Concentration Dependent Effect of Azadirachta indica (Neem) Seed Oil and Neem Bark extract on Planktonic and Established Biofilm Growth of Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

Azadirachta indica Juss (Neem) is well documented for its antimicrobial activity. The effect of varying concentrations (0.1 to 50% v/v) of Azadirachta indica derived neem seed oil (NSO), neem seed oil with tween 20 and neem bark extract was evaluated on planktonic, biofilm formation and mature biofilms of multiple drug resistant Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 25923 using the crystal violet assay and scanning electron microscopy. NSO showed antimicrobial activity at 25% v/v for P. aeruginosa but not S. aureus in zone of inhibition assay. Neem bark extract on the contrary showed antimicrobial activity against both the isolates at 50% v/v concentrations. Interestingly, in biofilm formation assay, low concentrations of NSO (3.5 to 0.2% v/v) induced biofilm formation while inhibition of both planktonic and biofilm was seen in concentration dependent manner from 12.5% v/v onwards. Complex of NSO and tween in comparison of NSO alone caused low induction in S.aureus biofilm formation, while inhibiting biofilm formation of P. aeruginosa at all the concentrations. In biofilm eradication assay, NSO induced biofilm of both P. aeruginosa (50 to 0.1%v/v) and S. aureus (50 to 3.13%v/v). Eradication effect of neem bark extract was found on P. aeruginosa biofilm in a dose dependent fashion from 50 to 20% v/v followed by 0.2 to 0.1%v/v concentration respectively. S. aureus biofilm were eradicated at 50 to 25%v/v concentrations. At low concentrations, both the neem derivatives induced biofilm mediated growth of the pathogenic organisms. The data also indicate that neem seed oil was more effective against Gram negative P. aeruginosa while neem bark extract was effective against Gram positive S. aureus. This study highlights the crucial but variable effects of concentration dependent effect of phytochemicals and their composition on biofilm induction as well as eradication, the primary growth form in clinical settings. This challenges the notion that all herbal products are safe as antimicrobial activities differ as per microbial growth modes. Hence, concentration dependent effect of medicinal plant derived products requires thorough investigation prior to their use as antimicrobial agents.