Effects of Neem Extracts on Formed Biofilm of Pseudomonas aeruginosa

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Over time, bacteria have been developing increasing resistance to commonly used antibiotics in medicine. To compound this issue, certain surface adherent bacteria can undergo a community response to form biofilms, a protective layer consisting of extracellular polymeric matrix that enhances resistance to antibiotics and host defenses. Biofilm association can confer bacteria with up to 1,000 times more antibiotic‐resistance. According to the NIH, up to 65% of all human infections and 80% of chronic infections are biofilm‐associated. P. aeruginosa, a common nosocomial pathogen exhibiting antibiotic resistance, is a known biofilm producer. Due to the prevalence of biofilms and their role in the antibiotic resistance of common nosocomial pathogens, it is important to look for antibiofilm agents. One understudied source of antibiofilm agents is plant derived compounds (phytochemicals). Of note, neem leaf extracts have been shown to inhibit biofilm formation in scientific literature. The purpose of my project was to identify the effect of neem extracts (bark, fresh leaf, dried leaf) on eradicating pre‐formed biofilm of P. aeruginosa (ATCC 27853) via a microplate biofilm quantitative assay. Bacteria were first inoculated into tryptic soy broth (TSB) and incubated (37°C, overnight). Afterwards, bacteria were density standardized (A590=0.2), transferred to microplates (polystyrene U‐bottom 24‐well), and incubated (37°C, overnight) to form biofilm. To isolate and treat the pre‐formed biofilm, microplates were washed (3 times, TSB), TSB was replaced, neem extracts were placed in the appropriate wells (a final concentration of 10% for each neem extract), and plates were incubated (37°C, 5 days). Afterwards, biofilm associated bacteria were killed with glutaraldehyde (10%) and microplates were washed again (3 times, dH2O). Subsequently, crystal violet (0.1% aqueous) was added to stain the biofilm. Excess dye was removed by washing (3 times, dH2O) and stained biofilm was solubilized (8% acetic acid). Solubilized biofilm was quantitated by A590 readings. Results indicated that all the neem extracts had no significant (p<0.05) effect on biofilm eradication for P. aeruginosa 27853. This suggests that neem extracts may be more effective on developing biofilm rather than formed biofilm. Further strains will be tested for neem effect on biofilm eradication.Support or Funding InformationFinancial support for VK was received from the DeNardo Education and Research Foundation.