Rose Cooper

The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds

Aims: To determine the sensitivity to honey of Gram‐positive cocci of clinical significance in wounds and demonstrate that inhibition is not exclusively due to osmotic effects.

Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins.

Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes.

Absence of bacterial resistance to medical-grade manuka honey.

Clinical use of honey in the topical treatment of wounds has increased in Europe and North America since licensed wound care products became available in 2004 and 2007, respectively. Honey-resistant bacteria have not been isolated from wounds, but there is a need to investigate whether honey has the potential to select for honey resistance. Two cultures of bacteria from reference collections (Staphylococcus aureus NCTC 10017 and Pseudomonas aeruginosa ATCC 27853) and four cultures isolated from wounds (Escherichia coli, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and S. epidermidis) were exposed to sub-lethal concentrations of manuka honey in continuous and stepwise training experiments to determine whether the susceptibility to honey diminished. Reduced susceptibilities to manuka honey in the test organisms during long-term stepwise resistance training were found, but these changes were not permanent and honey-resistant mutants were not detected. The risk of bacteria acquiring resistance to honey will be low if high concentrations are maintained clinically.

Manuka honey inhibits cell division in methicillin-resistant Staphylococcus aureus

OBJECTIVES The aim of this study was to investigate the effect of manuka honey, artificial honey and an antibacterial component (methylglyoxal) on cell division in methicillin-resistant Staphylococcus aureus (MRSA). METHODS Viability of epidemic MRSA-15 NCTC 13142 incubated with manuka honey, artificial honey and methylglyoxal was determined, and structural effects monitored by electron microscopy. Activity of murein hydrolase (a peptidoglycan-degrading enzyme implicated in cell separation, encoded by atl) was estimated by cell wall hydrolysis and zymography; expression of atl was quantified by real-time PCR. RESULTS Growth of MRSA was inhibited by 5%, 10% and 20% (w/v) manuka honey and 10% (w/v) artificial honey containing methylglyoxal, but not 10% (w/v) artificial honey. Statistically significantly increased numbers of cells containing septa and increased cell diameter (P < 0.001 and P < 0.001, respectively) were found in MRSA exposed to 5%, 10% or 20% (w/v) manuka honey, but not 10% (w/v) artificial honey with and without methylglyoxal. Intracellular activity of murein hydrolase was elevated in MRSA grown in 10% (w/v) artificial honey and at undetectable levels in MRSA treated with 10% (w/v) manuka honey. Increased atl expression was found in MRSA treated with 10% (w/v) manuka honey and 10% artificial honey containing methylglyoxal. CONCLUSIONS Enlarged cells containing septa were observed in MRSA exposed to inhibitory concentrations of manuka honey, suggesting that cell division was interrupted. These changes were not caused by either the sugars or methylglyoxal in honey and indicate the presence of additional antibacterial components in manuka honey.

