Isolation and characterization of lytic bacteriophages against Pseudomonas aeruginosa isolates from human infections in the north-west of Iran.

Background and
Objectives: With the emergence of Pseudomonas aeruginosa antibiotic-resistant strains, using the anti-bacterial potential of bacteriophages could be an effective approach to combat bacterial infections. Materials and
Methods: In this study, after evaluation of antibiotic sensitivity of 20 clinical bacterial isolates of Pseudomonas aeruginosa, isolation of lytic phages was performed against 15 isolates using double-layer agar overlay technique. Molecular analysis of isolated phages was carried out using EcoRV and HindIII endonucleases. Then, the host range of the phages was evaluated and the phage with the broadest host range (PPaMa1/18) was selected to morphological characteristics by TEM. Also, its one-step growth curve was determined and the stability of the phage to environmental parameters (temperature and pH) was evaluated.
Results: All isolates of Pseudomonas aeruginosa were resistant to five antibiotics. In total, 15 phages were successfully isolated from the sewage sources against each of 15 used bacterial isolates. Molecular analysis of the phages showed a high rate of genomic variation. In the morphological analysis of the selected phage (PPaMa1/18) using TEM, an icosahedral head with a size of 90 nm × 75 nm and a long contractile tail (215 nm) were observed which indicated its similarity to the Myoviridae family. The latent period of the PPaMa1/18 was about 20 minutes and its burst size was estimated to be 58 PFU/cell. Also, the PPaMa1/18 phage antibacterial activity was not significantly affected at pH 4-10 and temperature 4-40C°.
Conclusion: Findings demonstrated that isolated bacteriophage (PPaMa1/18) with wide host range, strong lytic effect and high stability can be used as a promising candidate for the treatment of antibiotic-resistant infections caused by Pseudomonas aeruginosa.

INTRODUCTION

Pseudomonas aeruginosa is one of the major causes of community-acquired infections, considered in 9–10% of nosocomial opportunistic infections (1, 2). P. aeruginosa is found in various environments including water and soil and also it can infect humans, animals, and plants (3, 4). This bacterium is one of the most important causes of malignant external otitis, endocarditis, meningitis, pneumonia, septicemia, bacteremia, urinary tract infections and surgical site infection, especially in people with immunodeficiency (4, 5).

Various strains of P. aeruginosa have biofilm formation capability. Under special circumstances, this bacterium can form tightly-packed structures called biofilms and prevent the entrance of antimicrobial agents into the bacterial cell (6). They have a wide range of virulence factors and a set of resistance mechanisms to antibiotics with highly adaptive features in different environments (7). P. aeruginosa can attack a host cell through its wide range of virulence factors and protect itself from the host’s defense (4).

Moreover, the innate and adaptive resistance mechanisms of P. aeruginosa provide high resistance to several antibiotics. These mechanisms include: reducing the permeability of cell coatings, efflux pumps, obtaining resistance genes through plasmids and transposons, and changing the expression and function of chromosomally encoded mechanisms through mutation (8, 9). Infections caused by P. aeruginosa are often severe and fatal due to low sensitivity to different types of antibiotics and the emergence of antibiotic resistance during therapeutic stages (10). The excessive use of antibiotics and also increasing of antibiotic-resistant strains has made critical challenges to treat infections caused by this bacterium (9).

Nowadays, phage therapy or the use of phages to deal with multi-drug resistant (MDR) bacteria is a new therapeutic strategy (11). Phages are viruses that contain a genome (DNA or RNA) surrounded by a protein coating (capsid) and can infect bacteria (12–14). These bacterial viruses are found remarkably in a variety of environments (15).

The major advantages of phages, in comparison with antibiotics, can be their high specificity to bacterial targets, cost-effectiveness, easy isolation, abundance in the environment, ability to remove biofilms, proliferation at the site of infection, minimal effect on natural flora and non-serious side effects in eukaryotic cells (12, 14, 16, 17). Various studies have shown the successful use and antimicrobial function of P. aeruginosa phages to cope with antibiotic-resistant infections in in vitro and in vivo studies (18–20).

