BLT Stages

Abstract bachelorproef 2016-2017: Development and optimization of a quantification method for cyclic lipopeptides produced by Pseudoalteromonas strains

This bachelor project will revolve around the development of a quantification method for cyclic lipopeptides (CLPs) produced by Pseudoalteromonas strains.

Antibacterial resistance is a growing issue in modern society. Therefore, there is a need for new sources of antibacterial products, and the search turned to other bacteria being potential producers of new antibiotics. Pseudoalteromonas is a marine bacterial genus notably found in bivalves such as oysters and mussels. These bacteria produce a large range of metabolites, such as CLPs. Through analysis and characterisation of CLPs, it was discovered that these lipopeptides possess antimicrobial properties. There is a need for a quantification method but since they are unusual natural products, no commercial standard is available.

First, several strains of Pseudoalteromonas were cultured in CD29. CD29 is a synthetic medium that provides optimal conditions for the production of CLPs and generates less interferences when separating or analysing. The strains produce several CLPs. They were separated with RP-HPLC-PDA after purification on a SPE cartridge. The next step was to analyse the collected fractions with RP-UPLC-MS (²) with three different methods: MS Scan, Single Ion Research and Multiple Reaction Monitoring. The cone and capillary voltages were optimized for the mono-charged CLP detection, and fragmentation patterns were obtained. After development, the method was tested on a strain cultured in a complex medium i.e. Marine Broth.

The optimization proved successful for detecting purified CLPs in CD29. The CLPs can be separated according to various mono-charged masses in SIR. MRM was not sensitive enough to detect the CLPs. Because the SIR method could not be applied to the Marine Broth medium due to interferences in the detection, further improvements need to be performed.

The method can be applied to purified samples for mono-charged masses, but the method needs to be assessed for di- or tri-charged ionic masses. It could prove useful to set parameters for each individual CLP to have a more specific detection for all three methods. SIR could be more sensitive and specific when set to a di-charged ionic mass. Higher collision energies could provide more specific fragments for each CLP in MRM.