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Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of ventilator-associated pneumonia (VAP). Patients with VAP have poorly functioning neutrophils, related to increased levels of the complement fragment C5a. The antibiotic linezolid has been useful in controlling MRSA-related VAP infections; however clinical benefit does not always correlate with antimicrobial effect, suggesting the possibility of immunomodulatory properties. Here the effects of linezolid on healthy and dysfunctional neutrophils (modelled by C5a-induced injury) was investigated. Functional assays (killing, phagocytosis, transmigration, and respiratory burst) were used to assess the effects of pre-, co- and post-incubating linezolid (0.4–40 mg/L) with healthy neutrophils relative to those with C5a-induced injury. C5a decreased neutrophil killing, and phagocytosis of MRSA. Furthermore, C5a significantly decreased neutrophil transmigration to IL-8, but did not affect respiratory burst. Co-incubation of linezolid significantly improved killing of MRSA by dysfunctional neutrophils, which was supported by concomitant increases in phagocytosis. Conversely linezolid impaired killing responses in healthy neutrophils. Pre- or post-incubation of linezolid prior or following C5a induced injury had no effect on neutrophil function. This study suggests that linezolid has immunomodulatory properties that protect human neutrophils from injury and provides insight into its mode of action beyond a basic antibiotic.

Human exposure to carbon black (CB) is inevitable due to its widespread applications in the medical, industrial and consumer sectors. With an ageing population, it is imperative that the effects of (nano)particle exposure in individuals with compromised immunity or infection are considered. Since barrier immunity provides the first line of defence against CB and the human skin and lung pathogen, Staphylococcus aureus, this work focuses on studying the impact of CB exposure upon compromised immunity during infection on human skin and lung epithelial cells in vitro. The principal aim of the work was to develop an epithelial cell model to characterise (co-)exposure to CB and S. aureus. The work used two human epithelial cell lines, HaCaT (skin) and A549 (lung), ELISA technology to assess the (pro-)inflammatory response, aseptic microbiology techniques to grow S. aureus and a Zetasizer, EDX spectroscopy, and both scanning and transmission electron microscopy (SEM and TEM) to characterise the CB under the conditions used in the study. Physicochemical characterisation of CB confirmed its shape, dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth, but in a biological media dependent manner. CB did not induce cytokines or antimicrobial peptides from skin and lung epithelial cells, when given alone. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both skin and lung epithelial cells. IL-6 and human beta defensin (hβD)-2 could only be detected when cells were stimulated with S. aureus. However, co-exposure to CB (100µg/ml) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus only induced levels). Furthermore, the same co-exposure induced significantly more hβD-2 (compared to S. aureus alone). The ability to detect pathogen responses to particle, in addition to epithelial responses to particle and pathogen is an advance on assessing cell responses under ‘healthy’ conditions and supports the need for developing exposure models under stressed or immunosuppressed conditions. This model will be useful for studying mechanisms of exposure in at-risk groups, including factory workers, the elderly and immunocompromised. Advanced models, that better represent human pathophysiology are essential for understanding cellular mechanisms of toxicity in the 21st Century.

Sepsis is defined as life threatening organ dysfunction caused by a dysregulated host response to infection, and is responsible for 52,000 deaths in the UK per year. Approximately 50% of sepsis episodes are related to bacteria where the Gram-negative bacteria Escherichia coli is a leading causative agent. Our previous work with the Hywel Dda Health Board has identified a high level of E. coli sepsis and in the current work, we aim to identify genetic (genes) and host biomarkers (e.g. IL-6) to discriminate E. coli sepsis isolates based on original source of infection. E. coli isolates (n=100) from blood cultures in patients with defined sources of infection (urinary, biliary, intra-abdominal or unknown) were used to investigate potential biomarkers using next generation sequencing, whole blood modelling and molecular microbiology phenotyping. Sequencing of isolates is underway. Growth curve analysis demonstrated that human serum could modulate E. coli growth to three phenotype groups; i) no growth, ii) retarded / decreased growth and iii) unaffected growth (compared to LB control). Whole blood modelling over 6 hours confirmed 4 hours to be the optimal time point to study IL-6 expression. Grouping isolates by source of infection showed that those from the urinary tract and ‘unknown’ sources produced significantly more IL-6 than E. coli K12. Completion of phenotyping will allow association studies to bacterial genotype. These results will help define new biomarkers associated with the host and genetic biomarkers associated with E. coli that will better predict and inform the diagnosis and treatment of sepsis.

