
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12274-8
10.1016/j.heliyon.2024.e36243
e36243
Research Article
Characterization and genomic analysis of PA-56 Pseudomonas phage from Istanbul, Turkey: Antibacterial and antibiofilm efficacy alone and with antibiotics
Damar Celik Damla a
Karaynir Abdulkerim b
Salih Dogan Hanife b
Bozdogan Bulent bc
Ozbek Celik Berna berna.ozbek@istanbul.edu.tr
d⁎
a Istanbul University Institute of Graduate Studies in Health Sciences Department of Pharmaceutical Microbiology, 34116, Beyazıt, Istanbul, Turkey
b Recombinant DNA and Recombinant Protein Research Center (REDPROM), Aydın Adnan Menderes University, Aydın, Turkey
c Medical Faculty, Department of Medical Microbiology, Aydın Adnan Menderes University, Aydın, Turkey
d Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Istanbul University, 34116, Beyazit, Istanbul, Turkey
⁎ Corresponding author. Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 34116, Beyazit, Istanbul, Turkey. berna.ozbek@istanbul.edu.tr
22 8 2024
15 9 2024
22 8 2024
10 17 e3624326 3 2024
8 8 2024
13 8 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Phages are ubiquitous in freshwater, seawater, soil, the human body, and sewage water. They are potent biopharmaceuticals against antimicrobial-resistant bacteria and offer a promising alternative for treating infectious diseases. Also, combining phages with antibiotics enhances the antibiotics' efficacy. This study focused on two Pseudomonas aeruginosa phages isolated from lake and sewage water samples and one of them selected for further investigation. Isolated phages PA-56 and PA-18 infected 92 % and 86 % of the tested 25 clinical Pseudomonas aeruginosa strains, respectively. PA-56 with strong activity was chosen for detailed characterization, antimicrobial studies, and genome analysis. Combining PA-56 with ciprofloxacin or meropenem demonstrated phage-antibiotic synergism and increased antibiofilm efficacy. Genome analysis revealed a GC ratio of 54 % and a genome size of 42.761 bp, with no virulence or antibiotic resistance genes. Notably, PA-56 harboured the toxin-antitoxin protein, MazG. Overall, this study suggests that PA-56 holds promise for future applications in industry or medicine.

Keywords

Pseudomonas aeruginosa
Bacteriophage
Biofilm
Phage genome
Phage-antibiotic synergism
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pmc1 Introduction

Since antibiotics were discovered, they have been the most effective approach to treating infectious diseases. Nevertheless, antibiotics have lost their effectiveness in recent years due to their overuse and misuse. Infectious diseases caused by antibiotic-resistant bacteria cause 0.7 million deaths worldwide yearly, which is projected to reach 10 million by 2050 [1]. Because of that, research on new alternative treatment approaches that can destroy antibiotic-resistant bacteria and eradicate their biofilms is urgently needed. Use of bacteriophages (phages) in treating infectious diseases is one of the alternative strategies [2].

Phages are bacterial viruses that infect target bacteria and are the most abundant biological constituents on the planet [3]. They can be found wherever bacteria exist, such as sewage, freshwater, seawater, soil, and the human body (saliva, blood, urine, sputum, faeces). Phages are obligate intracellular parasites that are natural killers of bacteria and act as self-replications that interact with bacteria with excellent specificity. Phage therapy uses phages to treat infectious diseases such as upper respiratory tract infections, burns, inflamed wounds and abscesses [4]. Besides, phages can be successfully used in many areas worldwide, such as agriculture, animal feed, and the cosmetics industry [5]. However, phages may have some limitations, such as being strain-specific and having a narrow host range.

Pseudomonas aeruginosa is a motile, opportunistic Gram-negative rod pathogen, mainly found in animal farms, hospitals, soil, slaughterhouses, sewage water and aquatic environments. P. aeruginosa is the primary cause of nosocomial infections, especially in immunosuppressed patients, ventilator-associated severe pneumonia, catheter-associated chronic infections, patients with cystic fibrosis, severe wounds and chronic lung infections [[6], [7], [8]]. The difficulties of the treatment of P. aeruginosa infections, their ability to form biofilms and their multidrug resistance have led to investigations of phages against this species [8,9]. Although many studies have previously reported the isolation of numerous Pseudomonas phages, there is still a lack of knowledge regarding host ranges, genomic characterization, antibiofilm activity of isolated Pseudomonas phages.

Researchers have shown that isolated phages can potentially treat P. aeruginosa infections, previously untreatable with conventional antibiotics [[10], [11], [12]]. So, the main objective of this study was to isolate novel lytic phages effective against clinical strains of P. aeruginosa from local wastewater sources in Istanbul, Turkey. Then, the lytic effects of the isolated phage on diverse clinical strains were investigated, and a comprehensive examination of its morphological, biological, and genomic characteristics was conducted. Furthermore, our investigation included the analysis of the isolated phage's antibacterial and antibiofilm effects, both when administered alone and in combination with antibiotics. The ultimate objective of our study was to reveal potential applications for effectively combating P. aeruginosa infections.

2 Materials and methods

2.1 Bacterial isolates, media, and routine culture

A total of 75 clinical strains (25 Escherichia coli strains, 25 P. aeruginosa strains and 25 Staphylococcus aureus strains) were used to assess the phage susceptibility in the study. The API20 NE, API20 E and API STAPH (Biomérieux, France) kits were used for the identification of tested bacterial strains. All the clinical strains were obtained from Istanbul University, Faculty of Pharmacy, Pharmaceutical Microbiology Laboratory, Turkey. Bacteria were cultured in Luria Bertani (LB) broth at 37 °C overnight. To cultivate phages LB agar (1.5 % w/v agar), LB-soft agar supplemented with agar (0.7 % w/v), calcium chloride (5 mM, Merck Laboratory, Germany) and Luria Bertani Broth (LB) were used.