In this study, 15 lytic phages were isolated separately against antibiotic-resistant isolates of P. aeruginosa from Maragheh sewage. After determining the range of phages hosting, one of the phages was chosen with the widest spectrum of hosts and named “selected Phage”. Then the morphology, burst size and other physiological characteristics of this phage were investigated.

MATERIALS AND METHODS

Identification of bacterial isolates of Pseudomonas aeruginosa.

Clinical samples were taken from hospitals of Tabriz, East-Azarbaijan, Iran. The collected samples were cultured on selective media (MacConkey agar, Blood agar, and EMB) and after incubation, bacterial cultures were evaluated for morphological and biochemical characteristics. Biochemical tests were performed including catalase, oxidase, IMVIC, SIM, TSI, and carbohydrate metabolism.

Antibiotic susceptibility of Pseudomonas aeruginosa isolates.

Antibiotic sensitivity of 20 isolates of P. aeruginosa from patients was evaluated to 12 different antibiotics. Antibiotic susceptibility patterns of isolates were determined by Kirby-Bauer disk diffusion method on the Muller-Hinton agar medium (Hi-Media, India) (21).

Different antibiotic discs (Padtan Teb, Iran) including penicillin (10 μg), amoxicillin (25 μg), gentamicin (10 μg), nalidixic acid (30 μg), tetracycline (30 μg), imipenem (10 μg), amikacin (30 μg), kanamycin (30 μg), tobramycin (10 μg), cefepime (30 μg), cefixime (5 μg), rifampicin (5 μg) were used in this study. After incubation time, the diameter of the non-growth holes was measured by the ruler and the isolates were classified into sensitive, semi-sensitive and resistant groups, according to the Clinical & Laboratory Standards Institute (CLSI) guidelines (22).

Isolation and purification of phages.

To isolate the phages against MDR P. aeruginosa isolates, samples were collected from the sewage of Maragheh city, East-Azarbaijan, Iran. The collected sewage samples were centrifuged for 5 minutes at 2500 rpm and the supernatant solution was then filtered through 0.45 μm syringe driven filters (Jet Biofil, Canada). Then, 5 ml of cultured P. aeruginosa (OD = 0.4 / 0.5) and 5 ml of 2× LB broth (Luria Bertani Broth) were added to 45 ml of the filtered solution and incubated for 24 hours at 37°C at 180 rpm (23). After incubation, 3 ml of chloroform was added to the suspension and centrifuged at 3500 rpm for 30 min at 4°C (The chloroform addition process was repeated to ensure purification of the phage) and finally, the supernatant was filtered through 0.45 μm syringe driven filters. Chloroform does not affect the performance of phages and results in the bacterial killing in a phage solution (23) with some minor modifications.

Evaluation of forming plaques with bacteriophages was carried out using Adams double layer agar method with minor modifications (23). 200 μl of the bacterial culture of P. aeruginosa and 100 μl of purified phage solution were mixed and the solution was incubated for 5 minutes to absorb the phage into the bacteria and then the phage-bacterial solution was added to a sterile tube containing 5 ml soft agar (0.7% agar). The mixture was transferred to the base agar surface and incubated for 24 hours at 37°C. The phages of formed plaques on the plate surface were collected and stored in SM buffer (NaCl 5.8 g/L MgSO4·7H2O 2 g/L, 1M Tris HCl (pH7.5) 50 ml/L and 2% gelatin 5 ml/L) (24).

Extraction and digestion of the genomic DNA of phages.

The genomic DNA of each isolated phage was extracted according to the kit (Phage DNA Isolation, Norgen, Canada) manufacturer’s instructions. HindIII and EcoRV restriction enzymes were used to digest the extracted DNA of the phages based on the manufacturer’s protocols (Thermo Fisher Scientific, USA). In summary, the DNA of the phages was digested for 5 hours at 37°C and electrophoresed on 0.8% agarose gel. After staining the gel with Safe Red (iNtRON Biotechnology, South Korea), the bands were visible when exposed to UV radiation.

Determining the host range of phages.

