Studies on use of bacteriophages in treating antibiotic-resistant infections of diabetic foot ulcers

Author: Legesse Kifelew

Kifelew, Legesse, 2020 Studies on use of bacteriophages in treating antibiotic-resistant infections of diabetic foot ulcers, Flinders University, College of Science and Engineering

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This work is in response to two severe problems confronting global health systems: diabetes and antibiotic resistance. Diabetic foot ulcers (DFUs) are open lesions that fail to heal in the foot of a diabetic patient. About one in four people living with diabetes develop diabetic foot ulceration during their lifetime. DFUs causes close to 90% of limb amputations among persons with diabetes. The 5-year mortality rate following foot amputation due to DFUs is up to 74%. Most DFUs become infected, and antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are often isolated from DFUs. The increasing failure of antibiotics prompts the development of bacteriophages ("phages") as alternative anti-infective agents.

An important aspect of bringing phages to the clinic is that they are made to high standards. We have access to phage cocktails AB-SA01, against S. aureus, and AB-PA01, against P. aeruginosa, both of which are made to current good manufacturing practice (cGMP) standard. This study sought to investigate the lytic efficacy of AB-SA01 and its three component phages against S. aureus, and AB-PA01 and its four component phages against P. aeruginosa clinical isolates. Because wound infections are often polymicrobial and contain bacteria in biofilm form, this work sought to examine the efficacy of phages in such a setting.

Findings in chapters 2 and 3 indicate that phage cocktails AB-SA01 and AB-PA01 and their components are strongly lytic on most clinical isolates in planktonic and biofilms states. Moreover, these phage cocktails produced significant biomass reduction in single-species biofilms similar or superior to antibiotics used as a positive control. The efficacy and host range of phage cocktails AB-SA01 and AB-PA01 and their components suggest the enormous potential of phages in the treatment of antibiotic-resistant S. aureus and P. aeruginosa infections.

Real-time monitoring using spectrophotometry, supported by the colony count method, demonstrates that the fluorescence and population density of mCherry S. aureus KUB7 and GFP P. aeruginosa PAO1 significantly decreased when treated by their phage cocktail alone and the mixture of the two phage cocktails, similar to gentamicin treatment, as detailed in chapter 4. The findings in chapter 4 show that AB-SA01 and AB-PA01 effectively lysed their hosts in the presence of non-susceptible bacteria, both in planktonic and biofilm states. Moreover, the findings demonstrate that mCherry- and GFP-based mixed-species microplate assay using spectrophotometry combines reproducibility, rapidity, ease of management, and compatibility with high-throughput screening.

The results of multidrug-resistant S. aureus infected diabetic mice wound treatment show that AB-SA01 effectively decreased the bacterial load and significantly improved wound healing, similar to vancomycin treatment. In contrast, wounds of saline-treated mice showed no healing, but expanded and became inflamed, ulcerated, and suppurating. No adverse effect related to the application of the phage cocktail was observed. Hence, the results suggest that topical phage cocktail treatment may be useful in treating antibiotic-resistant S. aureus infections.

Overall, this thesis shows that the lytic efficacy, broad host range, and significant biofilm biomass reduction capability, in single-species and mixed-species biofilms, of the phages, makes them suitable candidates for therapeutic use in the DFU setting. Further investigation is required to evaluate the clinical application and off-target effect of phages and phage resistance development.

Keywords: Antibiotic-resistant infections, Bacterial load, Bacteriophage, Diabetic foot ulcer, Mice skin wound, Mixed-species biofilm, Mixed-species infection, Pseudomonas aeruginosa, Resistance, Staphylococcus aureus, Susceptibility, Wound closure

Subject: Microbiology & Infectious Diseases thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Science and Engineering
Supervisor: Associate Professor Peter Speck