Comparison of solid‐phase cytometry and the plate count method for the evaluation of the survival of bacteria in pharmaceutical oils

Aim: To compare the survival of four bacterial strains (Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa) in pharmaceutical oils, including jojoba oil/tea tree oil, carbol oil, jojoba oil and sesame oil. Methods and Results: Oils were spiked with the test bacteria in a concentration of 10⁴ CFU ml^?1. Bacteria were extracted from oils with phosphate‐buffered saline containing 0·5% Tween 20. Aliquots of the pooled water layers were analysed by solid‐phase cytometry and plate counting. Plate counts dropped to zero for all test strains exposed for 24 h to three of the four oils. In contrast, significant numbers of viable cells were still detected by SPC, except in the jojoba oil/tea tree oil mixture and partly in sesame oil. Conclusions: Exposure of bacteria for 24 h to the two oils containing an antimicrobial led to a loss of their culturability but not necessarily of their viability. The antibacterial activity of the jojoba oil/tea tree oil mixture supersedes that of carbol oil. Significance and Impact of the Study: These in vitro data suggest that the jojoba oil/tea tree oil mixture more than carbol oil inhibits bacterial proliferation when used for intermittent self‐catherization.     

Influence of organic matter, cations and surfactants on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil in vitro

The effect of some potentially interfering substances and conditions on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil was investigated. Agar and broth dilution methods were used to determine minimum inhibitory and cidal concentrations of tea tree oil in the presence and absence of each potentially interfering substance. Activity was determined against Gram-positive and -negative bacteria, and Candida albicans. Minimum inhibitory or cidal concentrations differed from controls by two or more dilutions, for one or more organisms, where Tween-20, Tween-80, skim-milk powder and bovine serum albumin were assessed. These differences were not seen when assays were performed in anaerobic conditions, or in the presence of calcium and magnesium ions. The effect of organic matter on the antimicrobial activity of tea tree oil was also investigated by an organic soil neutralization test. Organisms were exposed to lethal concentrations of tea tree oil ranging from 1-10% (v/v), in the presence of 1-30% (w/v) dry bakers' yeast. After 10 min contact time, viability was determined. At > or = 1%, organic matter compromised the activity of each concentration of tea tree oil against Staphylococcus aureus and C. albicans. At 10% or more, organic matter compromised the activity of each tea tree oil concentration against Pseudomonas aeruginosa. Organic matter affected 1 and 2% tea tree oil, but not 4 and 8%, against Escherichia coli. In conclusion, organic matter and surfactants compromise the antimicrobial activity of tea tree oil, although these effects vary between organisms.     

Interactions between components of the essential oil of Melaleuca alternifolia

AIMS: This study compared the antimicrobial activity of Melaleuca alternifolia (tea tree) oil with that of some of its components, both individually and in two-component combinations. METHODS AND RESULTS: Minimum inhibitory concentration and time-kill assays revealed that terpinen-4-ol, the principal active component of tea tree oil, was more active on its own than when present in tea tree oil. Combinations of terpinen-4-ol and either gamma-terpinene or p-cymene produced similar activities to tea tree oil. Concentration-dependent reductions in terpinen-4-ol activity and solubility also occurred in the presence of gamma-terpinene. CONCLUSION: Non-oxygenated terpenes in tea tree oil appear to reduce terpinen-4-ol efficacy by lowering its aqueous solubility. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings explain why tea tree oil can be less active in vitro than terpinen-4-ol alone and further suggest that the presence of a non-aqueous phase in tea tree oil formulations may limit the microbial availability of its active components.     

Antifungal Activity of Nanocapsule Suspensions Containing Tea Tree Oil on the Growth of Trichophyton rubrum

