Anti-pseudomonad Activity of Manuka Honey and Antibiotics in a Specialized ex vivo Model Simulating Cystic Fibrosis Lung Infection

Abstract

Pseudomonas aeruginosa causes problematic chronic lung infections in those suffering from cystic fibrosis. This is due to its antimicrobial resistance mechanisms and its ability to form robust biofilm communities with increased antimicrobial tolerances. Using novel antimicrobials or repurposing current ones is required in order to overcome these problems. Manuka honey is a natural antimicrobial agent that has been used for many decades in the treatment of chronic surface wounds with great success, particularly those infected with P. aeruginosa. Here we aim to determine whether the antimicrobial activity of manuka honey could potentially be repurposed to inhibit pulmonary P. aeruginosa infections using two ex vivo models. P. aeruginosa isolates (n = 28) from an international panel were tested for their susceptibility to manuka honey and clinically relevant antibiotics (ciprofloxacin, ceftazidime, and tobramycin), alone and in combination, using conventional antimicrobial susceptibility testing (AST). To increase clinical applicability, two ex vivo porcine lung (EVPL) models (using alveolar and bronchiolar tissue) were used to determine the anti-biofilm effects of manuka honey alone and in combination with antibiotics. All P. aeruginosa isolates were susceptible to manuka honey, however, varying incidences of resistance were seen against antibiotics. The combination of sub-inhibitory manuka honey and antibiotics using conventional AST had no effect on activity against the majority of isolates tested. Using the two ex vivo models, 64% (w/v) manuka honey inhibited many of the isolates where abnormally high concentrations of antibiotics could not. Typically, combinations of both manuka honey and antibiotics had increased antimicrobial activity. These results highlight the potential of manuka honey as a future antimicrobial for the treatment of pulmonary P. aeruginosa isolates, clearing potential infection reservoirs within the upper airway.

Keywords: Pseudomonas aeruginosa; antimicrobial susceptibility testing; biofilms; cystic fibrosis; ex vivo model; manuka honey.

FIGURE 1

Minimum Inhibitory Concentrations (MICs) of P. aeruginosa Reference Panel (PaRP) isolates associated with Cystic Fibrosis (CF) to manuka honey (expressed as % [w/v]; A) and three clinically relevant antibiotics (expressed as μg/ml); ciprofloxacin (B), ceftazidime (C) and tobramycin (D). PaRP strains found to be sensitive/resistant to the antibiotics tested (B–D), based on EUCAST breakpoints (dashed horizontal line), are colored light/dark gray, respectively (this does not apply to manuka honey (A) where there is no EUCAST breakpoint).

FIGURE 2

Fractional Inhibitory Concentration Index (FICI) of sub-inhibitory manuka honey concentrations supplemented with one of three clinically relevant antibiotics; ciprofloxacin (A), ceftazidime (B) and tobramycin (C). The interaction between manuka honey and antibiotics were interpreted as ’synergistic (FICI < 0.5), “antagonistic” (FICI > 4.0), and “no interaction” (FICI > 0.5–4.0).

FIGURE 3

Viability of P. aeruginosa Reference Panel (PaRP) isolates grown in ex vivo Porcine Lung (EVPL) alveolar tissue for 24 h and treated with ASM (A), ciprofloxacin (B), ceftazidime (C), and tobramycin (D) supplemented with 32 and 64% w/v manuka honey for 16 h. Dashed horizontal lines indicate the detection level of the EVPL model. Error bars denote Standard Deviation.

FIGURE 4

Viability of three P. aeruginosa Reference Panel (PaRP) isolates; #1, #17, and #28, grown in ex vivo Porcine Lung (EVPL) bronchiolar tissue for 24 h and treated with ASM or clinically relevant antibiotics (Cip; Ciprofloxacin, Tob; Tobramycin, and Caz; Ceftazidime) supplemented with 32% and 64% (w/v) manuka honey for 16 h. Dashed horizontal lines indicate the detection level of the model. Error bars denote Standard Deviation. Varying levels of statistical significance denoted by ^∗, ^∗∗, and ^∗∗∗ (P≤0.05, 0.001, and 0.0001 respectively).

FIGURE 5

Scanning Electron Microscopy (SEM) images of P. aeruginosa Reference Panel (PaRP) isolate #11 (LES B58) recovered from ex vivo Porcine Lung (EVPL) bronchiole tissue samples grown in Artificial Sputum Media (ASM) with/without 2048 μg/ml ceftazidime and supplemented with/without 32/65% (w/v) manuka honey for 16 h. Images show bacteria cells (red arrows) and their subsequent inhibition with increasing antimicrobial concentrations. Scale bar = 20 μm.