Phage Therapy Research Project

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for 25-30% of all S. aureus infections in hospital settings and is largely untreatable due to multi-drug resistance to all penicillin’s and most β-lactams, the most prescribed antibiotic (Lakhundi & Zhang, 2018). Livestock-associated MRSA (LA-MRSA) strains are a result of overuse and misuse of antibiotics in agriculture and human contact. In 2004 the first recorded case of LA-MRSA was found in a 6-month-old child and her pig farmer parents (Bakker, Klaassen, Loeffen, Voss, & Wulf, 2005). The same strain colonised the family and a pig from the farm demonstrating that MRSA can be transmitted back and forth between human and animal which increases the number of multi-drug resistant S. aureus strains infecting humans.

In 1961 after one year of clinical use, epidemic strains of S. aureus evolved to become methicillin resistant (Jevons, 1961). Multi-drug resistance is constantly progressing with the first vancomycin-resistant S. aureus (VRSA) strain recorded in 2010 increasing to 52 strains today (Gould, 2010). Skin infections, osteomyelitis, meningitis, pneumonia, and abscesses are the most common hospital-associated MRSA (HA-MRSA) infections with a higher 30-day mortality rate of 25% compared to 11.4% in those with methicillin-susceptible S. aureus (MSSA) (Aratani et al). High global mortality rates of cancer patients with MRSA blood stream infections were also estimated. The findings were a 60-day mortality rate of 12% and a 6-month mortality rate of 43.2% (Dong, Li, Wang, Wang, and Zhuang, 2021).

2.2. Phage Therapy.

Increasing antibiotic resistance of multiple bacterial strains including MRSA calls for alternative treatments such as phage therapy. Phages are strain specific viruses that co-exist with the bacterial species they infect. They are the most abundant organism found naturally in the environment, animals, and the human body. S. aureus phages belong to the Siphoviride, Podoviridae and Myoviridae family which have an icosahedral capsid enclosing double stranded DNA and a non-contractile tail which attaches to the wall teichoic acid (WTA) on the bacterial host cell triggering phage DNA injection (Wolz & Xia, 2014). Phage genes encode proteins promoting survival such as integrase for recombination of phage and bacterial DNA and terminase to initiate phage DNA packaging. Other proteins act against the host cell such as endolysin which degrades peptidoglycan, lysing the cell and releasing replicated phages whereas, DnaI-binding protein and ORF240 inhibit host transcription, leading to cell death (Wolz & Xia, 2014).

Links have been found between prophage induction and disease. The gut mucosa is reported to have less Caudovirales phages in healthy patients than those with ulcerative colitis (Cao et al., 2019). The same was observed as far back as 1983 by Furuse et al. with 2.5% virulent phages in faecal samples of healthy individuals compared to 13-20% in those with leukaemia or respiratory diseases. This suggests a protective role within the body, regulating bacterial population and immune response according to a study by He et al. (2018) into S. aureus phage JS25 which may bind inflammatory mediators inhibiting release of proinflammatory cytokines.

Phage strains can be in combination, individually, or alongside antibiotics which not only reduces the risk of inducing resistance but may also re-sensitise pathogens to antibiotics. They have a narrow spectrum of activity and non-toxic effect on eukaryotic cells unlike antibiotics. A study by Biswas et al., reported an antibiotic dose 1000 times higher is needed to eradicate MRSA biofilms around prosthetic joints than is needed to treat MSSA infections. This would result in high levels of toxicity and secondary infections such as Clostridium difficile from microbiome disruption. Whereas a small phage dose can be administered as phages auto dose, with up to 100 phages replicating from one host cell. Phages lyse bacterial cells eradicating the infection but also their host for replication therefore both populations decrease.

2.3. Phage Bank.

The Citizen Phage Library (CPL) is an ongoing project aimed at collecting phages to increase accessibility to phages capable of being developed into therapies to treat multi-drug resistant pathogens (Citizen Phage Library, 2023). The patient from the Biswas et al. study received phage therapy to penetrate the MRSA biofilm around the prosthetic joint. A phage bank similar to the CPL was used to find a compatible phage by sequencing an S. aureus isolate from the patient and screening it against the library. Compatible phages were then introduced in vitro to the patients isolate and phage with highest inhibition of cellular respiration was selected for amplification, purification, therapy development and quality control testing. Successful phage treatment was also recorded by Brownstein, Fackler, Horne, Rodriguez, and Woodworth (2022). A patient with MRSA rhinosinusitis resistant to 5 antibiotics including the most effective and commonly prescribed vancomycin experienced toxic side effects after 2 years of unsuccessful antibiotic treatment. Phage therapy was administered on 2 occasions for recurring infection resulting in negative cultures after 2 weeks and 1 week.