This article was published Open Access in Immunity, Inflammation and Disease (online) available at http://dx.doi.org/10.1002/iid3.153

The susceptibility of the cell-wall free bacterial pathogens Ureaplasma spp. to Manuka honey was examined. The minimum inhibitory concentration (MIC) of Manuka honey for four Ureaplasma urealyticum and four Ureaplasma parvum isolates was determined. Sensitivity to honey was also compared to clinical isolates with resistance to tetracycline, macrolide and fluoroquinolone antibiotics. Finally step-wise resistance training was utilised in an attempt to induce increased tolerance to honey. The MIC was dependent on the initial bacterial load with 7.5 % and 18.0 % w/v honey required to inhibit U. urealyticum at 1 and 10 6 colour changing units (CCU), respectively, and 4.8 % and 15.3 % w/v required to inhibit U. parvum at 1 and 10 6 CCU, respectively. MIC values were consistently lower for U. parvum compared with U. urealyticum. Antimicrobial activity was seen against tetracycline resistant, erythromycin resistant and ciprofloxacin resistant isolates at 10 5 CCU. No resistance to honey was observed with fifty consecutive challenges at increasing concentrations of honey. This is the first report of the antimicrobial activity of Manuka honey against a cell-wall free bacterial pathogen. The antimicrobial activity was retained against antibiotic resistant strains and it was not possible to generate resistant mutants.

Staphylococcus pseudintermedius is an important member of the skin microbial community in animals and can cause opportunistic infections in both pets and their owners. The high incidence of antimicrobial resistance in S. pseudintermedius highlights that this opportunistic zoonotic pathogen can cause infections which require prolonged and intensive treatment to resolve. Manuka honey has proven efficacy against many bacterial pathogens and is an accepted topical treatment for infections in both veterinary and clinical practice, and so it is a particularly appropriate antimicrobial for use with zoonotic pathogens such as S. pseudintermedius. Here, we demonstrate that not only is manuka honey highly potent against novel multidrug-resistant S. pseudintermedius isolates, it also acts synergistically with clinically relevant antibiotics. In addition, manuka honey modulates S. pseudintermedius virulence activity, even at subinhibitory concentrations. In a clinical setting, these attributes may assist in controlling infection, allowing a more rapid resolution and reducing antibiotic use., Staphylococcus pseudintermedius causes opportunistic infections in dogs. It also has significant zoonotic potential, with the emergence of multidrug resistance leading to difficulty treating both animal and human infections. Manuka honey has previously been reported to inhibit many bacterial pathogens, including methicillin-resistant Staphylococcus aureus, and is successfully utilized in both clinical and veterinary practice. Here, we evaluated the ability of manuka honey to inhibit strains of S. pseudintermedius grown alone and in combination with antibiotics, as well as its capacity to modulate virulence within multiple S. pseudintermedius isolates. All 18 of the genetically diverse S. pseudintermedius strains sequenced and tested were inhibited by ≤12% (wt/vol) medical-grade manuka honey, although tolerance to five clinically relevant antibiotics was observed. The susceptibility of the isolates to four of these antibiotics was significantly increased (P ≤ 0.05) when combined with sublethal concentrations of honey, although sensitivity to oxacillin was decreased. Virulence factor (DNase, protease, and hemolysin) activity was also significantly reduced (P ≤ 0.05) in over half of isolates when cultured with sublethal concentrations of honey (13, 9, and 10 isolates, respectively). These findings highlight the potential for manuka honey to be utilized against S. pseudintermedius infections. IMPORTANCE Staphylococcus pseudintermedius is an important member of the skin microbial community in animals and can cause opportunistic infections in both pets and their owners. The high incidence of antimicrobial resistance in S. pseudintermedius highlights that this opportunistic zoonotic pathogen can cause infections which require prolonged and intensive treatment to resolve. Manuka honey has proven efficacy against many bacterial pathogens and is an accepted topical treatment for infections in both veterinary and clinical practice, and so it is a particularly appropriate antimicrobial for use with zoonotic pathogens such as S. pseudintermedius. Here, we demonstrate that not only is manuka honey highly potent against novel multidrug-resistant S. pseudintermedius isolates, it also acts synergistically with clinically relevant antibiotics. In addition, manuka honey modulates S. pseudintermedius virulence activity, even at subinhibitory concentrations. In a clinical setting, these attributes may assist in controlling infection, allowing a more rapid resolution and reducing antibiotic use.

