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iGEM HKUST 2023 - Cereulide Test Kit

Inspirations

      During the ideation stage of our concept, we came upon "fried rice syndrome." Food poisoning associated with eating fried rice is referred to as "fried rice syndrome" in layman's terms. Fried rice is typically made with pre-cooked or leftover rice. Inadequate cooking or storage methods significantly increase the danger of bacterial contamination, specifically by Bacillus cereus (B. cereus), which causes diarrheal or emetic symptoms when consumed.

      Fried rice is a popular dish in Hong Kong cuisine, and B. cereus contamination is a major public health risk. As a local team based in Hong Kong, we are committed to using synthetic biology to solve the local problem of rice contamination.

Background

Cereulide Poisoning cases

      Cereulide poisoning is a dangerous and potentially fatal foodborne illness caused by the bacteria B. cereus. The following cases highlight the importance of proper food handling and storage to prevent food poisoning, as well as the need for accurate, early diagnosis and treatment in cases of suspected food poisoning caused by B. cereus.

      Three cases, one happened in 2003, two happened 2008, are prominent cases of B. cereus poisoning. The youngest child in the 2003 case died barely 13 hours after consuming B. cereus-contaminated food in Leuven[1]. In 2008, a 20-year-old man died one day after consuming B. cereus-contaminated spaghetti that was five days old [2]. In another instance in 2008, three family members felt unwell after eating reheated fried rice, and the one-year-old brother died six hours later of severe encephalopathy. The two-year-old sister recovered after plasma exchange and hemodialysis, and their mother recovered with fluid treatment. B. cereus was isolated from leftover fried rice as well as the children's stomach contents, and serum cereulide was found in both of them[3]. According to post-mortem examination results, they died due to severe symptoms such as vascular congestion of the lungs, microvascular and extensive coagulation necrosis.

      Between 1973 and 1985, B. cereus was responsible for 17.8% of all bacterial food poisoning incidents in Finland and 11.5% in The Netherlands, suggesting its widespread distribution and capacity to cause foodborne illness[4]. B. cereus has remained a source of concern in recent years, ranking sixth among bacterial etiological agents responsible for documented foodborne outbreaks from 2009 to 2015 in the United States[5]. The seriousness of cereulide poisoning emphasizes the need for low-cost cereulide testing methods and heightened awareness among consumers and food workers.

Total rice consumption worldwide
Figure 1 Total rice consumption worldwide from 2008/2009 to 2022/2023[6]

Cereulide Toxicity Threshold To Human

      Cereulight test kit aims to detect food with unsafe level of cereulide for human consumption. Therefore, it is necessary to formulate an equation to quantify the toxicity level in food samples.

      Researchers tried to find the concentration of cereulide in food samples when food poisoning cases happened. A 2003 paper points out that poisonous food contained approximately 1.6ug/g of cereulide, implying the toxic dose in humans as 8 ug/kg body weight[7]. We decided to use this set of data in later calculations and experiments.

      To quantify the cereulide toxicity level of food samples to humans, we assume that cereulide is equally distributed in the rice and there is a linear relationship between T to r and m, where T is the toxicity level, r is the weight of rice samples, and m is the average body weight. In the linear relationship, T and m are directly proportional while T and r are inversely proportional:

\[T = k{m \over r}\]

      By substituting the value from the paper,

\[k = T{r \over m}\]

\[k = {1.6µg/g} \times {300g/60kg} = 8µg/kg\]

      We have the equation:

\[T = 8µg/kg \times {m \over r}\]

      Using this equation, we are able to calculate the cereulide concentration that leads to food poisoning when the weight of rice samples and the body weight of consumers vary.

Stakeholders

      The viewpoints of various stakeholders can offer insight into current food safety challenges as well as the scope of the problem we are addressing. By connecting a variety of stakeholders, such as government regulatory bodies, food suppliers, food quality auditors, restaurants, and NGOs, we can acquire more market insights and learn how to make our project relevant and feasible on technical grounds.

Problems

Temperature control to restrict the growth of Bacillus cereus
Figure 2 Temperature control to restrict the growth of Bacillus cereus[8]

      Improper food preparation and storage, such as leaving food at room temperature for an extended period of time, can allow B. cereus to grow and produce cereulide.

      Cereulide, frequently associated with rice or other starch-rich foods like pasta and potatoes, is an ionophore toxin generated by B. cereus that can cause emetic syndrome. Nausea, vomiting, and stomach cramps are symptoms of cereulide food poisoning. Because the toxin is created during bacterial growth in food, it is difficult to detect during processing and manufacturing[9]. B. cereus endospores in food can germinate quickly and create cereulide, especially when chilled or heated[10]. B. cereus spores are extremely resistant to cooking temperatures, making contamination prevention challenging. As a result, early identification of cereulide concentrations in food is critical for preventing food poisoning and guaranteeing food safety, particularly for heat-stable toxins such as cereulide.

Current methods

      Currently, there are two main methods to detect cereulide, fluorescent probes[11] and conventional high-performance liquid chromatography (HPLC) connected to a tandem mass spectrometer (LC-MS/MS)[12].

  • Fluorescent probes: Cereulide complexes with potassium ions, resulting in a decrease in JG76 probe fluorescence, which can be evaluated using fluorescent probe tests. This test is already the most convenient and portable method for detecting cereulide in food extracts. However, fluorescence titration of the sample and a benchtop fluorometer are required, making the test only suitable for use in laboratories by professionals.
  • LC-MS/MS: The sensitivity and specificity of LC-MS/MS are well recognized. However, the LC-MS/MS detection method is quite expensive, typically between $100 and $200 per sample, in terms of machine acquisition, daily operation, and routine maintenance. Preparation work that takes time is also required. This procedure is not suitable for users who do not have access to a well-equipped laboratory or who prefer to conduct testing outside of a laboratory setting.

