An In Vitro Model to Assess Early Immune Markers Following Co-Exposure of Epithelial Cells to Carbon Black (Nano)Particles in the Presence of S. aureus: A Role for Stressed Cells in Toxicological Testing.

The exposure of human lung and skin to carbon black (CB) is continuous due to its widespread applications. Current toxicological testing uses 'healthy' cellular systems; however, questions remain whether this mimics the everyday stresses that human cells are exposed to, including infection. Staphylococcus aureus lung and skin infections remain prevalent in society, and include pneumonia and atopic dermatitis, respectively, but current in vitro toxicological testing does not consider infection stress. Therefore, investigating the effects of CB co-exposure in 'stressed' infected epithelial cells in vitro may better approximate true toxicity. This work aims to study the impact of CB exposure during Staphylococcus aureus infection stress in A549 (lung) and HaCaT (skin) epithelial cells. Physicochemical characterisation of CB confirmed its dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth in cell culture media. CB did not induce cytokines or antimicrobial peptides from lung and skin epithelial cells, when given alone, but did reduce HaCaT and A549 cell viability to 55% and 77%, respectively. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both lung and skin epithelial cells. IL-6 and human beta defensin (hßD)-2 could only be detected when cells were stimulated with S. aureus with no decreases in cell viability. However, co-exposure to CB (100 µg/mL) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus alone) without further reduction in cell viability. Furthermore, the same co-exposure induced significantly more hßD-2 (compared to S. aureus alone). This work confirms that toxicological testing in healthy versus stressed cells gives significantly different responses. This has significant implications for toxicological testing and suggests that cell stresses (including infection) should be included in current models to better represent the diversity of cell viabilities found in lung and skin within a general population. This model will have significant application when estimating CB exposure in at-risk groups, such as factory workers, the elderly, and the immunocompromised.

A pilot study investigating the effects of a manuka honey sinus rinse compared to a standard sinus rinse on sino-nasal outcome test scores in cystic fibrosis patients.

BACKGROUND: People with cystic fibrosis (CF) are prone to bacterial respiratory infections; these are often antibiotic resistant, are difficult to treat, and impact on the quality of life and lung function. The upper respiratory tract can act as a reservoir for these pathogens, and as part of clinical care, sinus rinses are used to alleviate symptoms in the upper airway. We have developed a sinus rinse containing manuka honey, to identify whether it can help improve symptoms or reduce the bacterial load. METHODS: We will undertake a randomised controlled trial where 30 adults with CF will be recruited and randomised to either the control or intervention group. Both groups will follow a sinus rinse protocol for 30 days (± 7 days); the control group will use the standard of care rinse, and the intervention group will use a manuka honey rinse. Both groups will provide samples at day 0 and day 30. The primary outcome measure will be a change in the 22-item Sino-Nasal Outcome Test (SNOT-22) score. Secondary outcomes will include changes to quality of life (questionnaire), bacterial load/community composition, and sputum viscosity. DISCUSSION: This trial will look at the use of a manuka honey-infused sinus rinse solution on patients diagnosed with cystic fibrosis (CF) suffering with sinusitis; it will allow us to determine the efficacy of the manuka honey sinus rinse compared to standard rinse and will allow us to determine if molecular bacterial diversity analysis will provide in-depth information beyond the usual conventional microbiological. It will allow us to determine the feasibility of recruiting participants to this type of trial, allow us to check participant compliance with the protocol, and inform future studies. TRIAL REGISTRATION: Approval was obtained from the Research Ethics Committee Wales REC7 reference 18/WA/0319. Results of this study will be published at international conferences and in peer-reviewed journals; they will also be presented to the relevant stakeholders and research networks. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier NCT04589897 (retrospectively registered).

Contrasting effects of linezolid on healthy and dysfunctional human neutrophils: reducing C5a-induced injury.

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.

Antibacterial and Antivirulence Activity of Manuka Honey against Genetically Diverse Staphylococcus pseudintermedius Strains.

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.IMPORTANCEStaphylococcus 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.

Anti-pseudomonad Activity of Manuka Honey and Antibiotics in a Specialized ex vivo Model Simulating Cystic Fibrosis Lung Infection.

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.

Honey is potentially effective in the treatment of atopic dermatitis: Clinical and mechanistic studies.

INTRODUCTION: As manuka honey (MH) exhibits immunoregulatory and anti-staphylococcal activities, we aimed to investigate if it could be effective in the treatment of atopic dermatitis (AD). METHODS: Adult volunteers with bilateral AD lesions were asked to apply MH on one site overnight for seven consecutive days and leave the contralateral site untreated as possible. Three Item Severity score was used to evaluate the response. Skin swabs were obtained from both sites before and after treatment to investigate the presence of staphylococci and enterotoxin production. In addition, the ability of MH and its methanolic and hexane extracts to down regulate IL4-induced CCL26 protein release from HaCaT cells was evaluated by enzyme linked immunosorbent assay. Also, the ability of MH to modulate calcium ionophore-induced mast cell degranulation was assessed by enzyme immunoassay. RESULTS: In 14 patients, AD lesions significantly improved post MH treatment versus pre-treatment as compared to control lesions. No significant changes in the skin staphylococci were observed after day 7, irrespective of honey treatment. Consistent with the clinical observation, MH significantly down regulated IL4-induced CCL26 release from HaCaT cells in a dose-dependent manner. This effect was partially lost, though remained significant, when methanolic and hexane extracts of MH were utilized. In addition, mast cell degranulation was significantly inhibited following treatment with MH. CONCLUSIONS: MH is potentially effective in the treatment of AD lesions based on both clinical and cellular studies through different mechanisms. This needs to be confirmed by randomized and controlled clinical trials.

A demonstration of the susceptibility of clinical isolates obtained from cystic fibrosis patients to manuka honey.

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.

Proteomic and genomic analysis of methicillin-resistant Staphylococcus aureus (MRSA) exposed to manuka honey in vitro demonstrated down-regulation of virulence markers.

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.

Honey: a sweet solution to the growing problem of antimicrobial resistance?

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.

Manuka honey inhibits adhesion and invasion of medically important wound bacteria in vitro.

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.

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.

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.

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.

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.