This chapter explains how to plan and undertake several in vitro investigations shed light on bacterial interactions in the broader context of infectious disease. This includes co-aggregation, mixed biofilm, relative competitive indices and determining rates of mutation.

This chapter takes you through all of the practical considerations you will need to address before you enter the bacteriology laboratory, as well as the basic skills you are likely to need once you have started your work. From setting up the initial laboratory bench space through to assessing the risk of the work to be carried out, this chapter allows the reader to master basics such as aseptic pipetting, media preparation and filtering of solutions. It will also provide details on the units and calculations you will come across and the equipment that you will use in your day-to-day laboratory experience.

The information contained in Chapter 3 should enable to reader to prepare bacterial cultures for staining, set up and use a light microscope and calibrate the microscope to take measurements of bacteria. From simple stains to differential stains that can be used to identify bacteria, by the end of this chapter the reader will have the material required to choose an appropriate stain and microscope and troubleshoot some common problems encountered when first using these techniques. It also provides a brief overview of some of the more complex microscopes and techniques available if the reader wishes to move on to more advanced microscopy.

This chapter introduces and describes two in vivo invertebrate infection models. These are a useful first step beyond in vitro analysis. It describes how to set up these models and collect and present the data produced.

Pseudomonas aeruginosa causes problematic chronic lung infections in those suffering from cystic fibrosis. This is due to its antimicrobial resistance mechanisms and its ability to form robust biofilm communities with increased antimicrobial tolerances. Using novel antimicrobials or repurposing current ones is required in order to overcome these problems. Manuka honey is a natural antimicrobial agent that has been used for many decades in the treatment of chronic surface wounds with great success, particularly those infected with P. aeruginosa. Here we aim to determine whether the antimicrobial activity of manuka honey could potentially be repurposed to inhibit pulmonary P. aeruginosa infections using two ex vivo models. P. aeruginosa isolates (n = 28) from an international panel were tested for their susceptibility to manuka honey and clinically relevant antibiotics (ciprofloxacin, ceftazidime, and tobramycin), alone and in combination, using conventional antimicrobial susceptibility testing (AST). To increase clinical applicability, two ex vivo porcine lung (EVPL) models (using alveolar and bronchiolar tissue) were used to determine the anti-biofilm effects of manuka honey alone and in combination with antibiotics. All P. aeruginosa isolates were susceptible to manuka honey, however, varying incidences of resistance were seen against antibiotics. The combination of sub-inhibitory manuka honey and antibiotics using conventional AST had no effect on activity against the majority of isolates tested. Using the two ex vivo models, 64% (w/v) manuka honey inhibited many of the isolates where abnormally high concentrations of antibiotics could not. Typically, combinations of both manuka honey and antibiotics had increased antimicrobial activity. These results highlight the potential of manuka honey as a future antimicrobial for the treatment of pulmonary P. aeruginosa isolates, clearing potential infection reservoirs within the upper airway.

