Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography

The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA–MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA–MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method’s suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography–mass spectrometry (UPLC–MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide.

1. Strike bacterial strains on LB agar plates. Incubate plates overnight at 30 °C.
2. Incubate bacterial pre-cultures in 3 mL of LB broth at 30 °C for 16 to 18 h at 120 rpm.
3. Inoculate fresh cultures with a final inoculum of 10 3 CFU/mL in 100 mL of LB broth and incubate cultures at 120 rpm for 24 h at 30 °C, as described previously [1]. 4. Harvest cells by centrifugation at 8600 × g for 12 min at room temperature and discard supernatant. 5. Freeze pellets in liquid nitrogen for storage at −80 °C.
2. For each strain, weigh 1 g (wet weight) of cell material into a 50 mL tube.
3. Add 10 mL of ethanol absolute and resuspend the pellet by gently shaking or pipetting.
4. Extract overnight at room temperature on a rocking table. The screw cap should be covered by parafilm to avoid leakage. The samples should also be covered with aluminum foil to protect them from light.
5. On the next day, centrifuge the suspension for 12 min at 8600 × g at room temperature.
6. Filtrate the extracts to remove any remaining cell debris by using a syringe and PTFE filters (pore size of 0.2 μm). Aliquot the extracts to 2 mL for each strain and store them at −20 °C until use.
7. Concentrate extracts to 200 μL using a concentrator at 45 °C for about 1 h. Pool extracts from each strain into a single tube with a final volume of 1 mL.

Materials:
 Äkta™ pure 25M (GE Healthcare, Solingen, Germany) with UV monitor U9-M for triple wavelengths detection  Screw neck vials N9, 1.5 mL, 11.6 × 32 mm with N9 PP screw caps with red rubber (Machery -Nagel) Note: Prepare all solutions in advance. All solutions should be degassed in an ultrasonic bath before use. Make sure the Äkta™ pure 25M system is set-up according to the manufacturer's instructions (GE Healthcare). In this method, all percentage values of ethanol and acetonitrile refer to percentage of volume (% vol).
Procedure:  Pre-fill the 5 mL sample loop of the Äkta™ pure 25M system with 5 mL of the sample.
 Due to the highly hydrophobic character of cereulide, for the purification of cereulide a silica based C12 column (Jupiter ® 4 μm Proteo 90 Å, LC Column, size 250 * 4.6 mm (Phenomenex)) as reversed phase chromatography (RPC) and a pre-column SecurityGuard Cartridge (Phenomenex) as fast protein liquid chromatography (FPLC) system is recommended.

Method Settings:
 Reverse phase chromatography (RPC)  Inlet A1 and inlet B1. Unless stated otherwise, use the same inlets as mentioned in the method settings.
 UV absorption is simultaneously measured with the UV detector (UV monitor (U9-L), (GE Healthcare)) at 280 nm and 210 nm.
 Air sensor enables the air sensor alarm for the built-in air sensor at inlet A and B.
 Method based unit is column volumes (CV).
 To minimize gas formation in the system, use a flow restrictor.
 Set the flow rate to 0.5 mL/min (control the flow to avoid overpressure). Use always the same flow rate as mentioned in the method settings, if not stated otherwise.
 Column position is variable.
 Fractionation collector is only required for elution.
Note: In general PEEK tubing is ID 0.75 mm, OD 1/16ʺ (GE Healthcare). Change PEEK tubing from injection valve, column valve, column, UV monitor, and conductivity monitor to PEEK tubing of ID 0.25 mm OD 1/16ʺ (GE Healthcare) to build-up a higher pressure for cereulide purification.

Running conditions for toxin quantification:
 o Set flow rate to 0.5 mL/min and wash the column with 1 CV of 65% acetonitrile. Perform a linear gradient from 65% to 6.5% of acetonitrile within 2 CV.

 Equilibration step:
o Reseting of the UV monitor is recommended if the equilibration occurs before purification.
o Equilibrate column with 4 CV of 10% ethanol.

 Sample application:
o Apply sample directly to the column using the prefilled 5 mL capillary loop.