Team:Leiden/Safety
Safety
Our product has been developed to serve as a proof-of-concept product for the market, designed to measure bacterial cell stress in order to discover novel antibiotic combination therapies. In the future, we envisage our product to be mass produced and sold commercially. As such, the product needs to be safe to handle and distribute. We therefore need to consider a safe working environment for the manufacturers, the distributors and the consumers. This page will showcase our considerations regarding lab safety and security for each of these stakeholders.Biosafety requires guidelines, regulation and laws to prevent harm from dangerous lifeforms to the population and environment. In short, safety intends to “keep bad bugs away from people”.
Safety in the lab
Our bacterial strains are intended to express a color when they undergo stress, which will require us to genetically modify bacteria. Genetically modified organisms (GMO’s) could potentially
be dangerous as they can introduce their modified elements into the environment and affect the pathogenicity of an organism[1]. To minimize risks,
we used the non-pathogenic chassis Escherichia coli DH5α and Bacillus subtilis 168 to produce our proof-of-concept model and designed our
product to be solely used in laboratory settings. Escherichia coli DH5α[2] and Bacillus subtilis belong to risk group 1[3], which allowed us to
keep our experiments in ML-1 laboratories.
We performed our experiments in a fully equipped laboratory in the Sylvius Building of Leiden University during the summer months,
which was kindly provided to us by Kees Koops. The production of GMOs requires the use of hazardous substances. These substances are
dangerous due to their possibly explosive, poisonous or carcinogenic properties and unnecessary contact with humans or the environment should
always be prevented. To protect ourselves and our surroundings, we were trained to work safely in an ML-1 laboratory and act correctly in case
of emergencies by Marc Fluttert, the Biosafety officer of the Faculty of Biology of Leiden University. We performed all our research according
to the biotechnology regulations of biosafety for The Netherlands[4].
Safety during transportation
While the GMOs are kept from the environment in labs, they also need to be safely transported between labs. The GMOs of our proof-of-concept project
belong to category B; non-infectious biological substances, therefore the transportation needs to follow the packing instructions P904
(ICAO/IATA PI959)[5]. Our product needs to be labeled with the UN3373 label for road transport and UN3245 for flight transport according to the
ARD and IATA transportation laws.
The package itself needs to have triple containment: one or more leak-proof primary receptacle(s), a leak-proof secondary packaging, and an outer packaging of adequate strength for its capacity, mass and intended use. It has to have at
least one
surface with minimum dimensions of 100 mm × 100 mm. For the primary receptacle, we designed a 96-wells plate with a seal on top of each well, where each row of wells can be accessed and thrown away independently. The bacteria are freeze
dried to remove liquids and reduce further risk of leakage. The second and third stages of packaging are based on the MiniMailBox designs for UN3733 transportation, containing a leak-proof safety bag surrounded by bubble-wrap and an
absorbent holder and a cardboard box with the correct hazard information.
Despite the bacteria being freeze-dried, they could still form a treat during their transportation if the bacteria, acquainted with powdered minimal
medium came in contact with water. As we will include the minimal medium with the kit, we are also able to take safety considerations into account with the composition of
the medium. To decrease the chance that the bacterial cell strains in our kit will grow, we looked at the autotrophic qualities of our bacterial
strain DH5α. Our strain cannot grow without the amino acid arginine and the vitamin thiamine, thus they need to be provided in the medium for growth.
Therefore, during the development of our final minimal medium formula, we will consider to deprive the minimal medium in the wells of these two compounds and
provide them in a separate container, to be added when the bacteria are intended to be used.
Safety for the user
Once the product arrives on the bench of the customer, bacteria can be used in strips that are closed off separately by a seal. The strips can be thrown away in biohazard waste containers. We initially intended for customers to use our
product outside the lab in uninhabited areas to actively search for stressful substances. However, after the initial risk assessment with Cecile van der Vlugt and Korienke Smit from the Dutch National Institute of Health and Environment
(Rijksinstituut voor Volksgezondheid en Milieu, RIVM), we realized that our design would unnecessarily expose our environment to GMOs. This could give the organisms a chance to escape, evolve and adapt to an environment outside the
laboratory, where they could distort the equilibrium. We realized that we could still proceed with our system if the bacteria were kept in the lab, where researchers can test the compounds on our strains.
