Morphological Characterization and whole Genomeanalysis of Novel lytic Phages against Drug resistant Human Pathogens- An alternative approach to Antibiotic Therapy

Abstract

Introduction: Antimicrobial resistance – a condition when microbes are no longer sensitive to the drugs used to kill them – is a global concern and emergence of superbugs have threatened advances of modern medicine pushing us closer to post antibiotic era. Once easily treatable infections are now killing and alternatives to antibiotics are being explored. Among all alternatives, phage therapy – though used longest in clinics – is largely ignored by western world. Bacteriophages are viruses that infect bacteria but leave animal and plant cells unscathed. As MDR becomes a threat, interest in phage therapy is revitalized and now even US-NIAID lists ‘phage’ as research priority to address antibiotic crisis. Here, we explore availability of phages against MDR bacteria in our environment and assess their efficacy invivo followed by morphological identification. Methodology: Forty different drug resistant human pathogens representing 12 genera were collected from hospital after biochemical identification and antibiotic susceptibility test. Water sample from 5 holy rivers was screened for presence of lytic phages by double layer agar assay. Phages were purified by successively sub-culturing single plaque thrice & standard spot assay was employed to determine multiple host range. Most potent phages were confirmed by TEM. Whole Genome Sequence of three phages were then analyzed for presence of lytic proteins / enzymes than may be potential antibiotic candidate. Results: Thirty-four lytic phages - 16 E. coli, 13 Salmonella, 2 Shigella, 2 Klebsiella, 1 Citrobacter - were isolated. Except 2 Klebsiella phages all 32 phages showed multiple host range (lysed more than 1 strains). All Salmonella phages showed interspecific (S. typhi and S. paratyphi) lytic ability. Bacteriophage induced mutants were only observed against Klebsiella phages. All except Klebsiella phage completely lysed bacterial host up to five consecutive cycles. Whole genome sequencing of three phages revealed that they do not possess any ‘virulent’ bacterial genes within their genome. Conclusion: Our findings showed ‘phages against MDR bacteria’ that could be used therapeutically exists in real world and they are not extremely host specific as professed in scientific world. They’ve evolved to achieve broader host range and even possess interspecific lytic capability. Conclusively – we are not out of weapons to kill drug resistant pathogens yet, and co-evolving phages may be the ‘once and for all’ alternative to antibiotics.

Keywords: AMR, ABR, Alternative medicine, Bacteriophage, Holy rivers