Synergism of the Combination of Traditional Antibiotics and Novel Phenolic Compounds against Escherichia coli

Abstract

Pathogenic Escherichia coli (E. coli)-associated infections are becoming difficult to treat because of the rapid emergence of antibiotic-resistant strains. Novel approaches are required to prevent the progression of resistance and to extend the lifespan of existing antibiotics. This study was designed to improve the effectiveness of traditional antibiotics against E. coli using a combination of the gallic acid (GA), hamamelitannin, epicatechin gallate, epigallocatechin, and epicatechin. The fractional inhibitory concentration index (FICI) of each of the phenolic compound-antibiotic combinations against E. coli was ascertained. Considering the clinical significance and FICI, two combinations (hamamelitannin-erythromycin and GA-ampicillin) were evaluated for their impact on certain virulence factors of E. coli. Finally, the effects of hamamelitannin and GA on Rattus norvegicus (IEC-6) cell viability were investigated. The FICIs of the antibacterial combinations against E. coli were 0.281-1.008. The GA-ampicillin and hamamelitannin-erythromycin combinations more effectively prohibited the growth, biofilm viability, and swim and swarm motilities of E. coli than individual antibiotics. The concentration of hamamelitannin and GA required to reduce viability by 50% (IC₅₀) in IEC-6 cells was 988.54 μM and 564.55 μM, correspondingly. GA-ampicillin and hamamelitannin-erythromycin may be potent combinations and promising candidates for eradicating pathogenic E. coli in humans and animals.

Keywords: ampicillin; antibacterial agents; bacterial pathogenicity; erythromycin; synergistic effect.

Figure 1

Time-kill curves of Escherichia coli ATCC25922 after treatment with (A) HAMA-ERY combination, and (B) GA-AMP combination. Data were analyzed by one-way analysis of variance with F-test. Data represented as mean ± standard deviation of three independent experiments (n = 3). AMP, ampicillin; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin; MIC, minimum inhibitory concentration.

Figure 2

Effect of HAMA-ERY combination and GA-AMP combination on the ultrastructure morphology of Escherichia coli ATCC25922 cells. Escherichia coli cells treated with (A,F) no drug, (B) ERY (1× MIC), (C) HAMA (1× MIC), (D) HAMA (1× MIC) + ERY (1× MIC), (E) HAMA (½× MIC) + ERY (½× MIC), (G) AMP (1× MIC), (H) GA (1× MIC), and (I) GA (1× MIC) + AMP (1× MIC), (J) GA (½× MIC) + AMP (½× MIC), and examined the cells by scanning electronic microscope. Three independent experiments (n = 3) were performed. Representative images of bacterial cells treated with individual and combination antibacterials are shown, where the images are in 10,000× magnification. Yellow colored arrows indicate septa of binary fission. AMP, ampicillin; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin; MIC, minimum inhibitory concentration.

Figure 3

Effects of HAMA-ERY combination (A) and GA-AMP combination (B) on planktonic cells of Escherichia coli ATCC25922. Effects of HAMA-ERY combination (C) and GA-AMP combination (D) on biofilm cells of Escherichia coli ATCC25922. Data were analyzed by one-way analysis of variance with F-test. Data represented as mean ± standard deviation of three (n = 3) independent experiments. * Represents significance difference (P < 0.05) among the effects of individual drugs and combination drug within each concentration group. ** Represents significance difference (P < 0.05) compared with the untreated control group. AMP, ampicillin; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin; MIC, minimum inhibitory concentration.

Figure 4

Effects of HAMA-ERY and GA-AMP combinations on the viability of Escherichia coli ATCC25922 biofilm. The CLSM images of SYTO9- and propidium iodide- stained biofilms after treated with (A) [(a) no drug, (b) HAMA (1× MIC), (c) ERY (1× MIC), (d) HAMA (1× MIC) + ERY (1× MIC)], and (B) [(a) no drug, (b) GA (1× MIC), (c) AMP (1× MIC), (d) GA (1× MIC) + AMP (1× MIC)]. The biofilm cell viabilities were evaluated by using BacLight LIVE/DEAD stain (red: dead cell, green: live cell). In each combined image such as image (a) or (b) or (c) or (d), the top-left image segment shows only the SYTO9-stained green fluorescent cells (live cells), the top-right image segment represents only the propidium iodide-stained red fluorescent cells (dead cells), and the below-left and -right image segments show both the SYTO9-stained green fluorescent cells (live cells) and propidium iodide-stained red fluorescent cells (dead cells) in together. In each combined image, all image segments are shown as two-dimensional except the below-right image segment; only the below-right image segment is represented as three-dimensional. Representative images of Escherichia coli biofilm cells from three independent experiments (n = 3) are presented in this figure. Images shown in this figure are in 50× magnification. AMP, ampicillin; CLSM, confocal laser scanning microscope; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin; MIC, minimum inhibitory concentration.

Figure 5

Percentages (%) of biofilm-biomasses of Escherichia coli after treatment with (A) HAMA-ERY and (B) GA-AMP combinations. In each test group, biomasses of “Total biofilm” were thought to be 100%, and with this consideration the percent (%) biomasses of dead (Propidium Iodide-stained) biofilm and live (SYTO9-stained) biofilm were calculated. Data were analyzed by one-way analysis of variance with F-test. Data represented as mean ± standard deviation of three (n = 3) independent experiments. * Represents significance difference (P < 0.05) among the effects of individual drugs and combination drugs within each concentration group. ** Represents significance difference (P < 0.05) compared with the untreated control group. AMP, ampicillin; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin; MIC, minimum inhibitory concentration.

Figure 6

Representative images of swim and swarm zones of Escherichia coli ATCC25922 treated with HAMA-ERY and GA-AMP combinations. Representative images of three independent experiments (n = 3) are presented in this figure. Images shown in this figure are in 1× magnification. AMP, ampicillin; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin; MIC: minimum inhibitory concentration.

Figure 7

Effects of (A) HAMA-ERY and (B) GA-AMP combinations on the viability of Rattus norvegicus (IEC-6) cells. Data were analyzed by one-way analysis of variance with F-test. Data represented as mean ± standard deviation of three (n = 3) independent experiments. * Represents significance difference (P < 0.05) among the effects of individual drugs and combination drugs within each concentration group. ** Represents significance difference (P < 0.05) compared with the untreated control group. AMP, ampicillin; ERY, erythromycin; GA, gallic acid; HAMA, hamamelitannin.