Abstract
Aim
Staphylococcus aureus and Pseudomonas aeruginosa are ubiquitous pathogens often found together in polymicrobial, biofilm-associated infections. The mixed-species biofilm are significantly more resistant to antimicrobial treatment and are associated with failures. Bacteriophages present a promising alternative to treat biofilm-related infections due to their rapid bactericidal activity on multi-drug resistant bacteria. In this study, we assess the simultaneous or sequential application of phages and ciprofloxacin on the mixed-species biofilm in vitro.
Method
Ciprofloxacin was tested alone and in combination with Pyo-bacteriophage cocktail against P.aeurginosa ATCC 27853 and MRSA ATCC 43300 mixed-species biofilm. In order to evaluate the effect of combined treatment on biofilm-embedded cells, mature biofilms were grown on porous glass beads with MRSA (106 CFU/ml) and P.aeruginosa (103 CFU/ml) and incubated for 24h at 37° C in LB broth. The beads were then washed and placed in fresh LB in the presence of sub-eradicating titers/concentrations of phages and ciprofloxacin (corresponding to 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 × MBECbiofilm), respectively, simultaneous or in order (pretreated with phages for 3-6-12-24 hours) at 37°C. In all cases, heat flow produced by the viable cells still embedded in the biofilm was measured for 48 hours by isothermal microcalorimetry
Results
Simultaneous or sequential treatment with pyo-bacteriophage (105 and 106 PFU/ml) and ciprofloxacin, producing a synergistic effect resulting in the complete eradication of the biofilm was evaluated. When sub-eradicating concentrations of ciprofloxacin together with sub-eradicating titers of phages simultaneously used to treat mixed-species biofilm, a delay and/or reduction of heat flow produced by bacteria was observed. The same effect was seen when mix-biofilm was pre-treated with phages for 3 hours and 24 hours, respectively. However, antibiotic introduction after 6 and 12 hours resulted in a high synergistic eradicating effect with pyo-bacteriophage. The concentration of ciprofloxacin decreased dramatically from >512 μg/ml to < 16 μg/ml.
Conclusions
While MBEC of ciprofloxacin against mixed-species biofilm of Pseudomonas aeruginosa and Staphylococcus aureus was above drug concentrations reachable in clinical practice, the co-administration with bacteriophage strongly reduced the antibiotic doses needed to eradicate biofilm. There is a specific time delay in antibiotic introduction to reach the eradication of mix-species biofilm. These results have implications for optimal combined treatment approaches.
