Team:Nottingham/Description


Project

Description



How can we help food manufacturers improve their packaging and production processes, in order to prevent botulism outbreaks?

Project
Description

Project Aims

  • Use synthetic biology to develop a safe and reliable technology for monitoring Clostridium botulinum toxin production in food.
  • Reduce costs and risks associated with botulinum toxin testing during food packaging development trials.
  • Improve the accessibility of botulinum toxin testing for smaller food companies without the need to outsource to specialist laboratories.

Food-Borne Botulism

The toxin is produced by the anaerobic, spore-forming bacterium Clostridium botulinum (C. botulinum). It is the most potent toxin known to humankind. Indicatively:

  • 30 ng of orally ingested toxin is enough to kill a person.[1]
  • a single gram of purified toxin could potentially kill 1 million people.[2]

There are 7 distinct forms of botulinum toxin: Types A, B, E and rarely F cause human botulism, whereas types C, D and E cause illness in other mammals, birds and fish.[3]


Spores of C. botulinum are heat-resistant and widely ubiquitous in nature.[4] Under permissive conditions, they germinate, grow and elaborate toxins. Such conditions may be found when contaminated foods (e.g., foods with spore-containing raw ingredients) are packed anaerobically (e.g., in sealed plastic bags). Typically, lightly preserved, inadequately processed, home-canned or home-bottled food is associated with a higher risk of containing toxin.[1] Additionally, ready-to-eat food poses a big threat, given that pre-formed toxin would be consumed intact, having not had the chance to be destroyed by heating.


Outbreaks of botulism are rare but can result in high morbidity and mortality rates. Therefore, even a single case of foodborne botulism is sufficient to evoke a public health emergency, requiring rapid recognition and intervention. 197 outbreaks have been reported between 1920 and 2014 worldwide, with the majority of them (55%) occurring in the US.[5] According to the National Botulism Surveillance programme of the US Centres for Disease Control and Prevention (CDC), approximately 200 individual cases occur in the US each year and 10% are fatal.

Botulism Prevention in the Food Industry

Generally, food manufacturers adhere to stringent food processing and preservation guidelines thus negating the requirement for routine botulinum toxin testing. Sufficient heat-treatment, food acidity, sodium chloride concentration and low water activity are all common conditions that can be used to reduce the risk of C. botulinum growth and toxinogenesis.


However, when new foods and packaging processes are developed, it is often necessary to determine whether they offer adequate protection against the growth of C. botulinum. This is particularly crucial when a food composition or process deviates from the pre-defined food safety guidelines. In these instances, challenge testing must be performed.


Challenge testing is a well-established food safety and quality validation step and provides the most direct evidence of product safety and stability over shelf life. Challenge testing involves the deliberate inoculation of food products with C. botulinum, to determine whether toxin production occurs under expected storage conditions. For C. botulinum challenge tests, it is important to demonstrate that toxin formation is categorically prevented; solely performing viable cell counts is not enough, given that that botulinum toxin may be formed in food without a measured increase in bacterial viable count. Toxin presence may be verified via the mouse lethality bioassay and/or validated in vitro assays (e.g., toxin ELISA).[6] Unfortunately, in many cases, the mouse lethality assay has remained the standard test for the detection of botulinum neurotoxins, given that the complex components of food may interfere with in vitro test reactions and a suitable validated in vitro test may not be available.[7]


Figure 1. Challenge testing process.

Additionally, due to the rigorous safety requirements associated with C. botulinum (handling requires certified facilities and personnel), challenge testing is typically outsourced to specialist laboratories, rendering it an extremely expensive and time-consuming process. Expectedly, this is a discouraging factor for food companies that may have wished to explore novel and more eco-friendly packaging.

Project Description

The Nottingham iGEM 2019 project “NoTox” sought to apply a synthetic biology approach to generate a safe and accessible technology for the prediction of botulinum toxin in food without the need to handle toxigenic C. botulinum during challenge testing. We envision that our product could serve as a process aid and R&D tool for the food industry.


The technology would consist of a safe (non-toxinogenic) C. botulinum strain expressing instead of the toxin a suitable and easily detectable reporter. The strain should behave identically to wild type C. botulinum in terms of spore germination, growth and elaboration of toxin/reporter, hence be a suitable alternative in challenge testing. 


Although the lab our iGEM team was based at possesses a C. botulinum-dedicated facility, to be able to work with the organism, we would have to undertake a 6-month training programme. Therefore, to develop our reporter technology and obtain proof-of-concept in time for the Jamboree, we opted to using Clostridium sporogenes (C. sporogenes), a harmless relative of C. botulinum.


We aimed to engineer C. sporogenes to produce a suitable reporter under the control of the native botulinum toxin expression mechanism. Consequently, reporter expression was anticipated to mimic toxin expression. We chose acetone as a suitable reporter, detectable using electronic nose technology and a device that we designed and programmed ourselves. In parallel, we also used alternative protein-based reporters (e.g., GusA and FAST) for validation purposes. 

Figure 2. The technology behind Project NoTox.

Why C. sporogenes?

  • There is a 99% sequence similarity between sporogenes and C. botulinum according to 16s rDNA sequencing experiments.[8]
  • Both organisms are true clostridia sharing Gram positive and anaerobic physiologies, naturally associated with an ability to form hardy endospores.
  • sporogenes is already used as a safe surrogate strain in industry.[8]
Figure 3. Clostridium botulinum and Clostridium sporogenes.[9]

Acetone as a Reporter

Acetone is a volatile, generally innocuous gas that can be easily detected with a gas sensor, eliminating the need for food sampling and processing. Therefore, it would allow real time detection, even in complex food environments.

