Lack of drinking water and proper sanitation are the major causes of enteric infection leading to huge malnutrition and other health problems in developing nations. Solving the sanitation challenge in the developing world will require radically new innovations that are deployable on a large scale. We aim to reduce the bacterial load of waste water by developing bacteriophages from sewage against the enteric pathogens. Bacteriophages are the viruses which infect bacteria and kill them. They are very specific to the target bacteria and hence can safely be used to kill the bacterial pathogens without affecting the human host or any non-target organisms in waste water. Thus they can be used to treat human waste and waste water safely. We developed broad spectrum phages by simply filtering the sewage water from different regions of the Amritapuri campus through 0.22μm filter, enrich the filtrated virus stock against all the waste water microbes by culturing them in different enrichment media recommended for different classes of bacteria such as Vibrio, Shigella, sulphur reducers, coliforms and nutrient broth media. We isolated specific plaques from different bacterial colonies isolated from the waste water cultured on different enrichment media.
Recent evidences show that humans might be actively hosting the phages in their gut to combat the bacterial infections. We intend to explore the possible molecular entities of phages interacting with host extracellular matrix proteins such as fibronectin, heparin and gelatin (denatured collagen). We chose phages against E. coli to explore the interaction. The phage plaques from E. coli colonies were grown in liquid Luria Bertani (LB) media and filtered through fresh lysate was passed through the gelatin, fibronectin and heparin affinity columns to check for the interaction of the specific phages with the matrix proteins. The non-specific interaction was prevented by extensive washing (20 bed volume) with chromatography buffer (50mM Tris, 150mM NaCl, pH7.4). The bound phages were eluted with 1M NaCl followed by 3%DMSO and 1M NaCl which were separately plated to check the number of plaque forming units (PFU). DMSO eluted fractions from all three affinity columns gave more plaques than that of NaCl eluted fractions. Fibronectin binding DMSO eluted phage were re-grown and concentrated by either acetone precipitation or passing through fibronectin-affinity column followed by elution with 4M urea in chromatography buffer. They were then separated by SDS-PAGE and stained with Coomassie Blue followed by Silver stain.

Project Details
Sanjay Pal
Co Pi: 
NarraSaiSandhya, Sarath JS, VinuSankar
Start Date: 
Tuesday, January 1, 2013 to Thursday, May 30, 2013