Isolation and characterization of novel phage (Podoviridae ɸParuNE1) and its efficacy against multi-drug-resistant Pseudomonas aeruginosa planktonic cells and biofilm

Bacterial pathogen (Pseudomonas aeruginosa) could form biofilm that conveys multi-drug resistance. Bacteriophage as an alternative to antibacterial resistance is useful against biofilm complications. This study evaluated antibacterial and biofilm removal activities of lytic phage, specific against multi-drug-resistant clinical P. aeruginosa. The phage showed a wide range of pH (5–10) and heat (7–44 °C) stability. Electron microscopy showed ɸPauNE1 phage head (60 nm in diameter) and non-contractile tail (12 nm in length by 8 nm in width); hence, the family Podoviridae and the order Caudovirales. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed structured protein of 55 kDa and double-stranded DNA of 45 kb. The phage was species specific and had broad host range activity. It inhibited bacterial growth at multiplicity of infection (MOI) 1–0.000001 pfu/ml. Inhibition was maximal at both low (1 × 105) and high (1 × 109) bacterial CFU/ml. Biofilm removal test showed that the phage removed more than 60% cell biomass within CFU/ml of 1.5 × 108, 6.0 × 108 and l.0 × 109. Phage (ɸPauNE1) was unique and had broad host range activity. The phage exhibited strong bacteriolytic activity against biofilm forming multi-drug-resistant strains. It had no lytic effect on the heterogeneous strains and so a promising bioagent.


Background
Pseudomonas aeruginosa is a pathogen frequently implicated in healthcare-associated infections (HAIs), especially in critically ill and patients with low immunity [1]. It is a versatile pathogen with the ability to cause diverse range of infections such as superficial infections, systemic and life-threatening conditions with mortality rate of 40-60% [2]. Various studies reported biofilm formation of this organism as complication of human disease [3,4]. Biofilm-associated P. aeruginosa morbidity and mortality were reported mostly in hospitals, where they are one of the leading pathogens in cases like cystic fibrosis and infections within nursing homes [5]. The organism is notorious as a multiple antibacterial-resistant pathogen difficult to treat globally [6]. According to World Health Organization's "priority pathogens, " P. aeruginosa made first list among the catalogue of 12 bacterial species that pose the greatest threat to public health [7]. The implication of the WHO's report was that these bacteria have become resistant to a number of antibiotics, including carbapenems and the third-generation cephalosporin [7].
In fact, the organism has been reported to be intrinsically resistant to many antibiotics including newer β-lactam [8,9]. Consequently, scholars have severally attempted to describe the relative severity and complexity, many scholars have described it as "Extremely Drug Resistant" (XDR) [6]. Available clinical data suggest that high morbidity and mortality rates associated with antibacterialresistant P. aeruginosa infections are mainly nosocomial and in particular surgical wounds [1]. Usually, patients with this complication have prolonged hospital stay [1].
Globally, there is intensive search for newer antibacterial agents or alternative to the existing classes of antibiotics available for containment of public health pathogens. Phage technology appears to be one of such promising alternatives. Phage therapy is considered more beneficial than antibiotics because it is safer and more specific in activity [10,11]. Application of bacteriophages to treat/control bacterial infection is known as phage therapy [12]. The advantage of the use of phage over antibiotics arises from the fact that the mechanism by which phage cause death of the host (bacteria) cells is entirely different from the mechanisms of antimicrobials [12]. Another advantage of this technique is that the earth biosphere contains a very large number (≈10 31 ) of bacteriophages [13], although only few of these have been characterized [14]. Therefore, in an effort to find alternative to failing antibacterial agents, this study isolated and characterized novel lytic phage that could eradicate multi-drug-resistant clinical P. aeruginosa and evaluated their specificity and biofilm removal activities.

Isolation and identification of bacterial strain
The multi-drug-resistant P. aeruginosa isolate from teaching hospital was identified using conventional standard methods [15]. It was further identified with VITEK ® -2 automated system using Gram negative identification (GN ID) card according to the manufacturer's instruction (BioMerieux, France) [16]. The strain was cultivated and maintained in Luria-Bertani broth and agar (Miller's, USA), respectively, at 37 °C. Bacterial growth was monitored using turbidimeter at optical density of 600 nm (OD 600 ). An OD 600 of 0.6 corresponded to1 × 10 9 cells/ml was used in the study.

