Team:Oxford/Safety
Safety
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Pathogenicity
Oral administration of probiotic
Toxigenicity
Immune response
Choice of chassis
Colicins
Kill switch
AND gate (temperature and copper sensitive expression)
Spread of antibiotic resistance
Effective waste management
Bacteria become dangerous and must be killed
Bacteria enter sewage after secretion into faeces
Interaction with the gut microbiome
Exposure to the environment
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Further information Hover over a box on the flowchart to see more information!
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<input name="chassiText" id="interactionBodyText" type="hidden" value="As with any novel treatment, we must consider the potential effects of the treatment outside of the role we are designing it for. There is no sense is designing a wonderful therapeutic that cannot be taken due to severe, limiting side effects. We considered the potential pathogenicity, toxigenicity and immune response of our therapeutic.">
<input name="chassiText" id="interactionGutText" type="hidden" value="As our ultimate goal is for a therapeutic to be able to stably reside in the gut for significant periods of time, we had to consider how it might interact with the gut microbiome. This was a key safety aspect of our project: as the importance of the gut microbiome has become increasingly evident in recent years (1)( 2), and we do not want our probiotic to damagingly disrupt it. ">
<input name="chassiText" id="exposureEnvironmentText" type="hidden" value="Although we hope that our therapeutic would reside in the intestine, human faeces contain live gut bacteria, so there is the possibility that our live probiotic could escape the confines of the body and be released to the environment. We had to consider the potential impact of this when considering safety.">
<input name="chassiText" id="pathoText" type="hidden" value="Pathogenicity refers to the ability of a pathogen to cause disease. When investigating public perception on GMOs and their use as a possible medical treatment, some members of the public made clear to us that that they found the idea of ingesting bacteria “off-putting” and were concerned about safety - bacteria are usually viewed as “bad”. This tied into our safety research into the pathogenicity of a possible treatment.">
<input name="chassiText" id="toxicoText" type="hidden" value="Toxigenicity refers to the ability of a bacterium to produce a toxin. A unique feature of probiotic therapeutics is that they are live when administered, and hence have the potential to produce toxins in situ. A bacterial toxin has the potential to seriously harm a patient, so this was a serious safety concern that we considered as we designed our project.">
<input name="chassiText" id="immuneText" type="hidden" value="Although considered to be widely safe (3), certain risks are associated with the use of probiotics. These risks can vary depending on the bacteria and strain, but little is known about the extent to which they may affect a patient due to insufficient research into the safety of most probiotic strains.
It has been long known that probiotic bacteria can confer health benefits onto a consumer (4). One of the potential causes of this may be the ability of these bacteria to modulate the innate immune response (5). The long term effect of this manipulation on the host is difficult to predict, but there could potentially be an adverse effect on immune development. However, immune development occurs at a sufficiently early age that Wilson’s Disease is unlikely to have been diagnosed, and thus, treatments are unlikely to have been initiated. ">
<input name="chassiText" id="antiResText" type="hidden" value="Probiotic bacteria constitute a possible source of antibiotic resistance determinants. The large population of bacteria required to be ingested for use as a therapeutic probiotic may contain a reservoir of antibiotic resistance genes that could influence the presence and establishment of antibiotic resistance genes in the body (6). Should these genes be passed to pathogenic strains found in the intestine, the result on the health of the patient could be drastic. ">
<input name="chassiText" id="killSwitchProblemText" type="hidden" value="There are a variety of ways in which our bacteria may become dangerous, requiring a rapid end to the treatment. The treatment system may work differently than expected, e.g. too well - resulting in a copper deficiency, which is undesirable. However, this is not currently a concern within our project as our modelling suggest that the maximum theoretical degree of chelation is still below the excess level that is ingested by Wilson’s Disease patients. Alternatively, as bacteria are living organisms, with a relatively high replication rate, there is the possibility that dangerous mutations may occur that could eventually harm the patient. ">
<input name="chassiText" id="enterSewageText" type="hidden" value="We considered the possibility of bacteria escaping the confines of the body when developing our potential therapeutic, and the effect that this may have on the environment. ">
<input name="chassiText" id="pillText" type="hidden" value="When researching possible delivery method for our probiotic, we had to consider the risks associated with the different possible delivery methods. Probiotic bacteria have been shown, in rare cases, to have a severe pathogenic effect: resulting in conditions such as bacteremia (bacterial infection of the blood) and endocarditis (bacterial infection of the inner lining of the heart). All cases of these conditions are characterised by the presence of central venous catheters or feeding tubes (3). Hence, we decided that our therapeutic would have to be delivered orally, to reduce the risk to the patient. Following this we talked to patients (link) to determine in what form they would prefer their oral therapeutic to be delivered. ">
<input name="chassiText" id="chassiText" type="hidden" value="Chassis choice plays a key part in the safety and longevity of our potential probiotic. When choosing the chassis we ultimately wanted to test our chelation system in, we had to consider a variety of concerns. These included the pathogenicity, toxigenicity, possible presence of colicins, potential to spread antibiotic resistance, and the ease with which the treatment could be aborted if necessary.
