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

## TL;DR

This study shows that testing phage-antibiotic combinations on planktonic bacteria can predict antagonistic effects on biofilm-encased bacteria, but not synergistic ones.

## Contribution

The study introduces a novel screening method using planktonic bacteria to predict PAC efficacy on biofilms.

## Key findings

- Testing planktonic bacteria predicted antagonistic effects on biofilms with high accuracy (AUC = 0.95).
- Synergistic effects on biofilms were not reliably predicted from planktonic tests (AUC = 0.30).
- Multiple daily exposures to PAC were needed to achieve maximal biofilm reduction.

## Abstract

Phage-antibiotic combinations (PAC) may be effective in eradicating clinical, mostly biofilm-associated, infections. As the efficacy of PAC largely depends on the phage and 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.

Methicillin-sensitive Staphylococcus aureus (MSSA, clinical isolate) in mid-logarithmic phase was exposed to (sub)optimal doses of phage ISP combined with 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 with PAC compared to the most effective single agent (phage or antibiotic) 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.

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 (area under the curve (AUC) = 0.95), but did not predict synergistic effects (AUC = 0.30) after 3 days of exposure to the respective phage-antibiotic concentrations.

Together, PAC testing on planktonic bacteria provides a valuable first-line screening tool for clinicians treating biofilm-associated infections.