Detection and partial characterization of extracellular inducers of persistence in Staphylococcus epidermidis and Staphylococcus aureus

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci. Hypothesis/Gap Statement. Increases in persisters during mid log phase growth indicate that quorum-sensing factors might be produced by staphylococci. Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000. Methodology. Others have demonstrated a significant increase in persister numbers during mid log phase. Inducers of this mid log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD(600)s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications. Results. The optimal OD(600)s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis' PIF activity was decreased by storage at 4 degrees C but not at 20 degrees C (16 h), 37 degrees C (1 h) or 100 degrees C (15 min). S. aureus' PIF activity was decreased following storage at 4 degrees C (2 weeks) and after boiling at 100 degrees C for 5 min but not after incubation at 37 degrees C (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis. PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells. Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600-based PIF assay.

Automated summary

This study aimed to identify and partially characterize persistence-inducing factors (PIFs) from staphylococci by measuring optical density to determine when persister numbers increased. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were used to develop a PIF assay, revealing that CCFs from both species could induce persistence.