In vitro activity of Western Australian honeys and Manuka honey against clinically important yeasts.

With the steady rise in antifungal resistance amongst clinically important yeasts, antifungal drug discovery remains of the utmost importance. To determine the potential of some honeys as alternative antifungal agents, we quantified the antifungal activity of 12 Western Australian honey samples, two Manuka honey samples and an artificial honey against 10 yeast isolates including clinical and reference strains. Results showed that the tested honeys varied in activity, and yeasts species also differed in susceptibility, with minimum inhibitory concentrations (MICs) determined by broth microdilution ranging from 8% to >44% w/v honey. Honeys with the highest overall activity were derived from Blackbutt (Eucalyptus patens), Jarrah (E. marginata), and Karri (E. diversicolor). The optical density of each MIC microtitre plate was determined after incubation and showed that at relatively low concentrations of honey the growth of all yeasts was enhanced compared to the untreated control, whereas at and above approximately 12% w/v, honeys exerted a dose-dependent growth inhibitory effect, the extent of which varied by honey type. Time-kill studies with 64% w/v honey showed that all eight of the natural honeys tested had greater fungicidal activity than the comparator artificial honey. Our findings suggest that the specific nectar-derived phytochemicals present within each honey play an important role in antifungal activity, and support the notion that activity is due to a combination of factors including osmotic activity, hydrogen peroxide and phytochemical compounds. These data indicate that honey is worthy of further investigation as a potential therapeutic agent for superficial yeast infections.

Changes in antibacterial activity, colour, and hydrogen peroxide content of Western Australian Jarrah and Marri honeys after storage at different temperatures over time.

AIMS: This study aimed to evaluate the effects of storage and different temperatures on the antibacterial activity and physicochemical characteristics of several types of honey. METHODS AND RESULTS: Honeys stored for 16 weeks at 37 and 45°C showed significant declines in antibacterial activity determined by minimum inhibitory concentrations, the loss of hydrogen peroxide, decreases in honey pH, and increases in honey colour, with changes most pronounced at 45°C. In contrast, honeys stored for 16 weeks at ambient (∼22°C) and cold (4, -20, and -80°C) temperatures showed only minor changes. In a second set of 12 honeys stored for 16-32 months at ambient temperature and then 4°C, honeys showed minor changes in antibacterial activity, increases in colour, and decreases in pH. For a third set of 17 honeys stored for five years at ambient temperature, the honeys showed almost complete loss of hydrogen peroxide and were all significantly darker in colour, but showed varied changes in antibacterial activity. CONCLUSIONS: Heat was detrimental to the antibacterial activity of honeys, as was long-term storage at ambient temperatures for some honeys but not others.

Bioactivities and Phenolic Profiles of Honeys Derived from Plants of the Goldfields, Esperance and Wheatbelt Regions of Western Australia.

The aim of this study was to examine a collection of 79 honeys derived from plants endemic to several Western Australian unique bioregions for bioactivity and physicochemical characteristics. For physicochemical analyses, total phenolic content, high performance thin layer chromatography (HPTLC) fingerprints, pH, Brix, colour and hydrogen peroxide generation were examined. Brix (82.6±1.3) and pH (4.34±0.24) values were within expected ranges, whereas hydrogen peroxide levels determined using an o-dianisidine/horseradish peroxidase assay were relatively low, ranging from 0-244 µM. Antibacterial activity determined by the broth microdilution assay showed that Moort (Eucalyptus platypus) and Yate (Eucalyptus occidentalis) honeys had the highest overall activity with mean minimum inhibitory concentrations of 24.8 % and 25.1 % (w/v) honey, respectively. Yate honey also had the highest overall antioxidant activity (4.38±0.58 mmol Fe2+ /kg of honey), followed by Mallee honeys from various eucalypts, as determined by FRAP (Ferric reducing antioxidant power) and DPPH⋅ (2,2-Diphenyl-1-picrylhydrazyl) assays. This study identified new sources of honeys with potentially useful therapeutic properties from bioregions within Western Australia.

