Honey Has Broad-spectrum in vitro Antibacterial Activity
It is well documented that honey is effective against numerous pathogenic microorganisms including bacteria, fungi and viruses. A significant amount of early work focused on multifloral or pasture honeys and their efficacy against Gram-positive organisms, especially Staphylococcus aureus and its antibiotic-resistant variants. A total of 58 strains of coagulase-positive S. aureus isolated from infected wounds were found to be sensitive to both manuka honey and pasture honey.[19] Sensitivity remained consistent between the strains and inhibitory concentrations of honey were between 2 and 4% (w/v). Following on from this study, 18 wound isolates of methicillin-resistant Staphylococcus (MRSA) and seven wound isolates of vancomycin-resistant enterococci were found to be susceptible to two different types of pasture honey at concentrations of 10% (w/v) and below – data indicated that the antibiotic sensitive counterparts for these microorganisms were no more or less susceptible to treatment.[20]Staphylococcus epidermidis used to be regarded as a commensal member of the skin microbiota, but is now considered to be an opportunistic pathogen, often associated with the use of temporary or permanent invasive medical devices, such as endotracheal tubes or intravenous catheters. Methicillin-resistant variants of this species are beginning to emerge with serious consequences for effective treatment. Agar incorporation assays determined that this microorganism was also susceptible to both pasture and manuka honey treatment, suggesting that there might be potential to use medical grade manuka honeys prophylactically at the insertion sites for medical devices to try to limit the likelihood of infection developing.[21]
Wider ranging studies screened numerous microorganisms encompassing both Gram-negative and Gram-positive bacteria and fungi from a range of anatomical sites including the skin, GI tract, oral cavity and genitourinary tract.[22] Of these microorganisms, only Serratia marcescens and the pathogenic yeast Candida albicans were not inhibited by the presence of different honeys up to a concentration of 20% (w/v). Irish et al. revealed a mean inhibitory concentration of honey against Candida spp. of between 34.6 and 43.1% (w/v), and these studies never observed Candida colonization or infection in wounds treated with medical honey in adequate concentrations.[23]
It has been suggested that honey might be an efficacious treatment for gastric ulcers caused by Helicobacter pylori. Two studies have demonstrated that honey can inhibit growth of H. pylori at concentrations ranging from between 20% (w/v) up to 82% (w/v) in vitro.[24–26] However, in practice it is unlikely that honey would effectively 'cure' gastric ulcers since it is not known how long it would be in contact with organisms in the stomach or to what extent it would be diluted to ineffective levels by bodily fluids. Unlike antibiotic treatments given for gastric ulcers, honey is not a systemic therapy and therefore would have limited use. A small patient study was undertaken to clarify this in six volunteers with H. pylori gastric ulcers. Each individual was given a tablespoon of manuka honey four-times a day for 6 weeks. Following treatment, no improvement was seen and the patients still gave a positive result for H. pylori compared with six members of the control group who were given a conventional treatment.[20] This demonstrates the necessity for such treatments to undergo clinical trial prior to acceptance into clinical practice.
Cellular Targets for Honey
In terms of understanding the mechanisms of action of honeys against microbial pathogens, much research has been undertaken using manuka honey because sterile preparations are commercially available. It is abundantly evident that manuka honey has activity against a wide variety of pathogens in vitro. It has been observed that antibiotic-sensitive pathogens and their equivalent antibiotic-resistant strains are equally susceptible to treatment.[20,27] In methicillin-sensitive S. aureus and MRSA the primary mode of action has been demonstrated to involve interruption of the cell cycle, whereby bacteria fail to divide leading to an accumulation of arrested cells with fully formed septum.[28] The cleavage of the septum is normally controlled by autolysins, which digest peptidoglycan to produce two daughter cells; it has been recognized that in stalled cells treated with manuka honey, there is loss of autolysin activity.[29] In addition to this, it has been demonstrated that treatment with manuka honey leads to the downregulation of the universal stress protein, UspA, in MRSA, reducing the ability of bacteria to survive conditions of cellular and metabolic stress.[29]
The mode of action of manuka honey against Pseudomonas aeruginosa is quite different. Manuka honey-treated P. aeruginosa exhibit widespread structural damage leading to cell lysis and death.[28] Atomic force microscopy and fluorescent microscopy revealed large membrane blebs in honey-treated cells suggesting that manuka honey resulted in damage to the cell envelope.[30] This was verified by genomic analysis showing that manuka honey treatment mediated a reduction in the expression of OprF, an integral membrane protein that is paramount for the structural stability of the cell envelope in Gram-negative microorganisms.[30] It is possible that future studies will reveal target sites that are specific for Gram-positive or Gram-negative microorganisms; although preliminary evidence suggests that target specificity might be unique to particular genera or even species.