Phage isolation
The multi-drug-resistant P. aeruginosa was used to isolate and propagate specific phage. The PAO1 and Pa ATCC-27853 (Phage lab-Texas A&M, USA) were used for quality control. Bacteriophage was isolated from sewage (collected from sewage treatment center, College Station, Texas, USA) following a simple enrichment procedure [12]. Aliquot of sewage water aseptically collected from a sewage plant was centrifuged for 10 min at 8000 rpm in cold condition (4 °C) and then filter sterilized with 0.22µm pore-size membrane filters.

Phage enrichment
A 30 ml of double-strength tryptone soya broth (TSB), 30 ml of filter-sterilized sewage water and 100 µl of 24-h culture of test organisms (MDR clinical P. aeruginosa) were mixed in a sterile 250-ml conical flasks and incubated at 37 °C on 200 rpm thermo-shaker incubator (Dubuque, Iowa 52,001, USA) for 24 h. After incubation, the phage-host mixtures were centrifuged at 8000 rpm for 5 min at 4 °C and filtered through 0.22-µm pore-size membrane filter [12].

Spot titration
The phage was extracted from the filtrate by spotting 10 µl of the phage suspension onto the test bacterial lawn within aseptic zone. Top-agar plate was produced by mixing a 100 µl of 4-h old culture of test bacteria strain (MDR clinical P. aeruginosa: 1 × 10 9 ) with 4 ml of molten tryptone soya agar (TSA), and the suspension was dispensed uniformly over the surface of 20 ml of TSA plate. The inoculated top-agar was allowed to solidify. After incubation at 37 °C for 24 h, zones of lysis were monitored [13].

Production of microplagues
The isolated lysed phage from the spot plate was enumerated using double agar layer plaque assay technique [13]. Briefly, A 100 µl of tenfold serial diluted phage suspension and 100 µl of host bacteria (1 × 10 9 CFU/ml) were mixed with 4 ml of molten TSA. The suspension was dispensed uniformly over the surface of 20 ml of hard TSA in 96-mm-diameter plate. The plate was allowed to set and then incubated at 37 °C for 12 to 16 h. After incubation, the presence of plaques was observed by comparison with a negative control plate [13].

Phage purification and amplification
The phage was purified by three successive singleplaque subculture method until homogenous plague was obtained [12]. The purified phage was amplified using 100 µl of 10 -3 phage lowest dilution which produced 10 4 -10 5 PFU/ml on agar layer plate. After incubation, the phage was harvested with phage buffer solution (SM: 100 mM NaCl, 25 mM TrisHCl pH7.5, 8 mM MgSO 4, 0.01% w/v gelatin). The lysate was centrifuged at 8000 rpm for 10 min and then filter sterilized with 0.22-µm pore-size membrane filter. The phage lysate was stored at 4 °C [12].

Examination of phage morphology by Transition Electron Microscope (TEM)
The morphology of the phage was detected by negative staining with uranyl acetate; sample was examined by transmission electron microscopy at an acceleration voltage of 100 kV and at calibrated magnifications. Images were recorded digitally with a slow-scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM Software (Olympus Soft Imaging Solutions, Münster, Germany) [14].

Phage-host range determination
Fifteen clinical isolates of MDR P. aeruginosa, a laboratory strain of P. putida and control strain PA01, were used to determine phage-host range, using standard spot test. The sensitivity of bacterial strains was confirmed by appearance of lysis zones at the site where the phage suspension was spotted. The phage was tested against each bacterial strain in three independent replicates. The lysis was categorized as clear (+), turbid (c) or no reaction (−) [19].

Isolation of bacteriophage DNA
Purified phage (10 10 -10 11 PFU/ml) was treated with 10 µg/ml of DNase (New England Biolabs) at 37 °C for 30 min and precipitated with 5 ml of 3 mM NaCl in 30% polyethylene glycol (PEG). The pellet was re-suspended in 500 µl of buffer (5 mM MgSO 4 ). The mixture was treated with 1-ml purification resin in a mini column (Promega Wizard DNA purification kit, USA) and finally washed by adding 2 ml of 80% isopropanol. The DNA was eluted from the resin by adding 100 µl of sterile water at 80 °C and was centrifuged at 13,000 rpm for 1 min. The DNA was stored at − 20 °C [20].