Non-conventional antimicrobial and alternative therapies for the treatment of Clostridium difficile infection.

Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus that causes disease ranging from self-limiting diarrhoea to severe pseudomembranous colitis. C. difficile infection (CDI) commonly affects hospitalised patients and is increasingly identified in patients in the community with no hospital contact. For the last 15 years the incidence of CDI worldwide has been rising, especially in the northern hemisphere. The yearly average number of hospitalizations as a result of this disease is estimated to be over a quarter of a million per year in the United States alone. The main risk factor for CDI is exposure to antimicrobials that affect the gut microflora and, paradoxically, the most common treatments for CDI are the antimicrobials, metronidazole and vancomycin. However, the increasing frequency of highly virulent C. difficile strains, antimicrobial treatment failures, hospital outbreaks, patients with severe complications and cases with multiple recurrences have driven the search for new therapies. Several novel or popular complementary and alternative therapies are self-prescribed for treatment of other diarrheal diseases, and these may also be appropriate for treating CDI. In general, complementary and alternative medicine (CAM) is mainly used by patients when conventional therapeutic agents show limited success against C. difficile and other antimicrobial-resistant bacteria. Among these alternative approaches, a number of treatments, such as herbal remedies, are embraced less by pharmaceutical and medical professions. This review summarises current knowledge of non-conventional antimicrobial and alternative therapies for treatment of CDI. As the demand for non-conventional antimicrobial therapies increases, further studies are required in the field of CAM, especially natural products, for the treatment of CDI.

Antibacterial activity and chemical characteristics of several Western Australian honeys compared to manuka honey and pasture honey.

The physicochemical parameters and antibacterial activity of 10 Western Australian (WA) and two comparator honeys were determined. Honeys showed a pH range of 4.0-4.7, colour range of 41.3-470.7 mAU, methylglyoxal levels ranging from 82.2 to 325.9 mg kg-1 and hydrogen peroxide levels after 2 h of 22.7-295.5 µM. Antibacterial activity was assessed by the disc diffusion assay, phenol equivalence assay, determination of minimum inhibitory and bactericidal concentrations and a time-kill assay. Activity was shown for all honeys by one or more method, however, activity varied according to which assay was used. Minimum inhibitory concentrations for WA honeys against 10 organisms ranged from 4.0 to >32.0% (w/v). Removal of hydrogen peroxide activity by catalase resulted in decreased activity for several honeys. Overall, the data showed that honeys in addition to those derived from Leptospermum spp. have antimicrobial activity and should not be overlooked as potential sources of clinically useful honey.

Tea tree oil gel for mild to moderate acne; a 12 week uncontrolled, open-label phase II pilot study.

BACKGROUND: The efficacy, tolerability and acceptability of a tea tree oil gel (200 mg/g) and face wash (7 mg/g) were evaluated for the treatment of mild to moderate facial acne. METHODS: In this open-label, uncontrolled phase II pilot study, participants applied tea tree oil products to the face twice daily for 12 weeks and were assessed after 4, 8 and 12 weeks. Efficacy was determined from total numbers of facial acne lesions and the investigator global assessment (IGA) score. Tolerability was evaluated by the frequency of adverse events and the mean tolerability score determined at each visit. Product acceptability was assessed via a questionnaire at the end of the study period. RESULTS: Altogether 18 participants were enrolled, of whom 14 completed the study. Mean total lesion counts were 23.7 at baseline, 17.2 at 4, 15.1 at 8 and 10.7 at 12 weeks. Total lesion counts differed significantly over time by repeated measures anova (P < 0.0001). The mean IGA score was 2.4 at baseline, 2.2 at 4, 2.0 at 8 and 1.9 at 12 weeks, which also differed significantly over time (P = 0.0094). No serious adverse events occurred and minor local tolerability events were limited to peeling, dryness and scaling, all of which resolved without intervention. CONCLUSION: This study shows that the use of the tea tree oil products significantly improved mild to moderate acne and that the products were well tolerated.

