Inactivation of efflux pumps abolishes bacterial biofilm formation

Abstract

Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general.

FIG. 1.

EPIs significantly reduce biofilm formation by E. coli UTI strain 83972, E. coli wild-type strain F18, and K. pneumoniae i222-86. The EPIs thioridazine (T) (50 μg/ml), PAβN (50 μg/ml), and NMP (100 μg/ml) or combinations of thioridazine and PAβN (50 μg/ml each) or of thioridazine and NMP (50 and 100 mg/ml, respectively) were added to static cultures at the time of inoculation, and the amount of biofilm after 24 h was determined by crystal violet staining. (A) Addition of either thioridazine or PAβN significantly reduced biofilm formation up to 80%, and addition of the combinations significantly reduced biofilm formation up to 99% compared with controls without EPIs (P < 0.001, as determined by a paired two-tailed t test for at least three independent experiments). However, NMP alone did not have a significant effect. (B) Biofilm formation by E. coli UTI strain 83972-yfp in the presence of the EPIs thioridazine and NMP (50 and 100 μg/ml, respectively) was monitored using scanning confocal laser microscopy at 16 and 24 h postinoculation. In the presence of the combination of EPIs biofilm formation was reduced 54% at 16 h and 83% at 24 h. Scale bars = 30 μm. (C) Molecular structures of the three EPIs that were used to study bacterial biofilm formation.

FIG. 2.

Effects of the EPIs thioridazine, NMP, and PAβN on biofilm formation by S. aureus and P. putida. Thioridazine (T) (20 μg/ml), NMP (20 μg/ml), or PAβN (20 μg/ml) was added to static microtiter cultures of S. aureus 8324 and P. putida KTT2442 at the time of inoculation, and the amount of biofilm after 24 h was determined by crystal violet staining. Addition of thioridazine and addition of PAβN resulted in 50 to 70% and 40 to 50% reductions, respectively, in biofilm formation by S. aureus and P. putida compared with the biofilm formation in the absence of EPIs (*, P < 0.01, as determined by a paired two-tailed t test for five independent experiments).

FIG. 3.

Biofilm formation in the presence of antimicrobial agents in combination with the EPI NMP. (A) Biofilms of E. coli UTI strains 83972 and VR50, E. coli F18, and K. pneumoniae were significantly more sensitive to tetracycline in the presence of the EPI NMP. Tetracycline (100 μg/ml) was added to overnight biofilms that had been grown in media with and without NMP. After 2 h of exposure to tetracycline the biofilm density was determined by determining the number of CFU per microtiter well. In the presence of NMP (100 μg/ml) the killing effect of tetracycline was increased 2.6- to 7.5-fold (*, P < 0.01, as determined by a paired two-tailed t test for three independent experiments). (B) Biofilms of UTI strains 83972 and VR50 showed large increases in EtBr susceptibility in the presence of NMP. Overnight biofilms were exposed to EtBr (200 μg/ml) for 2 h after growth in media with and without NMP. The size of the viable biofilm was determined by determining the number of CFU per microtiter well. In the presence of NMP (100 μg/ml) the killing effect of EtBr was increased 13- and 7.5-fold for strains 83972 and VR50, respectively (*, P < 0.01, as determined by a paired two-tailed t test for five independent experiments).