Properties and stability of antimicrobial peptides - Polymyxins & Daptomycin
In this thesis, the analytical method, photodegradation, and inhibitory function of the peptidic antibiotics daptomycin, polymyxin B and E were investigated.
First, analytical methods with UHPLC using fluorescence detection were established involving more extensive method development for an online derivatization technique with AQC for the polymyxins.
Second, the antibiotics’ direct and indirect photochemical degradation behavior was investigated, including the decay in presence of reactive oxygen species (ROS) produced by dissolved organic matter (DOM), namely singlet oxygen (1O2), hydroxyl radicals (·OH), and triplet DOM (3DOM*). Findings show that additionally to direct photolysis, daptomycin was degraded by these indirect processes, as suspected by the presence of the reactive tryptophan and aniline moieties in its structure. The addition of DOM had an overall sensitizing effect on the degradation of all three antibiotics; the half-life of daptomycin decreased from 13.7 h for direct photochemical degradation alone to 6.7 h with DOM. The polymyxins were not degraded by direct sunlight, but showed a significant decay upon addition of DOM; the half-lives of polymyxin B and polymyxin E were 2.1 h and 1.6 h, respectively. The bimolecular reaction rate constants for singlet oxygen were assessed to be 1.6x107 M-1s-1, 7.2x107 M-1s-1 and 1.0x108 M-1s-1 for daptomycin, polymyxin E and polymyxin B, respectively. The bimolecular reaction rate constants with hydroxyl radicals were in the range of 1.0 – 1.8x1010 M-1s-1 for all three compounds. With measured steady state concentration of 3.4x10-13 M for singlet oxygen and 2.1x10-17 M for hydroxyl radicals, the relative contribution of each pathway to the overall decay in the presence of sunlight and DOM (SRFA, 10mgC/L) was assessed. Daptomycin was degraded to 48.7% by direct photochemistry, 18.9% by singlet oxygen, 0.7% by hydroxyl radicals, and a remaining 31.6% by other indirect photochemical processes. For polymyxin E and polymyxin B, the reaction with singlet oxygen contributed 19.9% and 37.4% to the overall degradation, which was unexpected, as no obvious reactive moieties could be identified in the structure. The impact of reaction with hydroxyl radicals was in the same range as daptomycin, with 0.3% for both polymyxins. The insignificant percentage of OH radical contribution can be explained by the low concentration of OH radicals formed. The remaining reactivity by other pathways can likely be allocated to reaction with the triplet excited ³DOM* and for the polymyxins possibly also to H2O2.
In a third part of the thesis, minimal inhibitory concentration (MIC) assays with E. coli and B. subtilis were performed to establish the applied method and reproduce MIC values reported in literature. Growth curves were evaluated with the Richards Model, which allowed to assess growth parameters and MIC values. Dose response curves were fitted with the Boltzmann model to assess EC50 values. For the gram-negative Escherichia coli, the MIC values for the polymyxins were in the same range as literature values, with 0.3125 – 0.625 μM for polymyxin E and 1.25 – 5 μM for polymyxin B. As expected, no influence of daptomycin on the growth of E. coli was observed, because it supposedly only acts on gram-positive bacteria. For the gram-negative Bacillus subtilis, the measured MIC values of 10 – 20 μM for daptomycin were two orders of magnitude higher than literature values. One reason for this discrepancy may be the lack of calcium ions which can increase effectiveness by factor 2 – 4 and should be tested further in the future. Despite the fact that polymyxin E and polymyxin B are not designed to be effective against gram-positive bacteria, MIC values of 10 – 15 μM and 6 – 8 μM, respectively, were assessed.