Purpose: In this work, changes in membrane integrity, membrane potential, internal pH (pHin), ATP synthesis and expression of the Cholera toxin gene, were determined in V. cholerae cells after exposure to extracts of edible and medicinal plants.
Methods: Preliminary antimicrobial properties of methanolic, ethanolic, and aqueous plant extracts were evaluated using the hole-plate diffusion technique. A micro-dilution method was used to determine the minimal bactericide concentrations (MBC) of extracts showing the higher antimicrobial activity. For the experiments, 10X MBC was used. Damage of V. cholerae membrane was evaluated using the LIVE/DEAD BacLight kit, cFDA-SE was used to measure internal pH, membrane potential was determined with DiBAC4(3) and ATP was measured using Enliten ATP detection kit. Expression of the Cholera toxin gene was determined by qRT-PCR.
Results: The methanolic extracts of basil, nopal cactus, sweet acacia, and white sagebrush, were the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Membrane integrity was affected by all selected extracts damaging 100% of the cells. The pHi showed a clear decrease from approximately pHi 7 to pHi 4. Membrane potential was affected; three plant extracts produced hiperpolarization and one extract depolarization. Cellular ATP production was reduced 55 to 89% depending on extract and strain tested. The expression of V. cholerae enterotoxin gene was not inhibited during the treatment. All extracts exerted profound physiological changes that lead to bacterial death.
Significance: These four plant extracts could be regarded as alternatives to control V. cholerae contamination in foods and in the treatment of diseases associated with this microorganism.