An important note we want to make is that even though we took serious measures to keep our GMOs from the public for their safety, this is a
precaution for the potential dangers of modified bacterial DNA. Synthetic and natural bacteria are not always immediately dangerous, and many
natural bacteria are vital to sustain our environment and maintain human health. Unfortunately, synthetic bacteria have obtained a bad reputation
for their potential danger since the early development stages of GMOs. To improve the relationship of the public with bacteria, we went to several
events in our area and gave a workshop where people of all ages could draw with non-pathogenic, non-GMO bacteria: Micrococcus roseus from mammalian skin and
Micrococcus luteus from the mammalian upper respiratory tract. Those who followed the workshop were allowed to take the bacteria home and see their drawing grow for a period of time. People can be afraid of the unknown, and with the
workshop we hope to have educated people that bacteria can be safe under the right precautions.
In the future, we hope that our product design will serve as a screening platform for researchers to help find new antibiotics that can keep us safe from harmful bacterial infections.
References
[1]: Keese P. Risks from GMOs due to horizontal gene transfer. Environ Biosafety Res. 2008; 7(3): 123-49. doi: 10.1051/ebr:2008014.
[2] DSMZ. Escherichia coli (Migula 1895) Castellani and Chalmers 1919. DSMZ [Internet]. 1992 [cited 2018 Oct 10]. Available from: https://www.dsmz.de/catalogues/details/culture/DSM-6897
[3] DSMZ. Bacillus subtilis subsp. subtilis (Ehrenberg 1835) Nakamura et al. 1999. DSMZ [Internet]. 1992 [cited 2018 Oct 10]. Available from: https://www.dsmz.de/catalogues/details/culture/dsm-402.html
[4] Rijksoverheid. Wetten en regels biotechnologie. Rijksoverheid [Internet]. 2018 [cited 2018 Oct 10]. Available from: https://www.rijksoverheid.nl/onderwerpen/biotechnologie/wetten-en-regels-biotechnologie
[5] UCCS. Packing instructions 959. EHS [Internet] December 2010 Biosafety. [cited 2018 Oct 10]. Available from: https://www.uccs.edu/pusafety/sites/pusafety/files/inline-files/IATA_pack_instr_959.pdf
Biosecurity involves procedures or measures designed to prevent spreading of biological research that can be used by those that want to endanger other people or the environment. By applying a secure working environment, we hope to “keep bad people away from bugs”.
Vulnerability scan
As researchers, we have to make sure that the data we collect and share in an open source fashion cannot be misused. Currently, there are no laws that guide researchers to work securely in the Netherlands. To make sure that our team did
work securely, we contacted the Bureau of Biosecurity, which is the national knowledge and information point on biosecurity for the Dutch government and those that work with high-risk biological material. From them we learned about the two
key areas to imply secure work: on the workplace and on our open source network.
We checked the biosecurity of our work with the vulnerability scan of Bureau Biosecurity and improved our work based on the results (Figure 1). From the report, we learned about the eight pillars of secure work and implemented that
into our research. First, we provided awareness within our team by informing ourselves about the employees of our institute that dealt with biosecurity and talked about the possible consequences of our design. We made sure that we always
knew which experiment each teammate had planned for that day in a morning meeting. In addition, all experiments were carried out in duo's according to the four-eyes principle. We were aided by our supervisors concerning transport of
packages, and our biosafety counselor helped us pick the correct companies and measures for secure transport. All information we collected from experiments were documented in an online notebook, called Scinote, were we could keep track of
each other's work each day without the potential danger of someone finding and looking into our book. Materials were accounted for with weekly checks to see if any unusual amounts were missing. Storage was kept to a minimum to keep an
overview of all materials. And lastly, no one without the appropriate documentation could enter our building or lab due to our always friendly doorkeeper Hein.