Binaphthyl-1,2,3-triazole peptidomimetics with activity against Clostridium difficile and other pathogenic bacteria.

Clostridium difficile (C. difficile) is a problematic Gram positive bacterial pathogen causing moderate to severe gastrointestinal infections. Based on a lead binaphthyl-tripeptide dicationic antimicrobial, novel mono-, di- and tri-peptidomimetic analogues targeting C. difficile were designed and synthesized incorporating one, two or three d-configured cationic amino acid residues, with a common 1,2,3-triazole ester isostere at the C-terminus. Copper- and ruthenium-click chemistry facilitated the generation of a 46 compound library for in vitro bioactivity assays, with structure-activity trends over the largest compound subset revealing a clear advantage to triazole-substitution with a linear or branched hydrophobic group. The most active compounds were dicationic-dipeptides where the triazole was substituted with a 4- or 5-cyclohexylmethyl or 4,5-diphenyl moiety, providing MICs of 4 µg mL(-1) against three human isolates of C. difficile. Further biological screening revealed significant antimicrobial activity for several compounds against other common bacterial pathogens, both Gram positive and negative, including S. aureus (MICs ≥2 µg mL(-1)), S. pneumoniae (MICs ≥1 µg mL(-1)), E. coli (MICs ≥4 µg mL(-1)), A. baumannii (MICs ≥4 µg mL(-1)) and vancomycin-resistant E. faecalis (MICs ≥4 µg mL(-1)).

Synthesis and antimicrobial activity of binaphthyl-based, functionalized oxazole and thiazole peptidomimetics.

Thirty two new binaphthyl-based, functionalized oxazole and thiazole peptidomimetics and over thirty five novel leucine-containing intermediate oxazoles and thiazoles were prepared in this study. This includes the first examples of the direct C-5 arylation of an amino acid dipeptide-derived oxazole. Moderate to excellent antibacterial activity was observed for all new compounds across Gram positive isolates with MICs ranging from 1-16 µg mL(-1). Results for Gram negative E. coli and A. baumannii were more variable, but MICs as low as 4 µg mL(-1) were returned for two examples. Significantly, the in vitro results with a fluoromethyl-oxazole derivative collectively represent the best obtained to date for a member of our binaphthyl peptide antimicrobials.

Inspiration from old dyes: tris(stilbene) compounds as potent gram-positive antibacterial agents.

Herein we describe the preparation and structure-activity relationship studies on range of stilbene based compounds and their antibacterial activity. Two related compounds, each bearing carboxylic acid moieties, exhibit good activity against several bacterial strains, including methicillin-resistant Staphylococcus aureus MRSA (ATCC 33592 and NCTC 10442). Compound 10 was most active against Moraxella catarrhalis with minimum inhibitory concentrations (MICs) of 0.12-0.25 µg mL(-1) and against Staphylococcus spp. with MICs ranging from 2-4 µg mL(-1). The derivative 17 showed increased activity with MICs of 0.06-0.25 µg mL(-1) against M. catarrhalis and 0.12-1 against Staphylococcus spp. This level of activity is similar to that reported for S. aureus for antibiotics, such as vancomycin, with MICs of ≤2.0 µg mL(-1) and clindamycin with MICs of ≤0.5 µg mL(-1). As an indicator of toxicity, 17 was tested for its ability to lyse sheep erythrocytes, and showed low haemolytic activity. Such results highlight the value of tris(stilbene) compounds as antibacterial agents providing suitable properties for further development.

Effects of Melaleuca alternifolia (tea tree) essential oil and the major monoterpene component terpinen-4-ol on the development of single- and multistep antibiotic resistance and antimicrobial susceptibility.

