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Use of the Phytochemical Neem as a Component of Combination Treatment Against Pseudomonas aeruginosa Biofilm

According to the NIH, 80% of chronic infections are caused by biofilm‐associated bacteria. Biofilm communities contribute to antimicrobic resistance by providing a physical extracellular barrier and exhibit metabolic diversity within bacterial subpopulations. Clinically, combination antibiotic strategies are used to provide high dosage treatments to successfully combat biofilm‐associated infections. Microscopically, biofilms of Pseudomonas aeruginosa have been visualized as mushroom‐like structures with two distinct subpopulations: a high‐metabolic “mushroom cap” outer population and a low‐metabolic “stalk” inner population. Further, the inner subpopulation consists of persister cells that are not killed by many therapies and contribute to chronicity of biofilm‐associated infections. It has been proposed that complete eradication of biofilm is achieved by targeting specific biofilm populations with antimicrobic combination of colistin and ciprofloxacin that target inner and outer subpopulations of biofilm respectively. However, colistin is sparingly used clinically because of high renal toxicity. Use of phytochemical alternatives instead of antibiotics is under assessment for treatment of biofilm‐associated infections. Extract from Azadirachta indica, or neem tree, is reported to have antimicrobic and antibiofilm effects on P. aeruginosa with low human toxicity. For these reasons, P. aeruginosa was grown (24 h) and microplates washed to remove non‐adherent cells. Remaining biofilm and cells were treated overnight with neem, colistin, ciprofloxacin or dual combinations. Finally, biofilm was removed from the substratum by scraping and aspiration, and bacterial viability was determined by colony forming unit (CFU/mL) enumeration. Results indicated that neem and ciprofloxacin in combination had an inhibitory effect (≤103 CFU/ml) similar to colistin and ciprofloxacin combination (≤102 CFU/ml) compared to growth and vehicle controls (both of which were 1012 CFU/ml). Colistin and neem combination showed no combined effect (107 CFU/ml) different from single treatment effects (108 and 1010 CFU/ml, respectively). These results suggest that neem extract targets the inner, low‐metabolic subpopulation of bacteria in biofilms and, for this reason, neem has potential to be a non‐toxic alternative to colistin for use therapeutically. When colistin or neem (that target the inner, low‐metabolic bacterial subpopulation) are used in combination with ciprofloxacin (that targets the outer, high‐metabolic subpopulation), the entirety of the biofilm may be targeted and prevent chronicity in biofilm‐associated infections.