2.2 Phage isolation

Phage PA-56 and PA-18 was isolated from the lake and sewage water of Istanbul/Turkey, respectively, in sterile conditions. The isolation, purification and enrichment of isolated phage was carried out as described previously [13]. Briefly, 10 mL of collected water sample was centrifuged (5000×g, 4 °C, 10 min) to remove cell debris. The supernatant was filtered using 0.22-μm pore filter into a sterile tube. To observe the filtrate for phage, the supernatant was spotted onto clinical P. aeruginosa strains in the top agar and then incubated at 37 °C overnight. The host bacteria expressing lytic zone was selected for the continuing studies as the indicator bacteria. Besides, for the phage enrichment, 10 mL of the sample was enriched with 100 μL of the overnight host bacteria in an equivalent volume of LB and incubated overnight at 37 °C. The enriched sample was centrifuged 5000×g (10 min, 4 °C) and filtered using 0.22-μm pore filter.

2.3 Purification of isolated phages

Phage purification was performed by the double layer agar method (single plaque assay) [14,15]. Briefly, serial dilutions of phage filtrate were conducted by Saline Magnesium (SM) buffer. Then, 200 μL of overnight indicator bacteria culture and 200 μL of each dilution were mixed in 4 mL soft agar (0.5 %, w/v), vortexed and poured onto top agar (1.5 %, w/v) and incubated overnight at 37 °C. On the next day, single plaques were selected and picked from the plates according to size and morphology. The selected plaques were placed into separate sterile tubes containing fresh LB broth inoculated with 200 μL of the overnight indicator bacteria and then incubated at 37 °C overnight to provide phage particles to diffuse into the LB broth. Phage was then harvested by centrifugation (5000×g, 10 min, 4 °C) and the supernatant was filtered through 0.22-μm sterile filter. For a final confirmation of purified phages, plaque purification process was repeated 5–6 times. The purified phages were stored at 4 °C for further experiments. The titer of each phage suspension was determined with double layer agar assay previously described. Briefly, 200 μL of diluted phage suspension and 200 μL of indicator bacterial culture were added into a 4 mL soft agar, vortexed and poured onto top agar. After solidification, the plates were incubated overnight at 37 °C. Following overnight incubation, the plaque forming unit (PFU/mL) of the phage was determined.

2.4 Host range analysis

A total of 75 clinical isolates (25 E. coli, 25 P. aeruginosa and 25 S. aureus) were used to determine the host range of the isolated phages (Table 1). The susceptibility of the bacteria and lytic activities of the phages were determined by the spot test as described [16,17].Table 1 Spot test results (%) of phages against various clinical strains.

Table 1Phages	Strains	Effectivities of phages by spot test (%)	
Positive	Negative	
Phage PA-56	All strains (n = 75)	43 %	57 %	
	P. aeruginosa strains (n = 25)	92 %	8 %	
	E. coli strains (n = 25)	24 %	76 %	
	S. aureus strains (n = 25)	12 %	88 %	
Phage PA-18	All strains (n = 75)	47 %	53 %	
	P. aeruginosa strains (n = 25)	86 %	14 %	
	E. coli strains (n = 25)	36 %	64 %	
	S. aureus strains (n = 25)	20 %	80 %	
(Negative) no lysis, (Positive) lysis.

2.5 Electron microscopy

Phage morphology was carried out by transmission electron microscopy as previously described [18]. The phage suspension (1012 PFU/ml) was concentrated at 25,000 g for 120 min d by ultracentrifugation. The supernatant was discarded and the pellet was resuspended gently with SM Buffer (Himedia). 10 μl of concentrated phage was taken on a 300-mesh formvar-coated copper grid. Negative staining was conducted with 2 % uranyl acetate solution. The visualization of phage morphology was performed at 120 kV by Transmission Electron Microscopy.

2.6 One-step growth curve

One-step growth curve experiment was performed, to determine the latent period and burst time of isolated phage. Briefly, host bacteria were inoculated into a 5 mL fresh LB media and incubated overnight at 37 °C. 0.1 ml of phage 108 PFU/mL was pipetted into a 1 mL logarithmic phase culture (108 CFU/mL; multiplicity of infection (MOI) = 0.1) and incubated for 10 min at 37 °C for the adsorption of phage. Subsequently, the culture (phage and host bacteria) was centrifuged for 5 min at 6000×g and the pellet was resuspended in LB broth for removing non-adsorbed phage. Then 0.1 ml samples were collected for 90 min at every 10 min interval. Phage titre was determined by the double-agar method. The latent period was defined as the time between adsorption of phages to bacterial cells and lysis of the bacteria. The rise period is the time from the end of the latent period until all phages exit the cell. The burst size of the phage was determined as the ratio of the final number of free phage particles to the number of bacterial cells infected during the plateu phase [13].

2.7 Multiplicity of infection (MOI)

The optimal MOI is defined as the MOI at which the highest phage progeny production is obtained. The optimal MOI was determined as previously described [19]. Briefly, culture of indicator strain, were infected with different MOIs of 0.001, 0.001, 0.01, 0.1, 1, 10 and 100 (PFU/CFU). After 10 min of adsorption, free phages were removed by centrifugation (5 min at 12.000×g, 4 °C), pellets were resuspended in 5 mL LB, and incubated at 37 °C for 4 h. The culture was centrifuged (5 min at 12.000 g 4 °C) and supernatant was filtered via a 0.22 mm pore-size filters. Phage titer was then analyzed by the double-agar layer method and the MOI value which gives the maximum phage yield was considered as the optimal MOI.