Manuka Honey Used to Heal a Recalcitrant Surgical Wound

This report documents the use of manuka honey to suc-cessfully treat a recalcitrant wound resulting from surgi-cal treatment of hidradenitis suppurativa. The wound hadfailed to heal during 3 years of treatment with conven-tional therapies and following four surgical procedures.After treatment with dressings impregnated with irradiat-ed manuka honey was initiated, the patient’s recurrentstaphylococcal infections ceased, and healing wasachieved within 4 months.In March 1999 a 38-year-old female with a recalci-trant surgical wound of 36 months’ duration requestedtreatment with topical honey. She had first been diag-nosed with hidradenitis suppurativa localised in the leftaxilla in January 1977. Her right axilla, inframammaryfolds and groin were unaffected, and she was otherwisefit and healthy. Abscesses in the left axilla were treatedwith incision and drainage in 1977, 1991 and, again, inMarch 1996. However, the wound that resulted from thesurgical treatment in 1996 failed to heal, and the patientcomplained of pain, limited movement and offensiveodour emanating from the wound. Wide excisions ofchronically indurated, recalcitrant tissue from the left ax-illa were performed in 1997 and 1998, but the woundstill failed to heal. A wide range of therapeutic dressingswas applied with limited effect.Recurrent wound infections complicated the healingprocess. Staphylococcus aureus(resistant to penicillinbut sensitive to methicillin, flucloxacillin, erythromycin,fusidic acid, cephalexin and gentamicin) was isolated onfive occasions, twice in combination with coryneformbacteria, and once with nonhaemolytic streptococci.Atypical mycobacteria were not sought. Antimicrobialtreatment regimens included systemic co-amoxiclav, ci-profloxacin, flucloxacillin, metronidazole, topical silversulphadiazine, silver nitrate and povidone-iodine. DuringSeptember and October 1997 a prolonged course of flu-cloxacillin combined with metronidazole was adminis-tered. Although healing progressed with this therapy, ab-normal granulation tissue developed and regression oc-curred within 10–14 days of treatment cessation.The patient’s shoulder was marginally abducted as aresult of scarring. In an attempt to reintroduce healthyundamaged skin to the area and to improve the range ofshoulder movement, the scarred area was excised andcovered by a rotational skin flap in November 1998. Ini-tially, the range of movement of the shoulder joint im-proved, but 3 months later the distal end of the flapbroke down and Staphylococcus aureuswas again isolat-ed. At this point (having endured 4 operations and 36months of failure to heal), the patient requested treat-ment with honey.Thus, in March 1999, co-amoxiclav was prescribed(375 mg thrice daily for 7 days) and Combine dressings(Smith & Nephew, UK) impregnated with 25–35 g activemanuka honey and sterilised by gamma radiation wereapplied to the infected wound and covered by absorbentpads. The manuka honey used had inhibitory activityagainst Staphylococcus aureusATCC 25923 equivalentto that of 13% (w/v) phenol, as determined by an in vitroassay with catalase added to exclude activity due to hy-drogen peroxide [1]. The patient was provided with hon-ey dressings and instructed to redress the wound at 24-hintervals, following showering. The wound was moni-tored and swabbed at weekly visits to the clinic.A noticeable improvement in the appearance of thewound was observed within 1 week of applying the hon-ey-impregnated dressings; it became smaller and less in-flamed, and the already healed, scarred area becamemore pliable. Bacteria were absent at 7 days, and the re-

Synergy between oxacillin and manuka honey sensitizes methicillin-resistant Staphylococcus aureus to oxacillin

OBJECTIVES Honey is an ancient wound remedy that has recently been introduced into modern clinical practice in developed countries. Manuka honey inhibits growth of methicillin-resistant Staphylococcus aureus (MRSA) by preventing cell division. In Gram-negative bacteria a synergistic interaction between honey and antibiotics has been suggested. We aimed to determine the effect of manuka honey on oxacillin resistance in MRSA. METHODS Inhibition of MRSA by manuka honey and oxacillin separately and in combination was tested by disc diffusion, Etest strips, serial broth dilution, chequerboards and growth curves. RESULTS Manuka honey and oxacillin interacted synergistically to inhibit MRSA. Manuka honey reversed oxacillin resistance in MRSA, and down-regulation of mecR1 was found in cells treated with manuka honey. CONCLUSIONS Microarray analysis showed that exposure of MRSA to inhibitory concentrations of manuka honey resulted in down-regulation of mecR1. Here we demonstrated that subinhibitory concentrations of honey in combination with oxacillin restored oxacillin susceptibility to MRSA. Other honey and antibiotic combinations must now be evaluated.

