A Hydrogel-Based in Vitro Assay for the Fast Prediction of Antibiotic Accumulation in Gram-Negative Bacteria

Abstract

The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living E. coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. Machine learning by random forest analysis of in vitro data revealed minimum projection area, molecular mass, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our data set of 27 antibiotics from different structural classes to reported MIC values of seven clinically relevant pathogens allowed to distinguish active from non-active compounds based on their low in vitro permeability and in particular to identify poorly permeable antimicrobial candidates before testing them on living bacteria.<br>

An Outer Membrane Vesicle‐Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability

“…In addition, we are aware that the differences in biophysical characteristics observed in the model membranes compared to those in whole cells have a high influence on permeability. Nevertheless, it has been demonstrated previously that the permeability through much simpler biomaterials qualitatively correlated with respective bacterial accumulation data, [ 20 ] demonstrating the high importance of sufficient antibiotic uptake to overall intrabacterial accumulation. ( Figure ) In this study, we found that in vitro permeability coefficients matched in bacterio accumulation best when the permeation model was made of OMV from E. coli BL21 DE3 or E. coli BL21 DE3 Omp8 (Figure 9A,B), while purely PL‐containing layers as obtained from liposomes failed to adequately match in vitro permeability with in bacterio accumulation.…”

“…Importantly, the porin‐blocking study was conducted with 15 × 10 −6 m norfloxacin while the standard permeation assay was conducted with 200 × 10 −6 m compound concentration as reported earlier. [ 20 ] Surprisingly the porin‐deficiency of the BL21 DE3 Omp8‐based model did not lead to a significant drop of permeation for the β ‐lactams, tetracycline and chloramphenicol. This was, however, the case for the liposome‐based models (Section S3, Supporting Information).…”

“…[ 9 ] and Richter et al. [ 20 ] Gray quadrants demarcate the zone of fast in vitro permeation and high in bacterio accumulation (upper right) or slow in vitro permeation and low in bacterio accumulation, respectively (lower left). Non‐colored quadrants represent zones of lacking correlation of permeation and accumulation.…”

“…[ 5–7 ] To help drug developers also optimize their structures toward better cell penetration and enhanced bacterial bioavailability, [ 8 ] various approaches have been undertaken, ranging from cell‐based assays, [ 9–13 ] vesicle swelling assays, [ 14,15 ] electrophysiological assays [ 16,17 ] to PAMPA‐like filter support‐based assays coated with different biomaterials. [ 18–20 ] Only the latter assay type currently offers the opportunity to be employed in high throughput screening. However, the so far employed materials either mimic non‐facilitated uptake [ 18,19 ] or mainly facilitated uptake [ 20 ] while employing either artificial PLs or physiologically irrelevant hydrogels.…”

“…[ 18–20 ] Only the latter assay type currently offers the opportunity to be employed in high throughput screening. However, the so far employed materials either mimic non‐facilitated uptake [ 18,19 ] or mainly facilitated uptake [ 20 ] while employing either artificial PLs or physiologically irrelevant hydrogels. By introducing natural bacterial membrane components such as LPS and porins into such an assay via OMVs, predictive capacity and strain specificity could be increased while the impact of several components on antibiotic translocation can be observed.…”