2.8 Thermal and pH stability

To determination of the pH and thermal stability of isolated phage was conducted as previously described [14]. The pH stability assay phage PA-56 was determined by a pH gradient pH 2-7-11 for 48 h. In brief, 100 μL of phage (1010 PFU/mL) was added to 900 μL media (SM buffer solution) at various pH range from 2 to 11, and incubated for 48 h at 4 °C. The phage titer was determined using the double layer method. Besides, the thermal stability of phage titer was determined at different temperatures for 120 min (40 °C, 50 °C, 60 °C, 70 °C, 80 °C and 90 °C) using the double-agar method.

2.9 Biofilm formation

10 clinical P. aeruginosa strains evaluated for biofilm-forming ability by themicrotiter-plate method. In brief, a loop of overnight strains was added in 5 ml TSB media supplemented with 1 % glucose and incubated for 24h at 37 °C. Then it was diluted in TSB media to adjust bacterial concentration of approximately 107 CFU/ml. Then 200 μl of suspension was transferred to the 96-well tissue culture microtiter plates and to incubate overnight at 37 °C. Positive control wells contained E. coli ATCC 25922 strain and negative control is medium alone. Following incubation, the wells was aspirated and washed gently three times with 200 μl of sterile physiological saline buffer and left to dry. Then, biofilms in the well was fixed for 15 min with 99 % methanol and left to dry. Subsequeintly biofilms were stained for 5 min with 0.1 % crystal violet per well. After dring the well, the crystal violet was resolubilized by f 95 % ethanol for 30 min on orbital shaker, and measured at OD595 nm. Biofilm formations were evaluated using the scale reported by Stepanovic et al. [20]. All tests were conducted three times and results were averaged.

2.10 MIC determinations, phage susceptibility and MBC determinations

Minimum inhibitory concentrations (MICs) of meropenem and ciprofloxacin were determined by the broth microdilution technique that conformed with the Clinical and Laboratory Standards Institute [21] against 2 clinical and one E. coli ATCC 25922 strain. Serial two-fold dilutions (range 16 to 0.008 mg/L) of used antibiotics were prepared in cation-adjusted Mueller Hinton Broth (CAMHB) for antibiotics. The turbidity of bacterial cultures was adjusted to the 0.5 McFarland standard. Then the inolucum was diluted in CAMHB to give a final bacterial inoculum approximately 5 × 105 CFU/ml and incubated at 37 °C for 18–24 h. Besides for the phage suscebtibility 20 μL of phage PA-56 (1010 PFU/mL) was added to 190 μL of logarithmic phase bacterial culture (∼106 CFU/mL) and then incubated at 37 °C for 24 h. The turbidity of phage-trated bacterial cultures were measured visiually. Minimum bactericidal concentrations (MBC) of tested antibiotics in this study was also determined. The experiment was performed by plating 10 μl suspension from each dilution on plates followed by overnight incubation at 37 °C. The lowest concentration showing no visible growth of the colonies was the MBC value of tested antibiotics [22].

2.11 Inhibition of biofilm formation and quantification of mature biofilm biomass using crystal violet staining

The ability of phage and tested antibiotics alone and in combination to inhibit biofilm formation determined as described previously [20]. For this experiment, 2 clinical isolates with the strongest biofilm production of strains were tested. Each of the tested clinical strains were incubated in TSB media +1 % glucose and with antimicrobials and phages (1010 PFU/mL) alone and in combination (meropenem or ciprofloxacin at (1x MIC, 1/10xMIC and 1/100xMIC) in microtiter plates overnight at 37 °C. Six wells were used for antibiotics and phages. The positive control was E. coli ATCC 25922 strains in TSB media without antibiotics and phages. Following incubation, each well were rinsed with PBS solution three times and measured at OD595 nm after crystal violet staining.

For quantification of mature biofilm biomass, biofilms of 2 clinical strains and E. coli 25922 ATCC were grown in 96 wells plate for 24 h. Biofilms (24-h-old) were rinsed with PBS, and treated with phage PA-56 (1010 PFU/mL) then incubated at 150 rpm for 24 h at 37 °C. After 24-h treatment, the biomass of biofilm was washed with PBS twice, and then stained with 0.1 % crystal violet (w/v) (200 μL). The biomass of biofilm was quantified by microplate reader [23].

2.12 Phage-antibiotic synergy

Synergism of the two antibiotics (meropenem and ciprofloxacin) and isolated phage was investigated by the MIC and MBC of tested antibiotics by broth serial dilution method [24]. Synergistic effect between the isolated phage (108 PFU/ml, with a MOI of 0.1) and the antibiotics was evaluated by MIC assay [25]. 25 μL of the tested meropenem and ciprofloxacin (ranging from 16 to 0.031 mg/L and 32 to 1 mg/L) respectively were put in microtiter plate. 25 μL of phage PA-56 108 PFU/ml were added to wells and then 50 μL of tested bacterial suspension (108 CFU/ml) were put in the wells. Synergism could be verified if MIC and MBC values got reduced after treatment of antibiotics and phage [26].

