Isolation and Characterization of Novel Bacteriophages Against Multidrug Resistant Enterobacter Cloacae as Alternatives to Antibiotics

Abstract

Enterobacter cloacae is a significant nosocomial pathogen, causing bacteremia and infections of the lower respiratory tract, urinary tract, and intra-abdominal cavity. Acquisition of antibiotic resistant genes due to increased pressure on antibiotics use has resulted in emergence of multidrug-resistant (MDR) Enterobacter species implicated in hospital acquired infections. Development of antibiotic regimens against MDRs is not at par with bacteria rates of resistance necessating the need for adoption of alternative strategies among them the use of bacteriophages (phages). The general objective of this study was to identify and characterize bacteriophages that can lyse gram-negative MDR E. cloacae bacteria with potential clinical applications. Three environmental water samples from each site were obtained from Kenyatta National Hospital (KNH) sewer, Chiromo River, Mathare River, Kibera slums and Zimmerman and fresh water (Lake Victoria). Host bacteria were obtained from the KEMRI-CMR repository. Spot test and plaque assay were used to screen and quantify the isolated bacteriophages. Isolated bacteriophages were tested for their stability at different temperatures (4, 25, 37, 60 and 90 °C) and pH (2.0, 4.5, 5.5, 7.5, 9.0, 11.5 and 13.0). Host range determination using 28 isolates of E. cloacae bacteria and one Staphylococcus aureus bacteria as a negative control was done. Whole genome sequencing was done using the Nanopore platform and results analysed using various bioinformatics tools like PhiSiGns programmes, GeneMark, REsFINDEr, among others. Nineteen (19) bacteriophages were isolated with all 19 phages lysing 12/28 (42.9%) E. cloacae bacteria isolates. The phages were stable at 4, 25 and 37 °C and 4.5, 5.5, 7.5, 9.0, and 11.5 pH. The selected 5 phages had dsDNA genomes with no genes associated with antibiotic resistance or toxicity. Three phages belonged to the family Autographiviridea/Studiervirinae while two could not be assigned. The study identified potentially suitable phage candidates for Enterobacter cloacae therapy due to their wide host range against endemic clinical isolates and their lack of genes associated with resistance and toxicity