This chapter introduces some widely used standardised models to assess the capacity for bacteria to form biofilms. Several different static biofilm models are described with suggestions for their application. In addition, a number of biofilm flow systems are described considering their uses and applicability for modelling bacterial infection.

This chapter will provide readers with basic knowledge needed to culture bacteria successfully in the laboratory. Highlighting the range of growth media available, this chapter should provide readers with confidence to choose appropriate media and culture conditions for experiments they wish to carry out. It will equip readers with an understanding of how to measure bacterial growth by a variety of methods, isolate individual bacterial colonies and use differential and selective media to gain basic information about their bacterial culture. There are hints and tips on common issues encountered in each technique described, which should ensure that any bacterial culture carried out is problem-free.

s This chapter explains how to plan and undertake several in vitro investigations that will shed light on bacterial interactions in the broader context of infectious disease. This includes co-aggregation, mixed biofilm, relative competitive indices and determining rates of mutation.

s This chapter will guide the reader through the main considerations and planning required to use simple, cell-based infection models. This includes preparing for tissue culture, choosing an appropriate cell line, examples of cell infection models and downstream analysis of data derived from such models.

Having introduced some of the concepts of quantitative polymerase chain reaction (Q-PCR) in the previous chapter, this chapter deals with some of the more practical considerations for setting up and running Q-PCR. It provides information about reagents, commercially available kits, and suppliers as well as suggesting some basic protocols that can be easily modified by the user. Additionally, it describes how to analyze data using the ΔΔCt method and how this can be converted into fold-changes to give an indication of differential gene expression, normalized to a reference or housekeeping control.

Polymerase chain reaction (PCR) can be utilized as a molecular tool for functional analysis of genes and can be used in concert with protein and gene expression to thoroughly describe the role of a given gene or genes. This chapter will describe how PCR can be used to prepare construct that are necessary to generate knockout mutations and how to assign function by complementing gene mutations using Kochs' molecular postulates. This chapter uses prokaryotes as a model for generating knockout mutations, and the procedures described will need to be adapted for single celled eukaryotes such as yeast and for more complex organisms, which are beyond the scope of this book.

This chapter provides practical advice on designing polymerase chain reaction primers and optimizing them. From using databases to identify genes and ordering primers, the reader is guided through the practical aspects of primer design and the common pitfalls. Guidance on how to test a new pair of primers is given along with a trouble-shooting guide.

This chapter will describe how polymerase chain reaction amplified genes or gene fragments can be used in cloning experiments for a number of applications including complementation, functional analysis of proteins, or proteins overexpression prior to purification by chromatography. The reader will be able to utilize the skills learned in Chapters 1–3 Chapter 1 Chapter 2 Chapter 3 and apply them to the techniques described here.

This chapter provides practical advice on what needs to be addressed before undertaking polymerase chain reaction (PCR). From keeping the workspace nuclease free to recipes and shopping lists; information that is vital know and understand before putting on a lab coat and entering the lab, is described. The reader is guided through the practical aspects of preparing to undertake PCR with examples of reagents provided as well as a list of common manufacturers of PCR reagents. Safety considerations are discussed and thought is given to equipment and reagents that will be required for analysis of the first PCR reaction.

This chapter explains how to analyze your template DNA and end point polymerase chain reaction (PCR) reaction using classical electrophoresis techniques as well as by spectrophotometry. It explains how to interpret what you see to determine yield and purity of the DNA/PCR product. This chapter also offers useful recipes for basic reagents necessary to carry out analysis of PCR by electrophoresis, which can be adapted to the user's needs.

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.

