A biofilm is a protection mechanism allowing bacteria to function as a single unit and conserve resources. Bacteria can break off from an established biofilm and then form more biofilm elsewhere. To initiate biofilm formation, bacteria emit different chemicals and this process is called quorum sensing (QS). Controlling or preventing biofilms is of significant importance in numerous industries as well as in healthcare. Quantitative measurement of quorum sensing chemicals is challenging due to their low concentration and varying chemical properties. Gram-negative bacteria communicate using variable chain length acyl-homoserine lactones (AHLs) and acyl-oxo-homoserine lactones (AOHLs). Microdialysis is a diffusion-based sampling technique that allows collection of unbound solutes. Collected dialysates can be quantified without the need of additional sample clean-up. In this work, microdialysis sampling technique was developed as a model to monitor QS chemicals from bacterial biofilms. The bacterial strain <i>Vibrio harveyi</i> was cultured in marine broth and incubated for 4 days at 30 °C. Microdialysis sampling was performed using CMA 20 probes with a 100 kDa molecular weight cutoff and 10 mm membrane. Biofilm formation on the probe membrane was confirmed using 0.1% safranin (figure 1). Dialysates from the planktonic bacterial broth were obtained at 0.5 µL/min for 48 hours using marine broth as a perfusion fluid. AHLs were extracted using a Bond-Elut PPL cartridge and quantified using LC-ESI-MS/MS in positive ion mode (figure 2). A wide variety of AHLs and AOHLs such as C4-AHLs, C6-AHLs, C8AHLs, C15-AHLs, C6-OXO-AHLs, C12-OXO-AHLs and C14-OXO-AHLs were possible to collect and quantify using microdialysis and LC-ESI-MS/MS. Moreover, the extraction efficiency of AHLs and AOHLs was determined to be in the range from 2-20 % and concentration around 600 ppb. Future work will focus on quantifying peptide-based QS chemicals from Gram-positive bacteria.

Microdialysis sampling of quorum sensing during biofilm formation

255th National Meeting (2018)

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