Abstract
Aim
Aim of this study was to evaluate the ability of Sb-1 to enhance the antibiotic activity (tested in combination) degrading the biofilm matrix (impairing the freely diffusion of antimicrobials) and specifically targeting “persister” cells (biofilm sub-population tolerant to most antibiotics and responsible for the infection recalcitrance) of methicillin-resistant Staphylococcus aureus.
Method
MRSA ATCC 43300 24h-old biofilm was treated for 18h with Sb-1 titers (from 104 to 106 pfu/ml). Biofilm matrix was evaluated by confocal laser scanning microscopy after staining with wheat germ agglutinin conjugate with Alexafluor488 (WGA488) to label exopolysaccharide matrix and Syto 85 to label bacterial cells. Persister status was induced using two different protocols: i) by exposing stationary phase S. aureus to 400 µg/ml carbonyl cyanide m-chlorophenylhydrazone (CCCP) in PBS for 3h at 37°C and ii) by treatment of 24h old biofilm with 512 µg/ml ciprofloxacin for further 24h at 37°C. Then, induced persister cells and non-induced controls (106 CFU/ml) were treated with 104 PFU/ml and 107 PFU/ml Sb-1 for 3h, followed by CFU counting. Alternatively, bacteria were washed and incubated in fresh BHI medium for the resumption of normal growth and the bacterial growth assessed after further 24 hours.
Results
Sb-1 showed a dose-dependent reduction of exopolysaccharide components of MRSA biofilm matrix at sub-inhibiting phage titers. With 106 PFU/ml Sb-1, no fluorescent signal related to WGA488 was detected, although bacterial viability was not impaired. Higher Sb-1 titer (107 PFU/ml) determined a strong reduction (ranging between 2.5 – 5 log CFU/ml) of persister cells. By contrast, in presence of 104 PFU/ml Sb-1, no reduction was observed in persister cells. However, persister cells pre-treated with 104 pfu/ml Sb-1 were completely killed when bacteria were inoculated after phage treatment in fresh medium, reverting to a normal-growing phenotype.
Conclusions
Due to its ability to degrade the MRSA exopolysaccharide matrix at sub-inhibitory concentrations and kill persister cells, directly at higher titers or indirectly with lower titers, Sb-1 phage is a valid therapeutic option to be used alone or in combination with current antibiotics for the successful eradication of methicillin resistant S. aureus biofilm associated with prosthetic joint infections.
