Manuka honey inhibits siderophore production in Pseudomonas aeruginosa


Study one

 Manuka honey inhibits siderophore production in Pseudomonas aeruginosa

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Materials and methods

Bacterial strains Pseudomonas aeruginosa reference strain ATCC 9027 (NCIMB 8626) and clinical isolates 867 and LE08 were used throughout the study. The clinical isolates were from wound swabs collected from patients with chronic wounds who attended an outpatient clinic at the University Hospital of Wales, Cardiff. All strains were cultured aerobically at 37°C in nutrient broth. To attain iron restriction, 2,2-dipyridyl was added to the media to achieve a concentration of 2 mmol l1 .

Manuka honey

Sterile (gamma irradiated) medical grade manuka honey (MedihoneyTM) was provided by Comvita in 50-g tubes and is available as a licensed, commercial medical device (Comvita, Berkshire, UK). It is supplied as a standardized, 100% pure honey derived from the Leptospermum scoparium plant in New Zealand.

Minimum inhibitory concentration

The minimum inhibitory concentration for manuka honey against the test bacteria was determined by serial dilution (0 – 50% w/v) in a total volume of 5-ml nutrient broth (Oxoid, Cambridge, UK) (according to British Society for Antimicrobial Chemotherapy methodology for determining minimum inhibitory concentration (MIC); Andrews 2011; Roberts et al. 2012). Cultures were incubated for 16 h at 37°C in aerobic conditions. Assays were carried out in triplicate on each of three separate occasions. Where combinations of manuka honey and 2,2-dipyridyl were tested, cultures were incubated with 2,2-dipyridyl at concentrations of 0, 1, 2 and 3 mmol l1 in media containing manuka honey equivalent to ¼ and ½ MIC (for each strain tested). Cultures were incubated as described above. MICreadings were taken using a Spectrostar Nano spectrophotometer at a wavelength of 620 nm.

Chrome azurol S assay

The method to detect siderophore production was based on that described by Schwyn and Neilands (Schwyn and Neilands 1987), cultures were grown under conditions of iron restriction by the addition of 2,2-dipyridyl (2 mmol l1 was found to be the optimum concentration for siderophore production; data not shown) as described above. Chrome azurol S assay (CAS) solution was used for quanti- fication of siderophores in culture supernatants. Cultures were centrifuged for 10 000 g for 10 min and mixed with the CAS reagent at a ratio of 1 : 1. After reaching equilibration, absorbance readings were measured at 620 nm. CASagar plates were prepared as described previously (Deng et al. 2006). Standardized suspensions (OD 05 at 620 nm) of Ps. aeruginosa were inoculated onto separate CAS-agar plates. This procedure was performed in triplicate, and plates were incubated at 37°C for 48 h. Callipers were used to measure the diameter (mm) of observable zones.

Results

A combination of iron restriction and manuka honey treatment impairs growth of Pseudomonas aeruginosa The MIC for the reference strain (ATCC 9027) was found to be 30% (w/v) for manuka honey, and 10% (w/v) for each of the clinical isolates (LE08 and 867) (data not shown). For ATCC, 9027 growth was inhibited as the concentration of 2,2-dipyridyl increased, and the reduction was statistically significant with each increment (P < 005, using Student’s t-test) (Fig 1a). With 1 mmol l1 2,2-dipyridyl, ½ MIC manuka honey completely inhibited growth; with 2 and 3 mmol l1 2,2-dipyridyl, both ¼ and ½ MIC completely inhibited growth (Fig. 1a). The same pattern of inhibition was observed for clinical isolate 867 (Fig. 1b). However, LE08 required 2 mmol l1 2,2-dipyridyl and above combined with ¼ and ½ MIC to completely inhibit growth (Fig. 1c). When supplemented with 200 lmol l1 ferric citrate, MICs were restored to that equivalent to honey treatment alone (data not shown). This suggests that manuka honey treatment in combination with 2,2-dipyridyl was more deleterious for microbial growth than either condition individually. Manuka honey at sublethal concentrations inhibits siderophore production Each strain of Ps. aeruginosa was grown under ironlimited conditions and assessed for siderophore production Jo aeruginosa strains studied. Cultures were equilibrated to OD 05 (A620 nm) prior to assay. Quantitative CAS assay showed 18- and 22-fold reductions in siderophore production by strain ATCC 9027 treated with ¼ and ½ MIC manuka honey, respectively (Fig. 2). The clinical strains 867 and LE08 showed similar results of 16- and 13-fold (¼ MIC) and 22- and 18-fold (½ MIC) reductions in siderophore production, respectively, following honey treatment. In each case, the reduction in siderophore production compared to the untreated control, at either ¼ or ½ MIC, was found to be statistically significant (P < 005). However, there was no statistically significant reduction in siderophore production between ¼ and ½ MIC. CAS-agar plate assays supported this data, showing a marked reduction in siderophore production evident as a smaller zone of yellow colouration on the blue CAS-agar, following honey treatment at both ¼ and ½ MIC for each strain studied (Table 1). Again a statistically significant reduction in zone size was evident for both ¼ and ½ MIC as compared to the untreated control, but not between ¼ and ½ MIC.