Abstract
In recent years, the emetic toxin cereulide, produced by Bacillus cereus, has gained high relevance in food production and food safety. Cereulide is synthesized non-ribosomal by the multi-enzyme complex Ces-NRPS, which is encoded on a megaplasmid that shares its backbone with the Bacillus anthracis pX01 toxin plasmid. Due to its resistance against heat, proteolysis and extreme pH conditions, the formation of this highly potent depsipeptide toxin is of serious concern in food processing procedures including slow cooling procedures and/or storage of intermediate products at ambient temperatures. So far, systematic data on the effect of extrinsic factors on cereulide synthesis has been lacking. Thus, we investigated the influence of temperature, a central extrinsic parameter in food processing, on the regulation of cereulide synthesis on transcriptional, translational and post-translational levels over the growth temperature range of emetic B. cereus. Bacteria were grown in 3°C interval steps from 12 to 46°C and cereulide synthesis was followed from ces gene transcription to cereulide toxin production. This systematic study revealed that temperature is a cardinal parameter, which primarily impacts cereulide synthesis on post-transcriptional levels, thereby altering the composition of cereulide isoforms. Our work also highlights that the risk of cereulide production could not be predicted from growth parameters or sole cell numbers. Furthermore, for the first time we could show that the formation of the recently identified cereulide isoforms is highly temperature dependent, which may have great importance in terms of food safety and predictive microbiology. Notably the production of isocereulide A, which is about 10-fold more cytotoxic than cereulide, was specifically supported at low temperatures.
Highlights
The incidence of foodborne intoxications caused by bacterial toxins has been steadily increasing over the last decade
Four emetic B. cereus strains were used: two wellcharacterized strains isolated in frame of food borne outbreaks, the emetic reference strain F4810/72, isolated from vomit and the strain F5881, isolated from Chinese takeaway fried rice, and, further two strains from food industrial environments, one strain isolated from wheat flour (B626) and one strain isolated from dehydrated puree with onions (AC01)
The influence of temperature on cereulide synthesis of B. cereus grown in LB medium was investigated photometrically at a wavelength of 600 nm (OD600)
Summary
The incidence of foodborne intoxications caused by bacterial toxins has been steadily increasing over the last decade. The number of reported food poisonings related to Bacillus cereus toxins has shown a steep increase from 2006 onward (Anonymous, 2009, 2015). Temperature Controls Synthesis of Cereulide related to the emetic B. cereus toxin cereulide, requiring hospitalization or even leading to death (Dierick et al, 2005; Posfay-Barbe et al, 2008; Ichikawa et al, 2010; Naranjo et al, 2011; Ehling-Schulz and Messelhäusser, 2012). Because of the high toxicity of cereulide and the high incidence rates up to 100%, usually observed in connection with outbreaks, accurate data on contamination sources and factors promoting toxin formation are urgently needed to prevent contamination and toxin production in food production processes. Toxin formation is of serious concern in food processing procedures that include slow cooling procedures and/or storage of intermediate products at ambient temperatures. Contaminations with cereulide or cereulide-producing bacteria have been reported from infant foods in Finland, various food products in Belgium and Bavaria as well as from ice creams in Germany (Shaheen et al, 2006; Rajkovic et al, 2007; Messelhäusser et al, 2010, 2014)
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