Boosting the Antibacterial Activity of Azithromycin on Multidrug-Resistant Escherichia coli by Efflux Pump Inhibition Coupled with Outer Membrane Permeabilization Induced by Phenylalanine-Arginine β-Naphthylamide

Abstract

The global spread of multidrug-resistant (MDR) bacteria increases the demand for the discovery of new antibiotics and adjuvants. Phenylalanine-arginine β-naphthylamide (PAβN) is an inhibitor of efflux pumps in Gram-negative bacteria, such as the AcrAB-TolC complex in Escherichia coli. We aimed to explore the synergistic effect and mechanism of action of PAβN combined with azithromycin (AZT) on a group of MDR E. coli strains. Antibiotic susceptibility was tested for 56 strains, which were screened for macrolide resistance genes. Then, 29 strains were tested for synergy using the checkerboard assay. PAβN significantly enhanced AZT activity in a dose-dependent manner in strains expressing the mphA gene and encoding macrolide phosphotransferase, but not in strains carrying the ermB gene and encoding macrolide methylase. Early bacterial killing (6 h) was observed in a colistin-resistant strain with the mcr-1 gene, leading to lipid remodeling, which caused outer membrane (OM) permeability defects. Clear OM damage was revealed by transmission electron microscopy in bacteria exposed to high doses of PAβN. Increased OM permeability was also proven by fluorometric assays, confirming the action of PAβN on OM. PAβN maintained its activity as an efflux pump inhibitor at low doses without permeabilizing OM. A non-significant increase in acrA, acrB, and tolC expression in response to prolonged exposure to PAβN was noted in cells treated with PAβN alone or with AZT, as a reflection of bacterial attempts to counteract pump inhibition. Thus, PAβN was found to be effective in potentiating the antibacterial activity of AZT on E. coli through dose-dependent action. This warrants further investigations of its effect combined with other antibiotics on multiple Gram-negative bacterial species. Synergetic combinations will help in the battle against MDR pathogens, adding new tools to the arsenal of existing medications.

Keywords: Escherichia coli; azithromycin; efflux pump inhibitor; phenylalanine-arginine β-naphthylamide; synergy.

Figure 1

Reduction in AZT MIC when used in combination with PAβN. (A) Fold reduction of AZT MIC in combination with different doses of PAβN (2–32 µg/mL). The fold reduction shown is the mean ± SD of all the strains regardless of the AZT MIC, while (B) shows the relation of AZT MIC (alone) with the genotypes of the strains with fold reduction in AZT MIC when used in combination with different doses of PAβN. n represents the number of strains per group.

Figure 2

Synergistic effect of PAβN with AZT, demonstrated by time-kill graphs. A selected E. coli strain (CDC AR-0346) is shown. Different synergistic combinations of PAβN (2–32 µg/mL) and AZT (1–32 µg/mL) were tested (A–E). PAβN and AZT (32 µg/mL) were used as controls in addition to an untreated growth control (GC). PAβN had no antibacterial activity when used alone, while synergistic combinations with AZT ≤ ½ X MIC killed the bacteria, depending on the concentration of PAβN used. The data shown represent the mean value of duplicates from two independent experiments ± SD.

Figure 3

Assessment of outer membrane permeability by 1-N-phenylnaphthylamine (NPN) assay in six selected E. coli strains. Data shown represent % of NPN uptake by bacteria treated with a serial dilution (0.5–128 μg/mL) of colistin, used as a positive control (A), and PAβN (B). Four selected bacteria treated with the synergetic combination of PAβN and AZT (concentrations reported between the brackets, respectively) at FICI in comparison with treatment with a single agent are shown in (C). ns represents a non-significant difference. The EC469 and EC500 strains were not included in figure (C) as synergy was not effective on these two strains, which carried both ermB and mphA, in contrast to the other strains, which carried the mphA gene only.

Figure 4

Transmission electron micrographs of E. coli strain EC477. Untreated cells (A) and cells treated with AZT—128 µg/mL (B) are shown with intact outer membranes (OMs) and clear periplasmic spaces (PS). Damage to the OM was obvious in bacterial cells treated with the positive control (colistin—2 µg/mL) (C), PAβN alone—64 µg/mL (D), and PAβN—64 µg/mL plus AZT—128 µg/mL (E). Magnified sections are shown below each figure to demonstrate an intact OM (A,B) versus a damaged OM (C,E) caused by the treatment. Magnification scale is 50–200 nm.

Figure 5

Transmission electron micrographs of E. coli strain EC500. Untreated cells (A) and cells treated with AZT—128 µg/mL (B) are shown with intact outer membranes (OMs) and clear periplasmic spaces (PS). Damage to the OM was obvious in bacterial cells treated with the positive control (colistin—2 µg/mL) (C), PAβN alone—64 µg/mL (D), and PAβN—64 µg/mL plus AZT—128 µg/mL (E). Magnified sections are shown below each figure to demonstrate an intact OM (A,B) versus a damaged OM (C,E) caused by the treatment. Magnification scale is 50–200 nm.

Figure 6

NPN efflux pump assay. Six selected bacterial strains (shown in the figure legend) were treated with serial dilutions of PAβN (A), colistin (B), or CCCP (C), either without adding glucose (marked with - g) or with glucose (marked with + g). The fluorescence level shown is after 5 min of incubation of bacteria pretreated with the test agents, after adding glucose to the appropriate cultures while keeping glucose-untreated cultures as comparators.

Figure 7

Change in NPN fluorescence over time, with and without glucose treatment, after exposure to PAβN (A–C), colistin (D,E), and CCCP (F). Fluorescence was recorded over 5 min of incubation time. Values are expressed as mean ± standard deviation. Untreated controls are shown in figure (A), and were not included in the other figures to avoid overlapping with other curves with low fluorescence levels. - g: without glucose; + g: with glucose.

Figure 8

Relative expression of acrA (A), acrB (B), and tolC (C) in selected bacterial strains treated with AZT, PAβN alone, and in combination. Some strains are marked with NS (no synergy between AZT and PAβN) or S (susceptible to AZT), while non-marked strains are those which were resistant to AZT and responded well to the synergistic action of PAβN.

Figure 8

Relative expression of acrA (A), acrB (B), and tolC (C) in selected bacterial strains treated with AZT, PAβN alone, and in combination. Some strains are marked with NS (no synergy between AZT and PAβN) or S (susceptible to AZT), while non-marked strains are those which were resistant to AZT and responded well to the synergistic action of PAβN.