Efficacy prediction of bacteriophage-antimicrobial antibiotic combinations against Staphylococcus aureus biofilms using planktonic bacteria

Marielle Verheul^1*Elizabeth Schonkeren-Ravensbergen²Eva Bus²Mark De Boer²Rob Nelissen³Bart Pijls³Peter Nibbering²

• ¹Leiden Center for Infectious Diseases (LUCID); Department of Orthopedics, Leids Universitair Medisch Centrum, Leiden, Netherlands
• ²Leiden Center for Infectious Diseases (LUCID), Leids Universitair Medisch Centrum, Leiden, Netherlands
• ³Department of Orthopedics, Leids Universitair Medisch Centrum, Leiden, Netherlands

Introduction: Phage-antimicrobial antibiotic combinations (PAC) may be effective in eradicating clinical, mostly biofilm-associated, infections. As the efficacy of PAC largely depends on the phage and antimicrobial antibiotic used, such combinations should be screened before their clinical application. Unfortunately, testing the efficacy of PAC on mature biofilms is laborious. This study aimed to assess whether the effects of PAC on biofilm-encased bacteria can be predicted by testing their effects on planktonic counterparts. Methods: Methicillin-sensitive Staphylococcus aureus (MSSA, clinical isolate) in mid-logarithmic phase was exposed to (sub)optimal doses of phage ISP combined with antimicrobials antibiotics targeting transcription, protein translation, the cell wall, and the cell membrane. These experiments were followed by studies assessing the effects of up to three daily exposures to PAC on MSSA within seven-day mature biofilms on metal implant mimics. An additional 2-log reduction or an increase in bacterial counts by with PAC compared to the most effective single agent (phage or antimicrobialantibiotic) was considered as synergy or antagonism, respectively. Receiver operating characteristic (ROC) curves were used to calculate whether the effects of PAC on planktonic bacteria were comparable to the effects on biofilm-encased bacteria. Results: The results for planktonic bacteria showed antagonism between ISP and rifampicin or gentamicin, synergism between ISP and ciprofloxacin, clindamycin, or flucloxacillin, and lack of interaction between ISP and daptomycin. Similarly, ISP combined with rifampicin or gentamicin displayed antagonism on biofilm-encased MSSA, whereas ISP and ciprofloxacin, clindamycin, flucloxacillin, or high-dose daptomycin induced synergy. Notably, two to three consecutive daily exposures to PAC were necessary to reduce biofilm-encased MSSA maximally. Testing PAC on planktonic bacteria predicted antagonistic effects on biofilms (AUC area under the curve (AUC) = 0.95), but did not predict synergistic effects (AUC = 0.30) after three days of exposure to the respective phage-antimicrobial antibiotic concentrations. Discussion: Together, PAC testing on planktonic bacteria provides a valuable first-line screening tool for clinicians treating biofilm-associated infections.