Effects of fatty acids in super critical fluid extracted fixed oil from Withania somnifera seeds on Gram-negative Salmonella enterica biofilms

Background: Biofilms are responsible for the growing resistance of bacteria to antibiotics, preventing the antibiotic accessibility to bacterial cells. As an alternative to antibiotics, fatty acids (FAs) have been explored for their antibiofilm activities due to their diffusible nature and ability to modulate the membrane fluidity of the bacteria. Fatty acids have been shown as anti-biofilm agents mostly for Gram-positive pathogens. Therefore, we chose to explore the anti-biofilm activity of FAs from Withania somnifera (L.) Dunal (Solanaceae) seeds fixed oil (WSSO) on the Gram-negative pathogen, Salmonella enterica. Biofilm formation in Salmonella spp enhances its resistance to antibiotics and host immune response, with consequent increase in its virulence and chronicity of infection. FAs from WSSO was reported for their effectivity against Psoriasis-like skin inflammation. Biofilms are implicated in Psoriasis pathology and anti-biofilm therapy can provide new treatment options. In view of its biological importance, we have explored the activity of WSSO against planktonic and biofilm forms of S. enterica. Purpose: The purpose of the current study is to evaluate the potentials of fixed fatty acids from an important medicinal plant (W. somnifera) as antibiofilm agents against a Gram-negative bacterium (S. enterica). Methods: Antibacterial activity of WSSO against planktonic form of S. enterica was evaluated through broth microdilution method. Antibiofilm activity in terms of prevention of biofilm initiation, inhibition of biofilm formation and disruption of mature biofilm were quantified through crystal-violet staining. WSSO induced loss of membrane integrity, and concomitant effect on S. enterica motility were assessed through quantification of intracellular potassium and nucleotide effluxes, and motility assays, respectively. Results: The minimal inhibitory concentration of WSSO required for 50% reduction in the planktonic bacterial load (IC50) was 5.78 mg/ml. Cells lost their motility when treated with WSSO at IC50. At a similar concentration of 6.28 mg/ml, WSSO disrupted mature biofilm of S. enterica. Bacterial cell membrane was compromised after treatment with 5.40 mg/ml WSSO as evident from potassium (K+) ion and nucleotide effluxes. Conclusion: WSSO not only prevented initiation of biofilm formation and but also efficiently disrupted mature biofilm of S. enterica. Taken together, these results indicated that WSSO has the potentials to be used as an alternative antibacterial agent against Gram-negative pathogenic S. enterica. The mode of action of WSSO seems to be linked to its ability to modulate membrane integrity and to restrain bacterial motility, most likely by its abundant and trace fatty acid contents.