Detection and quantification of Enterococcus faecalis RNPP-type quorum sensing peptides in bacterial culture media by UHPLC-MS.

ABSTRACT

Bacteria communicate with each other using quorum sensing; i.e. the production and sensing of signalling molecules. Enterococcus faecalis, a Gram-positive bacterium, employs peptides as quorum sensing molecules. These peptides have previously been isolated from culture media by elaborate, time and medium-consuming sample preparation approaches and specific bacteria-based bio-sensors. Here, a method for the detection and quantification of all nine currently reported E. faecalis quorum sensing peptides belonging to the RNPP family in bacterial cell culture medium was developed. The approach developed consists of solid-phase extraction (SPE) sample preparation followed by a UHPLC-triple quadrupole mass spectroscopic method, operated in Multiple Reaction Monitoring (MRM) mode. All nine peptides were quantified with a total analysis time below 90 min per sample and limited cell culture medium volumes of only 1 ml per sample. A method verification, performed in uniplicate, was carried out to obtain an idea of the method performance. The recovery varied between 19.9 and 119.0%, and the limit of detection is in the low nM range. Analytical stability, carry-over and dilution integrity were investigated and were acceptable. This method will be a useful tool in the investigation of the roles of the RNPP-type quorum sensing peptides in microbial processes.