This study examined the effect of subinhibitory Melaleuca alternifolia (tea tree) essential oil on the development of antibiotic resistance in Staphylococcus aureus and Escherichia coli. Frequencies of single-step antibiotic-resistant mutants were determined by inoculating bacteria cultured with or without subinhibitory tea tree oil onto agar containing 2 to 8 times the MIC of each antibiotic and with or without tea tree oil. Whereas most differences in resistance frequencies were relatively minor, the combination of kanamycin and tea tree oil yielded approximately 10-fold fewer resistant E. coli mutants than kanamycin alone. The development of multistep antibiotic resistance in the presence of tea tree oil or terpinen-4-ol was examined by culturing S. aureus and E. coli isolates daily with antibiotic alone, antibiotic with tea tree oil, and antibiotic with terpinen-4-ol for 6 days. Median MICs for each antibiotic alone increased 4- to 16-fold by day 6. Subinhibitory tea tree oil or terpinen-4-ol did not greatly alter results, with day 6 median MICs being either the same as or one concentration different from those for antibiotic alone. For tea tree oil and terpinen-4-ol alone, day 6 median MICs had increased 4-fold for S. aureus (n = 18) and 2-fold for E. coli (n = 18) from baseline values. Lastly, few significant changes in antimicrobial susceptibility were seen for S. aureus and S. epidermidis isolates that had been serially subcultured 14 to 22 times with subinhibitory terpinen-4-ol. Overall, these data indicate that tea tree oil and terpinen-4-ol have little impact on the development of antimicrobial resistance and susceptibility.

Candida albicans adhesion to human epithelial cells and polystyrene and formation of biofilm is reduced by sub-inhibitory Melaleuca alternifolia (tea tree) essential oil.

This study investigated the effects of the volatile terpene-rich oil from Melaleuca alternifolia (tea tree oil) on the formation of biofilms and the adhesion of C. albicans cells to both biotic and abiotic surfaces. Biofilm formation on polystyrene was significantly inhibited for 70% of the isolates at the lowest test concentration of 0.016% of tea tree oil (TTO) when quantified by XTT and 40% of isolates when measured by crystal violet staining. Adhesion to polystyrene, quantified by crystal violet staining, was significantly reduced for 3 isolates at 0.031%, 6 isolates at 0.062% and 0.125% and for all 7 isolates at 0.25% TTO. Reductions in adhesion were not due to loss of viability (at concentrations of ≤ 0.125%) or interactions between the TTO and polystyrene. Similarly, adhesion to buccal epithelial and HeLa cells was also significantly reduced in the presence of 0.016-0.062% TTO. Treatment with 0.125% TTO, but not 0.062%, decreased the cell surface hydrophobicity of C. albicans, indicating one potential mechanism by which adhesion may be reduced. These data demonstrate that sub-inhibitory TTO reduces the adhesion of C. albicans to both human cells and polystyrene, inhibits biofilm formation and decreases cell surface hydrophobicity.

Correlation of the antibacterial activity of commercial manuka and Leptospermum honeys from Australia and New Zealand with methylglyoxal content and other physicochemical characteristics.

Variation in the antibacterial potency of manuka honey has been reported in several published studies. However, many of these studies examine only a few honey samples, or test activity against only a few bacterial isolates. To address this deficit, a collection of 29 manuka/Leptospermum honeys was obtained, comprising commercial manuka honeys from Australia and New Zealand and several Western Australian Leptospermum honeys obtained directly from beekeepers. The antibacterial activity of honeys was quantified using several methods, including the broth microdilution method to determine minimum inhibitory concentrations (MICs) against four species of test bacteria, the phenol equivalence method, determination of antibacterial activity values from optical density, and time kill assays. Several physicochemical parameters or components were also quantified, including methylglyoxal (MGO), dihydroxyacetone (DHA), hydroxymethylfurfural (HMF) and total phenolics content as well as pH, colour and refractive index. Total antioxidant activity was also determined using the DPPH* (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing-antioxidant power) assays. Levels of MGO quantified in each honey were compared to the levels stated on the product labels, which revealed mostly minor differences. Antibacterial activity studies showed that MICs varied between different honey samples and between bacterial species. Correlation of the MGO content of honey with antibacterial activity showed differing relationships for each test organism, with Pseudomonas aeruginosa showing no relationship, Staphylococcus aureus showing a moderate relationship and both Enterococcus faecalis and Escherichia coli showing strong positive correlations. The association between MGO content and antibacterial activity was further investigated by adding known concentrations of MGO to a multifloral honey and quantifying activity, and by also conducting checkerboard assays. These investigations showed that interactions were largely additive in nature, and that synergistic interactions between MGO and the honey matrix did not occur.

