Safety and risks
Synthetic biology involves unknown factors and sometimes pathogenic organisms. In our project we have tested the effect of our bacteriocins on pathogenic organisms, which calls for some different precautions than when working with non-pathogenic organisms. We also use proteins from a secretory pathway of a toxin called alpha-hemolysin to transport PHB out of E. coli. The transport proteins have no pathogenic effect in a strain of bacteria, which does not produce the toxin, as our strain of E. coli. In order to secrete PHB through the pathway we biofuse a signal peptide from alpha-hemolysin with phasin. As the toxin is not part of the biofused protein, no special precautions are necessary.
We have also thought a lot about the safety of our final product: whether or not it is safe to use and safe for the environment. The results of our investigations can be found on this page, where we talk about working with pathogenic organisms, an upcoming problem with the environment and at last making a risk assessment of our project.
Staphylcoccus aureus (S. aureus) is a commencal, opportunistic pathogen. S. aureus is normally found on the skin, in the nostrils and throat. Any damages on the skin, such as wounds or on the mucosal barriers, allows the bacteria to interact with the blood, where it spreads to other parts of the body. Many strains of Staphylococcus demonstrate the ability to form biofilm matrices, which makes infections by these strains hard to treat. A biofilm matrix consists of exopolysaccharides, proteins, teichoic acids, lipids, extracellular DNA and the bacteria. The biofilm protects the bacteria, which makes them less vulnerable to antibiotics ARCIOLA, C. R., VISAI, L., TESTONI, F., ARCIOLA, S., CAMPOCCIA, D., SPEZIALE, P. & MONTANARO, L. 2011. Concise survey of Staphylococcus aureus virulence factors that promote adhesion and damage to peri-implant tissues. The International journal of artificial organs, 34, 771-780.HERMAN-BAUSIER, P., EL-KIRAT-CHATEL, S., FOSTER, T. J., GEOGHEGAN, J. A. & DUFRÊNE, Y. F. 2015. Staphylococcus aureus fibronectin-binding protein A mediates cell-cell adhesion through low-affinity homophilic bonds. MBio, 6, e00413-15.. Most places in the world, people have heard of Methicillin-resistant S. aureus (MRSA). It is divided into two major subgroups; hospital-associated-MRSA (HA-MRSA), and community-associated-MRSA (CA-MRSA) ARCIOLA, C. R., VISAI, L., TESTONI, F., ARCIOLA, S., CAMPOCCIA, D., SPEZIALE, P. & MONTANARO, L. 2011. Concise survey of Staphylococcus aureus virulence factors that promote adhesion and damage to peri-implant tissues. The International journal of artificial organs, 34, 771-780.. These two terms refer to where the infections are obtained HERMAN-BAUSIER, P., EL-KIRAT-CHATEL, S., FOSTER, T. J., GEOGHEGAN, J. A. & DUFRÊNE, Y. F. 2015. Staphylococcus aureus fibronectin-binding protein A mediates cell-cell adhesion through low-affinity homophilic bonds. MBio, 6, e00413-15.. We have worked with the following strains of S. aureus in the class II lab:
P. aeruginosa is an opportunistic Gram-negative rod-shaped bacterium, typically found on human skin. P. aeruginosa is found in an estimate of 10-20% of all hospital acquired infections WAGNER, Victoria E.; IGLEWSKI, Barbara H. P. aeruginosa biofilms in CF infection. Clinical reviews in allergy & immunology, 2008, 35.3: 124-134..
P. aeruginosa is intrinsically resistant to a variety of antimicrobials, and it produces a numerous virulence factors, including secreted factors such as elastase, proteases, phospholipase C, hydrogen cyanide etc WAGNER, Victoria E.; IGLEWSKI, Barbara H. P. aeruginosa biofilms in CF infection. Clinical reviews in allergy & immunology, 2008, 35.3: 124-134.. The bacteria is capable of forming biofilms, which is often associated with antimicrobial resistance, and studies indicates that bacteria that grow in biofilms are up to 1000 times more resistant to antimicrobial challenges versus planktonically grown cells WAGNER, Victoria E.; IGLEWSKI, Barbara H. P. aeruginosa biofilms in CF infection. Clinical reviews in allergy & immunology, 2008, 35.3: 124-134.. We have worked with the strain PAO1 of P. aeruginosa.
