1 Honey Research Unit, Department of Biological Sciences University of Waikato, Private Bag 3105, Hamilton, New Zealand; 2 Centre for Biomedical Sciences, School of Applied Sciences, University of Wales Institute, Cardiff, UK
Received 22 February 2005; returned 31 March 2005; revised 12 May 2005; accepted 15 May 2005
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Abstract |
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Methods: An agar incorporation technique was used to determine the minimum active dilution, with dilution steps of 1% (v/v) honey [or steps of 5% (v/v) of a sugar syrup matching the osmotic effect of honey]. The plates were inoculated with 10 µL spots of cultures of the isolates.
Results: The honeys were inhibitory at dilutions down to 3.6 ± 0.7% (v/v) for the pasture honey, 3.4 ± 0.5% (v/v) for the manuka honey and 29.9 ± 1.9% (v/v) for the sugar syrup.
Conclusions: Typical honeys are about eight times more potent against coagulase-negative staphylococci than if bacterial inhibition were due to their osmolarity alone. Therefore, honey applied to skin at the insertion points of medical devices may have a role in the treatment or prevention of infections by coagulase-negative staphylococci.
Keywords: invasive medical devices , antibiotic resistance , minimum active dilution , manuka honey , device-related infections
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Introduction |
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The severe consequences to patients with such infections demands effective strategies designed to minimize and eliminate infections.1 Although use of electric fields to improve antibiotic therapy, modification of polymeric biomaterial to reduce bacterial adherence and incorporation of antimicrobial agents into devices to reduce bacterial growth have been explored,1 an optimum solution has yet to be found.
Honey is increasingly being used in the management of infected wounds where conventional pharmaceutical products are failing, especially now that CE-marked sterile honey and honey-impregnated dressings are available;4 thus it is reasonable to consider prophylactic application of honey in situ at device exit sites.
Although susceptibility to the antibacterial activity of honey of other pathogens in vitro has been established,57 coagulase-negative staphylococci have not been tested. This study was undertaken to determine their susceptibility to honey in vitro. The antibacterial activity of honey varies not only between floral sources but even within one floral source,8 so representative honeys with median levels of activity were used. The antimicrobial activity in most honeys is due to the enzymic production of hydrogen peroxide, but honey from some Leptospermum species, such as manuka, can also have a high antimicrobial activity due to an unidentified phytochemical component:8 both types of activity were studied. Also, to distinguish these activities from any osmotic inhibition of bacteria, a syrup simulating honey was included in the study as a control.
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Materials and methods |
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The two natural honeys used were selected to be close to the median antibacterial activity for each type of activity, tested against Staphylococcus aureus (ATCC 25923):9 a manuka honey with non-peroxide activity equivalent to 16.8% (w/v) phenol and a pasture honey with hydrogen peroxide activity equivalent to 17.5% (w/v) phenol. The simulated honey was prepared by combining 38.4 g of fructose, 30.3 g of glucose, 1.3 g of sucrose, 8.6 g of maltose and 1.4 g of maltodextrin with 17.2 mL of distilled water.
Bacterial isolates
Isolates of coagulase-negative staphylococci were obtained from 18 Waikato Hospital patients. Cultures were isolated from midstream and catheter urines, peritoneal fluid, cerebrospinal fluid, breast aspirate, a peritoneal catheter tip and blood cultures. The isolates were identified using a range of biochemical and morphological techniques, and the Vitek automated bacterial identification instrument (McDonnell Douglas Health System Company).
The isolates were stored on Protect Bacterial Preserver Beads (LabSupply Pierce) at 70°C. We confirmed identity of the isolates to species level by means of BBL Crystal Gram-positive kits (Becton Dickinson N.Z.).
Microbiological materials
Tryptic soy broth (TSB) was obtained from Difco Laboratories. Nutrient agar was obtained from Scharlau Laboratories. Blood agar base was obtained from Merck Laboratories, and 5% sterile defibrinated sheep's blood (Life Technologies N.Z.) was added.