2.13 DNA extraction of phage PA-56 and restriction fragment length polymorphism (RFLP)

For the isolation of phage DNA, purified phage (1011 PFU/mL) was centrifuged (15.000×g, 120 min, 4 °C) to provide concentrated phage solution. The concentrated phage solution was treated with DNase and SM buffer (Thermo Fisher Scientific, USA) to obtain final concentration of this solution of 1 μg/mL, and incubated at for 45 min 37 °C. Nucleases were inactivated by incubation for 15 min at 70 °C. Proteinase K (Triogen, Turkey) was added to the to DNase-treated phage suspension to obtain final concentration of this solution of 20 μg/mL. Then the mixture was incubated at 56 °C for 45 min to eliminate the capsid proteins of phage and to diffuse the viral DNA. After that, the DNA was purified using phenol: chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol precipitation method. To determine RFLP, phage DNA was digested using HindIII enzymes and EcoRI. The electhophoresis of DNA was performed on 1 % agarose gel for 30 min and run at 100 V. DNA fragments were estimated using a Lambda-PstI marker (Thermo Fisher Scientific, USA) [27].

2.14 Library preparation and sequencing

The paired-end library of the phage genome was prepared from the isolated samples using Nextera DNA Prep Library Prep Kit (Illumina, San Diego, CA), and sequenced on an Illumina Nextseq 500 (Illumina, USA) platform with a 2 × 150 cycle [27].

2.15 Bioinformatic analysis

The quality of the raw data was evaluated using FastQC v0.11.9 (Babraham Bioinformatics) and Trimmo matic v0.36 for trimming low-quality bases, primers, and remnant adapters [28]. The reads were assembled using MEGAHIT v1.2.9 [29]. For annotation, RASTtk v1.073 annotation pipeline was used [30]. The existence of antibiotic resistance, and virulence genes on the phage genome was investigated using VirulenceFinder 2.0 and ResFinder 4.1 [31]. The genome comparison of PA-56 phage and other phages in the Genbank database were investigated using BLASTN. VipTree 3.3 was used to construct viral proteomic tree [32].

2.16 Statistical analysis

Statistical analysis was conducted using GraphPad Prism 6.0. One-way ANOVA tests were used to determine the statistical significance.

3 Results

3.1 Isolation and purification of phage

The two phages were isolated from the water samples in Istanbul/Turkey and they were named PA-56 and PA-18. P. aeruginosa 5619 and P. aeruginosa 8992 were the indicator strains of isolated PA-56 and PA-18 phages, respectively and the strains were used to determine their plaque morphologies. The plaques of phage PA-56 and PA-18 formed about 2.8 mm and 2.3 mm in diameter respectively and produced clear round plaques on bacterial hosts. The highest phage concentrations of PA-56 and PA-18 were 4.4 × 1015 and 3.4 × 109 PFU/ml respectively.

3.2 Host ranges analyses of phages

The host range analyses of isolated PA-56 and PA-18 phages were assessed on a panel comprising a total of 75 clinical bacterial strains, including 25 P. aeruginosa, 25 E. coli, and 25 S. aureus strains. In the section host range analyses of phages, the percentage values for the lytic effects of PA-56 and PA-18 on different bacterial strains show differences. PA-56 affects 92 % of 25 P. aeruginosa, 24 % of 25 E. coli, and 12 % of 25 S. aureus strains while PA-18 affects 86 % of P. aeruginosa, 36 % of 25 E. coli, and 20 % of 25 S. aureus strains (Table 1). The obtained results indicated that PA-56 possesses higher lytic activity against clinical P. aeruginosa strains. In this context, phage PA-56 was investigated for further studies such as more detailed characterization and antibacterial and antibiofilm activities.

3.3 Electron microscopy

Transmission Electron Microscopy (TEM) analysis indicated that the phage PA-56 belongs to the Caudoviricetes class, which is characterized by long tail fibers and isometric heads. The isolated phage PA-56 exhibits a distinct morphology with an icosahedral head measuring 48.83 nm in diameter and a long contractile tail ranging from 100.96 to 4.92 nm in diameter (Fig. 1).Fig. 1 Transmission Electron image of negatively stained phage PA-56. Purifed phage was negatively stained by 2 % (w/v) uranyl acetate and visualized by a TEM at a voltage of 120 kV.

Fig. 1

3.4 One-step growth curve and MOI

One-step growth assay was conducted to determine the burst size and latent time of the phage PA-56. The burst size and latent period of phage PA-56 was approximately 100 PFU/infected cells and 40 min respectively (Fig. 2). This result indicates that efficient and rapid development of phage PA-56 for bacterial inhibition.Fig. 2 One-step growth curve of phage PA-56. L: latent period, P: plateau phase, R: rise phase, B: Burst size (B = average P/average L). Latent period and burst size of phage PA-56 were 40 min and 100 pfu/infected bacterial host.

Fig. 2

MOI is defined as the ratio of phage production to bacterial host. The optimum MOI is the highest phage production obtained. Host bacteria were infected different MOI values and incubated for 4h at 37 °C. After the incubation total phage titers were calculated by double-layer agar. The optimal MOI of phage PA-56 was 0.1 (Fig. 3).Fig. 3 Multiplicity of infection (MOI) of PA-56 phage. The optimal MOI value of PA-56 was 0.1.

Fig. 3

3.5 Phage stability at different pH and temperatures

Ph and termal stability of phage PA-56 was determined according to phage titers under different conditions. For the pH stability, the phage PA-56 was kept between pH 2 Ph 7 and Ph 11 at 4 °C for 48 h. Compared to the initial titer of phage, phage viability was fundamentally unaffected at pH 7, while a tolerable decreased about 1 log was observed at pH 11 end of the second day. At Ph 2, titre of phage PA-56 was completely inactivated after 48 h. For the thermal stability of the phage PA-56 titer were determined at 4 °C, 25 °C, 37 °C, 45 °C, 65 °C and 90 °C for 120 min. PA-56 phage titre decreased about 6 log and about 7 log was observed at 65 °C and at 90 °C respectively. However, the concentration of phage PA-56 did not remarkable decreased during 120 min at 4 °C, at 25 °C, at 37 °C, at 45 °C (Fig. 4). Our results showed that the phage PA-56 still maintained its activity after incubation for 120 min at 65 °C and 90 °C and was resistant to high temperatures.Fig. 4 Temperature and pH stability on phage. (A) Biological stability of Pseudomonas phage PA-56 under different temperature conditions. (B) Biological stability of Pseudomonas phage PA-56 under different pH values.

