Identification of a novel benzimidazole that inhibits bacterial biofilm formation in a broad-spectrum manner

Abstract

Bacterial biofilm formation causes significant industrial economic loss and high morbidity and mortality in medical settings. Biofilms are defined as multicellular communities of bacteria encased in a matrix of protective extracellular polymers. Because biofilms have a high tolerance for treatment with antimicrobials, protect bacteria from immune defense, and resist clearance with standard sanitation protocols, it is critical to develop new approaches to prevent biofilm formation. Here, a novel benzimidazole molecule, named antibiofilm compound 1 (ABC-1), identified in a small-molecule screen, was found to prevent bacterial biofilm formation in multiple Gram-negative and Gram-positive bacterial pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, on a variety of different surface types. Importantly, ABC-1 itself does not inhibit the growth of bacteria, and it is effective at nanomolar concentrations. Also, coating a polystyrene surface with ABC-1 reduces biofilm formation. These data suggest ABC-1 is a new chemical scaffold for the development of antibiofilm compounds.

Fig. 1.

Chemical structures of ABC-1, 5-methoxy-2-[(4-methylbenzyl)sulfanyl]-1H-benzimidazole (A), and ABC-2, 2-[(4-chlorobenzyl)thio]-5-methoxy-1H-benzimidazole (B).

Fig. 2.

ABC-1 and ABC-2 inhibit biofilm formation by V. cholerae. V. cholerae was grown in LB medium for 8 h in the wells of MBEC plates with 10 μM ABC-1 or ABC-2 or an equivalent amount of DMSO. Biofilm was quantified by staining with crystal violet and elution with ethanol as described in the text. The results represent the means plus standard errors of the mean (SEM). Student's paired t test was used to determine the statistical significance (*, P < 0.05).

Fig. 3.

ABC-1 exhibits broad-spectrum antibiofilm activity. Cultures were incubated in the wells of MBEC plates, and biofilm formation was quantitated by staining with crystal violet and elution with ethanol as described in the text. ABC-1 was used at a concentration of 100 μM for the Gram-negative bacteria and 25 μM for S. aureus. The results represent the means plus SEM. Student's paired t test was used to determine the statistical significance of the treated versus untreated conditions (*, P < 0.05).

Fig. 4.

ABC-1 inhibits biofilm formation by V. cholerae and P. aeruginosa at nanomolar concentrations. A concentration response curve measuring the antibiofilm activity of ABC-1 was performed in duplicate. P. aeruginosa and V. cholerae are represented as circles and squares, respectively. The calculated IC₅₀ for P. aeruginosa is 45.9 nM with a 95% confidence interval (error bars) of 44.3 to 47.5. The IC₅₀ for V. cholerae is 32.3 nM with a 95% confidence interval of 14.1 to 74.4.

Fig. 5.

ABC-1 inhibits biofilm formation by multiple Gram-negative pathogens under flow conditions. Shown is a three-dimensional view of biofilm in flow cells through intensity mapping after 24 to 48 h of growth for V. cholerae (A and B), P. aeruginosa CF-145 (C and D), K. pneumoniae (E and F), E. amylovora (G and H), and S. boydii (I and J). (A, C, E, G, and I) Biofilm growth in the absence of ABC-1. (B, D, F, H, and J) Biofilm growth in the presence of ABC-1. The intensity mapping had the maximum value set at 250, and the maps were developed with five color layers. Additional layers of color indicate greater intensity of the signal.

Fig. 6.

ABC-1 inhibits biofilm formation by S. aureus under flow conditions. Shown is a three-dimensional view of biofilm in flow cells through intensity mapping of S. aureus Newman (A and B) and S. aureus USA300 (C and D). (A and C) Biofilm growth in the absence of ABC-1. (B and D) Biofilm growth in the presence of ABC-1.

Fig. 7.

ABC-1 inhibits biofilm formation on catheters. Biofilm formation by P. aeruginosa PA01 was determined macroscopically (A) and using CLSM (B) on a silicone catheter in the absence and presence of 100 μM ABC-1. The biofilms were grown for 48 h.

Fig. 8.

ABC-1 does not disperse preformed biofilms. Shown is a three-dimensional view of biofilm formation in flow cells through intensity mapping of K. pneumoniae (A and B) and P. aeruginosa CF-145 (C and D). (A and C) Untreated conditions. (B and D) ABC-1 was added after the biofilms had been allowed to form for 24 h. The images shown are 3 h after addition of ABC-1.

Fig. 9.

Coating of a surface with ABC-1 reduces biofilm formation. Cultures of V. cholerae and P. aeruginosa were allowed to form biofilms on the pegs of an MBEC plate that were coated with polymer and ABC-1 or polymer only or that were uncoated. Biofilm was quantitated by staining the pegs with crystal violet and elution with ethanol as described in the text. The results represent the means and SEM from at least three independent experiments. Student's paired t test was used to compare the biofilm growth on an uncoated surface to that on an ABC-1-coated surface (*, P < 0.05).