Stress adaptation under <i>in vitro</i> evolution influences survival and metabolic phenotypes of clinical and environmental strains of <i>Vibrio cholerae</i> 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 <i>Vibrio cholerae</i>. 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 <i>Galleria mellonella</i> 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 <i>V. cholerae</i>, which may explain the continuous evolution of El-Tor biotype strains toward variants with improved survival in the environment.IMPORTANCEHow <i>Vibrio cholerae</i>, the causative agent of cholera, survives during the periods between outbreaks remains a critical question. Using experimental evolution based on serial bacterial passages in culture media mimicking diverse environmental stress conditions, we investigated whether clinical and environmental isolates of <i>V. cholerae</i> develop changes in survival and in their metabolism. The evolved variants exhibited alterations in colony morphology, biofilm formation, and metabolism, including changes in the preferred use of carbon, nitrogen, phosphorous, and sulfur substrates. These changes were accompanied by various genetic modifications, notably in genes encoding second messenger molecules that regulate multiple biochemical pathways implicated in stress survival and increased pathogenic potential. Our results suggest a continuous evolution of <i>V. cholerae</i> strains toward variants displaying increased survival under environmental stress conditions that may also be encountered in the human host.