The aim of this study was to evaluate, for the first time, the antifungal efficacy of nanocapsules and nanoemulsions containing Melaleuca alternifolia essential oil (tea tree oil) in an onychomycosis model. The antifungal activity of nanostructured formulations was evaluated against Trichophyton rubrum in two different in vitro models of dermatophyte nail infection. First, nail powder was infected with T. rubrum in a 96-well plate and then treated with the formulations. After 7 and 14 days, cell viability was verified. The plate counts for the samples were 2.37, 1.45 and 1.0 log CFU mL^?1 (emulsion, nanoemulsion containing tea tree oil and nanocapsules containing tea tree oil, respectively). A second model employed nails fragments which were infected with the microorganism and treated with the formulations. The diameter of fungal colony was measured. The areas obtained were 2.88 ± 2.08 mm², 14.59 ± 2.01 mm², 40.98 ± 2.76 mm² and 38.72 ± 1.22 mm² for the nanocapsules containing tea tree oil, nanoemulsion containing tea tree oil, emulsion and untreated nail, respectively. Nail infection models demonstrated the ability of the formulations to reduce T. rubrum growth, with the inclusion of oil in nanocapsules being most efficient.     

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil)

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.     

Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil

AIMS: To investigate the in vitro antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. METHODS AND RESULTS: Activity was investigated by broth microdilution and macrodilution, and time kill methods. Components showing the most activity, with minimum inhibitory concentrations and minimum fungicidal concentrations of < or =0.25%, were terpinen-4-ol, alpha-terpineol, linalool, alpha-pinene and beta-pinene, followed by 1,8-cineole. The remaining components showed slightly less activity and had values ranging from 0.5 to 2%, with the exception of beta-myrcene which showed no detectable activity. Susceptibility data generated for several of the least water-soluble components were two or more dilutions lower by macrodilution, compared with microdilution. CONCLUSIONS: All tea tree oil components, except beta-myrcene, had antifungal activity. The lack of activity reported for some components by microdilution may be due to these components becoming absorbed into the polystyrene of the microtitre tray. This indicates that plastics are unsuitable as assay vessels for tests with these or similar components. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified that most components of tea tree oil have activity against a range of fungi. However, the measurement of antifungal activity may be significantly influenced by the test method.     

The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil)

Pseudomonas aeruginosa is less susceptible to the antimicrobial properties of tea tree oil than many bacteria and its tolerance is considered to be due to its outer membrane. Polymyxin B nonapeptide (PMBN), which has no antibacterial action, was used to permeabilize the outer membrane. The addition of PMBN to Ps. aeruginosa NCTC 6749 markedly increased this organism's susceptibility to tea tree oil and to its normally inert hydrocarbons, p-cymene and gamma-terpinene.     

Antimicrobial activity of certain natural-based plant oils against the antibiotic-resistant acne bacteria

The unceasing emerging of multidrug-resistant bacteria imposes a global foremost human health threat and discovery of new alternative remedies are necessity. The use of plant essential oil in the treatment of many pathogenic bacteria is promising. Acne vulgaris is the most common skin complaint that fears many people about their aesthetic appearance. In this work we investigated the antibacterial activity of some plant oils against acne-inducing bacteria. Three bacterial isolates were identified from Egypt, biochemically and by means of 16s rRNA gene typing, and were designated as Staphylococcus aureus EG-AE1, Staphylococcus epidermidis EG-AE2 and Cutibacterium acnes EG-AE1. Antibiotic susceptibility test showed resistance of the isolates to at least six antibiotics, yet they are still susceptible to the last resort Vancomycin. In vitro investigations of eleven Egyptian plant oils, identified tea tree and rosemary oils to exhibit antibacterial activity against the antibiotic-resistant acne isolates. Inhibition zones of 15 ± 0.5, 21.02 ± 0.73 and 20.85 ± 0.76 mm was detected when tea tree oil applied against the above-mentioned bacteria respectively, while inhibition zones of 12.5 ± 1.5, 15.18 ± 0.38 and 14.77 ± 0.35 mm were detected by rosemary oils. Tea tree and rosemary oils exhibited bacteriostatic and bactericidal activity against all the strains with MICs/MBCs ranging between 39-78 mg/L for tea tree oil and 39–156 mg/L for rosemary oil. All the isolates were killed after 4 and 6 h upon growing with 200 mg/L of tea tree and rosemary oils, respectively. Additionally, gas chromatography mass spectrometry (GC/MS) profiling identified and detected a variable number of antimicrobial compounds in both oils.     

