Team:UI Indonesia/Safety

SAFETY


Biosafety is one of the top priorities in this project. Our components have been checked previously based on the safety list that the IGEM committee imposed. We also received formal online training (due to the pandemic) and supervision from Institute of Human Virology and Cancer Biology (IHVCB) Universitas Indonesia. We do not conduct the wet lab experiment due to the pandemic, but nevertheless we realize that biosafety consideration is still a relevant aspect to this project. The bacteria that we are using are E. coli Nissle 1917 which possesses biorisk level 1, meaning it is non-pathogenic, and H. pylori which possesses biorisk level 2, which might cause treatable gastritis if accidental exposure were to occur. Since IHVCB UI has the capability up to biosafety level of BSL-3, there are no limitations in containing H. pylori research (minimal BSL-2).1,2


Agents Helicobacter pylori ATCC 49503 Escherichia coli Nissle 1917
  1. Proteinase-K transformed
  2. PGLA-AM1 transformed
Specimen Bacteria culture (liquid and semisolid/agar) Bacteria culture (liquid and semisolid/agar)
Risk group II (treatable human pathogen) I (not causing disease)
Pathogenicity Gastric mucosa colonization and destruction via virulence factors CagA and VacA, inducing inflammation and may develop into ulcer, gastritis, and stomach cancer (class I carcinogen) Non-pathogen, probiotic activity
Virulence Factors CagA and VacA enable mucosal colonization and immune suppression. Associated with adenocarcinoma Urease enables survival under low pH condition -
Hosts Primates (human and monkey), cats Human, animal (not specified)
Toxin Both vacA and cagA induce immunosuppression effect, causing prolonged inflammation and might lead to cancer -
Transmission Fecal-oral, food, cutlery, waterborne Fecal oral, food, waterborne
Environmental stability
  • Waterborne transmission indicate survivability in liquids
  • Coccoid can last a full year
 Unknown
Management
  • Diagnosis: sample culture, endoscopy, serum antigen, urease breath test
  • Prevention: prophylaxis antibiotics (clarithromycin, metronidazole, amoxicillin) and PPIs
  • Treatment: antibiotics, PPIs, and bismuth (refer to local guideline)
 N/A


In the experiment, we will be handling cultures of both bacteria. The bacteria will be grown in separate media, all of which contain ampicillin which is used to select competent cells. We will also incorporate other genetical parts, such as arabinose inducible promoter, ammonia inducible promoter, Proteinase K (protein digesting enzyme), and PLGa-AM1 (the main anti-microbial component). All of the aforementioned parts have no observed negative nor harmful effects on human, have been checked according to the IGEM registry of standard biological parts and are neither marked with a red flag nor fall into a category that is listed on IGEM white list (https://2021.igem.org/Safety/White_List).


During the process of this experiment, we have acknowledged several drawbacks that need to be addressed in the future research. The main drawback of this experiment is its inability to test the system inside a living being due to the nature of the experiment. Secondly, we were also facing the pandemic during this project, which disabled us from doing the wet lab. Apart from the experimentation processes, we also discovered several concerns about horizontal gene transfer. The plasmid that we used, pM2s2TsR, contains tetracycline resistance gene, TcR. The inserted part used in this project contains lytic-inducing genes. During the lysis, the content of the bacteria will be released to the environment, possibly creating a plasmid-rich environment. The free-plasmids might be uptaken by local flora in the gut, via transformation, which might create tetracycline resistance. This problem has been mentioned in our reference paper by Kan et al, though not clearly addressed.3 We hypothesize that with careful selection of competent cells, this problem can be minimalized. This is because our parts induce apoptosis in absence of tetracycline. During administration, there will be an absence of tetracycline inside the gut environment, which will render our plasmid unfavourable for survival. As a safeguard, our system also requires ammonium ions to induce lysis. The ions are vastly abundant in lower GI tracts, further solidifying the biosafety aspect of the system. However, as a potential threat, we recommend further research regarding this concern.


We are aware about the mutation potential of every bacteria that is used for therapeutic purposes. The risk is very small if any for probiotic bacteria to mutate and become pathogenic, especially in non critically ill patients with intact gastrointestinal lining. Limited study shows that there is evidence for translocation of probiotic bacteria to blood (bacteremia) in ICU patients along with some mutations. However the clinical significance is needed for further evaluation.4

Besides designing the kill switch for our secretion system, this approach also makes sure that minimal amounts of our engineered E. coli escape the stomach and advance to the intestine. If this happens, the proteinase-K as a potent protease will affect the digestion process by interfering with several enzyme activities. Furthermore, the PGLa-AM1 may disrupt the microbiota composition of the gut by its broad antibiotic activity.5

So, ensuring the bacteria does not mutate and escape the appropriate location of action is important and we design the kill switch system for these purposes. For further detail of our kill switch systems see the Project section.



  1. Blanchard TG, Nedrud JG. Laboratory Maintenance of Helicobacter Species. Curr Protoc Microbiol. 2006 Jan;CHAPTER:Unit8B.1.
  2. Non-pathogenic Escherichia Coli Strains Biological Agent Reference Sheet (BARS) | Environment, Health and Safety [Internet]. [cited 2021 Oct 2]. Available from: https://ehs.cornell.edu/research-safety/biosafety-biosecurity/biological-safety-manuals-and-other-documents/bars-other/non-pathogenic-escherichia-coli
  3. Kan A, Gelfat I, Emani S, Praveschotinunt P, Joshi NS. Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917. 2020;10(1):94-106.
  4. Yelin I, Flett KB, Merakou C, Mehrotra P, Stam J, Snesrud E, et al. Genomic and epidemiological evidence of bacterial transmission from probiotic capsule to blood in ICU patients. Nat Med. 2019 Nov;25(11):1728–32.
  5. Lei J, Sun L, Huang S, Zhu C, Li P, He J, et al. The antimicrobial peptides and their potential clinical applications. Am J Transl Res. 2019 Jul 15;11(7):3919–31.