Broad-Spectrum Antimicrobial and Antibiofilm Activities of Halogenated Anilines Against Uropathogenic Escherichia coli and ESKAPE Pathogens

Abstract

Uropathogenic Escherichia coli (UPEC) is one of the leading causes of nosocomial infections and urinary tract infections (UTIs), capable of inducing a spectrum of conditions ranging from acute bladder cystitis to chronic pyelonephritis. The virulence arsenal of UPEC includes factors, such as curli fimbriae, hemolysin, motility elements and metallophores. Moreover, UPEC can form biofilm-like communities and quiescent intracellular reservoirs within host tissues, contributing to recurrent and persistent infections. This study investigates the antibiofilm and antimicrobial activities of two halogen-substituted aniline derivatives, 4-bromo-3-chloroaniline (4B3CA) and 3,5-dibromoaniline (3,5-DBA), against UPEC. The compounds demonstrated minimum inhibitory concentrations (MICs) of 200 μg/mL for 4B3CA and 100 μg/mL for 3,5-DBA, with both exhibiting a biofilm inhibition IC50 value of 10 μg/mL. Additionally, these derivatives showed antibiofilm activity against ESKAPE pathogens. Treatment with 4B3CA and 3,5-DBA led to significant downregulation of UPEC virulence- and biofilm-related genes, including those involved in curli production, fimbrial adhesion, motility, iron acquisition, quiescent colony formation, and stress response. Interestingly, a mild upregulation of hlyA, csrA and uvrY was noted, alongside a marked downregulation of the adenylate cyclase genes cyaA and crp. These findings suggest that inhibition of adenylate cyclase activity may be a primary mode of action, leading to both antimicrobial and antibiofilm effects. The presence of halogen atoms in these compounds appears to enhance their binding affinity to adenylate cyclase through stabilising halogen bond interactions. Furthermore, 3D-QSAR analysis indicates that electrostatic favourability at the third and fourth positions of the aniline ring is critical for bioactivity. Finally, in silico ADMET profiling and cytotoxicity assessments using Caenorhabditis elegans suggest that these aniline derivatives hold promise as therapeutic candidates, warranting further investigation.

Keywords: E. coli; ESKAPE; UPEC; aniline; biofilm; halogen; virulence.

FIGURE 1

Crystal violet biofilm assays of UPEC and various pathogens in the presence of aniline and its derivatives (A). Biofilm OD₅₇₀ represents the absorbance of CV‐stained biofilm. Cell growth curves of UPEC in the presence of aniline and its derivatives, wherein cell growth OD₆₀₀ represents turbidity of the culture (B) and cell survival assay of UPEC in the presence of aniline and its derivatives (C), wherein log₁₀ (CFU/mL) represents the log values of the colony‐forming units per millilitre. Incubation time of agar plates is 24 h at 37°C. *p < 0.05, **p < 0.01 and ***p < 0.001 versus non‐treated controls. All data from the cell growth and cell survival assays were statistically significant, with p < 0.05 compared to non‐treated controls.

FIGURE 2

2D‐3D representation of UPEC biofilms in the presence of aniline and its derivatives at 50 μg/mL, and amoxicillin at 5 μg/mL (A). SEM of UPEC biofilms in the presence of 4B3CA, 3,5‐DBA, aniline and amoxicillin at concentrations of 200, 100, 400 and 10 μg/mL, respectively, for 24 h and 37°C (B). All SEM images were taken at 5 kV. The orange, yellow and blue scale bars represent 50, 10, and 2 μm, respectively. The red arrows indicate altered membrane morphology with cell elongation in comparison to non‐treated controls.

FIGURE 3

Swarming motility (A) and swimming motility (B) of UPEC in the presence of aniline derivatives, incubated for 24 h at 37°C. Fisher's exact test revealed a statistically significant association between compound treatment and motility inhibition (p < 0.05).

FIGURE 4

Congo red assay (A), rugose colony formation (B), hemolysis (C) cell surface hydrophobicity (D), prodigiosin assay (E), and procyanin assay (F). All the experiments were performed at 37°C for 24 h, except rugose colony formation, which was incubated for 5 days at 25°C. *p < 0.05, **p < 0.01 and ***p < 0.001 versus non‐treated controls. Fisher's exact test demonstrated a statistically significant association between compound treatment and the inhibition of curli production and colony rugosity (p < 0.05).

FIGURE 5

CAS agar assay incubated for 72 h (A), in vitro quiescence assay incubated for 36 h (B), quiescent colony diameters (C), and chalkophore assay incubated for 3 h (D), in the presence of aniline derivatives. All the experiments were performed at 37°C. *p < 0.05 and **p < 0.01 versus non‐treated controls. Fisher's exact test demonstrated a statistically significant association between compound treatment and the inhibition of siderophore production (p < 0.05).

FIGURE 6

Gene expression profiles of UPEC following treatment with 4B3CA (200 μg/mL) and 3,5‐DBA (100 μg/mL) for 2 h at 37°C. The rrsG gene was used as a housekeeping control. *p < 0.05, **p < 0.01 and ***p < 0.001 versus non‐treated controls.

FIGURE 7

Substitutions on positions 1 and 2 are electrostatically unfavourable for antimicrobial activity (A), substitutions on positions 3 and 4 are electrostatically favourable for antimicrobial activity (B) and scatter plot of predicted antimicrobial activity bin vs. activity bin (C). Substitutions on positions 1 and 2 are electrostatically unfavourable for antibiofilm activity (D), substitutions on positions 3 and 4 are electrostatically favourable for antibiofilm activity (E), scatter plot of predicted antibiofilm activity bin vs. activity bin (F) and Caenorhabditis elegans cytotoxicity assay of halogenated anilines (G). All data from the C. elegans cytotoxicity assay showed statistically significance compared to the non‐treated controls (p < 0.05).