DNA repair is essential for <i>Vibrio cholerae</i> growth on thiosulfate–citrate–bile salts–sucrose (TCBS) medium

Thiosulfate-citrate-bile salts-sucrose (TCBS) agar is a selective and differential media for the enrichment of pathogenic <i>Vibrios</i>. We observed that an exonuclease VII (<i>exoVII</i>) mutant of <i>Vibrio cholerae</i> 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 <i>exoVII</i> mutant. Suppressor mutations in DNA gyrase restored growth of the <i>exoVII</i> mutants on TCBS, suggesting that TCBS inhibits DNA gyrase similar to the antibiotic ciprofloxacin. To better understand what factors are important for <i>V. cholerae</i> to grow on TCBS, we generated a randomly barcoded TnSeq (RB-TnSeq) library in <i>V. cholerae</i> 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 <i>V. cholerae</i> similarly to the mechanism of action of fluoroquinolones, and overcoming this DNA damage is critical for <i>Vibrio</i> growth on this selective medium.IMPORTANCETCBS is often used to diagnose cholera infection. We found that many mutant <i>V. cholerae</i> 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, <i>V. cholerae</i> grown successively on TCBS agar develops resistance to ciprofloxacin.