Spot test was used to determine the host range of each of the isolated phages. The host range of each of the phages was determined by adding 5 μl of the desired phage (1012 PFU/ml) to the surface of the cultivated plates with each of antibiotic-resistant P. aeruginosa isolates. The plates were incubated for 12 hours at 37°C and the formation of plaques on the surfaces of the plates was evaluated (24). Phage with the broadest host range was selected for more detailed assays for determining its characteristics.

Transmission Electron Microscopy (TEM) of selected phage.

The purified suspension of selected phage was added to the surface of copper grades and negative staining with 2% uranyl acetate was performed. The extra color was adsorbed by paper filters and the collected phage samples were dried on copper grades. The morphology of the phage was observed by the Zeiss LEO 906 TEM at the voltage of 100 kV (Zeiss, Germany). The image was taken in Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Determination of one-step growth of the selected phage.

A one-step growth test was performed to determine the latent period and burst size of the selected phage using the method of Pajunen et al. (25). Selected phage at MOI = 0.01 was added to the culture of P. aeruginosa.

In order to absorb the phage to bacteria, the mixture was incubated for 15 minutes at 37°C. After incubation, the mixture was centrifuged for one hour at 4°C and the supernatant was discarded. Pellets containing bacterial contaminant phages were transferred to the fresh LB medium and incubated at a temperature of 37°C at 180 rpm. Then, the mixture was sampled at ten-minute intervals and the phage titer was evaluated using the double-layer agar method. Burst size and latent period were estimated from triplicate experiments ± SEM.

Temperature and pH stability of the selected phage.

The selected phage stability to different temperatures and pHs was assessed according to the method described by Bao et al. and Verma et al. with minor modifications. The tube containing 1 ml of the selected phage solution was incubated at 4, 10, 20, 30, 40, 50, 60, 70°C for one hour. After incubation at selected temperatures, the selected phage’s survival rate was evaluated using “soft agar overly” method (26). To determine the stability of selected phage to different pHs, nutrient broth culture media (Hi-Media, India) with pH 2, 4, 6, 8, 10, and 12 prepared using 1M HCl and 1M NaOH solutions and then, 1 ml of the selected phage solution was added to 9 ml of nutrient broth medium with adjusted pH and incubated for one hour at 37°C. After the completion of incubation, the selected phage’s survival rate was evaluated at each pH using the “soft agar overly” method (27). These experiments were performed three times, and the results are reported as the mean of three observation ± SEM.

RESULTS

Isolation and identification of bacterial isolates of Pseudomonas aeruginosa.

In this study, based on standard biochemical tests, 20 collected bacterial isolates from hospital samples were confirmed as P. aeruginosa.

Antibiotic susceptibility of 20 P. aeruginosa isolates obtained from clinical specimens was determined according to the CLSI 2017 guidelines, the results are shown in the Table 1. All isolates of P. aeruginosa were resistant to penicillin (10 μg), amoxicillin (25 μg), nalidixic acid (30 μg), kanamycin (30 μg) and cefixime (5 μg) antibiotics. And the resistance rate of P. aeruginosa isolates to cefepime (30 μg), imipenem (10 μg), rifampicin (5 μg), tetracycline (30 μg), tobramycin (10 μg), gentamicin (10 μg) and amikacin (30 μg) were 95%, 90%, 55%, 35%, 30%, and 15% respectively. Among these bacteria, 15 of the most resistant isolates to antibiotics were used for the isolation of lytic phages against. The susceptibility pattern of these isolates to various antibiotics was shown in Table 2.

Table 1.

Antibiotic susceptibility of P. aeruginosa clinical isolates from patients

Antibiotics	N=20
	R%	I%	S%
Penicillin (P)	100	0	0
Amoxicillin (AMX)	100	0	0
Gentamicin (GM)	30	0	70
Nalidixic acid (NA)	100	0	0
Tetracycline (TE)	40	0	60
Imipenem (IMP)	90	10	0
Amikacin (AK)	15	5	80
Kanamycin (K)	100	0	0
Tobramycin (TOB)	35	10	55
Cefepime (FEP)	95	5	0
Cefixime (CFM)	100	0	0
Rifampicin (RA)	55	0	45

R: Resistant, I: Intermediate, S: Sensitive

Table 2.