Our Engineered Reporter Strain

Our prototypical reporter strain was generated by integration of the toxin-expression machinery, namely botR with its native promoter (PbotR), onto the chromosome of C. sporogenes; botR encodes an alternative sigma-factor involved in the regulation of core genes associated with the botulinum neurotoxin complex.[10]


Thereafter, genes predicted to allow acetone production in our surrogate host, were placed downstream of the promoter associated with the expression of the botulinum neurotoxin complex (Pntnh; recognisable by BotR). The acetone production operon was arranged onto a shuttle plasmid. Transformation of our botR-expressing strain with the reporter plasmid permitted the production of acetone as a marker for neurotoxin production, without the need to handle toxigenic C. botulinum.


BotR-mediated gene transcription operates with exquisite specificity given that BotR can only recognise promoters with conserved non-canonical -35 (and to a lesser extent -10) recognition sequences, while activation of the respective promoters is only feasible in the presence of BotR.[11] Given that C. sporogenes does not encode BotR or BotR-like sigma-factors nor possesses native promoters that are recognised by it, BotR-mediated transcription should only occur when the reporter and botR-expression modules coexist in the same cell.

Figure 4. Clostridium sporogenes reporter strain.

Application of NoTox in the Real World

We feel that the technology we have developed has suitability as a process-aid to the food industry. Our product would allow for the safe prediction of botulinum toxin production following food manufacturing and packaging without the need to outsource for expensive challenge testing. Doing so would allow smaller food companies to ensure that their process generate foods with appropriate microbiological quality, as well as hopefully help reduce the number of mice used in lethality bioassays.

Further development of NoTox

Our prototypical C. sporogenes reporter strain has proven the concept of using BotR to marry neurotoxin production with volatile acetone production. In the future, we would like to see the principles of Notox carried forward into a more relevant model comprising a safe, toxin-null strain of C. botulinum, engineered to produce acetone. We would use the same mutagenesis approach described in our project (CRISPR/Cas9) to integrate the acetone production module (or other relevant reporter module) into the chromosome of C. botulinum. Crucially, we would replace the toxin gene (e.g., boNTA1 in a Type A strain), encoding the central toxigenic component of the botulinum toxin, with our volatile reporter construct.


The use of a native C. botulinum host would provide a more accurate model than our prototype. Similarly, the reporter would be integrated into the genome as opposed to being plasmid-borne. This would allow for a more accurate transcriptional and translational control, more indicative of toxin production. We would like to generate volatile reporter strains in toxin-null C. botulinum strains belonging to type I, II and III to ensure that our methodology translates into all three groups associated with foodborne botulism.[12]

Figure 5. Replacement of neurotoxin gene on C. botulinum with our our acetone-production operon.

Project Inspiration

Working at a world-class research institute specializing in the genus Clostridium, presented us with the opportunity to explore project ideas relating to obligate anaerobes and gas fermentation. After speaking with our supervisors about their research areas, we became interested in the commercial applications of C. botulinum, beyond its cosmetic uses. We also found out and were fascinated by the fact that our lab is one of the few facilities in the world that do active research in C. botulinum, having certified facilities and processes for the handling of this dangerous bacterium. As a consequence, we decided to focus our project on the infamous C. botulinum - the pathogen responsible for botulism. Our research led us to an understanding of the current testing and prevention methods for C. botulinum bacteria in food.

References

3. Dhaked, R.K., Singh, M.K., Singh, P., and Gupta, P., Botulinum toxin: bioweapon & magic drug. The Indian journal of medical research, 2010. 132(5): p. 489-503.
2. Peck, M.W., Clostridium botulinum and the safety of minimally heated, chilled foods: an emerging issue? J Appl Microbiol, 2006. 101(3): p. 556-570.
1. World Health Organization (WHO), WHO fact sheet on botulism. 2019 [accessed: 20.10.2019]; Available from: https://www.who.int/news-room/fact-sheets/detail/botulism.
4. Johnson, E.A. and Bradshaw, M., Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective. Toxicon, 2001. 39(11): p. 1703-22.
5. Fleck-Derderian, S., Shankar, M., Rao, A.K., Chatham-Stephens, K., Adjei, S., Sobel, J., Meltzer, M.I., Meaney-Delman, D., and Pillai, S.K., The Epidemiology of Foodborne Botulism Outbreaks: A Systematic Review. Clinical Infectious Diseases, 2017. 66(suppl_1): p. S73-S81.
6. Bradshaw, M., Marshall, K.M., Heap, J.T., Tepp, W.H., Minton, N.P., and Johnson, E.A., Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies. Appl Environ Microbiol, 2010. 76(2): p. 387.
7. Lindström, M. and Korkeala, H., Laboratory diagnostics of botulism. Clinical microbiology reviews, 2006. 19(2): p. 298-314.
8. Brown, J.L., Tran-Dinh, N., and Chapman, B., Clostridium sporogenes PA 3679 and its uses in the derivation of thermal processing schedules for low-acid shelf-stable foods and as a research model for proteolytic Clostridium botulinum. J Food Prot, 2012. 75(4): p. 779-92.
9. Eye of Science, SCIENCE SOURCE IMAGES[accessed: 15.10.2019]; Available from: http://images.sciencesource.com/feature/gallery225-eye-of-science.
10. Dupuy, B., Raffestin, S., Matamouros, S., Mani, N., Popoff, M.R., and Sonenshein, A.L., Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors. Mol Microbiol, 2006. 60(4): p. 1044-57.
11. Dupuy, B. and Matamouros, S., Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase σ-factors. Res Microbiol, 2006. 157(3): p. 201-205.
12. Hill, K.K., Xie, G., Foley, B.T., and Smith, T.J., Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins. Toxicon, 2015. 107(Pt A): p. 2-8.