海南油茶主要形态和经济性状及聚类分析

对海南澄迈等6个主要油茶分布县10个乡镇油茶资源主要形态和经济性状进行调查测定,比较其与高州油茶和普通油茶的差异,并进行聚类分析。结果表明:海南油茶种质资源在形态、茶油脂肪酸组成等方面具有多样性,海南油茶树体、花、果实以及叶片均小于高州油茶;海南油茶鲜出子率在19.84%~36.21%之间,干出仁率在49.64%~64.22%之间,种仁含油率为44.56%~52.21%;海南油茶子油的不饱和脂肪酸为87.58%~89.68%,亚麻酸含量低,仅为普通油茶的0~88.7%,而饱和脂肪酸显著高于普通油茶。聚类分析表明海南油茶同高州油茶和普通油茶具有显著区别,除海南博鳌资源外,其他海南油茶被划分为一类。本研究为海南省油茶资源分类以及资源开发提供了参考资料。     

Comparison of the effects in vitro of tea tree oil and plaunotol on methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus

The effects in vitro of tea tree oil (TTO) and plaunotol were examined by monitoring the growth of a standard strain of Staphylococcus aureus FDA 209P and of fourteen methicillin-susceptible strains of S. aureus (MSSA), together with twenty methicillin-resistant strains (MRSA). The minimum inhibitory concentrations (MIC) and the doses for 50% inhibition of growth (ID50) were determined by the micro-broth dilution (MD) method, and the broth dilution with shaking (BDS) method, respectively. The MIC of plaunotol for 50 and 90% of the MSSA and MRSA were assessed by the MD method, as 16 microg/ml and > or = 1,024 microg/ml, respectively. No antibacterial effects of TTO on MSSA and MRSA were detected by the MD method. The growth-inhibitory effects of TTO on S. aureus by the BDS method were examined, and it appeared that TTO was effective over a lower range of concentrations than previously reported. It seems that TTO is very effective in vitro against MSSA and MRSA at high concentrations but less effective below 40 microg/ml of TTO.     

Decreased Immunoreactivity of Hepatitis E Virus Antigen Following Treatment with Sakhalin Spruce (Picea glehnii) Essential Oil

The hepatitis E virus (HEV) causes a common infectious disease that infects pigs, wild boars, deer, and humans. In most cases, humans are infected by eating raw meat. Some essential oils have been reported to exhibit antiviral activities. In this study, in order to investigate the anti-HEV properties of essential oils, the immunoreactivities of HEV antigen proteins against the relevant antibodies were analyzed after the HEV antigens underwent treatment with various essential oils. The essential oils extracted from the tea tree, which was previously reported to exhibit antiviral activity, lavender, and lemon had strongly reduced activity. We found that treatment with the essential oil prepared from Sakhalin spruce was associated with the strongest reduction in immunoreactivity of HEV antigen protein(s) among the tested substances. The main volatile constituents of Sakhalin spruce essential oil were found to be bornyl acetate (32.30 %), α-pinene (16.66 %), camphene (11.14 %), camphor (5.52 %), β-phellandrene (9.09 %), borneol (4.77 %), and limonene (4.57 %). The anti-HEV properties of the various components of the essential oils were examined: treatment with bornyl acetate, the main component of Sakhalin spruce oil, α-pinene, the main component of tea tree oil, and limonene, the main component of lemon oil, resulted in a strong reduction in HEV antigen immunoreactivity. These results indicate that each main component of the essential oils plays an important role in the reduction of the immunoreactivity of HEV antigen protein(s); they also suggest that Sakhalin spruce essential oil exhibits anti-HEV activity. In a formulation with the potential to eliminate the infectivity of HEV in foodborne infections, this essential oil can be applied as an inactivating agent for meat processing and cooking utensils, such as knives and chopping boards.     

