Author: Anjali Bandara
Bandara, Anjali, 2025 Discovery and Characterization of Novel Lytic Bacteriophages from Sewage Against Multidrug-Resistant Staphylococcus aureus, Flinders University, College of Medicine and Public Health
Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.
Background: Antibiotic resistance in Staphylococcus aureus has become an extensive global health concern, especially with the spread of methicillin-resistant S. aureus (MRSA) infection. Conventional antibiotics are increasingly becoming ineffective, leading to longer hospitalizations and higher treatment costs. Bacteriophages (phages), viruses infecting and killing bacteria, have been investigated as potential alternatives to antibiotics in recent years. They are specific, self-replicating, and biodegradable. Sewage has been identified as a source of many bacteriophages because of the high concentration of bacteria and their viruses. Phage isolation from sewage could, therefore, bring new candidates for controlling antibiotic-resistant S. aureus infections. Phages have two potential lifecycles: lytic, in which they replicate and kill their bacterial host, and lysogenic, in which they co-exist with their host while integrating into its genome; in this study lytic hosts were sought.
Hypothesis/Aims: Hypothesis of this study is that phages that demonstrate lytic activity against antibiotic-resistant S. aureus strains can be isolated from sewage. The aim of this study was to isolate and characterize novel lytic bacteriophages from sewage that are effective against drug-resistant S. aureus.
Results summary: Bacteriophages were successfully isolated from sewage samples collected from the SA Water sewage treatment plant, Glenelg North. Clear plaques were observed on S. aureus strain 63-5222, indicating lytic phage activity. Examination using gel electrophoresis of extracted DNA confirmed the presence of intact phage DNA, and restriction digestion with enzymes SacI and PstI showed varying patterns, which reflected genomic variability. Host-range screening using direct spot test (DST) and efficiency of plating (EOP) showed strong lytic activity against various antibiotic-resistant S. aureus isolates. Among them, the phage Φ63-5222.PS had the highest titre (4 × 10¹⁰ PFU/mL) and was selected for further experiments.
Killing kinetics (OD₆₀₀) assays demonstrated immediate bacteriolysis at each multiplicity of infection (MOI), complete killing within 24 hours, and no regrowth. The time-kill assay demonstrated that Φ63-5222.PS killed bacteria within 2–4 hours, while its serially passaged counterpart, Φ63-5222.SP, maintained long-term bacterial suppression with no regrowth. An eclipse phase of about 40 minutes and a burst size of 25–27 PFU per infected cell, typical of efficient lytic S. aureus phages, were revealed by the one-step growth assay. The isolated phages demonstrated strong, rapid, and stable antibacterial activity; had a wide host range; and favorable replication characteristics. Serially passaged (trained) phage Φ63-5222.SP resulted in an increase in its killing potential and infectivity for the host. Thus, it is a good candidate for phage therapy.
Conclusion: This research successfully demonstrated that sewage is an excellent source for isolation of effective lytic bacteriophages against S. aureus. The isolated phages exhibited robust, rapid, and stable antibacterial activity, broad host range, and favorable replication characteristics. Trained phage Φ63-5222.SP exhibited enhanced killing potential and increased host infectivity, making it a suitable phage candidate for phage therapy. These findings define the potential for using environmental phages as safe, effective alternatives to antibiotics and provide a firm foundation for genomic and in-vivo studies that will confirm their clinical utility.
Keywords: Phage therapy, Staphylococcus aureus, Antimicrobial Resistance (AMR), Sewage, Multidrug-resistance (MDR), Diabetic Foot Ulcer (DFU)
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2025
School: College of Medicine and Public Health
Supervisor: ASSOCIATE PROFESSOR PETER SPECK