Sensitivity pattern of antibiotic-resistant P. aeroginosa isolates

P. aeruginosa isolates	Resistance
Isolate 1	P, NA, IMP, FEP, CFM, RA, AMX, K
Isolate 2	IMP, AMX, NA, P, TE, K, FEP, GM, CFM, TOB
Isolate 3	P, CFM, GM, NA, AMX, TE, IMP, AK, K, TOB, FEP, RA
Isolate 4	AMX, TE, IMP, NA, K, FEP, CFM, P
Isolate 5	NA, P, IMP, AMX, K, FEP, CFM, RA
Isolate 6	K, P, AMX, CFM, NA, IMP, FEP, RA
Isolate 7	P, AMX, GM, NA, TE, IMP, AK, K, TOB, FEP, CFM, RA
Isolate 8	NA, TE, IMP, P, K, FEP, CFM, RA, AMX
Isolate 9	P, NA, AMX, IMP, K, FEP, CFM
Isolate 10	P, RA, IMP, FEP, K, CFM, AMX, NA
Isolate 11	FEP, AMX, K, P, NA
Isolate 12	AK, NA, P, CFM, K, IMP, FEP, RA
Isolate 13	P, IMP, NA, AK, CFM, RA, K
Isolate 14	P, FEP, AMX, K, NA, CFM, RA, IMP
Isolate 15	IMP, AMX, NA, RA, K, P, FEP, CFM

P: Penicillin, AMX: Amoxicillin, GM: Gentamicin, NA: Nalidixic acid, TE: Tetracycline, IMP: Imipenem, AK: Amikacin, K: Kanamycin, TOB: Tobramycin, FEP: Cefepime, CFM: Cefixime, RA: Rifampicin

In this study, one phage was isolated against each of the 15 MDR P. aeruginosa isolates from the sewage of Maragheh city, East-Azarbaijan, Iran. Each of these phages formed clear plaques on the surface of plates inoculated with their host bacterial isolate, which two of the plates as shown in Fig. 1 (A and B).

Fig. 1.

(A and B). Plaque formation of lytic phages on double layer agar plates. Plaque assay of lytic phages on a lawn of MDR-P. aeruginosa isolates. A: Plaque assay of phage 6 on the lawn of MDR-P. aeruginosa isolate 6. B: Plaque assay of phage 7 (PPaMa1/18) on the lawn of MDR-P. aeruginosa isolate7.

Extraction and restriction endonuclease digestion of phage DNA.

Extraction of genomes of the 15 isolated phages was observed by electrophoresis on agarose gel (Fig. 2). The results showed that the genome of all phages was double-stranded DNA, but differences in the size of extracted DNAs were observed. Then, for genome analysis, the pure DNA of each phage was digested by two endonuclease enzymes HindIII and EcoRV. As shown in Fig. 3 (A and B) different restriction patterns obtained by using both of the enzymes, indicated a wide variation in the phage genomes sequences.

Fig. 2.

Genome of phages: 0.8% agarose gel. M shows 1 kb DNA Ladder (Cinagene, Iran) and Lane (1–15), show bands of phage DNA. Lane 7 shows band of phage PPaMa1/18 DNA.

Fig. 3.

A. Enzyme digestion analysis of P. aeruginosa phages DNA with HindIII. M:1 kb DNA Ladder (Cinagene, Iran), Lanes (1–15) contained: digested genome of P. aeruginosa phages with HindIII. Lane7: digested genome of phage PPa-Ma1/18 with HindIII. B. Enzyme digestion analysis of P. aeruginosa phages DNA with EcoRV. M:1 kb DNA Ladder (Cinagene, Iran), Lanes (1–15) contained: digested genome of P. aeruginosa phages with EcoRV. Lane7: digested genome of phage PPaMa1/18 with EcoRV.

Determination of the host range of isolated phages.

The host range of each phage against the P. aeruginosa isolates was determined by using spot test. The results of the evaluation of the host range of 15 phages were shown in Table 3. It indicated that Phage 7 (phage against isolate 7) has had the broadest spectrum of the host with an effect on 85.7% of P. aeruginosa isolates that was known as selected phage and was named PPaMa1/18.

Table 3.