Fig. 4

3.6 MIC, phage susceptibility and MBC

In our study, the MIC and MBC of ciprofloxacin and meropenem against 2 strong biofilm-forming P. aeruginosa strains were determined and the results are shown in Table 3. E. coli ATCC 25922 was the quality control strain. MIC of ciprofloxacin ranged from 0.008 ≥ 32 mg/L and meropenem ranged from 0.008 ≥ 16 mg/L. While P. aeruginosa-25 strain were found to be resistant to meropenem and ciprofloxin, P. aeruginosa-15 was found to be resistant only to ciprofloxacin (Table 3). PA-56 phage was found to be effective against all bacteria. When the results of the MBC experiments were evaluated, the MBC values of the 3 tested bacteria ranged from 0.031 to 16 mg/L for meropenem and 1–32 mg/L for ciprofloxacin (Table 4).Table 2 Biofilm formation abilities of P. aeruginosa clinical isolates.

Table 2Strains	Non-biofilm producer	Weak biofilm producer	Moderate biofilm producer	Strong biofilm producer	
P. aeruginosa (n = 10)	–	–	–	100 % (10)	

Table 3 MICs (mg/L) and MBCs (mg/L) of antibiotics, and phage PA-56 susceptibility against 2 clinical isolates and E. coli 25922 ATCC.

Table 3Strains	Meropenem	Ciprofloxacin	Phage PA-56	
MIC	MBC	MIC	MBC	
P. aeruginosa-25	16	16	32	32	Susceptible	
P. aeruginosa-15	16	32	1	32	Susceptible	
E. coli 25922 ATCC	0.008	0.031	0.008	1	Susceptible	

Table 4 Reduction in MIC values due to PAS among antibiotics (meropenem/ciprofloxacin) and phage on clinical strains.

Table 4Reduction in MIC (mg/L)	
Strains	Meropenem	Ciprofloxacin	Synergism	
P. aeruginosa-25	16 → 2	32 → 8	+	
P. aeruginosa-15	No change	No change	–	
E. coli 25922 ATCC	0.008 → <0.00001	0.008 →<0.00001	+	

3.7 Inhibition of biofilm formation and eradication of bacteria in mature biofilm with phage PA-56

Before biofilm inhibition studies firstly the biofilm forming abilities of tested 10 strains were determined and classified according to their biofilm measurements (Table 2). As a result, one standart E. coli ATCC 25922 which form moderate biofilm and two clinical strains of P. aeruginosa which form strong biofilms used to investigate the inhibition of biofilm formation of phage PA-56 alone and in combination with antibiotics (Fig. 5). The inhibiton of strains in the biofilm was examined using microtiter plate assay. OD595 values of wells treated were compared to positive control at 24 h post-inoculation.Fig. 5 Inhibition of biofilm formation of various bacterial strains by antibiotics (1xMIC, 1/10xMIC and 1/100xMIC) and phages alone and in combination. Asterisks show statistically significant differences (P < 0.05) in percentage biofilm biomass of the treated groups with antibiotic-phage combinations in comparison with antibiotics alone.

Fig. 5

According to our results, 1xMIC, 1/10xMIC and 1/100xMIC of meropenem prevented biofilm formation of meropenem and ciprofloxacin resistant-PA-25 strain by 48.54 %, 28.13 % and 25 % alone, respectively. PA-56 phage alone inhibited biofilm formation by 31.5 %. Combinations of meropenem with phage PA-56 inhibited biofilm formations by 58.54 %, 48.13 %, 54.21 % at 1xMIC, 1/10xMIC and 1/100xMIC concentrations respectively. 1xMIC, 1/10xMIC and 1/100xMIC of ciprofloxacin alone inhibited the biofilm formation by 36.46 %, 24.59 % and 30.39 %. Ciprofloxacin-PA-56 phage combinations increased the inhibition of biofilm formation by 38.46 %, 40.1 % and 37.33 % at 1xMIC, 1/10xMIC and 1/100xMIC. Biofilm formation of carbapenem-resistant P. aeruginosa-15 inhibited by 67.35 %–58.68 % at 1xMIC, 1/10xMIC and 1/100xMIC concentrations of meropenem. PA-56 phage alone prevented biofilm formation by 35.56 %. Combination of meropenem + PA-56 phage increased the biofilm inhibition by 77.46 %, 76.03 %, 63.18 % at 1xMIC, 1/10xMIC and 1/100xMIC. Carbapenem-resistant P. aeruginosa-15 strain biofilm formation, inhibited by 81.6 %, 70.99 %, and 60.83 %, at 1 x MIC, 1/10 x MIC and 1/100xMIC concentrations of ciprofloxacin alone, respectively. It was determined that when ciprofloxacin was combined with PA-56 phage, it prevented biofilm formation by 83.78 %, 80.12 %, and 71.56 % at 1xMIC, 1/10xMIC and 1/100xMIC. For E. coli ATCC 25922 strain, PA-56 phage inhibited biofilm formation by 10.57 %. Only 1xMIC of meropenem alone prevented biofilm formation of E. coli ATCC 25922 by 13.21 %. 1xMIC, 1/10xMIC and 1/100xMIC of ciprofloxacin alone prevented biofilm formation of this strain 54.74 %, 52.61 % and 45.07 % respectively. Ciprofloxacin-phage PA-56 combinations inhibited 50.77 %, 50.31 % and 45.85 % at 1xMIC, 1/10xMIC and 1/100xMIC respectively.

