# DNA repair is essential for Vibrio cholerae growth on thiosulfate–citrate–bile salts–sucrose (TCBS) medium

## TL;DR

This study shows that DNA repair is crucial for Vibrio cholerae to grow on TCBS agar, a common diagnostic medium, and that DNA-damaging effects of TCBS resemble antibiotic action.

## Contribution

The study reveals that TCBS agar causes DNA damage in V. cholerae and identifies DNA repair mutants that fail to grow on this medium.

## Key findings

- An exonuclease VII mutant of V. cholerae fails to grow on TCBS agar due to DNA damage.
- Bile acids in TCBS are primarily responsible for the toxicity to DNA repair mutants.
- DNA repair-deficient strains may be undetected on TCBS agar, affecting cholera diagnosis.

## Abstract

Thiosulfate–citrate–bile salts–sucrose (TCBS) agar is a selective and differential media for the enrichment of pathogenic Vibrios. We observed that an exonuclease VII (exoVII) mutant of Vibrio cholerae failed to grow on TCBS agar, suggesting that DNA repair mutant strains may be hampered for growth in this selective media. Examination of the selective components of TCBS revealed that bile acids were primarily responsible for the toxicity of the exoVII mutant. Suppressor mutations in DNA gyrase restored growth of the exoVII mutants on TCBS, suggesting that TCBS inhibits DNA gyrase similar to the antibiotic ciprofloxacin. To better understand what factors are important for V. cholerae to grow on TCBS, we generated a randomly barcoded TnSeq (RB-TnSeq) library in V. cholerae and have used it to uncover a range of DNA repair mutants that also fail to grow on TCBS agar. The results of this study suggest that TCBS agar causes DNA damage to V. cholerae similarly to the mechanism of action of fluoroquinolones, and overcoming this DNA damage is critical for Vibrio growth on this selective medium.

TCBS is often used to diagnose cholera infection. We found that many mutant V. cholerae strains are attenuated for growth on TCBS agar, meaning they could remain undetected using this culture-dependent method. Hypermutator strains with defects in DNA repair pathways might be especially inhibited by TCBS. In addition, V. cholerae grown successively on TCBS agar develops resistance to ciprofloxacin.