Dual use of our research
We share all the data collected in our research on this wiki, to provide fellow iGEM teams and researchers with information to help further research. However, this also means that we have to take into account dual-use research: research that could be misapplied to pose a threat to public health, animal health or the environment. Our product is intended to detect stress in bacteria, but this type of screening does not contain info on the effect on humans, animals or the environment. Our product can therefore not give insight on potential harmful substances for humans. We, and our advisors at RIVM and Bureau Biosecurity, do not think that there are immediate dual-use aspects on our product and our approach that can lead to biosecurity problems.
Safety considerations do not end with our kit reaching the bench of a researcher, but also include how we want to handle antibiotic resistance in the future, even if new antibiotics are developed. It is therefore essential to consider the social aspects (ethics) associated with our tool.
Introduction
Antibiotics allow us to fight bacterial infections and are therefore widely used as medication to treat and prevent such infections. These antibiotics are either lethal to bacteria or delay their growth by interfering with the bacterial cell wall or the cell contents. Unfortunately, bacteria can change the genetic composition within a bacterial colony to resist the lethal effects of antibiotics, known as antibiotic resistance[6]. Bacteria that have become resistant to one type of antibiotic need to be treated with a different type of antibiotic, for which the bacteria can eventually also develop resistance. This problem could be solved by continuing to find new antibiotics, but despite the greatests efforts of researchers, the latest class of antibiotics was discovered all the way back in 1987[7]. The lack of new antibiotics and the increasing number of resistant bacterial strains will cause all of our antibiotics to be rendered useless, which is why the World Health Organization (WHO) has declared antimicrobial resistance as a priority health issue and prompted its associated countries to think of solutions for multi-resistant bacteria.
Why is it important?
The primary concern with antibiotic resistance is the development of a strain that is resistant to all known antibiotics. Resistance occurs spontaneously through mutations that inhibit or work against the mechanism of the antibiotic, such as an efflux pump that pumps out the antibiotic particles. The genes of this mutated bacterium can be passed onto offspring or can be acquired by non-relatives through mobile genetic elements such as plasmids. This horizontal gene transfer can allow antibiotic resistance to be transferred among different species of bacteria [8]. It is therefore natural for antibiotic resistance to occur without the use of antibiotics. However, there is no selective pressure on these mutated bacteria and they form a small percentage of the entire colony. Once antibiotics are added, the majority of the bacteria will die and the resistant bacteria become the new majority of the bacterial population. The use of antibiotics is therefore directly linked to the amount of resistant bacteria, and the overuse of antibiotics could result in a serious acceleration of antibiotic resistant bacteria.
Are antibiotics regulated?
The regulation of antibiotics differs internationally. In some countries, people can buy antibiotics in drug stores, whereas other countries
require a doctor's prescription. The ease at which a doctor is willing to give a prescription is dependent on the guidelines provided by the
government and the social perception of antibiotics. Some countries allow the immediate use of antibiotics at the start of a standard infection,
whereas the guidelines in, for example, the Netherlands state that patients first have to wait to be allowed to recover on their own before they are
prescribed antibiotics. This both improves the immune system of the patients and decreases the use of antibiotics that could lead to antibiotic
resistance[11].
The type of antibiotic that is prescribed by doctors is based on reports of resistance rates in that country. To prevent the resistance to build up, doctors are usually advised to switch their most prescribed antibiotics between years. How
fast this switch is made is based on the antibiotic’s resistance rate.
Resistance rates are calculated by first determining the Minimum Inhibitory Concentration (MIC) value, which is the lowest dose of an
antimicrobial substance that inhibits growth[9]. The 0.5 MIC, 0.25 MIC and 0.1 MIC values are then used to assure that bacteria are still
alive at lower concentrations. The bacteria are subjected to a MIC value of 0.5 until they reached a desired concentration, diluted and repeated for
20 to 30 cycles. The difference in rate at which the bacteria grow after several cycles displays the rate of antibiotic resistance. MIC resistance
experiments are often followed by a test to see if the bacteria subjected to multiple cycles of 0.5 MIC can still induce infections.
