Exploring the Benefits of Metal Ions in Phage Cocktail for the Treatment of Methicillin-Resistant Staphylococcus aureus

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an important zoonotic pathogen worldwide. Infections due to MRSA are associated with higher mortality rates compared with methicillin-susceptible S. aureus. Meanwhile, bacteriophages have been shown to overcome the emergence of MRSA. Methods: Phage PHB22a, PHB25a, PHB38a, and PHB40a were isolated. Here, we evaluated the ability of a phage cocktail containing phages PHB22a, PHB25a, PHB38a, and PHB40a against MRSA S-18 strain in vivo and in vitro. Phage whole- genome sequencing, host-range determination, lytic activity, and biofilm clearance experiments were performed in vitro. Galleria mellonella larvae and a mouse systemic infection model to evaluate the efficacy of phage therapy in vivo. Results: The phage cocktail exhibited enhanced antibacterial and anti-biofilm effects compared to the single phage. Phage cocktail contained with [Ca.sup.2+]/[Zn.sup.2+] significantly reduced the number of viable bacteria (24-h or 48-h biofilm) by more than 0.81-log compared to the phage cocktail alone. Furthermore, we demonstrated that the addition of [Ca.sup.2+] and [Zn.sup.2+] phage cocktail could increase the survival rate of G. mellonella larvae infected with S. aureus by 10% compared with phage cocktail alone. This was further confirmed in the mouse model, which showed a 2.64-log reduction of host bacteria S-18, when [Ca.sup.2+] and [Zn.sup.2+] were included in the cocktail compared with the phage cocktail alone. Conclusion: Our results indicated that phage cocktail supplemented with [Ca.sup.2+]/[Zn.sup.2+] could effectively remove bacteria in biofilms and mice tissues infected with S. aureus. Keywords: methicillin-resistant Staphylococcus aureus, phage cocktail, metal ions, biofilm, phage therapy