The lytic spectrum of isolated phages on 15 P. aeruginosa isolates collected from hospitals of Tabriz, East-Azarbaijan, Iran

P.aeruginosa isolates	Phages
	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12	P13	P14	P15
Isolate 1	+	+	+	-	-	+	+	+	-	+	+	-	+	+	+
Isolate 2	-	+	-	+	-	-	+	+	-	+	+	+	-	-	+
Isolate 3	-	-	+	+	+	+	-	+	+	+	-	+	-	+	+
Isolate 4	+	-	+	+	+	-	+	+	+	-	-	+	+	+	-
Isolate 5	+	-	+	-	+	-	+	+	-	+	+	-	+	+	+
Isolate 6	-	+	-	-	-	+	+	-	+	+	-	-	-	-	+
Isolate 7	+	+	+	+	+	+	+	+	+	-	+	-	-	+	+
Isolate 8	+	+	+	+	+	-	+	+	-	+	+	+	+	+	-
Isolate 9	-	+	+	-	-	+	+	+	+	-	+	+	-	-	+
Isolate 10	+	+	-	+	+	+	-	-	+	+	-	-	+	+	+
Isolate 11	+	-	-	+	-	+	+	+	+	+	+	+	-	+	-
Isolate 12	-	-	+	-	+	-	+	+	-	+	-	+	-	-	-
Isolate 13	+	-	+	-	+	+	+	+	+	-	+	+	+	+	+
Isolate 14	+	+	+	+	-	+	+	-	-	-	-	-	-	+	-
Isolate 15	+	-	+	+	+	+	-	+	+	-	+	+	+	+	+

lysis; -: no lysis

P: Phage

P7: phage PPaMa1/18

phage PPaMa1/18 as a candidate for phage therapy, were used in subsequent analyses such as Transmission Electron Microscopy (TEM), evaluation of pH and temperature stability and, determination of one-step growth curve.

Transmission electron microscopy (TEM) of phage PPaMa1/18.

The image obtained from the TEM showed that phage PPaMa1/18 had a large, icosahedral head (dimensions were 90 nm × 75 nm) and a long contractile tail of 215 nm in length (Fig.4A and B). According to the International Committee on Taxonomy of Viruses (ICTV), this phage belonged to the Myoviridae family of the Caudovirales order.

Fig. 4.

Transmission electron micrographs of phage PPa-Ma1/18 negatively stained with uranyl acetate. The Scale bars represent (A) 50 nm and (B) 25 nm.

One-step growth curve of the phage PPaMa1/18.

The One-step growth curve in this study consisted latent phase, log phase, and stationary phase. Based on this curve, the latent period was estimated about 20 minutes and the burst size was calculated to be approximately 58 PFU/cell (Fig. 5).

Fig. 5.

One-step growth curve of phage PPaMa1/18 on P. aeruginosa. Data represent mean ± SEM from three triplicate experiments

Stability of phage PPaMa1/18 to pH and temperature.

In this study, survival rate of the phage PPaMa1/18 was evaluated at different temperatures. The results of thermal stability showed that the phage PPaMa1/18 was stable at the temperature range from 4°C to 40°C. In addition, at temperatures above 40°C, the phage’s titer dropped sharply so that at 70°C the phage completely deactivated (Fig. 6).

Fig. 6.

Thermal stability of phage PPaMa1/18 treated with different temperature for 1 h. Data represent mean ± SEM from three triplicate experiment.

The phage PPaMa1/18 stability to different pH values also investigated. The phage PPaMa1/18 had an appropriate lytic activity in pH ranges from 4 to 10. However, after an hour of incubation of the phage at pH 12, there was a significant reduction in the lytic activity of the phage. Another noteworthy point is that in pH 2, the phage was completely inactivated (Fig. 7).

Fig. 7.

pH stability of phage PPaMa1/18 treated with different pH for 1 h. Data represent mean ± SEM from three triplicate experiment.

