Abstract:

Background: Increased antibiotic resistance, particularly among Gram-negative bacteria to third-generation cephalosporins, is complicating and complicating clinical consequences. Bacteriophages are thought to be a good alternative for treating bacterial illnesses. The goal of this study was to figure out which Enterobacteriaceae strains were resistant to third-generation cephalosporins and how to treat them with virulent bacteriophages.

Objectives: In this study, the bacterial isolates were collected from diagnostic laboratories. The bacterial isolates were assessed for the antibiotic resistance profile, focusing on the phenotypic and genotypic methods. Effect of phage therapy was evaluated against the multi drug resistant pathogens in vitro and in vivo in C. elegans and G. mellonella model.

Methods: The antibiogram was assessed by disc diffusion method, and minimum inhibitory test. Further, the presence of resistant gene carbapenem-resistant genes
bla_NDM-1, bla_OXA-48, bla_IMP, bla_VIM, bla_KPC were studied. Phage was isolated against the resistant bacterial isolates. The bacteriophages against these clinical isolates were isolated from sewage water samples collected from Tamil Nadu, India. Phage isolation was performed using the enrichment method. All isolates were identified at the species level by 16S rRNA sequencing. All the phages were studied for their life cycle, genome analysis, and in vitro phage cocktail activity and in vivo efficacy in liquid-based assay in C. elegans and dosage analysis in G. mellonella.

Results: Out of 297 non-repetitive clinical isolates, 122 were Escherichia coli (41%), 76 Klebsiella pneumoniae (26%), 34 Enterobacter cloacae (11%). MIC results showed that 72% of the isolates were resistant to meropenem, and 28% to imipenem antibiotic. PCR results showed that 27 isolates carried bla_NDM, 5 isolates carried
OXA-181, and 1 bla_DIM. A totally of 3 phages were isolated against each genus of Enterobacteriaceae, that exhbited broad host range namely, Escherichia virus myPSH2311, Klebsiella virus myPSH1235 and Enterobacter virus myPSH1140. Microscopic observations (Transmission Electron Microscopy) suggested that the bacteriophages belonged to, Phieco32virus (Escherichia virus myPSH2311), Podoviridae (Klebsiella virus myPSH1235), and Myoviridae (Enterobacter virus myPSH1140). Whole Genome Sequence of three phages has been deposited in GenBank with the accession numbers MG976803 (Escherichia phage myPSH2311), MG972768 (Klebsiella phage myPSH1235), and MG999954 (Enterobacter phage myPSH1140). Treatment efficiency by in vivo method showed more than 80% efficiency.

Conclusion: All three phages were found to have the potential to infect the bacterial population that are resistant to two last-resort antibiotics meropenem and colistin.