A variety of bacteria colonise the human intestine, we wanted to choose the safest and most useful species for our probiotic. We spoke to an Professor of Evolutionary Biology, Kevin Foster, about the distribution of bacteria in the intestine and he advised us to investigate several well-studied probiotic species. We ultimately decided that our final chassis would be E. coli Nissle 1917, although we would carry out our experimentation in E. coli K-12 MG1655 for ease of testing.
E. coli Nissle 1917 is a non-pathogenic strain, reducing the risks associated with using a violent pathogen. However, it does produce “fitness factors” called colicins, which are toxic to other E. coli strains (7). This is advantageous to our treatment however, as we want our bacteria to successfully compete to remain in the gut. Furthermore, E. coli Nissle 1917 has actually been shown to successfully outcompete intestinal pathogens to form biofilms in the gut (8), meaning that our probiotic should be safe to consumer, and may actually have additional advantageous effects on patients. Furthermore, the ability of E. coli Nissle 1917 to form biofilms increases its long term persistence in the gut.
As the E. coli species includes enteropathogenic and extraintestinal pathogenic strains, the toxicological effects of E. coli Nissle 1917 strains, with relation to biosafety, have been extensively studied. It has been shown that it does not produce enterotoxin, cytotoxin or pathogenic adhesion factors. In addition, it does not display any immunotoxicity, blood serum resistance (reducing the possibility of sepsis) or uropathogenicity. It is considered non-toxic in conventional animals (8).
The E. coli Nissle 1917 strain also lacks antibiotic resistance to antibiotics commonly used against gram-negative enterobacteria (8). So if the treatment goes severely wrong, antibiotics such as ampicillin could be used to destroy the bacteria. The absence of antibiotic resistance plasmids in the strain means that the bacteria are unable to spread antibiotic resistance to other gut bacteria.">
<input name="chassiText" id="killSwitchSolutionText" type="hidden" value="Although we were unable to produce a bacterium containing a kill switch due to time restraints, we had originally designed our expression system in such a way that all potential risks were minimised. Our initial design was based on an AND gate, in which 2 specific conditions would need to be met to initiate the expression of our chelators. These 2 conditions would be copper concentration and temperature. By ensuring that both a specific copper concentration and temperature would need to be met to allow expression, we impose a safety mechanism on our bacteria that means that, outside of the gut when the temperature drops below 37 degrees, our bacteria would not act abnormally relative to an unmodified strain.">
<input name="chassiText" id="andGateText" type="hidden" value="IMAGE HERE.
An extension of the kill switch would be the use of the ‘Passcode’ kill switch (9). This kill switch is sensitive to 2 possible inputs. In order to permit cell survival, inputs 1 and 2 must be present, and input 3 must not be present. In the case of our therapeutic, inputs 1 and 2 could be copper concentration and temperature, as discussed above. Input 3 could be another molecule, which, if necessary, could be taken by the patient to initiate cell death.">
<input name="chassiText" id="wasteTreatmentText" type="hidden" value="The level of pathogenic bacteria in sewage sludge varies depending on the stabilisation procedures at different sewage treatment plants, with research suggesting that further optimisation of water purification methods are required (10). However, as we plan to use a non-pathogenic, well-studied probiotic bacterium as our chassis, this is not a concern for us. It is unlikely that our bacteria would pose a serious threat, especially as our chosen chassis has a limited ability to survive outside of the body.">
<input name="chassiText" id="colicinsText" type="hidden" value="E. coli produce 'fitness factors' called colicins, which are toxic to other E. coli strains.">
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