DISCUSSION

Many of the infections caused by Pseudomonas aeruginosa strains are highly resistant to antibiotic treatments. One of the reasons refers to the innate resistance of the bacteria but another important cause of this resistance is related to some other factors such as inappropriate use of various antibiotics (28). In recent years, with the rapid expansion of antibiotic-resistant bacteria and the decline of the pharmaceutical industries desire to invest in the production of new antibiotics, especially against Gram-negative bacteria due to increased research costs, finding effective and inexpensive therapy approaches for controlling bacterial infections is taken into consideration (29). In this regard, the isolation and characterization of lytic bacteriophages against clinical antibiotic-resistant isolates of Pseudomonas aeruginosa in different geographical area could be very helpful to combat these infections that threaten human healthcare recently. Then, we isolated and characterized lytic bacteriophage with antibacterial potential against P. aeruginosa isolates originated from north west of Iran as a candidate for phage therapy.

In our study, all of the isolates were resistant to at least five used antibiotics, also the rate of resistance to five antibiotics including cefixime, penicillin, amoxicillin, nalidixic acid, kanamycin was 100%. Antibiotic resistance to amoxicillin, kanamycin, tobramycin, gentamicin and, tetracycline was significantly higher than those reported by Pallavali et al. (23). Comparing to some studies in Iran, bacterial isolates in our study showed more resistance to imipenem, lower resistance to amikacin and gentamicin and similar resistance to cefepime (30). In another study, isolates of P. aeruginosa revealed 50%, 63.9%, 16.7%, 8.3%, and 16.7% resistance to imipenem, cefepime, amikacin, tobramycin, gentamicin, respectively which is lower than the current study (31). Brzozowski et al. reported lower rate of resistance to imipenem and cefepime and higher rate of resistance to tobramycin, amikacin, and gentamicin (32). Recently, phage therapy was studied as one of the renewed biologic approaches to combat multi-drug resistant bacteria in therapeutic fields. Wastewater is known to be a rich source of various microorganisms like bacteriophages (33) that can be exploited to control pathogenic bacterial infections. Lytic phages are preferable to phage therapeutics due to their rapid proliferation in host bacteria than lysogenic phages (34). P. aeruginosa lytic phages have been used in several studies to treat infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa effectively. The lytic phage PAXYB1 isolated from wastewater was used to infect P. aeruginosa strain PAO1 and other clinical isolates of MDR P. aeruginosa from different sources. The PAXYB1 was able to efficiently infect and kill clinical isolates of P. aeruginosa (35). The lytic phage AZ1 was isolated against the MDR clinical strain of P. aeruginosa-2995 and the activity of this phage was determined against P. aeruginosa-2995 in both planktonic cells and the biofilm. The phage showed desirable activity in the destruction of MDR P. aeruginosa-2995 of both planktonic cells and the biofilm. However, combination of phages was needed to complete elimination (36). In another work, bacteriophages isolated from sewage and evaluated against MDR-bacterial isolates (P. aeruginosa, S. aureus, K. pneumoniae and E. coli) from patient with septic wound infections. These phages demonstrated perfect lytic activity against the MDR bacteria causing septic wounds. Therefore, the authors suggested the phages as therapeutic options for treating septic wounds (23). In addition, P. aeruginosa phage isolated from sewage of Ilam had tremendous effects against a variety of clinical and general laboratory strains (ATCC15693) of P. aeruginosa (37). In our study, according to molecular analysis, highly genomic variation was observed using endonuclease enzymes among genomes of 15 isolated bacteriophages and just three and two genomes were not digested with HindIII and EcoRV endonuleases, respectively. In other studies, the digestion of isolated genomes of bacteriophage against Pseudomonas aeruginosa was reported by using some endonucleases (EcoRI and DraI) and also the insensitivity of the genomes to other enzymes (PstI and BamHI) was shown (38). Molecular analysis of the genomes of isolated P. aeruginosa phages in Kwiatek showed digestion of the genomes with EcoRI, HindIII, and SmaI enzymes but no digestion was observed by using BamHI (39). Genomic variety could be a good sign of possibility of isolating different effective bacteriophages against bacterial infections which could be helpful to introduce more effective packages against infections of P. aeruginosa. Rate of host range is one of the important factors to