Summary

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has now been described globally, as a clinically significant pathogen, particularly associated with skin and soft tissue infections, including abscesses, cellulitis and furunculosis. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of healthcare-associated HA-MRSA-related wound infection, as well as honey’s ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. Although previous studies have examined the antimicrobial activity of honey against HA-MRSA, such data are limited regarding the activity of honey against this emerging type of MRSA. CA-MRSA (n ¼ 6 isolates), was examined for its susceptibility to natural honey (n ¼ 3 honey produced from bees in Northern Ireland and one commercial French honey). Results demonstrated that all honey was able to reduce the cultural count of all CA-MRSA from approximately 106 colony-forming units (cfus) (mean ¼ 6.46 log10 cfu/g) to none detectable within 24 h of co-culture of separate CA-MRSA organisms individually with all four-honey types examined. Subsequent non-selective enrichment of honey demonstrated that inoculated honey remained positive for CA-MRSA until 72 h postinoculation, after which point no culturable organisms could be detected. This study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate if this antimicrobial activity has any clinical application.

Introduction

To date, there has been an extensive awareness of methicillin-resistant Staphylococcus aureus (MRSA) within healthcare facilities, particularly hospitals. More recently, there has been increased reporting of MRSA occurring in the community amongst healthy individuals who have no hospital association. The organisms responsible for these community cases are termed community-associated MRSA (CA-MRSA) and differ significantly from healthcare associated MRSA (HA-MRSA). Although all are S. aureus, they have distinct epidemiological and microbiological characteristics. CA-MRSA has recently emerged in the US as a clinically significant and virulent pathogen. It is associated with serious skin and soft tissue infections, particularly in young healthy individuals in the community and those who have no risk factors for acquisition of HA-MRSA.

Several reports have described this organism in individuals in prisons, military personnel, athletes (especially those involved in combat and ball sports, including rugby, American football, wrestling, fencing), male homosexuals and ethnic populations (native American Indians, Hawaiian islanders, Alaskan-native people). Risk factors for its acquisition include close physical contact, abrasion injuries and activities associated with poor communal hygiene (e.g. sharing towels). This organism is now emerging in several European countries, including the UK. While HA-MRSA cause heterogenous invasive infections, CA-MRSA is usually limited to skin and soft tissue infections, particularly folliculitis, pustular lesions and abscesses. Less commonly, CA-MRSA can cause severe and rapidly fatal infections such as necrotising pneumonia and necrotising fasciitis.

In order to minimize the potential development of further antimicrobial resistance ‘‘The Copenhagen Recommendations: Report from the Invitational EU Conference on The Microbial Threat’’ were published, which outlined the need for the development of ‘‘Novel principles for treating or preventing infections in humans and animals’’. Although honey has historically been known to have antimicrobial activity, to date, no reports have examined such activity against CA-MRSA. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of HA-MRSA-related wound infection, as well as honey’s ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. It was therefore the aim of this small study to examine the potential antimicrobial activity of natural honey against a collection of CA-MRSA organisms.

Materials and Methods

Natural honey samples were obtained from amateur bee-keepers in Northern Ireland. Down heather honey, a light-colored honey from Comber, Co. and an additional light-colored honey. In addition, a commercial French honey from the Suisse Normande was also included as a comparator honey.

Prior to inoculation with the CA-MRSA isolates, each honey was sterilized with gamma irradiation to a dose of 25 kGy. Irradiation was carried out using cobalt-60 (in a Gammabeam-650 irradiation unit; MDS Nordion, Kanata, Canada) at a dose rate of 15 kGy h and at an environmental temperature of 4 1C. Red Perspex dosimeters (AEA Technology, Harwell, UK) were attached to the outside of each sample receiving 25 kGy to measure the actual dose received by the samples. Following irradiation, the change in absorbance of the dosimeters was measured spectrophotometrically (Spectronic Unicam UV-500; Thermospectronic Inc., Rochester, NY, USA) at 603 and 640 nm, respectively, and their thickness was measured using a digital electronic micrometre (RS Components Ltd., Corby, UK). The corresponding dose received was then obtained from a calibration graph provided by the National Physical Laboratory (Teddington, Middlesex, UK). Following irradiation, all doses were checked for sterility by enumeration of honey (100 ml) on Columbia Blood Agar (CBA) (Oxoid CM0331, Basingstoke, UK) supplemented with 5% (v/v) defribinated horse blood, at 37 1C for 48 h.

CA-MRSA (n ¼ 6 isolates) were included in this study, including CA-MRSA ST35, 5134, 4388, 4266, 4526 and 5090. All isolates were confirmed as well characterized CA-MRSA organisms by the Staphylococcal Reference Laboratory, Health Protection Agency, Colindale, London. Isolates were initially cultured on CBA for 24 h at 37 1C. Under aseptic conditions, serial dilutions of each CA-MRSA isolate were prepared individually in 0.1% [w/v] peptone saline (PS) (Oxoid CM0733) and approximately 106 colony-forming units (cfu) of each CA-MRSA strain was inoculated separately into 5 g of each sterile honey variety, as detailed above. CA-MRSA inocula were thoroughly mixed into each honey and the honey was stored in the dark for 24 h at ambient room temperature (approx. 18–20 1C). At time points, t ¼ 0, 4, 8 and 24 h, approximately 1 g honey was removed and enumerated by preparing appropriate serial dilutions in PS and enumerating the remaining culturable organisms in triplicate on CBA, which was incubated at 37 1C for 24 h and the quantitative counts expressed as log 10 cfu/g honey. Following this, and in order to confirm when no CAMRSA remained culturable, each CA-MRSA inocula/ honey combination underwent a non-selective enrichment in nutrient broth (Oxoid CM1) (225 ml) at 37 1C for 24 h, followed by plating of 100 ml enrichment broth on CBA which was incubated at 37 1C for 24 h. Any resulting colonies were confirmed as MRSA by conventional phenotypic assays. Appropriate controls were established which included examining the persistence of each CA-MRSA strain in 0.1% PS, as detailed above, in the absence of honey.

