Isolation And Characterization Of Virulent Bacteriophages and their synergism with antibiotic against Multi-drug resistant klebsiella pneumoniae clinical isolates at Jimma medical center, southwest Ethiopia

Abstract

Background: The alarming rise of multidrug-resistant (MDR) Klebsiella pneumoniae poses a significant threat to global public health and has a considerable economic impact, necessitating the development of alternative therapeutic strategies. Bacteriophages are alternative therapeutic agents in the fight against such bacteria. Phages are host-specific and can target specific bacteria precisely; thus, a detailed understanding of phage-host interactions in the presence of antimicrobial drug is required for effective usage. This study contributes to the growing body of research supporting phage therapy as an effective solution in the face of rising antibiotic resistance. Objective: This study was conducted to isolate and characterize virulent bacteriophages against MDR K. pneumoniae clinical isolates and to assess the phages interaction with antibiotics and their effects on the bacterial biofilm eradication. Method: Laboratory based experimental study was used to conduct the study. Ten MDR K. pneumoniae isolates were selected, and three of them were taken randomly for use in the isolation and enrichment process of lytic phages. Four wastewater samples were collected at four different sites of Jimma medical center compound through one time collection approach. Three lytic phages were isolated on spot assay and further purified and quantified by plaque assay. The phages biological characteristics were studied using a spot and plaque assay. Checkerboard assay was utilized for synergy and biofilm disruption testes. Results: Three lytic phages; KpPhage57, KpPhage56, and KpPhage85 against MDR K. pneumoniae were isolated and phenotypically characterized. The KpPhage57, KpPhage56, and KpPhage85 virus particles yielded by the plaque assay were 1.80*109 PFU/ml, 1.60*108 PFU/ml, and 5.0*1010 PFU/ml, respectively. These phages were stable between a temperature range of 4°C to 50°C and a pH value of 4 to 11, with almost similar lytic activity and narrow host ranges, with KpPhage56 having a relatively broader host range. KpPhage56 and KpPhage85 (at a titer of 106 PFU/ml) showed synergetic interaction with Gentamicin (500µg/ml), having a fractional inhibitory concentration index (FICI) of 0.085 and 0.077, respectively, indicating promising candidate for future phage therapy. All the three phages effectively disrupted K. pneumoniae biofilm at multiplicity of infection (MOI) of 10. Conclusion: The lytic and synergistic efficacy of the isolated phages, in conjunction with gentamicin, demonstrates their significant potential as a compassionate treatment for patients afflicted with MDR K. pneumoniae and biofilm-associated infections, particularly in wound and catheter-associated cases especially in resource-limited settings like Ethiopia.