(WHS-P38) DETECTION OF PSEUDOMONAS AERUGINOSA CONCENTRATION VIA SYNTHETIC BIOSENSOR FOR QUORUM SENSING AUTOINDUCERS
Friday, May 17, 2024
7:30 AM – 5:00 PM East Coast USA Time
Purpose: The purpose of this study was to develop a real-time chronic wound infection monitoring sensor through the evaluation of a whole-cell Escherichia coli biosensor for the detection of N-(3-Oxydodecanoyl)-L-homoserine lactone (3OC12HSL), an important autoinducer in the quorum sensing network of Pseudomonas aeruginosa.
Methods: To simulate chronic wound fluid conditions in vitro, various concentrations of Lysogeny broth (LB) were combined with artificial wound fluid exudate (AWFE, Biochemzaone) to grow P. aeruginosa. This fluid was then used to determine ranges of 3OC12HSL detectable by our synthetic biosensor. P. aeruginosa was grown in varying concentrations of LB and AWFE, starting at 25% LB mixed with 75% AWFE and moving down to 5% LB and 95% AWFE; negative controls of (100% LB) and positive controls (100% AWFE) were also tested. A growth curve for the bacteria was generated via absorbance detection at 600nm. A crystal violet assay was also performed at 590nm for all media types. A synthetic gene network, including the lasR, amilCP (colorimetric reporter with blue color), and kanamycin resistance genes, were added to a DH5α E. coli host and selected by plating in LB agar + 50µM/mL kanamycin. The colonies were picked and grown overnight in LB with kanamycin. Cultures are then resuspended in fresh LB with kanamycin and added to 12 conical tubes. 10µM/mL of exogenous 3OC12HSL was added to the first tube and serially diluted by 1mL through the final tube (1:4 dilution each tube). The different concentrations and bacterial and media controls were plated in triplicate in a 48-well plate and read at 588nm, 600nm, and 700nm to quantify amilCP expression.
Results: Ten tests were performed for the differing media concentration growth curves in four replicates (n = 40) per concentration (total n = 280). A single-factor ANOVA and Tukey tests were used to determine which concentrations differed for biofilm analysis. There was a statistically significant increase in biofilm growth for the 100% LB condition compared to any other condition containing AWFE where p = 0.05. Additionally, biofilm growth decreased with increasing concentration of AWFE across all conditions where p = 0.05. Two tests were performed to detect exogenous 3OC12HSL in three replicates (n = 6). Currently, our designed biosensor has been able to detect concentrations of 3OC12HSL above 0.15nM/mL of 3OC12HSL.
Conclusions: The concentration of LB plays a significant role in P. aeruginosa growth and biofilm development. With future tests, we can predict which concentration accurately describes clinical samples. The synthetic biosensor can detect exogenous 3OC12HSL to about 0.15nM/mL in liquid cultures. Further testing will allow for more accurate quantification of 3OC12HSL toward real-time infection diagnosis.