Studies on Lytic Bacteriophages Recovered from Animal Waste: Isolation, Characterization and Therapeutic Potential against Pathogenic Bacteria
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Date
2008
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Chhattisgarh Kamdhenu Vishwavidyalaya
Abstract
In view of the global emergence of multidrug resistant strains of bacteria and strong requirement of a powerful weapon against them, the present study was conducted on isolation, characterization and evaluation of therapeutic potential of lytic phages recovered from wastewater samples from livestock farm of cow, buffalo, goat, pig, poultry including duck. A total of 75 samples were collected from different depths of the collection tank.
All the samples were primarily subjected to rapid detection methods viz. turbidity reduction method and streak plate method so as to detect lytic activity against bacteria; and then isolation of phage was done by Double agar layer (DAL) method. Result of initial screening indicated that recovery of phages was maximum from dairy cattle farm waste (90%), followed by buffalo farm waste (75%), pig farm waste (67%), goat farm waste (67%), poultry farm waste (50%) and least in duck rearing ponds (17%). Samples taken from bottom and middle layer were found more suitable for phage recovery than those samples obtained from surface. Using DAL method the primary isolation of phage was done on the two most common environmental bacteria namely B. subtilis (BsH) and E. coli (EH) total number of phage strains obtained were 35 and 6, respectively.
Physical and biological characterization of these phage isolates was done on the basis of plaque morphology, host range determination, effect of pH and temperature on viability. B. subtilis support the growth of more phages as compared to E. coli as indicated by more recovery of BsH than E. coli host specific (EH) phages.
For each bacteria, 3 different phage isolates namely BsHR1, BsHR2, BsHR3, EHR1, EHR2 and EHR3 were obtained on the basis of plaque morphology.
Phage isolates were tested against other bacterial genera i.e. Staphylococcus aureus, Salmonella spp, Pseudomonas aeruginosa, Klebsiella spp and Proteus spp. Results revealed that phage isolate EHR1 has widest host range while EHR2 is a narrow range phage isolate. BsHR1, BsHR2 and EHR3 were lytic against Pseudomonas aeruginosa and Salmonella spp but not against Staphylococcus aureus, Klebsiella spp and Proteus spp.
Phage isolates were investigated for their sensitivity to temperature based on ability of isolates to form plaques at 700C after exposure of 0, 1, 2 and 3 minute. Observation revealed that BsHR1, BsHR2 and EHR3 although viable for 2 min but were inactivated in 3 min. These isolates remain viable in pH range between 5 and 9. They were completely inactivated at pH values of 3 and 11.
From these isolates, high titer phage lysate/cocktail of phage was prepared so as to obtain phage concentration as 3x109 to 3x1012 PFU/ml.
Therapeutic potential of lytic phage isolates was evaluated by clinical trial conducted at Government Veterinary Hospital, Durg, Bhilai and Supela. A test group (10 animals) and a control group (10 animals) were formed. All animals were suffering from chronic wound infection which were not responding to antibiotic treatment. Swabs were taken for microbiological investigation and phage ‘cocktail’ using single dose and topical application was applied on wounds. In control group no phage treatment was given, but they were continued with antibiotic treatment.
In test group animals responded to phage treatment and recovery rate was 100%. Bacteria isolated from wounds were sensitive to phages in vitro. In swabs, obtained post-treatment, no bacteria were found at the site of wound.
The study is one step ahead to prove phage therapy as one of the effective antibacterial modality against pathogenic bacteria particularly those which are associated with chronic wound infections caused due to multidrug resistant bacteria. Methodology for isolation and characterization of phages helpful in characterization of phage isolates recovered from natural sources has also been discussed.
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