Isolation and Characterization of Therapeutic Lytic Phages against Multi-Drug Resistant Enterobacter Cloacae in Kenya
| dc.contributor.author | Mutai, Ivy Jepkurui | |
| dc.date.accessioned | 2025-03-07T06:44:23Z | |
| dc.date.available | 2025-03-07T06:44:23Z | |
| dc.date.issued | 2023-06 | |
| dc.description | A Thesis Submitted in Partial Fulfillment of the Requirement for the Award of the Degree of Master of Science (Biochemistry) in the School of Pure and Applied Sciences of Kenyatta University, June 2023 1.Anthony Kebira 2. Atunga Nyachieo | |
| dc.description.abstract | Nosocomial infections due to Enterobacter cloacae affect 7% of patients in high-income nations, whereas they affect 15% of patients in low- and middle-income countries. This has been attributed to the ability of E. cloacae to confer resistance on carbapenems. With the scarce information on surveillance data and mortality rates of E. cloacae in Kenya, a recent report described E. cloacae as the most predominant multidrug-resistant organism with a resistance rate of 42.9% in critically ill patients in national referral hospitals and 12.3% in neonates from a rural hospital. E. cloacae is a unit of the ESKAPE family. This is composed of key resistant bacterial pathogens of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and other Enterobacter species. With a 12% mutation rate, which is twice as high as those reported in other Gram-negative bacteria, E. cloacae has been referred to as one of the greatest mutators in a clinical environment. This has been attributed to its complexity and exponential evolution of phenotypic and genotypic characteristics. Following this rise in antimicrobial resistance, the use of bacteria-eating viruses, termed as bacteriophages (phages) has been recommended. This study determined the antibacterial profile of E. cloacae for phage isolation, evaluated the existence and therapeutic potential of lytic phages against the MDR E. cloacae, evaluated the phage's stability in different physicochemical properties, and determined their genetic characteristics. The phages were screened from environmental wastewater sourced from the informal settlements of Korogocho, Huruma, Kibera, Dandora, Kariobangi, and Mathare in Nairobi County. The identification and antimicrobial profiling of the bacteria was done through Gram staining, biochemical tests, and confirmation using the automated Vitek II Machine. Phages were screened from the individual samples using enrichment and spot test assays. The isolated phages were assessed for cross-reactivity on a panel of thirty clinical isolates using spot tests as part of a host range analysis. From host range analysis, the most potent phage from each source was selected for physicochemical properties studies and whole genome sequencing (WGS) using Oxford Nanopore Technology (ONT). According to antimicrobial tests, the isolate was resistant to six classes of antimicrobial agents that include: beta-lactams (penicillin, cephalosporins, monobactams, and carbapenems), chloramphenicol, tetracyclines, glycylcyclines, quinolones, and sulphonamides. The only antibacterial agents that worked were tigecycline (glycylcycline) and meropenem (carbapenem). Twenty-nine lytic phages were screened with 4 from Dandora, 5 from Huruma, 5 from Kibera, 5 from Kariobangi, 7 from Korogocho, and 3 from Mathare. The chosen phages remained steady from 4 -50ºC with a 5.1% (4/6) drop and a 1.8% (2/6) increase in phage titre at 50ºC respectively. After two weeks of incubation at 4°C, the phages were effective, with peak activity at 37°C (human body temperature) and lowest activity at -20°C. There was no phage activity at pH 2 but was active from pH 5-11 with optimal activity at pH 7.5. Furthermore, they were efficient with Ca2+ ion intensities of 0.002M and 0.015M. All phages' effectiveness decreased with time after UV exposure. All the phages (n=29) showed cross-reactivity against isolates of E. cloacae (n=30) during host range evaluation, with the highest potency being 67% and the lowest being 27%. In WGS, the phages were virulent, and none harboured AMR genes, virulence factors, toxins, plasmids, chromosomes, transfer (tRNA), ribosomal (rRNA), and lysogenic genes. This study revealed that Kenya has therapeutic super lytic phages that can be used to eliminate multidrug-resistant E. cloacae infections. This study recommends the need to explore on phage dosage and in vitro studies for safety and side effects. | |
| dc.identifier.uri | https://ir-library.ku.ac.ke/handle/123456789/29727 | |
| dc.language.iso | en | |
| dc.publisher | Kenyatta University | |
| dc.title | Isolation and Characterization of Therapeutic Lytic Phages against Multi-Drug Resistant Enterobacter Cloacae in Kenya | |
| dc.type | Thesis |