Team:SDU-Denmark/Proof

Proof of Concept

Bacteriocins – They work!

In the project we have designed biobricks, which contain the genes encoding a single bacteriocin or a a hybrid bacteriocin. We purified the bacteriocins using the IMPACT method and determined the respective concentrations using a Bradford standard protein assay with Bovine Serum Albumin (BSA) as a reference. In order to purify the bacteriocins, correct incorporation of the bacteriocin gene into the IMPACT vector pTXB1 was first key criteria. The picture below shows the result of colony PCR products. Well 1-3 + 5-7 + 13-14 corresponds to the bacteriocin Thuricin S incorporated into the plasmid pTXB1, marked by the red box in the above picture. The results show bands of similar length, thus verifying a successful ligation with Thuricin S inside the IMPACT vector pTXB1.

Let the purification begin!

Performing a MIC test tested the activity of the bacteriocins . Our MIC test shows inhibition of growth of the strains USA300 (S.aureus), CC398 (S.aureus), hetero-VISA (S.aureus) and Pseudomonas aeruginosa. The bacteriocins Lacticin Q and the hybrid Laterosporulin-Thuricin S did not elicit their activity towards P.aeruginosa. However the absence of inhibition could be due to a higher MIC value i.e. need of a higher concentration to inhibit growth, than used in the MIC test which still leaves the hybrid as a potential inhibitor of P.aeruginosa

Compared to traditional antibiotic the bacteriocins has similar or better effect . The bacteriocins shows inhibition of growth towards MRSA strains and P.aeruginosa at concentrations where the traditional antibiotics does not elicit an effect. The bacteriocins thus show promising results to support the idea of bacteriocins as being a supplement for traditional antibiotics of which multiresistent bacterias has evolved resistance against.

Synergistic effect of bacteriocins

Not only did the bacteriocins inhibit growth of the respective strains – the bacteriocins also showed synergistic effect. The synergistic effect is shown by a decrease in MIC when the bacteriocin Laterosporulin and Thuricin S are combined as a hybrid compared to their MIC value as single proteins. The hybrid LacticinQ-LacticinZ also shows a decreased MIC compared to Lacticin Q as a single acting bacteriocin. The absence of inhibition towards Pseudonomas aeruginosa  by Laterosporulin-Thuricin S when in a hybrid, compared to their single protein effect could be stated as a loss of effect. However the MIC values over all decrease towards the S.aureus strains indicating a stronger effect. The observation leaves the possibility of a gain of effect towards strains not tested in the MIC test. The gain of effect could be tested by use of other strains than tested towards and possible prove another characteristic of the designed hybrid bacteriocins. This leaves the opportunity to design an antimicrobial compound as needed.

Our bacteriocins face AMR - The bacteriocins could serve as a supplement for traditional antibiotics, which bacterias has evolved resistance towards as our purified bacteriocins inhibit the growth of methicillin resistant S. aureus strains. It could be hypothesized that a combination of traditional antibiotics with bacteriocins could elicit a synergistic effect towards strains that have evolved resistance towards the antibiotics thus leaving us with the opportunity to shift the balance of resistance and face the evolution of antimicrobial resistance (Dan, Y., M. P. Zacharof and R. W. Lovitt (2012)).