Aim

Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella.

Periprosthetic joint infection (PJI) remains one of the most devastating complications that can occur following total joint arthroplasty. Failure rate of standard treatment for PJI is estimated to be around 40% at two years post revision surgery. A major clinical challenge contributing to treatment failure and antibiotics tolerance is the biofilm formation on implant surfaces. Lytic bacteriophages (phages) can target biofilm associated bacteria at localized sites of infection by penetrating and disrupting biofilm matrices; furthermore, phage replication within the biofilm leads to high local concentrations resulting in a powerful therapeutic effect. The aim of this study is to test if phage cocktail has better antimicrobial effect than vancomycin or a single agent phage against biofilm forming MRSA clinical strain Staphylococcus aureus (S. aureus). S. aureus BP043 was utilized in this study. This strain is a PJI clinical isolate, methicillin resistant (MRSA) and biofilm-former. Three lytic phages, namely, 44AHJD, Team1 and P68, known to infect S. aureus, were tested for their efficiency against S. aureus BP043. The ability of the phages to eliminate S. aureus BP043 planktonic or biofilm cultures was tested either as singular phages or as a cocktail of the three phages. Planktonic cells were adjusted to ∼ 1×109 CFU/mL in tryptic soy broth (TSB) and each phage was added alone or as a cocktail at ∼ 1×109 PFU/mL with moi of 1 (a multiplicity of infection). Bacterial growth was assessed by measuring optical densities at 24hr and was compared to the control of S. aureus BP043 with no phage. BP043 biofilms was grown for 24hr on plasma sprayed titanium (Ti-6Al-4V) alloy disc surfaces. Mature biofilms were then treated with one of the three phages or a cocktail of the 3 phages for 24hr at ∼ 1×109 PFU/mL in TSB. Then, biofilms were dislodged, and bacterial survival was assessed by plating on tryptic soy agar plates. Survival in treated biofilms was compared to control biofilm that was exposed only to TSB. Planktonic cells growth in the presence of phage 44AHJD was reduced significantly (p <0.0001) after 24hr compared to the control. The other two phages did not show a similar pattern when used alone. The reduction in growth was more pronounced when the three phages were combined together (p <0.0001, compared to the control, p=0.011 3, 44AHJD alone versus 3 phages). Exposing BP043 biofilm to the phage cocktail resulted in more than three logs (CFU/mL) reduction in bacterial load residing in the biofilm while no effect was detected when either vancomycin or each phage was used solely. We have demonstrated that the usage of lytic phage cocktail contributes to better clearance of planktonic cultures of the S. aureus MRSA isolate. More importantly, viable bacteria in the biofilms that were grown on plasma sprayed titanium discs were reduced by more than 37% when a phage cocktail was used compared to using a single phage or vancomycin. This work is aimed at gathering preclinical evidence for using phage as a new therapeutic avenue to treat PJI