# Epoxytiglianes potentiate the activity of colistin against resistant Escherichia coli via modification of the bacterial cell membrane

## TL;DR

EBC-1013 enhances colistin's effectiveness against resistant E. coli by modifying the bacterial cell membrane.

## Contribution

EBC-1013 synergizes with colistin to combat colistin-resistant E. coli by altering the outer membrane.

## Key findings

- EBC-1013 synergized with colistin in 6/9 strains (FIC ≤ 0.5).
- EBC-1013 decreased bacterial hydrophilicity and increased membrane permeability.
- Combined EBC-1013/colistin treatment reduced biofilm biomass in resistant E. coli.

## Abstract

The unrestricted use of colistin in animal husbandry has led to the emergence of mobile plasmid-borne colistin resistance (mcr). The antimicrobial epoxytigliane, EBC-1013, has been shown to be effective in the topical treatment of biofilm-mediated infections in vivo. Hypothesizing that EBC-1013 interacts with the modified bacterial outer membrane of colistin-resistant Escherichia coli, we investigated its ability to potentiate the activity of colistin using fractional inhibitory concentration (FIC) index determinations against environmental mcr Enterobacteriaceae. Molecular dynamics (MD) simulations were used to visualize EBC-1013 interactions with the outer membrane in silico. Modification of outer membrane surface chemistry and adhesion properties was characterized using X-ray photoelectron spectroscopy (XPS) and hydrophobicity and bacterial membrane permeability assays. Anti-biofilm activity was quantified using confocal laser scanning microscopy (CLSM). Checkerboard assays revealed synergistic effects of EBC-1013 with colistin (FIC ≤ 0.5) in 6/9 strains. MD simulations demonstrated weak EBC-1013 interactions with colistin and the outer membrane. XPS suggested modification of the outer membrane in both colistin-sensitive and colistin-resistant E. coli, as well as the interaction of colistin and EBC-1013 with the phosphoethanolamine (pEtN)-substituted mcr lipid moiety. EBC-1013 binding significantly decreased bacterial hydrophilicity and potentiated membrane permeability induced by colistin (P < 0.05). In the biofilm model, potentiation was also evident, with a marked decrease in bacterial biomass in EBC-1013/colistin combined treatments (P < 0.0001) for colistin-resistant E. coli. The results highlight the ability of EBC-1013 to modify the bacterial membrane of colistin-resistant E. coli and potentiate the activity of colistin in the treatment of multidrug-resistant wound bacteria.

Resistance to colistin, an antibiotic of last resort for hard-to-treat infections, is on the increase. Therefore, the need to develop new antimicrobials to tackle antimicrobial resistance is of paramount importance. The epoxytiglianes represent an exciting range of molecules with a diverse range of biological effects in human and veterinary applications, including antimicrobial properties. In this study, we show how EBC-1013 interacts with the outer surface of colistin-resistant Escherichia coli cells, inducing chemical and structural changes to the cell wall, making it susceptible again to colistin treatment. This ability of EBC-1013 to enhance the activity of colistin against a range of colistin-resistant E. coli suggests that EBC-1013, alone (or as a combination therapy), has potential as a new treatment strategy to treat antibiotic-resistant bacterial wound infections and reduce antibiotic usage.

This study is registered with ClinicalTrials.gov as (Australian New Zealand Clinical Trials Register: ACTRN12624000544572).