We investigated also the effect of phage PA-56 alone on 24-h mature biofilms of 3 strains. According to our results, Phage PA-56 inhibited 45.24 % and 35.56 % of mature biofilm formation of P. aeruginosa-25 and P. aeruginosa-15 strains respectively. Besides PA-56 inhibited 20 % of mature biofilm formation of E. coli ATCC 25922. (Fig. 6).Fig. 6 Percentage mature biofilm biomass after treatment with phage PA-56 alone. Asterisks show statistically significant differences (P < 0.05) in percentage biofilm biomass of the treated groups in comparison with non-treated control.

Fig. 6

3.8 Determination of phage-antibiotic synergy (PAS) effect on tested strains

The MIC of tested antibiotics alone and in combination with phage PA-56 was studied for 3 strains. In the presence of the meropenem alone, P. aeruginosa strains had MIC values of 16 mg/L. However, phage-meropenem combinations exhibited synergism with 1 out of 2 strains and showed MIC value of 2 mg/L.

In the presence of ciprofloxacin alone, P. aeruginosa 25 strain had MIC value 32 μg/ml. When used in combination with phage PA-56 showed 2 fold reduction in MIC value. Phage PA-56 and ciprofloxacin did not show synergism against P. aeruginosa-15 strain (Table 4). Besides E. coli ATCC 25922 strain had MIC value 0.008 μg/ml, however after phage PA-56-meropenem sygergism this strains exhibited synergism and showed reduction in MIC values. Also reduction in MBC values of tested antibiotics was also observed for E. coli ATCC 25922 when combined of antibiotics and phages. All P. aeruginosa had the same MBC values after in combination with antibiotics (Table 5).Table 5 Reduction in MBC values among antibiotics (meropenem/ciprofloxacin) and phage on clinical strains.

Table 5Reduction in MBC (mg/L)	
Bacteria	Meropenem	Ciprofloxacin	
P. aeruginosa-25	No change	No change	
P. aeruginosa-15	No change	No change	
E. coli 25922 ATCC ATCCATCC	0.031→ <0.00001	1 → <0.00001	

3.9 Genomic DNA and restriction fragmentation of bacteriophages

The genomic DNA and restriction profile of the phage was purified and digested with EcoRI and HindIII restriction enzymes. As a result, we determined that PA-56 phage DNA was sensitive to both EcoRI and HindIII enzymes and fragments of various sizes (between 500 and 11973 bp) were formed (Fig. 7).Fig. 7 Phage DNA restriction profiles. (A) Phage genomic DNA. (B) Band profiles of phage PA-56 DNAs after cutting with EcoRI and HindIII restriction enzymes. M: Lambda-PstI marker.

Fig. 7

3.10 Sequence analysis and genomic characteristics of phage PA-56

The isolated DNA of Phage PA-56 was sequenced with NGS. The paired end library was trimmed and Fast Qc report of the library showed a total of 2236551 read pairs passed without poor quality and an average length of reads were 151.2 base pairs. The genome of phage is double-stranded. Its size 42.761 bp and G-C ratio is %54. In this genome 64 open reading frames (ORFs) were encoded and 29 (45 %) of them were assigned putative functions. Remaining ORFs were encoding hypothetical proteins. There are structural, DNA replication, lysis and packaging modules in the genome. The structural module includes encoding tail protein, 62 kDa structural protein, capsid, and head protein genes and colored as blue. The gene on the structural module tends to exist on the same DNA strand sequentially. The DNA replication module contains exonuclease, DNA helicase, DNA polymerase, DNA primase and colored as yellow and the genes belong to this module dispersed along the genome. The lysis module includes holin and Rz-like spanin which are colored as red. Toxin-antitoxin protein MazG and hypothetical proteins are shown in purple (Fig. 8). It was found that there is no tRNA encoding gene in the genome. There were no virulence and resistance genes detected in the genome. Additionally, no lysogeny-related gene was detected in the genome.Fig. 8 The genome map of Pseudomonas phage PA-56 with predicted 64 ORFs. Genes were colored based on their predicted functions. Lysis, red: structural, blue: DNA replication, yellow: hyphothetical, purple: The figure was created with (Snapgene viewer 5.3.2).

Fig. 8

3.11 Genome comparison

To find out similarities between Pseudomonas phage PA-56 to other phage genomes in the Genbank database, BLASTN analysis was performed. The BLASTN result showed that the PA-56 phage showed 97 % identity with Pseudomonas phage vB_PaeS_C1 (Fig. 10). The proteome comparison between PA-56 and Pseudomonas phage vB_PaeS_C1 genomes showed that there are 9 genes encoding hypothetical protein in PA-56 genome, not found in Pseudomonas phage vB_PaeS_C1 (Fig. 9).Fig. 9 The genome comparison of Pseudomonas phage PA-56 and Pseudomonas phage vB_PaeS_C1. The graphical representation was generated by Kablammo software. The darker color are high identity and lighter color are low identity.

Fig. 9

Fig. 10 VipTree analysis of Pseudomonas phage PA-56 and 10 similar phage genomes with the reference phages. (A) Pseudomonas phage PA-56 was represented with star. The outer ring shows bacterial host cell, and the inner ring shows the family of phage belong. (B) The taxonomic relationship for Pseudomonas phage PA-56 and its relatives showed that phage PA-56 belongs to the family Caudoviricetes and genus of Septimatrevirus.

