In the past decade, the number of medical cases involving multi-antibiotic resistant microbes has seen a steady increase, and misuse of antibiotics has repeatedly been mentioned as one of the major reasons for the arise of such organisms. This situation provided the inspiration for our project, which is to attempt to improve the effectiveness of antibiotics, so that we can apply less of it to achieve the same effect as we do now.
Our project name - E. trojan - has its origins rooted in the Trojan Horse found in the tale of the Trojan War. Similar to how the Greeks destroyed the city of Troy from within using a wooden horse filled with soldiers, our E. trojan also works to destroy an E. coli population that is under antibiotic stress from within through indole quorum-sensing disruption. [Top]
Indole has been proposed as a key signalling molecule for E. coli in mediating resistance to environmental stress. In particular, indole is found to stimulate passive resistance against antibiotics by triggering stress-response mechanisms inside cells, including increased expression of drug efflux pumps and oxidative-damage repairing systems.
In a normal E. coli population containing wild type and naturally resistant mutants, the mutants will supply indole to nearby cells that are under antibiotic stress. This is considered to be a 'charity' action as the mutant does so at a fitness cost to itself. Our aim is to introduce E. trojan into the population and proceed to degrade indole via an enzyme (T4MO) and remove the charity effect. If the charity effect is significant, removal of its presence will cause a notable increase in the effectiveness of antibiotics. [Top]
To study the interaction and population dynamics of the naturally-coexisting HR (high resistance) and LR (low resistance) E. coli strains under antibiotic selection pressure, the E. trojan - which is to be introduced to HR/ LR mixed culture - should harbor the T4MO gene to allow it to perform quorum sensing disruption between the two strains. In addition, the E. trojan should not possess a wide spectrum of antibiotic resistance as this would give it an inherent selective advantage, thus interfering with the relationship between MICs and indole degradation in our study.
To fulfill the above criteria, it becomes necessary for us to first construct a new, pre-E. trojan bacterial strain (E. CRAFT) that is capable of performing plasmid selection without antibiotics and consequently contains as few antibiotic resistance genes as possible. Due to some limitations of the various alternative (non-antibiotic) selection methods available today, we have designed E. CRAFT in a way that causes it to perform selection by imposing internal pressure of holding an essential gene for survival. Upon the completion of this precursor strain, the E. trojan can be easily constructed by transformation of the T4MO gene into it. In the long run, however, we hold a hope that this novel strain will prove to be a versatile, effective alternative to the widely- used antibiotic selection method.[Top]
Toluene-4-Monooxygenase (T4MO) is a three-component enzyme system that is naturally able to degrade a variety of hydrocarbons. In particular, its ability to oxidize small aromatic compounds has caught the attention of scientists looking for biological means of producing certain useful organic chemicals for industrial use, resulting in extensive research on modifying its activity and affinity.
For our project, a mutated form of T4MO (double mutations at G103L & A107G) was chosen as the candidate for indole degradation. While there were several possible candidates, this particular one was known to convert almost 50% of the indole into 7-hydroxyindole (7-HI). This is of great interest to us as 7-HI has been documented to inhibit biofilm formation in Enterohemorrhagic E. coli, and quite possibly for other strains of E. coli as well. Biofilm formation is a major element that reduces the efficiency of antibiotics due to reduced area of action, and hence we were keen to take advantage of this synergistic effect to improve our construct.[Top]
4.1 Soldiers need shields...maybe - Multidrug Efflux Pump (Bcr)
As our novel strain will have one of its essential genes maintained in a plasmid, a lot of uncertainties are present regarding its fitness. It is quite possible that it will be more vulnerable to antibiotics compared with wild type and thus will be selected against when cultured together with wild type E. coli. This would reduce the effectiveness of our construct and is not desirable. Hence we planed an additional component to be maintained in the plasmid, known as Bcr, which we hope will boost the resistance of E. trojan. You will find more details on its construction here.[Top]
 Bacterial charity work leads to population-wide resistance
Experiments and Results