Time to Biofilm Eradication Testing

A well-established biofilm colonization model (Kuhn et al., 2002) was employed to test eradication of pathogenic biofilms following different durations of exposure to antimicrobial agents. Briefly, 1 cm silicone disks were placed in 24-well flat bottom cell culture plates and exposed to 1 ml of human plasma overnight at 37°C. Biofilm was established on silicone disks by inoculating with challenge organism (1 ml of 5.5 × 10⁵ CFU/ml) and incubating shaking at 37°C for 24 h. All culture liquid was then removed and disks were washed shaking for 30 min in isotonic sterile saline to remove any remaining planktonic organisms. After washing, disks were exposed at 37°C for 30 or 60 min independently to 1 ml of each test. After exposure, any biofilm remaining on the surface of the silicone disks was assessed by disrupting biofilm via sonicating disks in 5 ml isotonic saline for 15 min. The resulting solution was serially diluted and quantitatively cultured onto Trypticase Soy Agar + 5% sheep blood (bacteria) or Sabouraud Dextrose Agar (yeast). Complete eradication of the biofilm requires a recovery of no viable colonies following treatment. Recovery of fewer viable organisms than from the control indicates partial eradication of the biofilm. Each time point for each organism for each disinfecting solution was tested with six replicates. To ensure eradication was complete (no surviving dormant or persister cells) from biofilms for which no viable colonies were recovered following the exposure to disinfecting solutions, regrowth experiments were conducted by first exposing biofilm-colonized disks to each experimental solution, then rinsing and subsequently transferring the disks to fresh broth and re-incubating for an additional 24 h. Following the 24 h regrowth interval, disks were then sonicated and cultured as indicated above to determine whether any organisms remaining embedded in the biofilm were still viable.

Antimicrobial solutions were prepared containing 1% PG (Sigma-Aldrich, St. Louis, MO, United States, <15% esterified) + either 0.1 or 0.4% CAP (Sigma-Aldrich, St. Louis, MO, United States). 0.1% CAP was selected because it was near the reported aqueous solubility limit for CAP (Hoerr et al., 1946). The emulsifying capacity of pectins for lipids has been reported near 40% or more (Liang et al., 2015), hence 1% PG and 0.4% CAP (40% of the PG concentration) were selected for testing since 0.4% CAP was well above the solubility limit of CAP and therefore presented the potential for increasing the effective concentration of CAP presented to microbes. MHB, 1% PG, 0.1% CAP, and 0.4% CAP were included as control solutions. For consistency, the pH of all test solutions, including MBH control, was maintained at pH 4.25 with sodium hydroxide or hydrochloric acid as necessary.