Activity of newest generation ß-lactam/ß-lactamase inhibitor combination therapies against multidrug resistant Pseudomonas aeruginosa.

Multidrug resistant (MDR) P. aeruginosa accounts for 35% of all P. aeruginosa isolated from respiratory samples of patients with cystic fibrosis (CF). The usefulness of ß-lactam antibiotics for treating CF, such as carbapenems and later generation cephalosporins, is limited by the development of antibacterial resistance. A proven treatment approach is the combination of a ß-lactam antibiotic with a ß-lactamase inhibitor. New ß-lactam/ß-lactamase inhibitor combinations are available, but data are lacking regarding the susceptibility of MDR CF-associated P. aeruginosa (CFPA) to these new combination therapies. In this study we determined MIC values for three new combinations; imipenem-relebactam (I-R), ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T) against MDR CFPA (n = 20). The MIC90 of I-R, CZA, and C/T was 64/4, 32/4, and 16/8 (all µg/mL), respectively. The susceptibility of isolates to imipenem was not significantly improved with the addition of relebactam (p = 0.68). However, susceptibility to ceftazidime was significantly improved with the addition of avibactam (p < 0.01), and the susceptibility to C/T was improved compared to piperacillin/tazobactam (p < 0.05) These data provide in vitro evidence that I-R may not be any more effective than imipenem monotherapy against MDR CFPA. The pattern of susceptibility observed for CZA and C/T in the current study was similar to data previously reported for non-CF-associated MDR P. aeruginosa.

A Comprehensive Survey of Phenolic Constituents Reported in Monofloral Honeys around the Globe.

The aim of this review is to provide a comprehensive overview of the large variety of phenolic compounds that have to date been identified in a wide range of monofloral honeys found globally. The collated information is structured along several themes, including the botanical family and genus of the monofloral honeys for which phenolic constituents have been reported, the chemical classes the phenolic compounds can be attributed to, and the analytical method employed in compound determination as well as countries with a particular research focus on phenolic honey constituents. This review covers 130 research papers that detail the phenolic constituents of a total of 556 monofloral honeys. Based on the findings of this review, it can be concluded that most of these honeys belong to the Myrtaceae and Fabaceae families and that Robinia (Robinia pseudoacacia, Fabaceae), Manuka (Leptospermum scoparium, Myrtaceae), and Chestnut (Castanea sp., Fagaceae) honeys are to date the most studied honeys for phenolic compound determination. China, Italy, and Turkey are the major honey phenolic research hubs. To date, 161 individual phenolic compounds belonging to five major compound groups have been reported, with caffeic acid, gallic acid, ferulic acid and quercetin being the most widely reported among them. HPLC with photodiode array detection appears to be the most popular method for chemical structure identification.

Antibacterial interactions between two monofloral honeys and several topical antiseptics, including essential oils.

