In Vitro Bactericidal Activity of An Environmentally Isolated Phage Cocktail Against a Characterized Recombinant Endolysin Lysecd7 on Clinical Bacterial Isolates

Abstract

Background: Antimicrobial resistance (AMR) is rapidly spreading, which is pushing humanity into a “post-antibiotic era” where most antibiotics are no longer effective. Scientists are researching bacteriophages as a potential therapy against ESKAPE pathogens, which are a problem in hospital settings, as a result of the development of bacterial resistance to antibiotics. This study assessed the antibacterial efficacy of recombinant LysECD7 and environmental bacteriophage cocktail on bacterial isolates. Materials and Methods: Environmental water and soil samples were examined for the presence of bacteriophages that were capable of killing the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterobacter cloacae. Utilizing environmental isolates, a bacteriophage cocktail was created and its ability to effectively lyse bacterial growth cultures in vitro was examined. A synthetically generated Myoviridae lytic enzyme named LysECD7 was cloned, produced, and demonstrated wide bactericidal efficacy against gram-negative bacterial pathogens. The bacteriophage cocktail and endolysin’s (0.203mg/ml) antibacterial activity were assessed using a spot assay and an antibacterial activity assay in broth culture. Phage and endolysin bacteria samples were taken and their OD600 levels were checked every 30 minutes for phages and every 30 seconds for endolysin. Results: The average antibacterial activity of isolated bacteriophages was 43.58%, as opposed to the recombinant endolysin’s 31.48%. Bacterial absorbance declined steadily with each subsequent phage replication cycle, however, after six hours, there was a minor improvement in bacterial growth. The Gram-negative E. coli ATCC 25922 and E. cloacae NCTC 134565 exhibited the highest activity for both treatments, while the S. aureus and P. aeruginosa had the lowest activity. Conclusion: A single dose of low endolysin concentration had a very significant antibacterial activity, bacteriophage treatment of bacterial isolates varied greatly per each species under the same conditions. Under the same circumstances, bacteriophage and endolysin activity change within bacterial isolates, demonstrating that activity is independent of concentration. Lysin activity was highest in E. coli and lowest in the other bacterial species, but it should be emphasized that lysin and bacteriophage activity and efficacy differ with each treatment.f