What Is the ‘Minimum Inhibitory Concentration’ (MIC) of Pexiganan Acting on Escherichia coli?—A Cautionary Case Study

Abstract

We measured the minimum inhibitory concentration (MIC) of the antimicrobial peptide pexiganan acting on Escherichia coli, and report an intrinsic variability in such measurements. These results led to a detailed study of the effect of pexiganan on the growth curve of E. coli, using a plate reader and manual plating (i.e. time-kill curves). The measured growth curves, together with single-cell observations and peptide depletion assays, suggested that addition of a sub-MIC concentration of pexiganan to a population of this bacterium killed a fraction of the cells, reducing peptide activity during the process, while leaving the remaining cells unaffected. This pharmacodynamic hypothesis suggests a considerable inoculum effect, which we quantified. Our results cast doubt on the use of the MIC as 'a measure of the concentration needed for peptide action' and show how 'coarse-grained' studies at the population level give vital information for the correct planning and interpretation of MIC measurements.

Binary Encoding of Random Peptide Sequences for Selective and Differential Antimicrobial Mechanisms

“…This is directly relevant to the biology of AMPs,which act within their proteolytic lifetime,also minutes.Despite the two mixtures being equally effective in binding to bacterial cells during the first hour of incubation ( Figures 3B and S6), only FK peptides kept bacterial growth at negligible rates over the first several hours (up to 11-12 hi nF igure 3C). [35] These findings indicate that the homochiral sequences are stronger or faster membrane disrupters,b ut are equally subject to inoculum-related depletion typical of antibiotics. [35] These findings indicate that the homochiral sequences are stronger or faster membrane disrupters,b ut are equally subject to inoculum-related depletion typical of antibiotics.…”

Neutron Reflectivity as a Tool for Physics-Based Studies of Model Bacterial Membranes

“…Although the detailed analysis of this data is ongoing (Titmuss 2013), we find that the floating bilayer undergoes a large-scale structural rearrangement, depicted schematically in Fig. 16.4, at a sub-phase concentration of peptide that is comparable to the minimum inhibitory concentration (MIC) determined for this peptide in Edinburgh (Jepson 2014;Jepson et al 2016). The challenge to the data analysis is that the fluctuations mean that the apparent Gaussian roughness of the layers corresponding to the molecular sub-units (lipid head groups and lipid tails), are greater than the thickness of the lipid head group region.…”

“…We have investigated the interactions of these floating bilayers with the antimicrobial peptides pexiganan, which has been the subject of microbial killing assays in the School of Physics and Astronomy at the University of Edinburgh (Jepson 2014;Jepson et al 2016). Although the detailed analysis of this data is ongoing (Titmuss 2013), we find that the floating bilayer undergoes a large-scale structural rearrangement, depicted schematically in Fig.…”

“…We would typically flow a 10 mL volume of peptide solution (at *μM concentration) through the flow cell, so the bilayer would be exposed to 10 À9 moles of peptide; hence overall there would be a peptide to lipid ratio of P=L $ 1. How that compares in detail to microbial assays, depends on how that assay is performed (Jepson 2014;Jepson et al 2016), but it is not unreasonable (Melo and Castanaho 2012).…”

“…Whilst the planar geometry and coarse-graining are inherent in the measurement, there are two subtle but important advantages in the reflectivity experiment: the coarse-graining is only applied to interpret the data, our bilayers and monolayers are built from real lipids, and so by definition have the correct force-field; and our bilayers and monolayers span areas of 10 cm 2 , so they are free to fluctuate, bend and buckle. For this reason, we view neutron reflectivity as providing a bridge across the reality gap between microbial growth studies, such as those described by Jepson and Poon (2014), Jepson et al (2016) and molecular dynamics simulations such as described by Carr (2015).…”