Fig. 10

3.12 Phylogenetic analysis

Phylogenetic tree of Pseudomonas phage PA-56 and other phages was shown in Fig. 10. Viptree analysis was conducted to construct a proteomic tree utilizing genome-wide sequence similarities. Circular and rectancular trees of phage PA-56 were visulized and its location in tree were showed. According to this, the taxonomic results showed that phage PA-56 belong to the family of Caudoviricetes and a genus of Septimatrevirus. It has been shown that the host of PA-56 phage belongs to the phylum of Pseudomonadota (Proteobacteria), which also includes P. aeruginosa strains.

4 Discussion

Increased misuse of antibiotics in agricultural and health sectors has caused the emergence of antibiotic resistance all over the world, and this threatens human health by decreasing the efficacy of antibiotic treatment. Phages used as antibacterial agents to combat antibiotic resistance are considered an effective solution against antibiotic-resistant microorganisms.

According to the World Health Organization, P. aeruginosa is the second most important species of 20 antibiotic-resistant bacteria, and new antimicrobials are urgently needed [33]. P. aeruginosa has a strong ability to develop biofilms that indicate antibiotic resistance. Phage therapy is a promising antimicrobial approach against P. aeruginosa strains [32]. The present study investigated lytic P. aeruginosa phages isolated from various water samples. Similar reports have shown that Pseudomonas phages were isolated from water samples, a good source for phages in different locations [34]. When co-plated with phages and their susceptible host bacteria, plaques are produced with different morphology on bacterial cells. Clear plaques, turbid edges, clear centres plaque, and plaque with a halo are common plaque types. The isolated phages PA-56 and PA-18 produced medium clear plaques with no halo zone.

For the potential use of phages in treatment or industrial application areas, virulent phage candidates with a broad lytic spectrum should be preferred instead of phage candidates with lysogenic and narrow lytic activity due to lysogenic phages can not kill bacteria and also can transfer virulence or antibiotic resistance genes to bacteria (transduction). Besides, the sensitivity of phages to physicochemical factors such as thermal stability, pH, lytic spectrum effect, adsorption rate, host range analysis, and one-step growth analyses are important factors for the characterization of phage candidates. Host-range experiments indicated that PA-56 phage has a wide host range with lytic activity against 92 % of P. aerguinosa strains, 24 % of E. coli strains and 12 % of S. aureus strains. PA-18 has a narrow lytic activity and host range against P. aeruginosa strains according to phage PA-56. Because of that, PA-18 was not included in further studies due to their narrow host ranges/lytic spectra. A phage isolated by Marashi et al. against P. aeruginosa phage lysed 62.5 % of 40 P. aeruginosa strains [35]. The results of host range are crucial parameters when selecting phages for therapeutic uses [36]. Abdelghafar et al. isolated phage vB_PaeP_PS28, exhibiting a wide host range against tested P. aeruginosa strains (72.2 %) [37].

In addition to characterization based on biological properties, the stability of isolated phages under stress conditions should also be evaluated for therapeutic or industrial applications. Phage should tolerate a wide range of pH as a therapeutic agent. In this study, the thermal and pH of phage PA-56 were tested. According to our results, PA-56 was stable at pH 7 for 48 h. However, at pH 2, phage completely lost its lytic activity, and at pH 11, there was a 2-log reduction in phage titer. Besides, the thermal stability of the isolated phage was tested. PA-56 phage was fully stable at temperatures between 4°C and 45 °C. However, at temperatures 65 °C and 90 °C, phage PA-56 lost its lytic activity after 120 min of incubation. In the study of Abdelghafar et al. the isolated lytic P. aeruginosa phage showed high stability over pH ranges and temperatures [37]. Following the present results, phages with tails have been indicated to be more durable under severe conditions, including pH and temperature changes [38,39]. Our results are similar to previous studies of phages against P. aeruginosa strains.

The optimal MOI value of the PA-56 phage was 0.1. Low MOI reduces the production cost of phage production [40]. Growth parameters of phages, such as latent, rise period and burst size, provide essential biological information about new phage isolates [41]. The PA-56 phage has a latent period of 40-min and a burst size of 100 PFU/infected cells. When we look at other studies on Pseudomonas phages, Forti et al. showed similar results for P. aeruginosa phage with a burst size of 100 PFU/CFU and a latent period of 20 min [42]. Fei et al. isolated a P. aeruginosa phage, which was found to have a latent period of approximately 40 min [43]. Our results are found to be similar to previous studies of P. aeruginosa phages.

Biofilm production of P. aeruginosa strains is essential for their survival and resistance to antimicrobials [44]. Recent studies reveal that bacteria colonising humans for a long time tend to form biofilms after a while, thus becoming resistant to human immune system responses and antibiotic treatments, causing chronic infections. For this reason, studies on developing new treatment strategies such as antibiofilm-effective antibiotics, phages and antimicrobial photodynamic effects to prevent biofilm formation continue worldwide [45]. In our study, the biofilm formation inhibitory effect of the tested antibiotics and phages, alone and in combination, was investigated against two clinical P. aeruginosa strains. According to our results, antibiotics alone and in combination with phage PA-56 had potent inhibitory effects on P. aeruginosa biofilm formations. Phage PA-56 showed 35.56 % inhibitory effect against P. aeruginosa biofilms alone. The synergistic effects of phages and meropenem have been studied previously against P. aeruginosa–S. aureus dual-species biofilm [46]. Henriksen et al. reported there is a synergism between phages and ciprofloxacin to prevent the biofilm formation of P. aeruginosa [47]. In the study conducted by Chang et al., in 2019, phage PEV20 and ciprofloxacin combinations were investigated to inhibit P. aeruginosa biofilms isolated from cystic fibrosis patients [23]. Additionally, the antibiofilm effect of PA-56 phage was investigated against E. coli ATCC 25922 strain. PA-56 phage inhibited biofilm formation by 10.57 % of this strain. Meropenem and PA-56 combinations showed an effect similar to meropenem alone, but the antibiofilm activity caused by the phage alone was not observed. However, when ciprofloxacin-PA-56 combinations were evaluated, they showed a lower antibiofilm effect than the antibiofilm effect of PA-56 phage alone.