Two different New Zealand honeys (manuka and kanuka honey) and clover honey were found to exert quite distinct alterations to growth dynamics and cellular morphology for Bacillus subtilis, Escherichia coli, S.aureus and P. aeruginosa.[31] Each microorganism exhibited a unique 'response profile' when exposed to inhibitory levels of these honeys. B. subtilis, S. aureus and E. coli had greatly extended lag phases when exposed to sublethal doses of each honey and growth was completely abrogated at concentrations exceeding 8–16%. During the extended lag phase, bacterial cells were also significantly shorter in length than usual. By contrast, the extended lag phase was not observed for P. aeruginosa and much higher concentrations of honey were required to completely inhibit growth; however, this is no surprise since P. aeruginosa is inherently more resistant to antimicrobial treatments than the other microorganisms tested. Interestingly, P. aeruginosa cells appeared longer than usual, perhaps a reflection of the cell envelope damage that was likely to be occurring. For both B. subtilis and S. aureus, the chromosome appeared condensed following honey treatment and did not exhibit the characteristic looped conformation seen during normal chromosome replication.[31] This was not the case for either E. coli or P. aeruginosa, adding weight to the hypothesis that at least some cellular targets, might be broadly specific for Gram-positive or Gram-negative microorganisms.
In E.coli, the multifactorial action of manuka honey has been highlighted by use of transcriptome analysis. This confirmed that many significant changes in gene regulation occur, including those involved in stress response, as previously seen in S. aureus.[32] In total, 2% of the genome was upregulated and 1% dowregulated, broadly these clustered into groups associated with stress response and proteins synthesis, respectively. Also noted in this study was an inability to induce resistance to honey in conditions that could rapidly produce resistance to antibiotics. The observed lack of resistance might be attributed to the fact that, unlike many antibiotics, honey has more than one target site and so the likelihood of acquiring resistance to multiple sites, although not impossible, is greatly reduced.
Honey as an Antivirulence Therapy
It is becoming increasingly apparent that honey impacts on the virulence of bacterial pathogens in addition to affecting cellular structure and metabolism. This is an attractive attribute for an antimicrobial, and studies investigating novel ways of treating bacterial infection are beginning to focus on antivirulence treatments rather than traditional bactericidal or bacteriostatic remedies. The major advantage is that antivirulence formulations do not afford the same selective evolutionary survival pressure that promotes the emergence of resistance. Three recent studies have described the mechanism by which honey inhibits quorum sensing and virulence.[33–35] Notably, the concentrations of honey that mediated this effect were far below the MIC. E. coli O157:H7 biofilm formation was disrupted by sublethal doses of honey, which was found to be mediated by repression of quorum sensing genes. Concurrently, a reduction in the expression of genes encoded on the locus of enterocyte effacement and curli genes (csgBAC) was observed; both of these operons are known to play a significant role in the virulence of this organism.[33] Similarly, subinhibitory concentrations of honey impaired quorum sensing in P. aeruginosa by reducing the expression of the las and rhl regulons and the transcriptional regulator MvfR.[35] The perturbation of these regulatory networks resulted in a reduction in expression of associated virulence factors, clearly demonstrating the global impact that honey has on the bacterial cell at the regulatory level. N-acyl-homoserine lactone production is also significantly reduced in Erwinia carotovora, Yersinia enterocolitica and Aeromonas hydrophila in response to chestnut honey, much like E. coli biofilm formation was also impaired, reiterating the close association between these two processes.[34] Biofilm formation and expression of virulence factors are fundamental to the successful colonization and subsequent pathology of many bacterial infections; thus, impairing this process could make honey an excellent prophylactic treatment.
One group of virulence factors that are known to be regulated by quorum sensing in numerous pathogenic microorganisms are the siderophores. These iron-chelating molecules are central to bacterial proliferation in the host environment, providing pathogens with a source of iron and iron acquisition itself is inherently linked to virulence. Pyocyanin, pyochelin and pyoverdin are all utilized by P. aeruginosa to sequester iron from the human host (pyoverdin and pyochelin are siderophores, whereas pyocyanin appropriates iron from transferrin).[36] Recently, in three different strains of P. aeruginosa, honey treatment mediated a marked reduction in siderophore production, although the assays used did not make it possible to distinguish between the different structural groups of siderophore. Much like previous studies into virulence regulation by honey, this effect was mediated by sublethal doses as low as 5% (w/v) which equated to quarter of the MIC.[37] Reduced production of pyocyanin in response to honey treatment was concomitant with a reduction in expression of genes involved in quorum sensing, demonstrating the global impact that altered gene expression can have on the expression of virulence factors.[35] Certainly the reduced capacity of pathogenic microorganisms to obtain iron from their host is detrimental to both colonization and subsequent progression of infection. Moreover these mechanisms demonstrate that honey functions via two independent mechanisms, being both bactericidal and antivirulent making it an attractive antimicrobial whose multifaceted action is not likely to promote resistance (Figure 1).
Figure 1.
The multiple effects of honey can be assigned to individual subgroups, but collectively exert a combined effect against numerous different types of microorganisms.
Future Microbiol. 2013;8(11):1419-1429. © 2013 Future Medicine Ltd.
