Phage Therapy for Treatment Of Multiple Drug Resistant Organisms

Abstract

In the hunt of phages for Gram-negative multiple drug resistant organisms, we have collected lytic phages. These were characterized by identification of genome nucleic acids and proteins profile of capsid. However present study was proceed with the phages isolated form urine of athlete lady. Microbiological examination of urine sample revealed the presence of E. coli as etiologic agent in urinary tract infection (UTI) patient. Filter sterilized urine produced enornomous amount of phages as indicated by plaque assay on the lawn of two local strains of Pseudomonas aeruginosa, P5 and P6. Electron micrograph of the lysates prepared from urine in Ps5 and Ps6 strains indicated the particles similar to Siphovirideae and Myoviradeae respectively. Absence of Pseudomonas in urine sample during active disease and absence of phages in convalescent patient’s urine was significant features that refer the association of these phages to E. coli. Urea induced E. coli lysate was used to raise lysates in P5 and P6 strain of Ps. aeruginosa called U9P5 and U9P6. Small clear and large clear plaques were exhibited on the lawn of P5 and P6 by these lysates. P5S and PS6 lysates were raised in P5 and P6 from single small plaque each on the lawn of P5 and P6 respectively. Similarly PL5 and PL6 lysates were produced in respective hostfrom single large plaque each. Genomes of these Фs were identified as dsDNA. DNA restriction analysis of all four lysates revealed very similar restriction pattern. Hybridization with DNA probe of PS6 and PL5 has indicated the homology in the DNA from these lysates. Methylated and unmethylated CpG motifs were identified in genome DNA of these phages. To test the immuno- stimulatory effects mice and rabbits were vaccinated with killed cell of S. typhi with or without fragmented genome DNA of these phages. In addition adjuvant efficacy of CpG motifs was compared with mineral oil. In antisera, types of antibodies were determined by immunoelectrophoresis and Western blotting. ELISA was done for Quantative analysis, have shown IgG immunoglobulin was produced in BALB/c mice and rabbit when fragmented DNA was used as adjuvant. In antisera raised by Ф DNA vaccine, strong and specific immunogenicity has been attributed due to the presence of CpG motifs. Difference in immune response in animals immunized by Ф DNA vaccine was detected that may be associated with the frequency of CpG island in the DNA. Ps. Ф DNA has additional CpG sites, compare to E.coli Ф DNA and this may enhance immunopotentional of its CpG motifs. PS5 phage has shown cross reactivity with HCV antisera. In order to confirm the cross reactivity of phage and antisera, the 100 sera samples from the HCV +ve patients were tested byELISA on coated plates with HCV antigen, phage and Ps. and have exhibited significant cross reaction with phage lysates. In order to determine genomic relation, PCR products raised by HCV specific primers were sequenced and aligned with HCV genome. BLAST, Gene locater and QuickAling software have shown homology with the 5’-UTR, 3’-UTR and NS3 regions of different HCV genotypes. A range of primers pair corresponding to different antibiotic resistance trait were used to see whether this phage is a carrier of any antibiotic resistant trait. ClustalW, BLASTn, BLASTx, ScanProsite and Swiss-Prot analysis of sequenced PCR products have shown that this phage carries VanA cassette implicated to vancomycin resistance. Van S & R primers have shown homology with histidine kinase and DNA-binding response regulators respectively. Interestingly Ps Ф genome was found to contain a composite transposon which was evolved from the combination of IS1216 and 1546 transposons. These transposons were associated with Gram-positive bacteria i.e. Enterococci. VanB gene primers highlighted seven signature sequence on phage genome correspond to Walker B motifs of ABC-transporter proteins of Enterococcus faecalis V583 in addition phage genome has reflected the presence of some functional domain i.e. SAM which is commonly present in eukaryotic proteins. Our studies provide the strong evidence that lytic life cycle of thisphage has therapeutic implications, not only for superficial infection, but effective for systematic infections as well. It is interesting to note that CpG motifs of phage DNA may have potential to enhance efficacy of antimicrobial therapies supported by DNA vaccin