introduce appropriate candidates for phage therapy. Phage PPa-Ma1/18 showed the highest host range against 87% of used isolates that was in accordance to the study of Garb et al. which showed high rate of lytic effects of the phage JG02 (84%) on P. aeruginosa clinical isolates (40). In another study, broad spectrum of isolated bacteriophages against P. aeruginosa isolates was confirmed (41). Also, the determination of host range analysis in a study showed that 51.4% of the P. aeruginosa bacterial strains investigated were susceptible to phage BrSP1 (42). Large burst size and short latency period are two significant features for bacteriophages in phage therapy and phage propagation has a closely relationship with the burst size (43). Then, large burst size of a bacteriophage is a clear advantage to select a phage as an antibacterial agent. Based on one-step growth characteristics, the latent period of the phage PPaMa1/18 was short, that is desirable for phage therapy but its burst size was approximately low. In spite of lower rate of burst size of PPaMa1/18, strong lytic efficiency and also broad spectrum of its lytic effect on clinical isolates can select it as an appropriate candidate to phage therapy. Selecting a potentiated bacteriophage for phage therapy needs to be regarded to different factors not just one characteristic. There are reports of significant changes in the latent period and the burst size of Pseudomonas aeruginosa phages. In some researches, Pseudomonas aeruginosa phages with longer latent periods and larger burst sizes have been introduced such as phage PAXYB1 (latent period of about 30 minutes and burst size approximately 141 PFU/cell) and phage PPA-ABTNL (latent period of about 35 minutes and burst size approximately 110 PFU/cell) (35, 24). In other reports, some phages with shorter latent period and larger burst sizes have been introduced such as phage ø/PSZ1 (latent period of about 12 minutes and burst size approximately 100 PFU/cell) and phage ø/PSZ2 (latent period of about 10 minutes and burst size approximately 100 PFU/cell) (44). Differences in the latent period and the burst size of phages can also be due to differences in the medium, environmental factors (pH and temperature), and host cell (45). In TEM morphological analysis, PPaMa1/18 was belonged to Myoviridea family of the Caudoviridals order (head size of 90 nm × 75 nm and a long contractile tail of 215 nm in length), which was similar to several other Pseudomonas aeruginosa phages such as MPK1, JG024 with the head diameter of 80 nm × 75 nm and tail length of 130 nm, SL1 (head diameter: 70 nm and length tail: 130 nm), SL2 (head diameter: 120 nm and length tail: 170 nm), C11 (head diameter: 65 nm and length tail: 122 nm), vB_paeM-10 (head diameter: 90 nm × 91 nm and length tail: 175 nm) in previous studies (46–48, 40, 49). The phages as candidates for phage therapy should be more stable in different environmental conditions (50). PPaMa1/18 showed high survivability in different temperatures and different pH values. High rate of lytic effects between temperature 4–40°C and pH range of 4 to 10 was determined. The phage was completely deactivated at 70°C and pH 2. Inactivation of phage at pH 2 may be due to denaturation of proteins in acidic conditions (51). Denaturation of virus particles and other irreversible damages are shown due to high temperatures. Also, aggregation process can cause the decrease of phage concentration in acidic solutions (52). Different stability of phages in different temperature and pH conditions have been shown in several studies. Didamony et al. showed that Pseudomonas aeruginosa phages ø/PSZ1 and ø/PSZ2 were stable in a temperature range of 30–50°C and were inactivated after incubation for an hour at 60°C. This indicates that the phage PPaMa1/18 is more stable than phages ø/PSZ1 and ø/PSZ2 at higher temperatures (44). In another report, the phage AZ1 was stable at temperature range of 37–65°C and its activity decreased with increasing temperature and it was completely deactivated at 80°C, indicating that AZ1 phage is more stable at higher temperatures comparing to our results (36). Two specific phages against Pseudomonas aeruginosa P2S2 and P. aeruginosa P5U5 exhibited significant stability within the pH range of 4 to 11 (53).

CONCLUSION

According to our results, using lytic phages can be potentiated for combatting infections due to Pseudomonas aeruginosa bacteria. The high rate of genomic variation of isolated phages refers to the abundance of lytic phages even in the same area. Because of the wide host range and effective lytic activity and likewise high stability of the phage PPaMa1/18 in various environmental conditions (temperature and pH), it can be used as a promising candidate for the treatment of antibiotic-resistant infections caused by Pseudomonas aeruginosa.