2.1 Isolation and identification of bacterial strain
The multi-drug-resistant P. aeruginosa isolate from teaching hospital was identified using conventional standard methods . It was further identified with VITEK®-2 automated system using Gram negative identification (GN ID) card according to the manufacturer's instruction (BioMerieux, France) . 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 (OD600). An OD600 of 0.6 corresponded to1 × 109cells/ml was used in the study.
2.2 Antibiotic sensitivity test
Antibiogram was conducted using VITEK®-2 system (BioMerieux, France) . The antibiotic susceptibility test packs (AST GN590 card) of VITEK®-2 system for Gram negative organisms were used. The antimicrobial test card contained 19 (ampicillin, ampicillin/sulbactam, cefotaxime, cefpodoxime, cefoxitin, ceftazidime, cefuroxime, cefuroxime/axetil, ciprofloxacin, gentamycin, imipenem, levofloxacin, meropenem, nitrofurantoin, piperacillin, piperacillin /tazobactam, tetracycline, tobramycin and trimethoprim /sulfamethoxazole) antibiotics for Gram negative bacteria in each panel. Pseudomonas aeruginosa CLI-22 was characterized as multi-drug resistant (MDR) according to CLSI guidelines .
2.3 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 . 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 0.22-µm pore-size membrane filters.
2.3.1 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 .
2.3.2 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 × 109) 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 .
2.3.3 Production of microplagues
The isolated lysed phage from the spot plate was enumerated using double agar layer plaque assay technique . Briefly, A 100 µl of tenfold serial diluted phage suspension and 100 µl of host bacteria (1 × 109 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 .
2.3.4 Phage purification and amplification
The phage was purified by three successive single-plaque subculture method until homogenous plague was obtained . The purified phage was amplified using 100 µl of 10–3 phage lowest dilution which produced 104–105 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 MgSO4, 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 .
2.4 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) .
2.5 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 (−) .
2.6 Isolation of bacteriophage DNA
Purified phage (1010–1011 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 MgSO4). 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 .
2.6.1 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 . The phage structural protein was determined by separating purified phage preparation on a sodium dodecyl sulfate polyacrylamide gel as previously described .
2.7 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 × 105 pfu/ml) in a series of tubes containing 900 µl of SM buffer (100 mM NaCl, 25 mM Tris HCl, 8 mM MgSO4, 0.01% w/v gelatine) with pH 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 .
2.8 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 MgSO4, 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.
2.8.1 One-step growth curve
The growth of phage was investigated by determining the phage latent period, burst time and burst size as previously described . 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 × 108 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.
2.9 Phage bacteriolytic activity in vitro experiment
An optical density (OD600) of 0.4 (2 × 109 CFU/ml) broth culture of test bacteria (MDR clinical P. aeruginosa) strain was inoculated into double-strength LB broth supplemented with 1% of glucose. A sterile 96-well micro-titer plate with flat bottom (Greiner bio one, Germany) was filled with 100 µl of inoculated medium in such a way to get 1 × 109 CFU/well (MOI 1) in the columns 1–3, 1 × 107 CFU/well (MOI 0.1) in columns 4–6, 1 × 105 CFU/well (MOI 0.01) in columns 7–9, and 1 × 103 CFU/well (MOI 0.001) in columns 10–12. However, the columns in row H were the control, and the wells in H1, H4, H7 and H11 were alternatively filled with 100 µl of sterile double-strength LB broth with glucose and 100 µl of sterile phage buffer. The remaining wells in row H were filled with100µl of sterile phage buffer solution and appropriate bacterial CFU/well without phage. Phage dilution in SM buffer solution was added (100 µL) into the wells, from row A-G to obtain 1 × 107–1 × 101 PFU/well (MOI: 1-000001). The plate was incubated in 120 rpm thermo-shaker at 37 °C for 18 h. After incubation, 50 µl of 0.1% filter-sterile 2,3,5-triphenyltetrazolium chloride (TTC) (Alfa Johnson Matthey Company, Lancaster, USA) was added into each well (final concentration 200 µg/ml, 50 µg/well) and incubated for additional 3 h. Absorbance was read at 540 nm, using a micro-titer plate reader (PerkinElmer Wallac EnVision 2104 Multilabel Reader, USA) .
2.9.1 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 OD600 0.1, 0.3 and 0.5. The test was determined as previously described .