BACKGROUND: Honey has broad spectrum antibacterial activity against clinically important organisms and may be suitable for treating superficial bacterial infections. However, very little data are available describing potential interactions between honey and other topically applied agents such as antiseptics or essential oils. METHODS: Interactions between pairs of antibacterial agents were investigated by performing checkerboard assays and determining the fractional inhibitory concentration indices (FICIs). Interactions between the two monofloral honeys marri (from Corymbia calophylla) and manuka, and the antiseptic agents benzalkonium chloride, chlorhexidine digluconate, silver (I) nitrate, tea tree oil, and Eucalyptus polybractea oil were investigated against Staphylococcus aureus ATCC® 43300 and Pseudomonas aeruginosa ATCC® 27853. RESULTS: Additive or indifferent interactions (FICI 0.5-2) were observed for all combinations against both organisms tested, with the exception of chlorhexidine and honey. Chlorhexidine and marri honey showed an antagonistic relationship against S. aureus (median FICI 2.00, range 1.25-4.83). Similarly, chlorhexidine and manuka honey showed antagonism against S. aureus (median FICI 2.33, range 2.00-2.67). CONCLUSIONS: With the exception of chlorhexidine, these data indicate that honey does not interfere with the antimicrobial activity of the tested agents, and that honey may be suitable for combination therapy with other topically applied antibacterial agents for treating superficial bacterial infections.

In vitro antibacterial activity of Western Australian honeys, and manuka honey, against bacteria implicated in impetigo.

Impetigo is a contagious skin disease caused by Staphylococcus aureus and Streptococcus pyogenes. Without treatment, impetigo may be recurrent, develop into severe disease, or have serious, life-threatening sequelae. Standard treatment consists of topical or systemic antibiotic therapy (depending on severity), however, due to antibiotic resistance some therapies are increasingly ineffective. In this study we evaluated the potential for honey as an alternative treatment for impetigo. A broth microdilution assay in 96-well microtitre trays was used to determine the minimum inhibitory concentrations (MICs) of six monofloral honeys (jarrah, marri, red bell, banksia, wandoo, and manuka), a multifloral honey and artificial honey against S. aureus (n = 10), S. pyogenes (n = 10), and coagulase-negative staphylococci (CoNS) (n = 10). The optical density (OD) of all microtitre tray wells was also determined before and after assay incubation to analyse whether sub-MIC growth inhibition occurred. Jarrah, marri, red bell, banksia, and manuka honeys were highly effective at inhibiting S. aureus and CoNS, with MIC50 values ranging from 4 to 8% w/v honey. S. pyogenes was also inhibited by these same honeys, albeit at higher concentrations (8-29% w/v). Wandoo and multifloral honeys had the least antibacterial activity with MICs of >30% (w/v) for all isolates. However, OD data indicated that sub-MIC concentrations of honey were still partially restricting bacterial growth. Our pre-clinical data indicate that honey may be a potential therapeutic agent for the routine treatment of mild impetigo, and we suggest that clinical trials would be appropriate to further investigate this.

An investigation of the suitability of melissopalynology to authenticate Jarrah honey.

This study reports on the analysis of eleven Jarrah (Eucalyptus marginata) honeys, of which nearly half (n = 5) were re-classified as Blackbutt (E. patens) honey on the grounds of the predominant flower pollen identified by melissopalynology. Based on a comprehensive analysis of the honeys' physico- and phytochemical characteristics and antioxidant activity data, taking into account pH, electrical conductivity, refractive index and Brix values as well as moisture content, individual fructose and glucose content and derived fructose to glucose ratio alongside total phenolic content and antioxidant activity determined by the DPPH assay, no statistically significant difference was found amongst the eleven honeys classified by pollen analysis into two honey groups, 'Jarrah' or 'Blackbutt'. This study therefore draws into question the value of melissopalynology as an analysis tool to authenticate Jarrah honey.

Differential gene expression in Daphnia magna suggests distinct modes of action and bioavailability for ZnO nanoparticles and Zn ions.