Determination of minimum active dilution of honey
Prior to testing, each isolate was cultured from preserver beads by inoculating two beads into 9 mL of TSB and incubating for 16 h at 37°C. Cultures obtained were diluted with TSB to obtain 23 x 107 cfu/mL, the minimum to produce confluent growth at inoculation positions.
The minimum active dilution of each honey for each of the clinical isolates was determined by an agar incorporation technique. Nutrient agar was made up at double strength, measured out into 25 mL aliquots and autoclaved. To prepare the plates it was melted and tempered in a 50°C water-bath until poured. Solutions of the two natural honey samples (at a concentration of 20% v/v) and the simulated honey (at a concentration of 70% v/v) were prepared in sterile de-ionized water immediately prior to performing an assay and diluted with different volumes of sterile de-ionized water to give double the final concentration required in a volume of 25 mL. These solutions were then also tempered at 50°C, then each mixed with one of the 25 mL lots of double-strength nutrient agar. The various agarhoney mixtures were then poured into duplicate Petri dishes.
A dilution series with honey concentrations in the range 110% (v/v) final honey concentration, in 1% increments, was used for the susceptibility assays for the natural honeys, and in the range 535% (v/v) final honey concentration, in 5% increments, for the simulated honey. Duplicate control plates of nutrient agar with no honey were included in each susceptibility assay to confirm the viability and density of the cultures.
Samples (10 µL) of each culture were inoculated onto the agar plates in three rows of three spots using an eight-channel auto-pipettor with tips attached to channels 1, 3 and 5, to obtain nine strains inoculated per plate as evenly spaced spots. Duplicate plates were inoculated and assays were repeated on two subsequent days, with fresh subcultures on each occasion.
The inoculated plates were incubated at 37°C for 16 h, and then growth, partial inhibition or complete inhibition was recorded at each inoculation position. The minimum active dilution was taken to be the lowest concentration of honey at which bacterial growth was completely inhibited, and the mean value for the minimum active dilution was calculated from the six replicates for each isolate.
Analysis of variance was carried out using GenStat (Lawes Agricultural Trust).
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Results |
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Discussion |
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These findings show that coagulase-negative staphylococci are very similar to S. aureus5,6 in their susceptibility to honey of similar antibacterial potency and more susceptible than Pseudomonas aeruginosa7 and Enterococcus species.6 Thus, they can be expected to be controlled by honey in vivo since there are many reports of honey rapidly healing wounds infected with S. aureus and pseudomonads.4 The results show that honey could be diluted by exudate up to 20-fold and still inhibit the growth of coagulase-negative staphylococci. Honey, therefore, would be suitably active for both therapeutic and prophylactic application. The Leptospermum honey in licensed products on sale for wound care in Australia, Europe and New Zealand4 has a standardized level of antibacterial activity close to that of the honey samples used in this study, and so the results are relevant to clinical usage.
There are other advantages in applying honey to the traumatized tissue around medical devices. Its anti-inflammatory activity can be expected to prevent serous exudates, which can provide a medium for bacteria to colonize.10 Also, its physical properties provide moist conditions ideal for healing and it has a stimulatory action on growth of wound repair tissues.10 Furthermore, unlike other antiseptics it has no harmful effects on tissues, the slow enzymic production of hydrogen peroxide giving about one thousandth of that in a 3% hydrogen peroxide solution.10
The development of honey in the form of a rubbery gel that can be moulded to conform to any shape4 will further increase the practicality of use with medical devices beyond that with the honey-impregnated dressings currently available. It remains for further clinical evaluation to be tried.
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Transparency declarations |
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Acknowledgements |
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References |
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2 Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative staphylococci. Clin Microbiol Rev 1994; 7: 11740.[Abstract]
3 Vandenesch F, Eykyn SJ, Etienne J. Infections caused by newly-described coagulase negative staphylococci. Rev Med Microbiol 1995; 6: 94100.
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