Statistical Analyses

Statistical analyses were performed where appropriate employing the Student t-test, where a probability value of po0.05 (5%) was considered significant.

Results and discussion

Quantitative counts of all CA-MRSA declined rapidly in all honey varieties tested, and culturable bacterial organisms were not detectable after 24 h of co-culture, as shown in Table 2. Survival of each CA-MRSA strain in the controls (without honey) remained unaltered (+0.04 log cfu/ml) and did not demonstrate any decline in bacterial numbers. Mean CA-MRSA declined by log 0.53 cfu/g in all honey varieties after 4 h, by 1.18 log cfu/g after 8 h and were not detectable after 24 h. There was no significant difference (p40.05) between survival of any of the CA-MRSA isolates or with the honey varieties examined, although the highest kill of CA-MRSA occurred in the Mournes honey. At 24 h co-culture, where no detectable organism was observed on enumerative plates, each honey/CA-MRSA combination was enriched in nutrient broth to demonstrate the potential survival of viable cells that were not detected by the enumeration method. Enrichment broths were positive for the presence of culturable CA-MRSA until 72 h co-incubation, after which time no culturable CA-MRSA were detected in any CAMRSA strain/honey combination.

The continued global rise of HA-MRSA within healthcare facilities coupled with the relatively recent emergence of CA-MRSA within the community, in combination with multi-resistance and evolving antibiotic resistance, respectively, merits an examination of alternative treatment regimens for these organisms, particularly associated with skin and soft tissue infections. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of HA-MRSA-related wound infection, as well as honey’s ease of topical application, makes the current study timely and of interest to healthcare practitioners involved with in wound management.

The medicinal and antimicrobial properties of honey in relation to wound treatment has been recognized for approximately 4500 years, where for instance, Prince Hal was treated with rose honey by John Bradmore, a London surgeon, relating to a facial wound, sustained at the battle of Shrewsbury in 1403. More recently, there has been a renewed medical interest in exploiting these antimicrobial properties, particularly in relation to wound management, with the publication of approximately 83 papers to date on the employment of honey in the treatment of various infections.

Previously, a small number of case studies have examined the antimicrobial activity of honey against MRSA organisms.

In the first study, MRSA was isolated from a recalcitrant surgical wound of a 80-year-old man, following a split skin graft harvested from his upper arm. Following application of Manuka honey tulle dressing, the wound healed within 2 weeks.

Additionally, a 64-year-old man had a postoperative infection to a radical forearm flap donor site, grafted with an abdominal full-thickness skin graft, which resolved with Manuka honey tulle and local debridement of eschar after 5 weeks treatment.

In a third case, a 47-year-old woman presented with a 2 month history of a painful ulcer over the right lateral malleolus, from which MRSA was isolated. Following commencement of Manuka honey, less MRSA was isolated at day 7 and at day 14 and at subsequent visits, no MRSA could be cultured from the ulcer.

Although these reports describe Manuka honey, as an active component in the resolution of wounds, no in vitro susceptibility data were presented to demonstrate the in vitro activity of the honey preparations against the MRSA isolated in situ. It was therefore the aim of the current study to examine the in vitro activity of various honey against CA-MRSA, which has not been reported to date with this organism.

In these studies, gamma-irradiated honey was employed for two reasons. Firstly, we wished to perform the bacteriological analyses in pure culture, using non-selective media, such as CBA, as the employment of a S. aureus selective medium, such as Baird–Parker medium, would have imposed a further stress/hurdle on potentially sub-lethally damaged organism due to the activity of the honey against these organisms and thus potentially gain a false (high) report of its antimicrobial activity. Secondly, food-grade honey produced for human consumption, although a shelf-stable product, is not a sterile product. In a previous study by our group, employing the same honey specimens, analyses of these honey products demonstrated the presence of various Bacillus spp.

In this study, seven samples of honey and related materials (propolis) were examined microbiologically and were demonstrated to have total viable counts (TVC) ranging from o100 to 1700 cfu/g. No yeasts or filamentous fungi were isolated from the honey materials and several bacterial isolates were identified using 16S rDNA and automated sequencing techniques, yielding two different genera (Paenibacillus and Bacillus), as well as four Bacillus species, namely Bacillus pumilus, B. licheniformis, B. subtilis and B. fusiformis, with B. pumilus the most frequently identified species present.

Therefore, healthcare practitioners, nurses and alternative therapists need to carefully note that food-grade honey is not a sterile product, which may be naturally contaminated with several organisms, that if applied to wounds, may actually act as an inoculum of potentially pathogenic skin pathogens to the lesion. Therefore, when advocating their use, practitioners should consider the employment of honey preparations that have been sterilized employing g-irradiation, so that no residual micro-flora remains and acts as potential inoculants of bacterial organisms.

In conclusion, this study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate the mechanism and components of such activity and whether this antimicrobial activity has any clinical application for the treatment of CA-MRSA skin and soft tissue infections.