Zinc oxide nanoparticles (ZnO NPs) are being rapidly developed for use in consumer products, wastewater treatment, and chemotherapy providing several possible routes for ZnO NP exposure to humans and aquatic organisms. Recent studies have shown that ZnO NPs undergo rapid dissolution to Zn(2+), but the relative contribution of Zn(2+) to ZnO NP bioavailability and toxicity is not clear. We show that a fraction of the ZnO NPs in suspension dissolves, and this fraction cannot account for the toxicity of the ZnO NP suspensions to Daphnia magna. Gene expression profiling of D. magna exposed to ZnO NPs or ZnSO(4) at sublethal concentrations revealed distinct modes of toxicity. There was also little overlap in gene expression between ZnO NPs and SiO(x) NPs, suggesting specificity for the ZnO NP expression profile. ZnO NPs effected expression of genes involved in cytoskeletal transport, cellular respiration, and reproduction. A specific pattern of differential expression of three biomarker genes including a multicystatin, ferritin, and C1q containing gene were confirmed for ZnO NP exposure and provide a suite of biomarkers for identifying environmental exposure to ZnO NPs and differentiating between NP and ionic exposure.

Cationic Peptidomimetic Amphiphiles Having a N-Aryl- or N-Naphthyl-1,2,3-Triazole Core Structure Targeting Clostridioides (Clostridium) difficile: Synthesis, Antibacterial Evaluation, and an In Vivo C. difficile Infection Model.

Clostridioides (also known as Clostridium) difficile is a Gram-positive anaerobic, spore producing bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are inadequate, expensive, and limited, and thus inexpensive drug treatments for C. difficile infection (CDI) with improved efficacy and specificity are urgently needed. To improve the solubility of our cationic amphiphilic 1,1'-binaphthylpeptidomimetics developed earlier that showed promise in an in vivo murine CDI model we have synthesized related compounds with an N-arytriazole or N-naphthyltriazole moiety instead of the 1,1'-biphenyl or 1,1'-binaphthyl moiety. This modification was made to increase the polarity and thus water solubility of the overall peptidomimetics, while maintaining the aromatic character. The dicationic N-naphthyltriazole derivative 40 was identified as a C. difficile-selective antibacterial with MIC values of 8 µg/mL against C. difficile strains ATCC 700057 and 132 (both ribotype 027). This compound displayed increased water solubility and reduced hemolytic activity (32 µg/mL) in an in vitro hemolysis assay and reduced cytotoxicity (CC50 32 µg/mL against HEK293 cells) relative to lead compound 2. Compound 40 exhibited mild efficacy (with 80% survival observed after 24 h compared to the DMSO control of 40%) in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease.

Development and validation of a new microplate assay that utilises optical density to quantify the antibacterial activity of honeys including Jarrah, Marri and Manuka.

The phenol equivalence assay is the current industry-adopted test used to quantify the antibacterial activity of honeys in Australia and New Zealand. Activity is measured based on the diffusion of honey through agar and resulting zone of growth inhibition. Due to differences in the aqueous solubilities of antibacterial compounds found in honeys, this method may not be optimal for quantifying activity. Therefore, a new method was developed based on the existing broth microdilution assay that is widely used for determining minimum inhibitory concentrations (MICs). It utilises the four organisms Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and an optical density endpoint to quantify bacterial growth. Decreases in bacterial growth in the presence of honey, relative to the positive growth control, are then used to derive a single value to represent the overall antibacterial activity of each honey. Antibacterial activity was quantified for a total of 77 honeys using the new method, the phenol equivalence assay and the standard broth microdilution assay. This included 69 honeys with undisclosed floral sources and the comparators Manuka, Jarrah (Eucalyptus marginata), Marri (Corymbia calophylla), artificial and multifloral honey. For the 69 honey samples, phenol equivalence values ranged from 0-48.5 with a mean of 34 (% w/v phenol). Mean MICs, determined as the average of the MICs obtained for each of the four organisms for each honey ranged from 7-24% (w/v honey). Using the new assay, values for the 69 honeys ranged from 368 to 669 activity units, with a mean of 596. These new antibacterial activity values correlated closely with mean MICs (R2 = 0.949) whereas the relationship with phenol equivalence values was weaker (R2 = 0.649). Limit of detection, limit of quantitation, measuring interval, limit of reporting, sensitivity, selectivity, repeatability, reproducibility, and ruggedness were also investigated and showed that the new assay was both robust and reproducible.