Determination of the Minimal Inhibitory Concentration
In the following the three substances Nor-Pyochelin, Pyochelin and Desferrithiocin will be named siderophores in order to enhance readability.

To analyse the effect of our siderophores and siderophore-analogs we conducted several Minimal Inhibitory Concentration (MIC) assays.

For this purpose, we prepared dilution series of the siderophores complexed with gallium overnight (Ga-complex), siderophores with gallium added right before ( +Ga(NO3)3 ) and siderophores without gallium. For the first MIC assay the siderophores and gallium were mixed in the relation 2:1. The dilution series ranged from 50 µM to 0.195 µM.
The siderophores were solvated in absolute ethanol. In order to be able to differentiate between the toxic effect of the ethanol and the toxic effect of the siderophores, a dilution series of ethanol was prepared that contained the same amount of ethanol as the siderophore solutions.
In parallel we used a gallium dilution series to show that gallium alone has not the same effect compared to a complex with the siderophore.
As positive control we used two wells with bacteria only and no additional substances and as a negative control two wells with pure medium.
These controls were carried out in all following MIC assays.

To keep the assay as standardized as possible we used Müller Hinton medium (Roth) provided by the Heisig research group, which is the standard medium for MIC assays. In the following the pipetting scheme and a picture of the first MIC assay are shown.

The expected value for MIC lies around 12.5 µM as indicated in the literature (Hier Paper quoten Pyochelin Potentiates the Inhibitory Activity of Gallium on Pseudomonas aeruginosa) could not be replicated, although a slight inhibitory effect is visible with the gallium complexed siderophores. After a whole day of trouble shooting it was found that the missing effect is probably based on the fact that the Müller Hinton medium contains bovine-infusion which also contains bovine serum. The serum contains a protein called lipocalin 2 which binds siderophores (like yersiniabactin) and is also a part of human serum to fend of bacterial infections.

The next MIC was conducted with the same dilution series but in LB medium. The pipetting scheme and the picture are shown below.

The MIC conducted in L.B. medium did not show the expected effect either. After several discussions it was found that a miscommunication occurred, referring the binding relation of siderophores to gallium ions. The used complexes were formed with a relation 2:1 siderophores to gallium. This relation is wrong and in the following MIC - assays the correct relation of 1:1 siderophores to gallium was used. With free siderophores in iron-containing L.B. medium the iron supply P.aeruginosa was warranted.

After eliminating this mistake, the MIC was repeated with new, correct gallium-siderophore-complexes and a different dilution series ranging from 400 µM to 1.563 µM. The dilution series of the gallium complexes were parallel duplicated on a second 96-well plate and two new concentrations between 12 µM and 6.25 µM were added. In the following the new pipetting schemes and the associated pictures are shown.

In this assays the siderophore-gallium-complexes show no significant effect on the cell growth. One explanation could be that the L.B. medium contains enough iron, that the P.aeruginosa can maintain their iron uptake and are not depending on gallium loaded siderophores.

To eliminate that source of trouble it would be necessary to conduct the MIC-assays in an ion free medium, or a medium with nearly the iron concentrations, present in physiological infectious conditions. In lack of such media and the lack of time to order those, the MIC was conducted again in M9 (minimal medium) and for completeness replicated in L.B. medium.

The pipetting schemes and pictures of the assays are shown in the following.

In the assays conducted in L.B. medium again no significant effect is visible as it was observed before.

In the minimal M9 medium an inhibiting effect is visible, but strongly relativized by the ethanol control dilution. In figure 14 and 16 can be observed that nor-pyochelin-gallium-complexes have no significant inhibitory effect on cell growth. In contrast the pyochelin-gallium complex inhibits the cell growth at a concentration of 50 µM (figure 14) but the number cannot be determined as MIC. Therefore the assay has to be replicated several times.

The main reason for the low effect should be the iron-containing medium the assays were conducted in. Another reason could be that in the chemical synthesis stereoisomeres were produced which could probably not as effective.

MTT-Assay to determine the toxicity of siderophore-Gallium-complexes on human lung epithelial cells

The MTT-assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a standard method to quantify the viability of cells. The yellow MTT is uptaken by the cells and metabolised in the mitochondria. The metabolized product forms blue crystals which are no longer able the cross the cell membrane. The production of blue chrystals is directly proportional for the cell vitality.

To test the toxicology of the siderophores Cystic Fibrosis Bronchial Epithelial (CFBE) cells were used because in case of P.aeruginosa the application site of siderophores would be the respiratory tract.

The cells were incubated 24 hours with the siderophores.