Restriction endonuclease enzyme digestion patterns
Specific volume (1 µl of 200-400 ng/µL) of DNA was digested with 0.5 µl of EcoR1, EcoRv, Bam H1, Pst1, Hind III, Bgl II restriction endonucleases (Fermenters, USA). The solution was incubated at 37 °C for 24 h. The DNA fragments generated were subjected to electrophoresis at 100 V for 1 h 30 min in TBS buffer and in a 1% agarose gel stained with 7.5 µl of ethidium bromide. The DNA in the gel was visualized under UV trans-illuminator. One hundred base pair DNA ladder markers were used as standard to measure the size of DNA fragment [21]. The phage structural protein was determined by separating purified phage preparation on a sodium dodecyl sulfate polyacrylamide gel as previously described [22].

Stability of the Phage under different (pH) values
The phage stability in the pH was determined by mixing 100 µl volume of phage suspensions (1 × 10 5 pfu/ml) in a series of tubes containing 900 µl of SM buffer (100 mM NaCl, 25 mM Tris HCl, 8 mM MgSO 4 , 0.01% w/v gelatine) with p H ranging from 1 to 10 (1.5, 3, 5, 7, 9 and 10). The mixture was incubated at 37 °C for 1 h, after which the phage suspension was immediately serially diluted in SM buffer (pH 7.5), and phage titer was determined by double-layer agar plate method for every treated sample. The phage survival rates were expressed as percentages of viable phage in suspensions [23].

Phage binding rate at specific temperatures
The binding rate of phage was estimated using the protocol previously described [22,23] with some modification.
Three replicate experiments were conducted to estimate adsorption rates. The cell concentrations at the beginning and the end of each experiment were determined and used separately for the estimation of each replicate adsorption rate. The influence of temperature on the adsorption rates of phage on the host cell was determined by incubating the phage infected culture at 7, 15, 25, 37 and 44 °C for 30 min in SM buffer (100 mM NaCl, 25 mM TrisHCl, 8 mM MgSO 4 , 0.01% w/v gelatine) at multiplicity of infection 0.01. After incubation, mixture was centrifuged, the supernatant was assayed for unabsorbed phage (double-layer plaque titration) and the count was compared with the titer of a control without cells. Phage adsorption rates were expressed as percentage of adsorbed phage in relation to the initial phage counts.

One-step growth curve
The growth of phage was investigated by determining the phage latent period, burst time and burst size as previously described [24]. Twenty milliliters of the host cell (MDR clinical P. aeruginosa) were incubated at 37 °C until the optical density (OD 600 nm ) was 0. 6; the culture was concentrated by centrifugation at 12,000 rpm for 5 min. The concentrate was mixed with equal volume of 500 µl LB broth and 500 µl phage (3.5 × 10 8 pfu/ml). The phage adsorption rate was determined a minute after the mixture. The mixture was centrifuged at 12,000 rpm for 2 min to remove non-adsorbing phage. The resulting pellet was suspended in 100 ml LB broth and kept at 37 °C. Samples were taken at five-minute intervals up to 90 min, and immediately tenfold serial dilution was carried out, and the phage titer was estimated through double-layer soft agar method.

In vitro determination of phage removal of biofilm
The efficacy of phage to remove biofilm formed by MDR clinical P. aeruginosa was investigated using the protocol as previously demonstrated with modification. The test bacteria were cultured overnight in three different media (LB, Muller Hinton broth, and TSB) at 37 °C. The strain of P. aeruginosa known as PA ATCC was used as control.
The host and the control bacteria were diluted to OD 600 0.1, 0.3 and 0.5. The test was determined as previously described [22].