In the struggle against infectious diseases, it is important to discover antibiotic-phage combinations, which are used to prevent the spread of antimicrobial resistance, increase the effects of clinically ineffective antibiotics, reduce the dose and the side effects of antibiotics used, and use as adjunctive therapeutic agents in the treatment of some diseases. Considering the studies in the literature little is known about the synergistic relationship between phages and antibiotics. Phage-antibiotic synergy reduces antibiotics' MIC and MBC values by reducing the antibiotic dose requirement in treatment [48]. Our study investigated the synergistic effects between the antibiotics (meropenem/ciprofloxacin) and the isolated phage PA-56. The results of our study show that phage-antibiotic combinations have therapeutic potential in combating infections caused by clinical strains. In a similar study, a synergistic effect of phage-antibiotic (amikacin/meropenem) combination was observed against planktonic and biofilm-forming cells of P. aeruginosa strains when phage alone, antibiotic alone, and phage-antibiotic combinations were compared [25]. Therefore, the antibiotic-phage combination can play an important role as an adjuvant treatment in cases where the microorganism is highly resistant to antibiotics or when antibiotics have minimal penetration into the infected area [49]. The results from previous in vitro studies recommend using the tested combinations of antibiotics and phages against Gram-negative bacteria, e.g. P. aeruginosa, Burkholderia cepacia, or Citrobacter spp. B. cepacia phages were studied in combination with ciprofloxacin, meropenem and tetracycline for phage-antibiotic synergy [34].

Phages with tails and a double-stranded DNA genome, which is a member of the order Caudovirales, probably form the predominant entity in the biosphere [50]. TEM images were examined for the morphology of PA-56 phage in our study. According to this, it showed that the PA-56 phage belonged to the class of Caudoviricetes. Holins, spanins and endolysins are biomolecules responsible for the lytic lifestyle in phages. These lysis proteins act on the inner membrane, outer membrane or peptidoglycan structure and ensure the separation of highly protected bonds [51]. Phage PA-56 was found to have holin and Rz-like spanin genes. Although tRNA was absent in Pseudomonas phage PA-56, it demonstrated a strong lytic effect. No lysogeny-related genes (excisionase, integrase, etc.) were in the phage genome. The absence of lysogeny-related genes in the PA-56 genome indicates, the phage has a lytic lifecycle. It was found that PA-56 phage has toxin-antitoxin protein MazG. Toxin-antitoxin (TA) systems can be found in some microorganisms and contain genes that inhibit cell growth and when overexpressed, result in programmed cell death [52,53]. The MazEF TA system is the first chromosomal TA system responsible for cell death identified in E. coli. MazG is a toxin-antitoxin protein located in the third open reading frame in the MazEF operon genome [54]. The functions of the MazG of the PA-56 phage should be studied and is currently under investigation in our laboratories.

There is a difference between the GC content of phage PA-56 and its host. The GC content of phage PA-56 is 54 %, and the GC content of P. aeruginosa hosts are between 66.6 %. This difference can be explained by the characteristic of the lytic phage. While lytic phages have larger genomic distances between the bacterial host genome, lysogenic phages have smaller genomic distances [9]. Kwan et al. hypothesized that GC contents between P. aeruginosa strains and their phages could be due to lateral gene transfer to P. aeruginosa strains or other characteristic features of the viruses, such as replication-related genes and variations on DNA polymerase [55].

5 Conclusions

In the present study, lytic Pseudomonas phages PA-18 and PA-56 capable of infecting clinical strains of P. aeruginosa strains were isolated and characterized. PA-56 phage showed higher lytic activity against P. aeruginosa strains compared with PA-18 phage. The isolated Pseudomonas phage PA-56 possesses properties such as antibacterial activity, strong antibiofilm, stability under neutral-alkaline conditions and moderate temperatures such as 4 °C, 25 °C, 37 °C, and 45 °C, high burst size, short latent period. Additionally, PA-56 phage has lysis proteins and there are no virulence or antibiotic resistance genes. To our knowledge, the toxin-antitoxin protein MazG has been detected in a Pseudomonas phage for the first time. Therefore newly isolated phage PA-56 is promising and considered as potential therapeutic phages.

Data availability statement

Not applicable.

CRediT authorship contribution statement

Damla Damar Celik: Writing – original draft, Visualization, Methodology, Investigation, Data curation. Abdulkerim Karaynir: Writing – original draft, Visualization, Methodology, Investigation. Hanife Salih Dogan: Writing – original draft, Visualization, Methodology. Bulent Bozdogan: Writing – original draft, Visualization, Supervision, Methodology, Investigation. Berna Ozbek Celik: Writing – review & editing, Writing – original draft, Supervision, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Research fund of the University of Istanbul (Istanbul, Turkey). Project-number: TDK-2021-37374, Ethics committee approval number: 2020/789.
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