The purpose of this study was to investigate the effects of manuka honey on the structural integrity of Pseudomonas aeruginosa ATCC 27853. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of manuka honey for P. aeruginosa were determined by a microtitre plate method, and the survival of bacteria exposed to a bactericidal concentration of manuka honey was monitored. The effect of manuka honey on the structure of the bacteria was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). The MIC and MBC values of manuka honey against P. aeruginosa were 9.5% (w/v) and 12% (w/v) respectively; a time-kill curve demonstrated a bactericidal rather than a bacteriostatic effect, with a 5 log reduction estimated within 257 min. Using SEM, loss of structural integrity and marked changes in cell shape and surface were observed in honey-treated cultures. With TEM, these changes were confirmed, and evidence of extensive cell disruption and lysis was found. Manuka honey does not induce the same structural changes in P. aeruginosa as those observed in staphylococci. Our results indicate that manuka honey has the potential to be an effective inhibitor of P. aeruginosa.

The purpose of this study was to investigate the effect of manuka honey on Staphylococcus aureus in order to identify the intracellular target site. The mode of inhibition of manuka honey against S. aureus NCTC 10017 was investigated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the effect of time on viability. Structural changes were observed by scanning (SEM) and transmission electron microscopy (TEM) of cells suspended for 4 h at 37 degrees C in 0.05 mM Tris buffer containing 10% (w/v) manuka honey and were compared to cells in buffer alone or buffer containing 10% (w/v) artificial honey (to assess osmotic damage). A bactericidal mode of inhibition for manuka honey on S. aureus was established. Marked structural changes in honey-treated cells were seen only with TEM, where a statistically significant increase in the number of whole cells with completed septa compared to untreated cells were observed (P0.05). Structural changes found with TEM suggest that honey-treated cells had failed to progress normally through the cell cycle and accumulated with fully formed septa at the point of cell division without separating. Sugars were not implicated in this effect. The staphylococcal target site of manuka honey involves the cell division machinery.

Antimicrobial resistance (AMR) is an increasing clinical problem precipitated by the inappropriate use of antibiotics in the later parts of the 20th Century. This problem, coupled with the lack of novel therapeutics in the development pipeline, means AMR is reaching crisis point, with an expected annual death rate of ten million people worldwide by 2050. To reduce, and to potentially remedy this problem, many researchers are looking into natural compounds with antimicrobial and/or antivirulence activity. Manuka honey is an ancient antimicrobial remedy with a good track record against a wide range of nosocomial pathogens that have increased AMR. Its inhibitory effects are the result of its constituent components, which add varying degrees of antimicrobial efficacy to the overall activity of manuka honey. The antimicrobial efficacy of manuka honey and some of its constituent components (such as methylglyoxal and leptosperin) are known to bestow some degree of antimicrobial efficacy to manuka honey. Despite growing in vitro evidence of its antimicrobial efficacy, the in vivo use of manuka honey (especially in a clinical environment) has been unexpectedly slow, partly due to the lack of mechanistic data. The mechanism by which manuka honey achieves its inhibitory efficacy has recently been identified against Staphylococcus aureus and Pseudomonas aeruginosa, with both of these contrasting organisms being inhibited through different mechanisms. Manuka honey inhibits S. aureus by interfering with the cell division process, whereas P. aeruginosa cells lyse in its presence due to the reduction of a key structural protein. In addition to these inhibitory effects, manuka honey is known to reduce virulence, motility, and biofilm formation. With this increasing in vitro dataset, we review the components and our mechanistic knowledge of manuka honey and how manuka honey could potentially be utilized in the future to impact positively on the treatment of microbial, resistant infections.\ud\udKeywords: Staphylococcus aureus, Pseudomonas aeruginosa, biofilm, antibiotic resistance

