Sub-MIC concentrations (2.5 μg/ml) of peptides DJK-5 and DJK-6 were used. Inhibition of biofilm development was tested by immediately adding peptide into the flow-through medium of the flow cell apparatus and then monitoring biofilm formation for 3 days. Eradication conditions involved waiting two days before addition of either peptide into the flow-through medium. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).

Sub-inhibitory concentrations of peptides DJK-5 and DJK-6 in combination with antibiotics prevented biofilm development of Gram-negative bacteria. Inhibition of biofilm development was tested by immediately (at the beginning of biofilm growth at day 0) adding peptide plus antibiotic into the flow-through medium of the flow cell apparatus and then monitoring biofilm formation for 3 days. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions). The top FIC combinations of peptide + antibiotic (determined in checkerboard assays) were used.

Bacteria were grown in flow cells and treated at day 2 of biofilm formation with peptide, antibiotic, or the combination of both. The top FIC combinations of peptide + antibiotic (determined in checkerboard assays) were used (as in ). In some cases, at the concentrations selected, the activity of the peptides led to complete eradication of the flow cell biofilms. Thus, we decreased the levels of peptide used, which lowered the FIC values (see on the right hand side of panels) compared to the checkerboard assay results shown in . Specifically, in (A) 0.8 (μg/ml of DJK-5 (instead of 6.4 (μg/ml) was used in combination with tobramycin vs A. baumannii. In (B), 0.5 (μg/ml of DJK-6 were used instead of 2 (μg/ml combined with imipenem vs. A. baumannii, 1 (μg/ml of DJK-6 (as opposed to 2 (μg/ml) was used in conjunction with ciprofloxacin vs S. enterica, and 0.5 (μg/ml of DJK-6, instead of 2 (μg/ml, was used in combination with tobramycin vs K. pneumoniae. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).

(A) Anti-biofilm peptides DJK-5 and DJK-6 at 1 (μg/ml led to the absence of (p)ppGpp accumulation as revealed by TLC separation of guanine nucleotides extracted from intact cells as described in Experimental Procedures. (B) D-enantiomeric peptides RI-1018 and DJK-5 led to complete disappearance of (p)ppGpp more potently than L-peptide 1018. (C) D-enantiomeric peptide DJK-6 exhibited increased ability to trigger the degradation of pre-formed (p)ppGpp compared to RI-1018 and RI-JK6. Twenty (μg/ml of each of the three peptides were used.