    • Solution

          We intend to create a user-friendly whole-cell biosensor platform capable of detecting the presence of cereulide in food samples, making it better suited for usage in food processing and manufacturing facilities, as well as food safety authorities.

          This biosensor platform is based on genetically modified Bacillus subtilis. The lab-on-a-chip device integrates several laboratory operations, including cell immobilization, cell culturing, and cell germination on a single device. When harmful quantities of cereulide are identified, the biosensor emits a red signal, whereas a green signal shows safe levels of the toxin. Our goal is to assure food safety and quality, which will benefit both end users and the food sector as a whole.

    Figure 3 Whole-cell biosensor in a lab-on-a-chip device[13]

          Our goal is to assure food safety by enabling food establishments to take immediate action to prevent the distribution of contaminated products, and by increasing inspection frequency. Our test kit would enable users for primary food safety testing a few times a week or even daily if the risk of emetic toxin is high based on risk estimation. Moreover, the routine Food Surveillance Programme in Hong Kong follows the Microbiological Guidelines for Food, which regulate the B. cereus level, but it does not include specific detection of cereulide.[14] By introducing cereulide detection, we can add another layer of protection to ensure food safety and prevent contaminated products from being distributed. Additionally, our project has the vision of raising public awareness about the risks of emetic toxin food poisoning and promoting safe food handling practices to prevent foodborne illnesses.

    References

      [1] Dierick, K., Van Coillie, E., Swiecicka, I., Meyfroidt, G., Devlieger, H., Meulemans, A., Hoedemaekers, G., Fourie, L., Heyndrickx, M., & Mahillon, J. (2005). Fatal family outbreak of Bacillus cereus-associated food poisoning. Journal of clinical microbiology, 43(8), 4277–4279. https://doi.org/10.1128/JCM.43.8.4277-4279.2005
      [2] Naranjo, M., Denayer, S., Botteldoorn, N., Delbrassinne, L., Veys, J., Waegenaere, J., Sirtaine, N., Driesen, R. B., Sipido, K. R., Mahillon, J., & Dierick, K. (2011). Sudden Death of a Young Adult Associated with Bacillus cereus Food Poisoning. Journal of Clinical Microbiology, 49(12), 4379-4381. https://doi.org/10.1128/JCM.05129-11
      [3] Shiota, M., Saitou, K., Mizumoto, H., Matsusaka, M., Agata, N., Nakayama, M., ... & Hata, D. (2010). Rapid detoxification of cereulide in Bacillus cereus food poisoning. Pediatrics, 125(4), e951-e955.https://doi.org/10.1542/peds.2009-2319
      [4] Kotiranta, A., Lounatmaa, K., & Haapasalo, M. (2000). Epidemiology and pathogenesis of Bacillus cereus infections. Microbes and Infection, 2(2), 189-198. https://doi.org/10.1016/S1286-4579(00)00269-0
      [5] Dewey-Mattia, D., Manikonda, K., Hall, A. J., Wise, M. E., & Crowe, S. J. (2018). Surveillance for Foodborne Disease Outbreaks — United States, 2009–2015. MMWR Surveillance Summaries, 67(10), 1-11. https://doi.org/10.15585/mmwr.ss6710a1
      [6] Shahbandeh, M. (2023). Total global rice consumption 2008/09-2022/23. photograph. https://www.statista.com/statistics/255977/total-global-rice-consumption/
      [7] E L Jääskeläinen, V Teplova, M A Andersson, L C Andersson, P Tammela, M C Andersson, T I Pirhonen, N-E L Saris, P Vuorela, M S Salkinoja-Salonen. (2003). In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus. https://pubmed.ncbi.nlm.nih.gov/14599471/
      [8] CHONG. (2014). Food Safety Focus (97th Issue, August 2014) – Incident in Focus Bacillus cereus in Processed Food. Centre for Food Safety. https://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_97_01.html
      [9] Agata, N., Ohta, M., & Yokoyama, K. (2002). Production of Bacillus cereus emetic toxin (cereulide) in various foods. International journal of food microbiology, 73(1), 23–27. https://doi.org/10.1016/s0168-1605(01)00692-4
      [10] Kranzler, M., Stollewerk, K., Rouzeau-Szynalski, K., Blayo, L., Sulyok, M., & Ehling-Schulz, M. (2016). Temperature exerts control of Bacillus cereus emetic toxin production on post-transcriptional levels. Frontiers in Microbiology, 7, 1640.https://doi.org/10.3389/fmicb.2016.01640
      [11] García-Calvo, J., Ibeas, S., Antón-García, E. C., Torroba, T., González-Aguilar, G., Antunes, W., González-Lavado, E., & Fanarraga, M. L. (2017). Potassium-Ion-Selective Fluorescent Sensors To Detect Cereulide, the Emetic Toxin of B. cereus, in Food Samples and HeLa Cells. ChemistryOpen, 6(4), 562–570. https://doi.org/10.1002/open.201700057
      [12] International Organization for Standardization. (2017). Microbiology of the food chain — Quantitative determination of emetic toxin (cereulide) using LC-MS/MS (ISO Standard No. 18465:2017). https://www.iso.org/obp/ui/#iso:std:iso:18465:ed-1:v1:en
      [13] Schulz-Schönhagen, K. (2019). Bacillus subtilis biosensors: Engineering a living material sensor platform (Doctoral dissertation, ETH Zurich). https://doi.org/10.3929/ethz-b-000389191
      [14] HKSAR Government . (2022, May 3). CFS finds excessive Bacillus cereus in fried rice sample. The Government of Hong Kong Special Administrative Region Press Release . Retrieved June 29, 2023, from https://www.info.gov.hk/gia/general/202205/03/P2022050300730.htm