# Functionally distinct mutations within AcrB underpin antibiotic resistance in different lifestyles

## TL;DR

The study shows how different mutations in the AcrB protein help Salmonella resist antibiotics depending on their growth environment.

## Contribution

The research identifies distinct AcrB mutations (R717L and Q176K) that confer antibiotic resistance in different lifestyles and explains their functional mechanisms.

## Key findings

- R717L mutation in AcrB reduces steric barriers for drug transit in planktonic Salmonella.
- Q176K mutation increases cefotaxime binding energy in biofilm-grown Salmonella.
- R717L is found in global Salmonella isolates, indicating clinical relevance.

## Abstract

Antibiotic resistance is a pressing healthcare challenge and is mediated by various mechanisms, including the active export of drugs via multidrug efflux systems, which prevent drug accumulation within the cell. Here, we studied how Salmonella evolved resistance to two key antibiotics, cefotaxime and azithromycin, when grown planktonically or as a biofilm. Resistance to both drugs emerged in both conditions and was associated with different substitutions within the efflux-associated transporter, AcrB. Azithromycin exposure selected for an R717L substitution, while cefotaxime for Q176K. Additional mutations in ramR or envZ accumulated concurrently with the R717L or Q176K substitutions respectively, resulting in clinical resistance to the selective antibiotics and cross-resistance to other drugs. Structural, genetic, and phenotypic analysis showed the two AcrB substitutions confer their benefits in profoundly different ways. R717L reduces steric barriers associated with transit through the substrate channel 2 of AcrB. Q176K increases binding energy for cefotaxime, improving recognition in the distal binding pocket, resulting in increased efflux efficiency. Finally, we show the R717 substitution is present in isolates recovered around the world.