# Measuring Communication in Microbial Biofilms in Response to Antibiotics, Phytochemicals and Stressors

## TL;DR

This study introduces a high-throughput method to measure microbial communication in biofilms and how it responds to antibiotics, phytochemicals, and stressors.

## Contribution

A novel high-throughput assay system for measuring microbial communication in biofilms using bioluminescent biosensors.

## Key findings

- QSMs induced MWCBs at 1 h and 4 h but were inhibited at 24 h at high concentrations.
- Curcumin and DIM inhibited quorum sensing in biofilms without causing disintegration.
- Antibiotics and H2O2 inhibited MWCBs at high concentrations but activated them near MIC, possibly via H2O2 generation.

## Abstract

A high-throughput assay system is developed for measuring communication in microbial biofilms in a 96-well microtiter plate format. In this assay, bioluminescent microbial whole cell biosensor systems (MWCBs) for quorum-sensing molecules (QSMs) are embedded into biofilms, and their response to chemical cues relevant for bacterial communication is assessed. For measuring the response to stress, a sigma factor 54 (σ54, RpoN)-dependent MWCB was developed. Biofilms generated in this platform were exposed to gradients of communication signals (QSMs such as N-acetyl-homoserine lactones (AHLs), 3,5- dimethylpyrazin-2-ol (DPO), or phytochemicals that can act as natural quorum-sensing inhibitors (QSIs) such as curcumin or 3,3′-diindolylmethane (DIM)), and the response pattern was monitored. Further, the regulatory role of stressors such as oxidants (H2O2) or antibiotics (ciprofloxacin, trimethoprim/sulfamethoxazole) on the communication response is assessed. QSMs induced the MWCBs at 1 h and 4 h in biofilms, but high concentrations inhibited them at 24 h. Curcumin and DIM at higher concentrations lead to inhibition of quorum sensing in biofilms after 4 h and 24 h, but this is not followed by biofilm disintegration. H2O2 above 0.002% efficiently inhibited the MWCB activities and led to biofilm disintegration. At lower concentrations of H2O2, we observed induction of MWCBs. The antibiotics inhibited the MWCB activity at concentrations above their minimal inhibitory concentration (MIC), but this did not necessarily lead to disintegration of the biofilm. Like low concentrations of H2O2, the antibiotics activated the MWCBs at concentrations close to their MIC, possibly as a result of H2O2 generated during their bactericidal action. Interestingly, the induction of communication in response to antibiotics can be quenched by iron chelators, suggesting involvement of H2O2 and free radicals generated by the Fenton reaction. We hypothesize that the observed response to these stressors reflects increased communication in the biofilm, possibly enhancing tolerance and increasing survival.