Antibiotic sensitivity of host cell
The P. aeruginosa studied was 100% resistant to some β-lactam (ampicillin and ampicillin/sulbactam) and cephalosporin (cefpodoxime, cefuroxime and cefuroxime/axetil) tested. It was 97% resistant to cefoxitin and 95% to cefotaxime, tetracycline and trimethoprim/ sulfamethoxazole, respectively. However, carbapenem was the most effective antibacterial agent being 98% susceptible. The susceptibility pattern of the tested strain to antibiotics is shown in Table 1.

Isolation and host range of ɸParuNE1 phage
The phage specific to MDR clinical P. aeruginosa was isolated from sewage water. The phage was designated ɸParuNE1. It exhibited potent lytic activity against MDR clinical P. aeruginosa isolate with clear large to medium plaques that measure 1-2.5 mm in diameter (Fig. 1). The titer was 1 × 10 11 PFU/ml. The phage was stable during long-term storage at 4 °C in phage buffer solution (100 mM NaCl, 25 mM TrisHCl pH7.5, 8 mM MgSo 4, 0.01% w/v gelatine).

Phage morphology and family affiliation
The appearance of the phage by transmission electron microscope is shown in Fig. 2. The nature of the nucleic acid and the morphology of the phage showed icosahedral capsid of 60 nm in diameter, and a stubby, non-contractile tail measuring 8 nm. The phage was assigned to the family Podoviridae on the basis of its morphology.

Phage genome isolation and protein analysis
The genome of the ɸParuNE1 phage as detected on 1% agarose gel was a double stranded DNA due to its sensitivity to restriction endonucleases EcoRI, EcoRV and Hind III. The DNA size was 45 kb (Fig. 3A) and was sensitive to digestion by EcoRI, EcoRv and Hind III except Pst1enzymes. Distinct fragments were obtained when the DNA was digested with these enzymes. The EcoRv enzyme yielded six fragments, while EcoRI and Hind III enzymes yielded four fragments, respectively (Fig. 3B). The SDS-PAGE revealed one predominant polypeptide with a molecular mass of 43 kDa (Fig. 3C).

Phage host range
The host range test had nine MDR P. aeruginosa susceptible to ɸParuNE1 phage, while others including laboratory stain of P. putida and control strain of PA01 were  Table 2.

Phage binding rate at various temperatures
The phage showed varied adsorption rate at different temperature ranges. A higher number (57%) of ɸPauNE1 phage adsorbed at 44 °C, 47% at 15 °C, 42% at 37 °C, while 40% adsorbed at 7 °C; however, a lowest number (37%) adsorbed at 25 °C. Phage was able to absorb within the temperature range of 7-44 °C, their adsorption rates were considerably higher at 15 °C, reaching maximum at 44 °C (Fig. 4).

Growth characteristic
The latent phase for ɸParuNE1 was longer (Fig. 6) and estimated to be 28 min. The number of phages in the culture increased suddenly as the phage released the progeny. The ɸParuNE1 had burst size of 300 phage per infected cell.

