/mL at 0, 4, 8, and 12 h, respectively ( Figure 8B). Scanning electron microscopic images also confirmed the total disruption of pre-formed biofilms at 12 h of treatment with phage vB_PseuP-SA22, as shown in Figure 8C. activity of vB_PseuP-SA22 was noticeable at an MOI of 0.1. The total biomass of biofilm showed a significant reduction (p < 0.001) at 4, 8, and 12 h. The viable bacterial cell count inside the biofilm was also significantly reduced (p < 0.001) throughout the incubation period, resulting in 2.25, 1.25, 0.34, and 0 log10 CFU/mL at 0, 4, 8, and 12 h, respectively ( Figure 8B). Scanning electron microscopic images also confirmed the total disruption of pre-formed biofilms at 12 h of treatment with phage vB_PseuP-SA22, as shown in Figure  8C.

Discussion
At present, nosocomial pathogens such as P. aeruginosa pose a serious public health threat worldwide due to their resistance to a variety of antimicrobial agents [66]. According to a study conducted by the World Health Organization in 2016, CRPA was ranked second among 20 antimicrobial-resistant bacterial species for which new antibacterials are urgently required [9]. To date, scientists and clinicians all over the globe have worked

Discussion
At present, nosocomial pathogens such as P. aeruginosa pose a serious public health threat worldwide due to their resistance to a variety of antimicrobial agents [66]. According to a study conducted by the World Health Organization in 2016, CRPA was ranked second among 20 antimicrobial-resistant bacterial species for which new antibacterials are urgently required [9]. To date, scientists and clinicians all over the globe have worked intensively to find promising alternative antimicrobial approaches (e.g., phage therapy) and combinatorial therapies [67,68]. Phage therapy against Pseudomonas spp. has considerably improved over the past decade [69]. Nevertheless, little is known regarding CRPA-specific phages [32].
In this study, we isolated a phage with potential lytic ability against CRPA from wastewater. vB_PseuP-SA22 has an icosahedral head and short, non-contractile tail, which is typical of podophages. Similar reports have indicated that multiple phages active against MDR P. aeruginosa were isolated from wastewater in different geographical locations [31,70]. Wastewater, in general, comprises a wide variety of micro-organisms due to contamination from fecal and hospital wastes [71], and wastewater is a good source of phages against multiple antibiotic-resistant bacterial strains [72].
The whole-genome sequence of the vB_PseuP-SA22 revealed that its genome is a 45,458 bp long double-strand DNA sequence with 56 ORFs. The genome does not carry any harmful genes, such as those linked with lysogeny, antibiotic resistance, toxins, or other factors associated with the virulence of the host bacterium. This suggests that vB_PseuP-SA22 can be considered as a virulent phage, a potential therapeutic agent against CRPA. The lysis cassette of vB_PseuP-SA22 encodes for holin (encoded by ORF35). Holin is a small phage-encoding protein that incorporates into the cell membrane, forms large holes and transports endolysin across the membrane [73]. Phages likely lyse cells to release their progeny through the holin-endolysin lytic system, which is common within almost all dsDNA phages [74]. The vB_PseuP-SA22 genome encodes for three tRNAs. There is evidence suggesting that lytic phages contain a greater number of tRNAs than temperate phages [75]. Most temperate phages (e.g., E. coli phage P4, P22, or Lambda) lack tRNA genes. Phages play a major role in horizontal gene transfer within bacteria. tRNA genes, however, are crucial housekeeping genes that are expected to be least susceptible to lateral gene transfer [76]. When these genes are deleted, rates of protein synthesis and burst sizes are lowered. Yet, why some phages possess tRNAs remains a mystery [77].
The stability of phages in a wide range of thermal and pH conditions is known to be a very crucial factor for the optimal replication of the viral particle in the host bacteria [78]. According to this study, vB_PseuP-SA22 displayed stability in a pH range of 4.0 to 12. Extreme basic or acidic pH could considerably influence the infectivity of most phages, and researchers have suggested that these situations cause the denaturation of phage proteins and, subsequently, loss of their viability [79]. Previous works have verified that most tailed phages are stable in a pH range of 5.0 to 9.0 [80,81], similar to the results obtained in this study.
A spot assay was employed to determine the host range of the isolated phage. However, the clear zone created in the tested bacterial strains sometimes is associated with bacteriocins, which might be present in the prepared suspension [82]. Hence, the spot assay result was further confirmed by plaque assay. In this line, vB_PseuP-SA22 was found to be lytic for a broad range of bacterial strains tested.
The bacterial killing assay indicated that the highest growth rate was recorded at an MOI of 0.1. On the other hand, normal growth was observed without phage (control), and the cell concentration kept increasing steadily throughout the incubation period. Bacterial reduction assay is one of the principal factors in determining the applicability of newly isolated phages to be used in phage therapy. The lytic kinetics showed a radical decline in bacterial growth from 4 to 8 h post-infection. However, except for 0.1 MOI, a slight increase in bacterial growth was noticed at all the other MOIs. According to previous reports, this result may be due to the development of phage-resistant mutants following infection; this characteristic is regarded as a drawback of phage therapy [83].
The biofilm reduction assay indicated that administration of vB_PseuP-SA22 led to a significant reduction (p < 0.001) in the biofilm biomass. In this assay, the growth of the host bacteria found in the biofilm was also significantly reduced (p < 0.001). Guo et al. have reported that two Myoviridae phages, vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), inhibited the growth of the P. aeruginosa strain PAO1 at low MOI levels, and showed good performance both in eradicating pre-formed biofilms as well as in preventing biofilm formation [84]. Similarly, in a study by Vukotic et al. [85], the A. baumannii phage ISTD produced 2 and 3.5 log reductions in a biofilm-associated bacterial cell and planktonic cell count, respectively [85]. In another study, five P. aeruginosa phages isolated from domestic sewage showed good performance regarding the degradation of biofilms formed in the endotracheal tubes [86]. A high biofilm reduction potential has been reported by Yuan et al. when using the anti-P. aeruginosa phage vB_PaeM_LS1 at 8 h post-infection [87]. Latz et al. have reported that three phages-phiKZ-like, PB1-like, and LUZ24-like-suppressed the biofilm as well as the planktonic form of MDR P. aeruginosa [30].
Several studies have indicated that the antibiofilm activity of phages is mainly associated with the enzymes that they encode. Polysaccharide depolymerases are phage-encoded polysaccharide-degrading enzymes that are highly specific and mainly associated with the tail structure of the virions, such as tail spikes, tail fibers, tail tubular, or base plate proteins [88]. They have a specific binding site with respect to capsular polysaccharides, lipopolysaccharides, or exopolysaccharides of the host bacteria. According to a recent report by Knecht et al., the biofilms of P. aeruginosa can be destabilized by alginate-specific tail spike proteins [15].

Conclusions
In conclusion, we successfully isolated a lytic bacteriophage targeting CRPA from samples collected from a wastewater treatment plant in Jeddah, Saudi Arabia. vB_PseuP-SA22 presented a wide host range and remained active in a wide range of temperatures and pH conditions. Its high burst size, a lack of toxic, virulent, and/or antibiotic-resistant genes in its genome, together with its antibiofilm activity against the tested host strain, are all crucial features of vB_PseuP-SA22, demonstrating its promise as an alternative therapeutic agent in treating infections associated with multidrug-resistant P. aeruginosa infections. Therefore, further clinical studies and other comprehensive in vivo examinations are necessary to investigate the therapeutic properties of the isolated phage against MDR P. aeruginosa. In addition, the combined effect of vB_PseuP-SA22 with other lytic bacteriophages may also be assessed in order to optimize its use.