The isolation and assessment of the antimicrobial activity of bacteriolytic and bactericidal endolysins of enterobacteriaceae infecting bacteriophages

Abstract

Pathogenic bacteria pose a significant threat to the health and well-being of vulnerable populations, including newborns and young children, individuals with compromised immune systems as well as those hospitalized or recovering from surgery. The worldwide burden of communicable diseases driven by pathogenic bacteria continues to be a pressing global health issue, with a substantial impact on mortality rates. The presence of antimicrobial resistance further complicates the treatment of bacterial infections. The changing patterns of antimicrobial resistance have greatly decreased the survival rate of individuals with bacterial infections. Irresponsible stewardship of antibiotics, coupled with a lack of knowledge on the use of antibiotics by the public has further fueled the development of antimicrobial resistance. Furthermore, the World Health Organization's Review on Antimicrobial Resistance states that, if the current trend in antimicrobial resistance continues, it can lead to a staggering 10 million deaths per year by 2050. Given that antimicrobial resistance is a globally pressing issue, new and innovative strategies and treatments are required to combat the burden posed by antimicrobial resistance. Bacteriophage lytic enzymes offer a promising solution to curb the issue of antimicrobial resistance. Aim The primary aim of the present study was to evaluate the bacteriolytic and bactericidal activity of bacteriophage proteins against Enterobacteriaceae and Staphylococcus aureus. Methodology In the present study, the Escherichia T4 Bacteriophage along with its host, Escherichia coli (DSM No 613, ATCC 11303, respectively; Leibniz Institute DSMZ) formed the basis of the work. After reconstitution in nutrient broth, the E. coli was cultured on nutrient agar (1.5%) in a 90 mm petri dish, to which nutrient agar (0.6%) containing the T4 bacteriophage was added to enumerate the bacteriophage. Escherichia coli and bacteriophage T4 were then co-cultivated in one litre of nutrient broth and the salting out method and column chromatography was used to isolate the biologically active proteins from the nutrient broth and proteomic analysis was performed to confirm the presence of the endolysin protein. Liposomes were created using the thin film hydration method and 1 ml of purified protein in phosphate buffer was encapsulated within the liposomes. Liposome formation was confirmed by Transmission electron microscopy and successful encapsulation of the protein was confirmed by breaking open 200 µl of liposomes and performing the Bradford protein quantification assay. Antimicrobial activity of T4 proteins and T4 protein loaded liposomes was evaluated using either a spot lytic assay or microdilution bioassay. In the latter, 100 µl of the bacteriophage protein solution (maintained in PBS) or 50 µl of the liposomal protein were serially diluted two-fold with 100 µl and 50 µl of sterile distilled water, respectively, yielding concentrations ranging from 205.68 µg/ml to 3.22 µg/ml. These samples were tested against three Gram-negative organisms, Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli and two Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis. Results Bacteriophage T4 proteins demonstrated varying levels of effectiveness in inhibiting the growth of the bacteria assessed in this study. For instance, in a microdilution assay, while the protein was able to inhibit the growth of Staphylococcus aureus at a concentration of 12.87 µg/ml and Escherichia coli at 43.70 µg/ml, it was incapable of influencing the growth of Enterococcus faecalis, Proteus mirabilis and Klebsiella pneumoniae at the highest concentration tested (205.68 µg/ml). Notably, in every instance, the liposomes loaded with T4 protein were more effective at inhibiting the growth of the bacteria. Molecular interactions were determined through protein-protein modeling that revealed that the endolysin protein from bacteriophage T4 was capable of binding with strong affinity to penicillin binding proteins from S. aureus. Conclusion The results of this study demonstrated the capability of T4 protein loaded liposomes to interact with and inhibit the growth of important bacteria linked to the BPPL as identified by the WHO, providing a pathway for the development of novel drugs aimed at combating these devastating bacteria