Discussion
Multi-drug resistance is a global public health challenge. The rates of resistance in P. aeruginosa are increasing worldwide [25,26]. Besides, the remarkable ability of P. aeruginosa to form biofilms in many environments renders antibiotic treatments ineffective, thereby promoting chronic infectious diseases and mortality [27][28][29]. Consequently, public health experts and researchers are in search of newer antibacterial agents including phage therapy as demonstrated in this study and so some other reports [30]. Bacteriophages are in abundance in aquatic environments and play useful role in controlling their host populations [31]. Hence, it could be used to eliminate microbial contaminants that pose threats to public health. Phages have been successfully demonstrated for therapy in some bacterial diseases for example, cases of chronic otitis [32], burn wounds [33], chronic lung infection and biofilm-associated cystic fibrosis [34]. This report presents species specific phage which is likely one of the few reported cases of phage lytic to this strain as there were no reports of lytic phage to all P. aeruginosa species. Thus, the underlying aim is to identify and characterize novel bio-phage that show a high specificity and  a narrow range against newly emerging and re-emerging MDR P. aeruginosa strains. The ɸParuNE1 phage was isolated from sewage water. This supports earlier report that sewage is quite rich in nutrient for the survival of diverse microorganisms and phages are usually found in association with their host bacterial species [35,36]. The ɸParuNE1 phage was highly lytic, giving clear plaques of an average diameter of 1-2 mm. It showed a very narrow host range, infecting only pseudomonas species among the bacteria tested. These include P. aeruginosa CLI 22, P. aeruginosa 61, P. aeruginosa 20, P. aeruginosa 25, P. aeruginosa 33 and P. aeruginosa 64. This is in line with the previous report that phages are very specific to their host cell receptors [37].
Pseudomonas aeruginosa grows in temperature range from 10 to 44 °C, with optimal temperature around 35 °C [42]. Accordingly, it is expected that the highest adsorption rates of its phage should be within this range and this agrees with this report. Previous findings have reported that bacteriophages may vary in both their thermal and pH stability [36]. This phage (ɸParuNE1), therefore, showed very high and almost identical adsorption at both 7 and 37 °C; being considerably higher at 15 °C, reaching maximum at 44 °C. This has demonstrated high tolerance to a wide range of temperature (7-44 °C). This finding was in agreement with earlier observation [24]. Similarly, our data on ɸParuNE1 phage viability at various pH values showed good stability over a wide (pH 3 to 10) pH range. The phage was inactivated at lower pH values of 1.5 but showed maximum stability at pH 10. This finding is in agreement with earlier report that most phage thrive well at pH range of 5 to 9 under physiological conditions [36]. Survival of phages at neutral and alkaline pH in contrast with acidic pH has been reported [38]. They are said to be less stable in acidic environments since it denatures their proteins [39]. Accordingly, the examined ɸParuNE1 phage could be considered for topical application for skin and soft tissue wound treatment and intravenous application, with skin and blood, pH being around 5.5 and 7.4, respectively. Oral administration of this phage formulation may not be efficient since the stomach pH is usually acidic.
The growth curve of ɸParuNE1 phage showed a latent period of 28 min and a burst size of 300 virons per cell. Similarly, a latent time of 21 min and a burst size of 320 virons per cell was reported for the Myoviridae MJ1 phage against E. coli [40], while a latent time of 28 min and a burst size of 300 virons per cell was reported for Myoviridae WZ1 phage against Shigella dysenteriae [41]. In contrast, a previous report [39] showed a small burst size of 13 virons and high latent time period of 31 min, indicating that both latent time and burst size vary among different bacteriophages; the differences may be associated with their size and nature of the envelope. The later therefore, underscore the importance of certain parameters such as inoculation timing, phage absorption rate to the host, and burst size as critical factors to be considered for the success of formulation of bio-phage therapy. According to electron micrograph, ɸParuNE1 phage is placed within the family Podoviridae since the genome is double stranded and also sensitive to restriction endonucleases that cut only dsDNA. This observation agrees with earlier report that all Podoviridae possessed dsDNA as genetic material [40]. The examination of in vitro phage lytic efficacy showed that ɸParuNE1 phage attained more than 60% of bacterial reduction at MOI 1, 0.1, 0.01, and 0.001, respectively, for both bacterial planktonic growth and growth in the form of biofilm. The maximal bacterial lysis was observed during 16-h time period. Inhibition occur at both higher and lower MDR clinical P. aeruginosa CFU/ml of 10 9 , 10 5 and 10 3 . The MDR clinical Pseudomonas aeruginosa formed biofilms in all the media used. The phage almost eliminated biofilm in all MOI and reduced biofilms for more than 60% with MOI 0.1 at high cfu/ml of host cell strain, hence indicating the impressive potential of this phage for use in phage therapy. Accordingly, the phage shows very good potential as P. aeruginosa biofilm formation control agent. Few studies have demonstrated the ability of phage to reduce cell density of P. aeruginosa biofilms in line with this report [27]. However, source of bacterial isolate may vary. This study may have confirmed the potentials of phage as a possible bioagent to control P. aeruginosa biofilm that presently constituting public health threat.

Conclusion
ɸParuNE1 virulent phage is effective in the eradication of MDR clinical P. aeruginosa planktonic culture and biofilms with some remarkable properties. It possessed a latent time period and burst size suitable for bacteriophage therapy. With these potential characteristics, the ɸParuNE1 phage promising candidate for phage therapy against P. aeruginosa infection and contamination and therefore recommend that this potential be further explored.