Integration and Potential Application Ability of Culturable Functional Microorganism in Oil Tea Camellia

Oil tea Camellia is a major woody oil plant, which has a positive influence on alleviating the contradiction between supply and demand of edible oil in China. Microbial fertilizer for Oil tea Camellia is urgently needed in current production, and it is of great significance to improve the yield and quality. Culturable functional microorganisms are the basis of research and development for microbial fertilizer. In this study, culturable microorganisms which had abilities of antagonism, growth promotion, phosphorus solubility, nitrogen fixation and drought resistance, were integrated from oil tea literature. And the strains potential application ability were evaluated in terms of functionality, safety and adaptability, culture characteristics, suitable conditions and colonization or infection ability of strains. The results showed that the strains with strongest antagonistic ability were Bacillus amyloliquefaciens D2WM and Bacillus subtilis Y13. Beauveria bassiana BbTK-01 and Metarhizium anisopliae FJMa201101 had the strongest insect resistant ability. Glomus versiforme and Glomus intraradices can promote oil tea fastest growth. Phosphorus dissolving ability of Bacillus aryabhattai NC285 and Burkholderia cepacia 6-Y-09 were strongest. The strains with strongest Nitrogen fixing ability were Azomonas N7-3 and Sphingobium B7-7, and the strains with strongest improving drought resistance ability were Glomus versiforme and Glomus intraradices. Comprehensive evaluation of strains showed that Bacillus subtilis Y13 and Azomonas N7-3 had a good applied potential ability. This study would save time-consuming of isolate, purify and identify repetitively for the researchers of functional bacteria of oil tea Camellia. Meanwhile it provides a research basis for selecting targeted strains and constructing the combination of functional strains, therefore provides data support for fertilizer efficiency.     

广西部分地区野生茶树遗传关系EST-SSR标记分析

为了明确广西野生茶树种质资源的遗传背景,该研究从广西的宁明县、金秀县、苍梧县收集到14份地方野生茶树种质资源,以17个国家级茶树良种作为参照,采用EST-SSR分子标记技术,探讨了广西这三个地方野生茶树与国家级茶树良种间的亲缘关系以及广西地方茶树自身的遗传多样性。结果表明:15对EST-SSR引物共检测到68个等位基因,平均每个引物可扩增出4.53个,其中多态性位点为60个,多态性比率达88.2%。平均观测杂合度、平均期望杂合度、平均Shannon信息指数分别为0.42、0.55和0.97。PIC值在0.23~0.74之间,平均为0.52,多态性较好。遗传相似系数在0.53~0.9之间,平均值为0.71,31份供试材料在遗传相似系数为0.71分为5组群,76%参照品种聚在A组群,而广西本地的野生茶树资源则主要分布在B、C、D、E组群。利用该研究中的4对核心引物即可将31份供试材料全部区分开,挑选其中10个多态性较好的等位位点进行编码,构建31份供试种质的DNA分子指纹图谱。这表明广西野生茶树资源与国家级茶树良种间遗传差异较大、亲缘关系较远、遗传基础宽、多样性非常丰富,可作为茶树育种的亲本或开展茶树功能基因研究的材料。     

Control of the chewing louse Bovicola (Werneckiella) ocellatus in donkeys,using essential oils

Infestations by lice can be a significant clinical and welfare issue in the management of large animals. The limited range of commercial pediculicides available and the development of resistance have led to the need to explore alternative louse management approaches. The results of in vitro and in vivo trials undertaken to control populations of the donkey chewing louse, Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae) using the essential oils of tea tree (Melaleuca alternifolia) and lavender (Lavandula angustifolia) are reported here. Results of contact and vapour bioassays showed that 5% (v/v) tea tree and lavender oils resulted in > 80% louse mortality after 2 h of exposure. On farms, separate groups of 10 donkeys sprayed with 5% (v/v) tea tree and lavender oil as part of their usual grooming regime showed significant reductions in louse numbers compared with a control group (0.2% polysorbate 80 in water). These findings indicate that tea tree and lavender essential oils can provide clinically useful levels of control of B. ocellatus when used as part of a grooming routine and suggest that with further development could form the basis of an easy to apply and valuable component of a louse management programme for donkeys.     

Antimicrobial activity of the essential oil of Melaleuca alternifolia

Melaleuca alternifolia has been used for medical purposes since Australia was colonized in 1788. Melaleuca alternifolia is commonly called tea tree, although this vernacular name is also given to many other species in the Leptospermum and Melaleuca genera. A small tree, it grows up to 5 m in height, has papery bark and narrow, tapered leaves up to 20 mm in length and flowers in summer. Melaleuca alternifolia is unique to Australia and its natural habitat is a relatively small area around the Clarence and Richmond rivers in the north-east coastal area of New South Wales where the terrain is generally low lying and swampy. The essential oil of M. alternifolia , or tea tree oil. has enjoyed increased medicinal use in recent years. It is a pale yellow viscous liquid with a distinctive pungent odour and is composed of a complex mixture of monoterpenes, 1-terpinen-4-ol, cineole and other hydrocarbons (Peña 1962).     

Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia

Tea tree oil, or the essential oil of Melaleuca alternifolia , is becoming increasingly popular as a naturally occurring antimicrobial agent. The antimicrobial activity of eight components of tea tree oil was evaluated using disc diffusion and broth microdilution methods. Attempts were also made to overcome methodological problems encountered with testing compounds which have limited solubility in aqueous media. After assessing media with and without solubilizing agents, the disc diffusion method was used to determine the susceptibility of a range of micro-organisms to 1,8-cineole, 1-terpinen-4-ol, ρ-cymene, linalool, α-terpinene, γ-terpinene, α-terpineol and terpinolene. While the disc diffusion method lacked reproducibility, it was considered useful as a procedure for screening for antimicrobial activity. Terpinen-4-ol was active against all the test organisms while ρ-cymene demonstrated no antimicrobial activity. Linalool and α-terpineol were active against all organisms with the exception of Pseudomonas aeruginosa. Minimum inhibitory and minimum cidal concentrations of each component against Candida albicans, Escherichia coli and Staphylococcus aureus were determined using a broth microdilution method. Modifications to this method overcame solubility and turbidity problems associated with the oil components and allowed the antimicrobial activity of each of the components to be quantified reproducibly. There was reasonable agreement between minimum inhibitory concentrations and zones of inhibition. These results may have significant implications for the future development of tea tree oil as an antimicrobial agent.     

Residual and ovicidal efficacy of essential oil‐based formulations in vitro against the donkey chewing louse Bovicola ocellatus

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N‐lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.     

Effect of antimicrobial activity of Melaleuca alternifolia essential oil on antagonistic potential of Pleurotus species against Trichoderma harzianum in dual culture

Melaleuca alternifolia (tea tree) essential oil was investigated for its “in vitro” ability to control Trichoderma harzianum, a fungal contaminant that causes extensive losses in the cultivation of Pleurotus species. The antifungal activity of M. alternifolia essential oil and antagonist activities between Pleurotus species against three T. harzianum strains were studied in dual-culture experiments on an agar-based medium in which different concentrations of essential oil were incorporated. M. alternifolia essential oil at a concentration of 0.625 μL/mL, inhibited T. harzianum mycelial growth by 5.9–9.0%, depending on the strain. At the same concentrations P. ferulae and P. nebrodensis stimulated mycelial growth by 5.2–8.1%. All strains of T. harzianum were antagonistic to the Pleurotus species in the control. When essential oil was added to the substrate cultural, the antagonistic activity of T. harzianum against the Pleurotus species was weak (0.0625 μL of essential oil) or non-existent (0.125 μL of essential oil). M. alternifolia essential oil could be an alternative to the synthetic chemicals that are currently used to prevent and control T. harzianum in mushroom cultivation.     

西双版纳不同林茶复合生态系统碳储量

为了探明上层遮荫树种对茶园碳储量的影响,根据所建立的茶园上层树种及茶树的生物量模型估算了不同林茶复合生态系统的生物量,结合植物、土壤样品碳含量的实测值,对西双版纳州勐海县4种茶园组合模式及纯茶园的碳储量进行了分析。结果表明:樟树+茶、樟-杉+茶2种组合模式的碳储量分别比纯茶园碳储量(223.442t·hm-2)高22.701、3.871t·hm-2,而4种遮荫树种+茶、6种遮荫树种+茶2种组合模式的碳储量则分别比纯茶园低10.828、5.717t·hm-2。各茶园总碳储量以土壤的碳储量所占比例最大,达91.8%～96.0%,随上层树种数量的增加而降低,并在4种遮荫树种+茶组合模式达到最低;而植物体的碳储量仅占总碳储量的4.0%～8.2%,呈现随上层树种数量增加而先增加后降低的趋势。表明西双版纳的人工茶园复合态系统具有很强的碳储存能力。