While doctors are aided by guidelines, the general population is not aware that these regulations exist, and many regard antibiotics as the remedy
for any infection, and they think that they can use antibiotics without negative consequences. Unfortunately, resistant bacteria are not restricted
by borders and the current trend in traveling and flights spreads resistant bacteria quickly. It is therefore of vital importance that we make
society aware of antibiotic resistance and to Read more about how we helped make the public aware of antibiotics on
our
Education & Public Engagement page
educate them about the dangers of antibiotic misuse and overuse.
Current developments against antibiotic resistance
As we cannot provide new antibiotics forever, we need to find alternative methods to fight infections and the development of antibiotic resistance. One of the strengths - but also the weakness - of antibiotics is its highly effective lethal mechanism on a common attribute of bacteria, which causes lethality in many different bacterial strains. Unfortunately, not all bacteria that are killed by an antibiotic are dangerous, some are even vital for human health.
Bacteriophages
One possible treatment is to specifically target pathogenic bacteria using bacteriophages, also called phages. Phages infect bacteria and either incorporate their viral genome into the host genome, replicating as part of the host (lysogenic cycle), or multiply inside the host cell before releasing new phage particles (lytic cycle). One major advantage is that these intracellular, self-replicating entities can exert their killing effect with minimal damage to the microbiome, which also reduces the chance of opportunistic infections. Phages also tend to be more successful than antibiotics to kill bacteria when there is a biofilm covered by a polysaccharide layer. Unfortunately, phages are not the ultimate solution. Phages are, due to their specificity on a subset of a bacterial species, intrinsically narrow spectrum agents. This has the disadvantage that an infection that could be solved with one or two antibiotics, would need dozens of phage strains to cover the likely sources. Additionally, the phages can provoke an immune response that would challenge a second treatment with phages. The research that is currently available for broad-spectrum phages is still unsubstantial, but phages could still replace antibiotics for specific types of infections such as diabetic foot ulcers and ear infections.
Pro- and prebiotics
Similarly to phages - where opportunistic infections are prevented by specifically targeting the pathogenic bacteria to decrease their number -
we could add bacteria that belong to a healthy microbiome, in order to make them compete with the pathogenic bacteria and reduce their relative number[12]. Moreover, probiotics can work in synergy with prebiotics. Prebiotics are
non-digestible dietary carbohydrates that travel to the colon
and are able to selectively stimulate the growth and activity of beneficial (probiotic) bacteria, together they are often called synbiotics due to their synergistic effect. However, the addition of probiotic strains to a microbiome holds
the risk that these probiotics also carry antibiotic resistance genes themselves, and share them with the pathogenic bacteria through horizontal gene transfer. Moreover, probiotics administration can bring unexpected adverse effects with
the production of toxins by the microorganisms themselves.
While the aforementioned methods are promising, they will unlikely replace all antibiotics because they all have specific limitations. We could therefore look at alternative methods to combat antibiotic resistance that strengthen existing
antibiotics, to enable them to attack
resistant bacteria. Alternatively, we could interfere with the mechanisms that promote resistance, rather than attempting to kill the bacteria.
Quorum sensing inhibitors
One such method is quorum sensing inhibitors. Quorum sensing is a molecular mechanism of bacteria to communicate through small extracellular molecules. In response to these molecules, the bacteria make biofilms to protect themselves. These biofilms make it more difficult for antibiotics to perform their antimicrobial effect. To prevent the formation of biofilms, inhibitors of quorum sensing can be administered to compete with the receptors that sense the small extracellular molecules, inhibit the signal synthesis or interfere with the degradation of quorum sensing signals[10]. One of the benefits of quorum sensing inhibitors is that mutated bacteria are likely to be surrounded by inhibitor-sensitive bacteria, and receive insufficient signals to express their quorum sensing regulon and their surroundings will not produce enough signals to allow the resistant bacteria to thrive. Quorum sensing inhibitors are still in the proof of concept phases and their efficiency has to be demonstrated in genetically and phenotypically diverse clinical isolates. Similarry, efflux pump inhibitors are proposed as a promising strategy to create new antibiotic drugs, and enable the reuse of old antibiotics against which resistant bacteria have formed. However, this technique requires further characterization.
