Bacteriocin purification

Successful cloning of the bacteriocins

Cloning into the IMPACT vector pTXB1 were shown by gel electrophoresis. A successful transformation were verified by a colony PCR with specially designed primers for each bacteriocin. The results were verified according to the theoretical expected length of the bacteriocin with small overhangs (1-4 nucleotides) which is seen in the current figure. In the figure the ladder XX is used.

FIGUR INDSÆTTES

Figur tekst; Results of a colony PCR originating from respective colonies from transformation of K2018011/pTXB1/E.coli Top10, K2018012/pTXB1/E.coli Top10 and K2018014/pTXB1/E.coli Top10. From right; Well 1-3= K2018011, Well 4 = K2018014, Well 5-7 = K2018011, Well 8 = K2018014, Well 9-12 = K2018012, Well 13-14 = K201801

The above figure shows the result of colony PCR products originating from respective colonies from transformation of K2018011/pTXB1/E.coli Top10, K2018012/pTXB1/E.coli Top10 and K2018014/pTXB1/E.coli Top10. Well 1-3 + 5-7 + 13-14 corresponds to the bacteriocin K2018011/pTXB1/E.coli Top10 (Thuricin S), marked by the red box in the above picture. The results show bands of similar length, thus showing a successful ligation of Thuricin S into pTXB1.

Determination of bacteriocin concentration

We purified the bacteriocins using the IMPACT method. Link her til ”Experiments”. To assay the concentration of our purified bacteriocins, we used a Bradford standard protein assay to compare measured absorbance of our bacteriocin to the relative absorbance of known standard concentrations.

A protein assay of known concentrations of BSA (Bovine serum albumine) according to the measured absorbance is shown below.

The respective concentrations are calculated according to y = 0,0011x + 0,0202.

Thus the protein concentration (x) is x = (y-0,0202)/0,0011.

The measured absorbance and the calculated protein concentrations are shown in the table below in µg/m L and ng/mL

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Bacteriocin effect towards S. Aureus MRSA and P. aeruginosa

To assay the effect of our purified bacteriocins we performed a MIC test following SOP0025_v_1.

The bacteriocins were dissolved by a two-fold dilution in water from well A-G having the largest concentration possible at column A. The maximal concentration is the half of the calculated concentration of the bacteriocin as the proteins were dissolved by 2-fold when the respective bacteria cultures were added. We used different MRSA strains to test the effect of our bacteriocins in order to state the possibility for our bacteriocins as substitute for traditional antibiotic of which the bacteria has evolved resistance towards. Following strains where tested:

•  MRSA CC398 – a methicillin and tetracyclin resistant Staphylococcus aureus isolated from a patient, which have gained it from a pig. The MRSA CC398 is called pig-MRSA which is found in many pigs in the Scandinavian and is a huge contributor to the resistance development.
• Hetero - VISA – a Staphylococcus aureus strain. The strain is capable of growing at vancomycin concentration of >4mg/L., thus have gained relative vancomycin resistance. This is due to thickened walls of the strain, which is suggested to catch the vancomycin before entry. Conly, J. M., & Johnston, B. L. (2002).
• USA300 – the Staphylococcus aureus strain is a community associated MRSA and is a common cause of skin infections. http://mbio.asm.org/content/6/2/e00054-15.abstract
• Pseudonomas aeruginosa – the strain is commonly spread at hospitals thus given rise to re-hospitalization of people with open wound infection, which we would like our bacteriocins to face.

The MIC plates were performed with respective concentrations. The concentrations in the table indicate final concentrations i.e. the concentration of the bacteriocin in the well after adding bacterial culture. Maximal concentration without dilution of bacterial culture is indicated in the table. An example of the bacteriocin K2018010/Laterosporulin concentration value plated in the MIC wells is shown below.

MIC test

To validate growth ore inhibition we use background absorbance from dH2O+Mueller Hinton (MH) Media + 10 % deviation as a reference value.

