Antibiofilm and Antimicrobial Activities of Chloroindoles Against Uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC) is a nosocomial pathogen associated with urinary tract infections and expresses several virulence factors that cause recurring infections and cystitis of the bladder, which can lead to pyelonephritis. UPEC uses different types of extracellular appendages like fimbriae and pili that aid colonization and adherence to bladder epithelium and can form persistent biofilm-like bacterial communities that aid its survival after the deployment of host immune responses. We investigated the antibiofilm, antimicrobial, and antivirulence properties of three indole derivatives namely, 4-chloroindole, 5-chloroindole, and 5-chloro 2-methyl indole. All the three chloroindoles had MICs of 75 μg/ml and inhibited biofilm formation by an average of 67% at 20 μg/ml. In addition, they inhibited swarming and swimming motilities, which are essential for dissemination from bacterial communities and colonization, reduced cell surface hydrophobicity, and inhibited indole production and curli formation. Gene expression analysis showed all three chloroindoles significantly downregulated the expressions of virulence genes associated with adhesion, stress regulation, and toxin production. A 3D-QSAR analysis revealed substitutions at the fourth and fifth positions of the indole moiety favored antimicrobial activity. Furthermore, these chloroindoles potently inhibited biofilm formation in other nosocomial pathogens and polymicrobial consortia.

Keywords: E. coli; UPEC; biofilm; chloroindoles; indoles; virulence.

Figure 1

(A) Crystal violet quantification of biofilm formation in UPEC in the presence of indoles, wherein biofilm OD₅₇₀ represents absorbance of CV-stained biofilm, (B) crystal violet biofilm dispersal assessment of UPEC in the presence of indoles, wherein biofilm OD₅₇₀ represents absorbance of CV-stained biofilm, (C) cell growth curves of UPEC in the presence of indoles, and (D) 3D representation of UPEC biofilms in the presence of select indoles at concentrations 50 and 75 μg/ml. *p < 0.05 vs. non-treated controls.

Figure 2

Scanning electron microscopy of UPEC biofilms in the presence of indoles (50 μg/ml) on nitrocellulose membranes. The red arrow represents cell membrane damage and shrinkage visualized at ×15,000 magnification. Yellow and blue scale bars represent 10 and 2 μm, respectively.

Figure 3

(A) Swarming and (B) swimming motility assay results for UPEC in the presence of indole, 4CI, 5CI, or 5CMI. Mean (C) swarming and (D) swimming motility halo diameters of indole and the three derivatives. *p < 0.05 vs. non-treated controls.

Figure 4

Congo red curli assay of UPEC in the presence of indoles at concentrations of 25 and 50 μg/ml. White scale bars represent 1 cm.

Figure 5

(A) Extracellular indole concentrations of UPEC in the presence of indole derivatives, (B) intracellular ROS production levels in UPEC in the presence of indoles or the positive control H₂O₂, and (C) Cell surface hydrophobicities of UPEC in the presence of indoles. *p < 0.05 vs. non-treated controls.

Figure 6

Gene expression profiles in the presence of indoles. *p < 0.05 vs. non-treated controls. The rrsG gene was used as a housekeeping control.

Figure 7

(A) Scatter plot of pMIC vs. predicted pMIC for the 73 indole derivatives, predicted by 3D-QSAR PLS factor 5 model. (B) 3D representation of positive (blue) and negative (red) positions of the indole moiety for MIC activity.

Figure 8

Crystal violet quantification of biofilm inhibition by the three chloroindoles of (A) Pseudomonas aeruginosa PAO1, (B) Acinetobacter baumannii ATCC 17978, (C) Candida albicans DAY 185, (D) Staphylococcus aureus ATCC 6538, and (E) a polymicrobial biofilm (S. aureus ATCC 6538, C. albicans DAY 185 and UPEC) grown in media comprised of equal parts of Luria-Bertani (LB), nutrient broth (NB), and potato dextrose broth (PDB) broth. Chloroindoles concentrations used are 0, 20, 50, and 100 μg/ml. The incubation time for all the conditions is 24 h at 37°C. *p < 0.05 vs. non-treated controls.

Figure 9

(A) Confocal laser scanning microscopy (CLSM) images of polymicrobial biofilms (S. aureus ATCC 6538, C. albicans DAY 185, and UPEC) grown in the presence of the three indole derivatives at 100 μg/ml, (B) COMSTAT analysis of polymicrobial biofilms (S. aureus ATCC 6538, C. albicans DAY 185, and UPEC), (C) SEM images of polymicrobial biofilms (S. aureus, UPEC, and C. albicans) grown in the presence of the three indole derivatives at 100 μg/ml. Red, yellow, and green arrows represent UPEC, C. albicans, and S. aureus, respectively. *p < 0.05 vs. non-treated controls. Orange, yellow, and blue scale bars represent 100, 10, and 3 μm, respectively.

Figure 10

Graphical illustration of the pathogenesis of UPEC, host immune responses, and the antibiofilm and antivirulence activities of 4-chloroindole, 5-chloroindole, and 5-chloro 2-methyl indole against UPEC.