Pseudomonas and Burkholderia pose a significant health threat to people with chronic respiratory conditions; the resistance inherent in these bacteria indicates that new antimicrobial strategies are required. Susceptibility of 56 strains of P. aeruginosa and 55 strains of Burkholderia to manuka honey, tobramycin and colistin using microbroth dilution and E strip was determined. MICs of antibiotics with honey were determined to search for synergistic combinations against two representative strains of each genus. All strains exhibited susceptibility to honey ≤10 % (w/v); mean susceptibility of Burkholderia (4.6 % w/v) was lower than P. aeruginosa (7.3 % w/v). Synergistic or additive combinations were found with all four strains tested. Combinations of manuka honey with antibiotics can be used to lower the MIC need to successfully inhibit both P. aeruginosa and B. cepacia. The use of honey as a combination agent may be possible for the management of P. aeruginosa and B. cepacia.

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen. Its resistance to multiple antibiotics and its prevalence in healthcare establishments make it a serious threat to human health that requires novel interventions. Manuka honey is a broad-spectrum antimicrobial agent that is gaining acceptance in the topical treatment of wounds. Because its mode of action is only partially understood, proteomic and genomic analysis was used to investigate the effects of manuka honey on MRSA at a molecular level. Methods Two-dimensional gel electrophoresis with dual-channel imaging was combined with matrix-assisted laser desorption ionization–time of flight mass spectrometry to determine the identities of differentially expressed proteins. The expression of the corresponding genes was investigated by quantitative PCR. Microarray analysis provided an overview of alterations in gene expression across the MRSA genome. Results Genes with increased expression following exposure to manuka honey were associated with glycolysis, transport and biosynthesis of amino acids, proteins and purines. Those with decreased expression were involved in the tricarboxylic acid cycle, cell division, quorum sensing and virulence. The greatest reductions were seen in genes conferring virulence (sec3, fnb, hlgA, lip and hla) and coincided with a down-regulation of global regulators, such as agr, sae and sarV. A model to illustrate these multiple effects was constructed and implicated glucose, which is one of the major sugars contained in honey. Conclusions A decreased expression of virulence genes in MRSA will impact on its pathogenicity and needs to be investigated in vivo.

Resistance to antibiotics continues to rise and few new therapies are on the horizon. Honey has good antibacterial activity against numerous microorganisms of many different genera and no honey-resistant phenotypes have yet emerged. The mechanisms of antimicrobial activity are just beginning to be understood; however, it is apparent that these are diverse and often specific for certain groups or even species of bacteria. Manuka honey has been most thoroughly characterized and is commercially available as a topical medical treatment for wound infections. Furthermore, since most data are available for this honey, there is a considerable focus on it in this review. It is becoming evident that honeys are more than just bactericidal, as they impact on biofilm formation, quorum sensing and the expression of virulence factors. With this in mind, honey represents an attractive antimicrobial treatment that might have the potential to be used alongside current therapies as a prophylactic or to treat wound infection with multidrug-resistant bacteria in future.

Aim: To characterize the effect of manuka honey on medically important wound bacteria in vitro, focusing on its antiadhesive properties. Materials & methods: Crystal violet biofilm assays, fluorescent microscopy, protein adhesion assay and gentamicin protection assay were used to determine the impact of manuka honey on biofilm formation, human protein binding and adherence to/invasion into human keratinocytes. Results: Manuka honey effectively disrupted and caused extensive cell death in biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes. Sublethal doses of manuka honey inhibited bacterial adhesion to the fibronectin, fibrinogen and collagen. Manuka honey impaired adhesion of laboratory and clinical isolates of S. aureus, P. aeruginosa and S. pyogenes to human keratinocytes in vitro, and inhibited invasion by S. pyogenes and homogeneous vancomycin intermediate S. aureus. Conclusion: Manuka honey can directly affect bacterial cells embedded in a biofilm and exhibits antiadhesive properties against three common wound pathogens.

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.

Antimicrobial agents have been used with varying success in the treatment of wound infections for thousands of years. Without knowing the identity of wound pathogens, ancient civilizations develope...

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.