New antibiotics
As mentioned before, no new antibiotic classes have been found since 1987. A possible reason for this is researchers having been shortsighted. For instance, substances are only tested for their antimicrobial qualities based on their
lethality or growth inhibiting function. Some substances that are harmful to a bacteria, but do not kill the bacteria and could therefore be ignored, as they are not detected through regular screening methods. We intend to visualize the
stress that is induced on the bacteria, as well as show what type of stress is initiated by the substance. The stressful compounds that are found through this method can be combined to find a synergistic combination which is lethal to
bacteria. Alternatively, the substances can be used to increase the lethality of existing antibiotics to use them in lower concentrations.
To show that such an approach is feasible, we used three commonly used antibiotics and combined them with a Read more about our
research about adjuvant compounds for existing bacteria at our Results page stressful compound we discovered,
L-ascorbic acid. In all three cases the lethal dose (MIC value) was lowered, which indicates that we can use smaller concentrations of the compound
to induce lethality. The smaller concentrations of antibiotics and the attack from multiple sites potentially hinder the development of resistance.
Are antibiotics a technical fix?
Antibiotics are not the tool to solve infections forever, but merely our best weapon to fight back. Every time the bacteria improve their “shield”, we find a way to strengthen our weapon. The improvements could therefore be regarded as merely an engineering attempt to solve a problem that was previously solved by earlier interventions, also known as a technical fix. We first heard this term from Robert Zwijnenberg, professor of Art and Science Interactions at Leiden University, who trained in civil engineering and philosophy. He emphasized that researchers are too focussed on finding these fixes, and that we should consider how far we want to go with our improvements.
Conclusion
Altogether, antibiotic resistance forms a treat to humans worldwide, and the inappropriate use in one location can affect people globally. The formation of dangerous resistant bacteria could be reduced by properly Read more about how we helped make the public aware of antibiotics on our Education & Public Engagement page educating people about the overuse of antibiotics. Aside from the lack of awareness, one of the biggest setbacks in our fight against infections has been the lack of new antimicrobial treatments. As an alternative to current developments, we propose a method that Read more about our project on our Project Description page combines stressful substances to work in synergy with current antibiotics. With this, the dosage of antibiotics can be lowered and resistance might occur less frequently.
References
[6] World Health organisation. Antibiotic resistance. WHO [Internet]. 2018 [cited 2018 Oct 10]. Available from: http://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance
[7] Read AF et al. Antibiotic resistance management. Evol Med Public Health. 2014;2014(1):147.
[8] Neiditch, MB et al. Regulation of LuxPQ Receptor Activity by the Quorum-Sensing Signal Autoinducer-2. Molecular cell 2005; 18.5: 507-18.
[9] Stokkou S et al. Impact of minimal inhibitory concentration breakpoints on local cumulative bacterial susceptibility data and antibiotic consumption. BMC Res Notes 2014; 7: 603.
[10] Heilmann S et al. Why do bacteria regulate public goods by quorum sensing?—how the shapes of cost and benefit functions determine the form of optimal regulation. Front. Microbiol. 2015; 6:767. doi: 10.3389/fmicb.2015.00767
[11] Yang JH et al. Antibiotic-Induced Changes to the Host Metabolic Environment Inhibit Drug Efficacy and Alter Immune Function. Cell host & Microbe 2017; 22(6): 757-765.
[12] Ouwehand AC et al. Probiotic approach to prevent antibiotic resistance. Ann Med. 2016; 48(4): 246-55.