Scored as NO growth; Abs < OD dH2O/MH Media + 10 %

Scored as growth; Abs > OD dH2O/MH Media + 10 %

The absorbance of dH2O/Mueller Hinton Media = MANGLER VÆRDI

Reference value; Abs dH2O/MH Media · 0,1 =

Below is shown an example of the MIC plate for K2018010/Laterosporulin. The picture states a visual validation of the inhibition of growth.

FIGUR INDSÆTTES

Figure text: Visual MIC test results of K2018010/ Thuricin S. Well A(1-10) contain 33,5 µg/mL as final maximal concentration. A 2-fold dilution is performed in well B-G(1-10). Well H(1-10) does not contain bacteriocin, but contain bacterial culture. Well A-H(11-12) is BLANK.

MIC – OD values

A mean was calculated for the measured OD values for each bacteriocin, acting toward the respective strain. The MIC was estimated according to the lowest concentration inhibiting growth according to the reference value listed above.

The table shows the respective MIC values according to the tested strains and the bacteriocins they were exposed to. N.I = No inhibition. Thus no inhibition were shown at the respective maximum concentrations.

The MIC values are visualized in the bar-chard below. The bacteriocin eliciting the strongest effect is indicated by the lowest bar – thus the lowest MIC value.

 FIGUR INDSÆTTES 

In the above figure the lowest bar – thus the lowest MIC value indicates the bacteriocin eliciting the strongest effect. Thuricin S effect all of the strains at a concentration of 16,6 µg/mL. Lacticin Q shows to affect only the Staphylococcus aureus strains at a MIC value of 22,2 µg/mL. Laterosporulin shows inhibition of growth towards all of the strains with the lowest MIC being towards the Staphylococcus aureus strains, USA300 and MSSA CC398, with a MIC at 16,8 µg/mL. Even though the laterosporulin as a single bacteriocin effect Pseudomonas aeruginosa, the Laterosporulin-Thuricin S hybrid does not elicit effect towards P.aeruginosa. However it is seen that the MIC values towards hVISA, USA300 and CC398 (13,1 µg/mL, 13,1 µg/mL, 6,6 µg/mL respectively) are lower than the MIC value found in their single bacteriocin form (Thuricin S MIC 16,7 µg/mL and Laterosporulin MIC 16,8 µg/mL). This indicates a synergistically effect of hybrid bacteriocins compared to their single effect.

Lacticin Q shows no inhibition towards Pseudomonas aeruginosa. However the absence of inhibition could also be due to a higher MIC value i.e. need of a higher concentration to inhibit growth than used in the MIC test. The hybrid LacticinQ-LacticinZ is seen to inhibit the growth of all of the above strains with a MIC equal to 7,6 µg/mL against the S. Aureus strains and a MIC corresponding to 15,2 µg/mL towards P.Aeruginosa. In addition the MIC is decreased compared to the MIC estimated for LacticinQ alone (22,2 µg/mL). This could be due to the presence of LacticinZ. However LacticinZ is not tested as a single bacteriocin and a direct comparison cannot state a decreased MIC for LacticinZ but only a decreased MIC for LacticinQ. Two bacteriocins working in concert (a hybrid) seem to contribute to a decrease in MIC and thus a stronger effect indicating synergy. It should however be noted that the hybrid Laterosporulin-Thuricin S “looses” its effect towards Pseudomonas aeruginosa when in a hybrid, compared to their effect as single proteins.

Replicating MIC

The MIC plates were incubated for 28 hours with equal OD value for each bacteria strain, thus equal amount of bacteria were added. For replicating the MIC experiment consider the respective generation times of the bacterial strains used. The generation times could diverge between the bacterial strains thus representing different conditions, which is reflected in the OD measured. Thus the OD between the strains cannot be compared, but the MIC values can. Furthermore instead of using duplicates, multiple replications could have been made for specification of the OD value. Thus instead of calculating a mean value a median could be calculated comparing more OD values. By use of duplicates pipette errors cannot be neglected. A new MIC test could therefore be performed with multiplicates.