# Molecular self-assembly mediates the flocculation activity of benzimidazole derivatives against E. coli

## TL;DR

Certain benzimidazole compounds cause E. coli to form flocs through self-assembly, offering a new method for bacterial immobilisation.

## Contribution

A novel flocculation mechanism via supramolecular self-assembly of bisbenzimidazole derivatives in E. coli is identified.

## Key findings

- Bisbenzimidazole derivatives induce rapid flocculation in E. coli in a structure-dependent manner.
- Flocculation is mediated by supramolecular fibres formed by self-assembly, not by extracellular matrix secretion.
- High-resolution microscopy revealed a dense fibrillar network within flocs.

## Abstract

Bacterial flocculation is a process in which bacteria aggregate to form cloudy, flake-like clusters known as flocs. While this phenomenon is commonly associated with water treatment, it also has interesting industrial applications, particularly as a method for cell immobilisation. Escherichia coli, extensively employed in industrial processes, typically does not possess inherent flocculation ability. In this study, we found that certain bisbenzimidazole derivatives can rapidly induce flocculation in E. coli (K-12 MG1655) in a structure-dependent manner. Among others, high-resolution microscopy (SEM, fluid AFM) revealed a dense fibrillar network within the flocs, initially suggestive of an extracellular matrix. Mechanistic investigations demonstrated that this phenomenon cannot be linked to the secretion of extracellular polymeric substances (EPS). Our findings suggest that flocculation arises from the self-assembly of bisbenzimidazole derivatives into supramolecular fibres that anchor to bacterial membranes. These results uncover an atypical flocculation process distinct from charge neutralisation or EPS-mediated pathways, broadening the potential applications of bisbenzimidazole derivatives in bacterial immobilisation.

The online version contains supplementary material available at 10.1038/s41598-025-13837-z.