Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group

Abstract

Enterotoxins produced by different species of the Bacillus cereus group, such as cytotoxin K1 (CytK1) and non-haemolytic enterotoxin (NHE), have been associated with diarrhoeal food poisoning incidents. Detection of CytK1 is not possible with commercial assays while NHE is recognised by an immunological kit (TECRA) that does not specifically target this protein because it is based on polyclonal antibodies. It is evident that the lack of suitable tools for the study of enterotoxins hampers the possibilities for accurate hazard identification and characterisation in microbial food safety risk assessment. We applied matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for the detection of CytK1 and NHE produced by pathogenic strains of the B. cereus group using protein digests from 1D gel electrophoresis. Secretion of CytK1 and two of the three components of NHE was confirmed in supernatants of different B. cereus cultures. For each protein, we introduce biomarkers that could be used for the screening of food poisoning or food/environmental isolates that can secrete enterotoxins. For example, tryptic peptides of 2,310.2 and 1,192.5 Da (calculated mass) can be indicators for CytK1 and NheA, respectively, although a simultaneous detection of other enterotoxin-specific peptides is recommended to assure the presence of a toxin in an unknown sample. Comparison of MALDI-TOF/MS with the TECRA kit showed that our methodological strategy performed well and it had the competitive advantage of specifically detecting NheA. Therefore, MALDI-TOF/MS can be successfully incorporated into risk assessment procedures in order to determine the involvement of strains of the B. cereus group in foodborne outbreaks, including the recently described cytK1 producing species, Bacillus cytotoxicus.

Advanced Mass Spectrometry

“…MALDI-TOF Detection of Listeria monocytogenes from selective enrichment broth [130] MALDI-TOF Identification and differentiation of foodrelated bacteria [131] MALDI-TOF Identification of lactobacilli isolated [132] MALDI-TOF Detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group [134] UHPLC-QqTOF Analysis on crude and wine-processed Siegesbeckia pubescens [135] MALDI-TOF Optimization of experimental and modeling parameters for the differentiation of beverage spoiling yeasts [136] MALDI-TOF Identification and differentiation of brewery isolates of Pectinatus sp.…”

Mass spectrometry in food proteomics: a tutorial

“…As an example, cytotoxin K1 (CytK1) and nonhemolytic enterotoxin (NHE) are two toxic compounds produced by pathogenic strains of the B. cereus group, for which no commercial assays (CytK1) or specific immonulogical kits (NHE) are available. In these cases, alternative tools, such as MS, for their detection and identification are highly desirable …”

Emerging Instrumental Methods for Antimicrobial Resistance and Virulence Testing

“…An MS-based assay for the quantification of S. aureus enterotoxin A (SEA) in serum has been developed [47]. MALDI TOF MS has been applied for detection of two enterotoxins-cytotoxin K1 (CytK1) and nonhaemolytic enterotoxin (NHE)-produced by species of the Bacillus cereus group [51]. After spiking the sample with full-length isotopically labeled SEA as an internal standard, immunoaffinity-based sample preparation is combined with tandem MS selected reaction monitoring of SEA-specific tryptic peptides to achieve 352 pg/mL limit of detection.…”