Synergism between Medihoney and rifampicin against methicillin-resistant Staphylococcus aureus (MRSA)

Abstract

Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.

Figure 1. Enhanced antibacterial activity of rifampicin-honey combination treatment against S. aureus.

(A) Time-kill curves for S. aureus NCTC8325 in CaMHB. Bacteria were incubated in 7% Medihoney, 0.2 µg/ml rifampicin, or both. A growth control using just CaMHB is included as indicated. Rif: rifampicin; *: below detection limit (<50 CFU/ml). (B) Filter discs containing 4 µg of rifampicin were placed on S. aureus NCTC8325 spread on TSA plates containing no honey (TSA), 5% sugar solution (sugar), 5% manuka honey, or 5% Medihoney. The shown plates were incubated at 37°C for 24 h. Red arrows denote rifampicin resistant colonies that appeared on the TSA and sugar control plates, but not on Medihoney or manuka honey plates. Minimum inhibitory concentrations of break-through colonies (clones 1–3) were determined against Medihoney and rifampicin (see Fig. 4). (C) Sensitivity of different S. aureus strains to rifampicin and honey using the agar disc diffusion assay. Diameter (in mm) of zones of inhibition around 4 µg-impregnated rifampicin discs on TSA plates without honey (red bars), and in the presence of either 5% sugar solution (blue bars), 5% manuka honey (green bars) or 5% Medihoney (black bars).

Figure 2. Growth curves of S. aureus NCTC8325 in CaMHB.

Bacteria were incubated with (A) 70 µg/ml MGO, 0.2 µg/ml rifampicin, or both, or with (B) 70 µg/ml MGO (in CaMHB with 7% sugar solution, MGO^S), 0.2 µg/ml rifampicin, or both. A growth control using just CaMHB is included as indicated. Rif is rifampicin.

Figure 3. Reversal of oxacillin resistance but not rifampicin resistance in S. aureus by Medihoney.

Oxacillin resistant MRSA RPAH18 and rifampicin resistant clone 1 (Fig. 1) were streaked out on TSA plates containing no honey (TSA), 5% sugar solution (sugar), or 5% Medihoney. Inhibition zones around filter discs containing 4 µg rifampicin (rif) or 4 µg oxacillin (oxa) were measured after incubation at 37°C for 24 h.

Figure 4. Susceptibility of break-through colonies to rifampicin and Medihoney.

Break-through colonies growing in the zone of inhibition around the rifampicin disc on TSA plates (clone 1 and clone 2) and TSA plates with 5% sugar solution (clone 3) were selected (see Fig. 2A). Overnight cultures were prepared in CaMHB and minimum inhibitory concentrations (MIC) of rifampicin (A) and Medihoney (B) were determined. WT is wild type S. aureus NCTC8325.