Effect of manuka honey on the expression of universal stress protein A in meticillin-resistant Staphylococcus aureus

Staphylococcus aureus is an important pathogen that can cause many problems, from impetigo to endocarditis. With its continued resistance to multiple antibiotics, S. aureus remains a serious health threat. Honey has been used to eradicate meticillin-resistant S. aureus (MRSA) strains from wounds, but its mode of action is not yet understood. Proteomics provides a potent group of techniques that can be used to analyse differences in protein expression between untreated bacterial cells and those treated with inhibitory concentrations of manuka honey. In this study, two-dimensional (2D) electrophoresis was combined with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to determine the identities of proteins whose levels of expression were changed at least two-fold following treatment with manuka honey. Protein extracts were obtained from cells grown in tryptone soy broth (with or without manuka honey) by mechanical disruption and were separated on 2D polyacrylamide gels. A protein was isolated in gels prepared from untreated cell extract that was absent from gels made using honey-treated cell extract. Using MALDI-TOF MS, the protein was identified as universal stress protein A (UspA). Downregulation of this protein was confirmed by real-time polymerase chain reaction (PCR), which showed a 16-fold downregulation in honey-treated cells compared with untreated samples. This protein is involved in the stress stamina response and its downregulation could help to explain the inhibition of MRSA by manuka honey.

Manuka honey is bactericidal against Pseudomonas aeruginosa and results in differential expression of oprF and algD.

The presence of Pseudomonas aeruginosa in cutaneous wounds is of clinical significance and can lead to persistent infections. Manuka honey has gained ground in clinical settings due to its effective therapeutic action and broad spectrum of antibacterial activity. In this study, the effect of manuka honey on P. aeruginosa was investigated using MIC, MBC, growth kinetics, confocal microscopy, atomic force microscopy and real-time PCR. A bactericidal mode of action for manuka honey against P. aeruginosa was deduced (12 %, w/v, MIC; 16 %, w/v, MBC) and confirmed by confocal and atomic force microscopy, which showed extensive cell lysis after 60 min exposure to inhibitory concentrations of manuka honey. The inability of honey-treated cells to form microcolonies was demonstrated and investigated using Q-PCR for three key microcolony-forming genes: algD, lasR and oprF. The expression of algD increased 16-fold whereas oprF expression decreased 10-fold following honey treatment; lasR expression remained unaltered. These findings confirm that manuka honey is effective at inducing cell lysis and identify two targets, at the genetic level, that might be involved in this process.

Biofilms in wounds: a review of present knowledge

Following confirmation of the presence of biofilms in chronic wounds, the term biofilm became a buzzword within the wound healing community. For more than a century pathogens have been successfully isolated and identified from wound specimens using techniques that were devised in the nineteenth century by Louis Pasteur and Robert Koch. Although this approach still provides valuable information with which to help diagnose acute infections and to select appropriate antibiotic therapies, it is evident that those organisms isolated from clinical specimens with the conditions normally used in diagnostic laboratories are mainly in a planktonic form that is unrepresentative of the way in which most microbial species exist naturally. Usually microbial species adhere to each other, as well as to living and non-living surfaces, where they form complex communities surrounded by collectively secreted extracellular polymeric substances (EPS). Cells within such aggregations (or biofilms) display varying physiological and metabolic properties that are distinct from those of planktonic cells, and which contribute to their persistence. There are many factors that influence healing in wounds and the discovery of biofilms in chronic wounds has provided new insight into the reasons why. Increased tolerance of biofilms to antimicrobial agents explains the limited efficacy of antimicrobial agents in chronic wounds and illustrates the need to develop new management strategies. This review aims to explain the nature of biofilms, with a view to explaining their impact on wounds.

Improving Antibiotic Activity against Wound Pathogens with Manuka Honey In Vitro

Following the discovery of synergistic action between oxacillin and manuka honey against methicillin-resistant Staphylococcus aureus, this study was undertaken to search for further synergistic combinations of antibiotics and honey that might have potential in treating wounds. Fifteen antibiotics were tested with and without sublethal concentrations of manuka honey against each of MRSA and Pseudomonas aeruginosa using disc diffusion, broth dilution, E strip, chequerboard titration and growth curves. Five novel antibiotic and manuka honey combinations were found that improved antibacterial effectiveness in vitro and these offer a new avenue of future topical treatments for wound infections caused by these two important pathogens.