Amino Acid Inhibition of Pseudomonas aeruginosa Biofilms: Implications for Delayed Wound Healing.

摘要

Chronic non-healing skin wounds impact approximately 6.5 million patients within the United States and account for more than ;Inhibition of bacterial biofilm formation by amino acids was described for several gram positive bacterial species. However, we show that tryptophan significantly arrests P. aeruginosa biofilm development and causes partial dissolution of existing biofilms. The goals of this dissertation included: characterizing P. aeruginosa biofilm inhibition by tryptophan and potential signaling pathways responsible for this effect; developing a delayed skin wound healing model using pre-formed P. aeruginosa biofilms; and assessing in vivo benefit of tryptophan in full thickness murine skin wounds.;Tryptophan inhibited P. aeruginosa biofilm formation on various substrates in a dose dependent manner with an equimolar ratio of D- and L-tryptophan performing best. Tryptophan significantly increased P. aeruginosa swimming motility, possibly increasing dispersion resulting in less biofilm formation. Incubating P. aeruginosa mutants defective in quorum sensing (lasI/lasR, rhlI/rhlR, and pqsA) with tryptophan did not arrest biofilm growth. A precursor for the Pseudomonas Quinolone Signal (PQS) autoinducer showed increased production with tryptophan exposure. Mutants for tryptophan metabolism (kynA ) lacked this signal, while PQS mutants showed the same signal with or without tryptophan. This suggests that amplifying production of precursors for PQS may also explain biofilm inhibition by tryptophan.;We also developed a novel delayed healing skin wound model by applying pre-formed P. aeruginosa biofilms directly on to full thickness wounds in mice. Significant clinical infections developed within 3 days and lasted at least 21 days. Wound closure occurred 6 -- 9 days later than control wounds. Inhibition of P. aeruginosa biofilms on the dressing with tryptophan, prior to addition of a biological dressing to the wounds, showed beneficial effects on healing and wound closure. This novel and robust model of delayed wound healing will provide investigators with an in vivo model for testing anti-biofilm and wound healing treatments.