# Characterization of adaptive evolution strains for the development of triclosan resistance in Agrobacterium tumefaciens C58

## TL;DR

This study shows how a soil bacterium evolved resistance to triclosan and became cross-resistant to other antibiotics, highlighting risks of environmental antimicrobial resistance.

## Contribution

The study identifies a key mutation in the triR gene as a mechanism of triclosan resistance and reveals cross-resistance to other antibiotics in evolved strains.

## Key findings

- HDR-20a strain developed a missense mutation in the triR gene, leading to overexpression of a TCS-specific efflux pump.
- HDR-12a showed transcriptomic changes in ABC transporters and metabolic genes, though their role in resistance is unclear.
- Both evolved strains exhibited cross-resistance to chloramphenicol and erythromycin.

## Abstract

Agrobacterium tumefaciens, a soil bacterium, was used as a model organism to study the mechanisms of triclosan (TCS) resistance in environmental bacteria. Adaptive laboratory evolution tests were performed to select TCS-resistant strains by challenging a wild-type (WT) strain with increasing concentrations of TCS (8, 12, 16, and 20 µg/mL). Two high-dose-resistant strains, HDR-12a and HDR-20a, were isolated and used for detailed examination. In comparison to the minimum inhibitory concentration of the WT strain (10 µg/mL), HDR-12a (20 µg/mL), and HDR-20a (32 µg/mL) showed increased resistance to TCS. Whole-genome sequencing and transcriptomic analysis performed to identify mechanisms underlying the different degrees of TCS resistance among the two evolved A. tumefaciens strains revealed a nucleotide base change (missense mutation, Asn157Thr) in the transcriptional repressor triR gene as the key mechanism of TCS resistance in HDR-20a. This change reduced the DNA-binding ability of TriR, causing overexpression of the triABC operon that encodes the TCS-specific efflux pump. In contrast, HDR-12a had no mutation in the triR gene. HDR-12a exhibited transcriptomic changes in several genes involved in ATP-binding cassette (ABC) transporters and in the metabolism of sulfur, fatty acids, and carbohydrates. However, it remains unclear whether these transcriptomic changes are directly responsible for TCS resistance in HDR-12a. Both the TCS-adapted strains also showed increased resistance to chloramphenicol and erythromycin. Overall, these results demonstrate that TCS pollution in environmental hotspots can select for adaptive and cross-resistant bacteria.

TCS is widely used as a preservative and disinfectant in many personal healthcare products. TCS is subsequently released into aquatic and terrestrial environments. The emergence and spread of multidrug-resistant pathogens from the use of antimicrobials like TCS and the misuse of antibiotic drugs now pose a serious global public health threat. Understanding how resistance develops has implications for preventing the emergence of antimicrobial resistance. The adapted TCS-resistant strains showed cross-resistance to chloramphenicol and erythromycin. This study provides insight into how environmental exposure to triclosan can drive adaptive and cross-resistance mechanisms in a soil bacterium, highlighting its relevance to environmental antimicrobial resistance and public health risk.