Overview. The present study was conducted in several steps, including P. aeruginosa identification and culture; bacteriophage characterization, reharvesting, and titration; enumeration of phage morphology and host range; P. aeruginosa biofilm formation on catheter; phage-biofilm interaction in vitro and in vivo; and identification of colony forming unit (CFU) and phage forming unit (PFU) counts after phage therapy of biofilms. The study was approved by the authors’ Institute Ethics Committee and the Institute Animal Ethics Committee for research. Care of the laboratory animals was provided according to the Indian Council of Medical Research Guidelines for use of Laboratory Animals in Medical Colleges 2001.22
P. aeruginosa identification and culture. Multidrug-resistant strains of P. aeruginosa were procured from stock culture collected from the Burn Unit of the authors’ University Hospital. One hundred (100) strains from stock were streaked on Muller Hilton agar plates (Hi media, India) and incubated overnight. Smears were prepared on a glass slide from 1 loopful of bacterial colony, Gram-stained and observed under microscope, and subjected to oxidase and catalase tests. For further experiments, the bacterial strains were grown in Luria Bertani (LB) broth at 37˚ C and harvested while in the exponential phase of the growth cycle. The harvested bacteria were washed with saline (0.85% NaCl in distilled water) and resuspended in saline to achieve 108 CFU/mL.
Phage characterization and re-harvesting and titration. Eleven (11) phages against P. aeruginosa were obtained from Bacteriophage Research Lab (BRL) of the Microbiology department of the University. The phages had been isolated from samples of various bodies of water including rivers, ponds, and sewers. The processing and reharvesting of the phages were done using the double agar overlay plaque assay technique,23 and PFU was measured. Small-scale concentration of Banaras Hindu University (BHU) bacteriophages was performed by spreading phages on the top-agar layer containing the respective host bacterium. The BHU bacteriophage titer was analyzed as described by Adams.24 Phages were characterized on the basis of their lytic profile, and a dendogram was obtained (see Figure 1), which depicts the level of similarity between the different phages used. When the 11 BHU phages were screened against the 40 MDR strains of P. aeruginosa, 11 strains were found to be lysed by 9 or more phage strains (see Table 1). These 11 strains then were re-subjected for testing against the BHU phages, leading to the conclusion P. aeruginosa strain 10958 is lysed by all the BHU phages; it was selected for further experimentation.
Enumeration of phage morphology and host range. Polyethylene glycol-precipitated BHU bacteriophage particles were subjected to transmission electron microscopy for viral morphology. The host range specificity and lysis efficiency in screening tests against different P. aeruginosa strains were measured. Eleven (11) strains, which showed lysis by 9 or more phages, were rechecked by repeating the process.
P. aeruginosa biofilm formation on catheter sections. P. aeruginosa biofilms were formed on 0.5 cm, presterilized polyurethane catheter sections (6 French, single lumen) according to the method described by Cerca et al25 with some alterations. Longitudinally cut open catheter sections were placed in a conical flask containing 6 mL of yeast peptone dextrose medium. A culture (10 µl) of P. aeruginosa strain 10958 with an optic density of 2.0 at 600 nm (corresponding to approximately 1.79 × 108 cells/mL) was added and incubated at 37˚ C on an orbital shaker for 48 hours at a constant speed of 120 rpm with the change of medium every 12 hours. The number of cells present on these catheter sections was assessed after 48 hours of biofilm formation in order to estimate the infective dose. CFU counts varied from 2 x 106 to 6 x 106. Presence of P. aeruginosa biofilm was confirmed in vitro after crystal violet staining of the catheter sections and viewed under oil immersion microscopy (100x) and biochemical analysis (oxidase and catalase positive).
Phage-bacterial interaction in vitro. Biofilm were infected with a cocktail of 11 BHU phages (100 µl each). The catheter sections with biofilm were immersed twice in phosphate buffer saline (PBS) and placed in microcentrifuge tubes with 0.5 mL of tris-magnesium sulphate-gelatin (TMG) buffer and 0.5 mL of phage cocktail solution in a concentration of 107 PFU/mL. The microcentrifuge tubes were incubated at 37˚ C. Control experiments were performed at the same conditions with the catheter sections after immersion in PBS in new microcentrifuge tubes with 0.5 mL of TMG and 0.5 ml of saline.
Phage-bacterial interaction in vivo. Male albino mice (25 ± 6 g) procured from the central animal house of the authors’ Institute were acclimatized in laboratory conditions for 7 days as per Indian Council of Medical Research guidelines.22 They were housed at 25˚ C, 45%–55% relative humidity, and 10:14 hours of light-dark cycle and had free access to rat pellets and water ad libitum. The animals were anesthetized with intraperitoneal ketamine (75 mg/kg). The back was shaved and disinfected with 0.5% chlorhexidine in 70% alcohol. A 10-mm transverse skin incision was made and a subcutaneous pocket was created between the skin and panniculus. Two catheter sections (with or without biofilm) were implanted in the subcutaneous pocket (see Figure 2). The incision was closed with interrupted 3-0 nylon stitches and disinfected with 0.5% chlorhexidine in 70% alcohol. The number of catheter sections to be used was assessed from a pilot study in which increasing numbers of catheter sections were implanted in 5 mice (ie, the first mouse had 1 catheter section while the fifth mouse had 5 sections). All developed pus formation in the subcutaneous pocket, but the first mouse showed complete resolution of pus, while the fourth and fifth mice perished after 48 hours of infection. The second and third mice showed pus formation without resolution for a week. Therefore, it was decided 2 catheter sections were optimum for the experiment.
The animals were randomly allocated into 4 groups of 6 mice each. In group A, the skin incision was followed by the creation of a subcutaneous pocket and 10 µl of sterile normal saline (NS) was injected daily into the pocket for 10 days. Group B had an incision with pocket creation in which uninfected catheter sections were placed; this group also had NS injections every day. In group C, catheter sections containing biofilm were placed in the pocket and the animals received daily NS injections. In group D, everything was similar to group C except instead of NS, 10 µl of the phage cocktail solution containing 107 PFU/mL phages was injected in the subcutaneous pocket.
After 10 days, the animals were anesthetized and after local disinfection the subcutaneous pockets were explored and wounds were examined macroscopically for signs of infection. Catheter sections were removed in an aseptic technique and washed twice with PBS and analyzed for bacterial and phage colony counts.
CFU and PFU counts after phage therapy. After washing twice with PBS, the catheter sections were put in microcentrifuge tubes containing 0.5 mL of NS solution. The tubes were thoroughly mixed (vortexed 4 × 30 seconds) and serial dilutions were immediately performed in NS solution for CFU counts and in TMG buffer for PFU counts. For CFU counts, the samples were immediately plated on Mueller Hinton agar plates, and for PFU counts samples were immediately plated using the method described above for phage titration.
Data collection and statistical analysis. The data generated from phage titration, bacterial colony counts, and phage colony counts both in vitro and in vivo were retrieved by a laboratory assistant blinded to the study. The statistical program SPSS version 16.0 (SPSS Inc, Chicago, IL) was used for data entry and analysis. An independent sample t-test was used to compare categorical variables between 2 groups. Analysis of variance (ANOVA) along with post-hoc Bonferroni test was done to compare variables in more than 2 groups. A P value <0.05 was considered statistically significant.