Since we perform tests on pathogenic strains of bacteria it is necessary to work in a class II lab, which require special safety precautions, such as wearing special shoes and making sure no biomass leaves the lab. Despite even our best efforts to minimize the spread, people could get colonized by potentially harmful bacteria. MRSA is known to colonize the nostrils of people working with it and then they unknowingly take the bacteria out of the lab and can possibly infect other people.
The idea of manipulating microbes into producing materials we desire from waste products can be considered beneficial, as we exclude the harvesting and reduce the depletion of the resources of the Earth. However, the fact that a material is produced in microorganisms does not mean that the product can not be ecologically harmful. This is why it is crucial to ensure that materials made from microbes can be degraded, either through a metabolic pathway or a relatively fast chemical decay. The silk and bacteriocins, in our project, are combined in a protein matrix and does not pose an ecological threat as proteases or pH in the gut would degrade the silk or bacteriocins.
Traditional plastic polymers are synthesized from fossil materials and are not degradable through metabolic pathways or decay processes that match the disposing rate of plastic into the environment. This leads to accumulation of plastic into the environment, where estimates suggest that 10 % of all produced plastics end up in the marine environment BARNES, David KA, et al. Accumulation and fragmentation of plastic debris in global environments. Philosophical Transactions of the Royal Society B: Biological Sciences, 2009, 364.1526: 1985-1998.. Detrimental effects to the food chain has been observed as a result of this accumulation COLE, Matthew, et al. The impact of polystyrene microplastics on feeding, function and fecundity in the marine copepod Calanus helgolandicus. Environmental science & technology, 2015, 49.2: 1130-1137.. PHB degrade through metabolic pathways as a carbon source in various natural occuring bacteria. Considering the environment, the replacement of traditional plastics with biodegradable versions reduces the negative impact we are currently employing on various ecosystems.
There are several studies on bacteriocins that shows that they do not affect human cells; i.e. the skin- and blood cells. We also know that the bacteriocin Nisin has been used for around 50 years in the dairy production and this has not yet shown negative consequences.
PHB is also already beginning to be used as a replacement for conventional plastic products, e.g. in water bottles and lunch boxes. The synthetic spider silk is both immune neutral, biocompatible and biodegradable and ongoing research shows great potential in different uses of spider silk TOKAREVA, Olena, et al. Recombinant DNA production of spider silk proteins. Microbial biotechnology, 2013, 6.6: 651-663.
The most significant risk we take is to face the general uncertainty that is present when working with genetically modified organisms. They are still ‘new’ in the world, and some people fear the long term consequences. Other possible damages are that there is no proof of that the use of bacteriocins as a therapeutic treatment will decrease antibiotic resistance. This hypothesis is based on predictions formed by knowledge about bacterial evolution. The use of bacteriocins in therapeutics is new and therefore does not exhibit any conclusive data on how - or if - they will have a detrimental effects on the health of an individual. If bacteriocins become commercially applied, we are left with only speculations of their large-scale impact on various ecosystems. The bacteriocins in Bacto-Aid are incorporated between the silk proteins and are in theory unable to move, but they will have contact with the blood vessels. We do not have any research on whether or not they could detach and travel with the blood stream and cause an unwanted effect (i.e. immuneresponses) in the human body, e.g. cross the blood-brain barrier or affect the hormonal balance.
Besides the possible biological damage to patients, there are also possible economical and social damages. Social injustice could potentially be increased since Bacto-Aid and bacteriocins most likely will be more expensive than traditional antibiotics at first. The production price of bacteriocins are lower than the production price of traditional antibiotics, but the sales price will probably be higher due to cost and demand. The pharmaceutical companies that produce the bacteriocins also have to pay for FDA approval and the clinical trials, and these costs have to be earned back when the sales begin. This could potentially lead to a greater distance between the high and low economic classes of the society. It is also likely that the access will be limited at first until the production has been effected enough for the cost to decrease.
Bacteriocins as therapeutics is not thoroughly researched yet and like all new pharmaceutical drugs being tested there is a lack of knowledge about the long term consequences. The biggest problem seems to be that we do not have much research on the effect in vivo. Some research in the use of bacteriocins in vivo in mouse models shows promising results with antimicrobial effect and no harm of the animal. This seems to be promising news for Bacto-Aid INGHAM, Aaron, et al. The bacteriocin piscicolin 126 retains antilisterial activity in vivo. Journal of Antimicrobial Chemotherapy, 2003, 51.6: 1365-1371..