Stress adaptation under in vitro evolution influences survival and metabolic phenotypes of clinical and environmental strains of Vibrio cholerae El-Tor

Bacterial adaptation to stress can lead to phenotypic variants with diverse levels of niche competitiveness, pathogenicity, and antimicrobial resistance. In this work, we employed experimental evolution to investigate whether exposure to various stress conditions results in new phenotypic and metabolic properties in clinical and environmental strains of Vibrio cholerae. Our findings revealed the emergence of variants with metabolic and genetic variations and enhanced survival under stress compared to the parental isolates. Phenotypic changes in the evolved variants included colony morphology, biofilm formation, and the appearance of proteolytic and hemolytic activities. The variants demonstrated metabolic changes in the preferred use of carbon, nitrogen, phosphorous, and sulfur substrates, while the genetic changes included single nucleotide polymorphisms (SNPs), breakpoints, translocations, and single nucleotide insertions and deletions. Mutations in genes encoding EAL and HD-GYP domain-containing proteins correlated with increased biofilm formation and different colony morphotypes. The combined analysis of the metabolic and genomic data pointed to pathways implicated in stress survival. The environmental strains were generally more pathogenic than the clinical strains in the Galleria mellonella infection model prior to the experimental evolution, and these differences did not change in the evolved variants. This study highlights the contribution of stress conditions as drivers for the evolution of genetic modifications and metabolic adaptation in V. cholerae, which may explain the continuous evolution of El-Tor biotype strains toward variants with improved survival in the environment.