In vitro and in vivo efficacy of tea tree essential oil for bacterial and yeast ear infections in dogs / Uso in vitro e in vivo do óleo essencial de Melaleuca (Melaleuca alternifolia) em otites bacterianas e por leveduras

Otitis externa is a common complaint in dogs. Bacteria and yeasts are commonly involved and may perpetuate inflammatory reactions inside the ear canal. Otoscopy, cytological examination of secretion and microbiological culture embody forms of diagnosis. Cytology also has great use in accessing treatment evolution. Therapy usually consists of cleaning ear canals and subsequent use of antibiotics or antifungal products. As some of them may cause hypersensitivity and even ototoxicity, searching for new pharmacological bases is currently necessary and justifies this study, which aimed to evaluate in vitro and in vivo efficacy of tea tree essential oil for bacterial and yeast ear infections in dogs. Twenty-eight dogs from a particular shelter in Cuiabá (Mato Grosso, Brazil), presenting clinical signs of otitis externa, were enrolled in this clinical trial. In all of them, clinical and cytological evaluations, as well as culture and susceptibility testing of the affected ears were carried out. From each dog, one ear was treated with 5% tea tree essential oil lotion and the other with standard otic formulation, according to the type of infection (bacterial, yeast or both). In vitro susceptibility testings of all ear cultures, to the same drugs used in treatment, were also carried out. Culture results showed 62.5% bacterial and fungal infection, 33.9% bacterial infection and 3.6% fungal infection, from the 56 ear samples collected. The most common microorganisms isolated were Staphylococcus intermedius, Staphylococcus aureus, Proteus mirabilis and Malassezia pachydermatis. Gram-positive bacteria were susceptible to gentamycin in 60.5% and resistant in 16.3% of the samples. Five percent tea tree essential oil formulation produced a 5mm clear zone of inhibition around the disks in one of the 63 samples evaluated. Pure (100%) tea tree essential oil formulation produced a 10mm clear zone of inhibition around the disks in four of the 63 samples evaluated, a 9mm zone in three samples, an 8mm zone in 16 samples, a 7mm zone in seven samples, a 6mm zone in two samples and there was no clear zone in 31 samples. Inhibition zones were produced by strains of Staphylococcus intermedius, Staphylococcus hyicus, Corynebacterium sp., Proteus mirabilis and Enterobacter sp. tea tree essential oil ear solution significantly induced remission of clinical signs both in bacterial and yeast ear infections. It also reduced as much Malassezia pachydermatis ear infection as the nystatin solution used in this study, while gentamycin solution showed better antibacterial effect. More studies should be conducted to evaluate in vitro diffusion properties of tea tree essential oil. Good antimicrobial spectrum and the absence of adverse reactions confirm the importance of developing a tea tree formulation as an alternative therapy for ear infections in dogs.(AU)

Otite externa é queixa frequente em cães. Bactérias e leveduras estão comumente envolvidas e podem perpetuar as reações inflamatórias dentro do canal auditivo. Dentre as formas de diagnóstico, encontram-se a otoscopia, o exame citológico da secreção e a cultura microbiológica. Citologia também tem grande utilidade no acesso à evolução do tratamento. A terapia consiste de limpeza dos canais auditivos e posterior utilização de antibióticos ou produtos antifúngicos. Como alguns antimicrobianos utilizados no tratamento podem causar hipersensibilidade e até mesmo ototoxicidade, a busca por novas bases farmacológicas justifica a existência deste estudo, que teve como objetivo avaliar in vitro e in vivo a eficiência do óleo de Melaleuca alternifolia em otites bacterianas e fúngicas de cães. Vinte e oito cães, de um abrigo particular, apresentando sinais clínicos de otite externa, foram incluídos neste estudo clínico. Todos passaram por avaliação clínica, citologia e cultura de material das orelhas afetadas. De cada animal, uma orelha foi tratada com óleo de Melaleuca 5% e a outra com formulação ótica padrão, de acordo com a afecção (bacteriana, fúngica ou mista). As culturas também foram submetidas a testes de susceptibilidade in vitro aos mesmos agentes utilizados no tratamento in vivo. Os resultados da cultura mostraram 62,5% de infecção mista (bacteriana e fúngica), 33,9% de infecção bacteriana e 3,6%, de infecção fúngica a partir das 56 orelhas. Os micro-organismos mais isolados foram Staphylococcus intermedius, Staphylococcus aureus, Proteus mirabilis e Malassezia pachydermatis. As bactérias GRAM-positivas foram sensíveis à gentamicina em 60,5% e resistentes em 16,3% das amostras. A formulação com 5% de óleo essencial de Melaleuca produziu uma zona de inibição de 5mm em torno dos discos em uma das 63 amostras avaliadas. A formulação pura (100%) do mesmo produto produziu uma zona de 10mm de inibição em quatro das 63 amostras analisadas, uma zona de 9 mm em três amostras, uma zona de 8mm em 16 amostras, uma zona de 7mm em sete amostras, uma zona de 6mm em duas amostras e não havia nenhuma zona clara em 31 amostras. Zonas de inibição foram produzidas por estirpes de Staphylococcus intermedius, Staphylococcus hyicus, Corynebacterium sp., Proteus mirabilis e Enterobacter sp. Clinicamente, a formulação com o óleo essencial de melaleuca induziu significativamente uma melhora nas manifestações, tanto nas otites bacterianas quanto nas causadas por leveduras, sendo estatisticamente semelhante ao tratamento com nistatina (nas otites por levedura), mas menos eficaz que a solução de gentamicina nas otites bacterianas. Mais estudos devem ser realizados para avaliar as propriedades de difusão in vitro do óleo essencial de melaleuca. O bom espectro antimicrobiano, a boa resposta clínica e a ausência de reações adversas confirmam a possibilidade de desenvolvimento de formulação ótica com o óleo essencial de melaleuca, como uma alternativa para a terapia de infecções do ouvido em cães.(AU)

Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment

Abstract The evolution of microorganisms resistant to many medicines has become a major challenge for the scientific community around the world. Motivated by the gravity of such a situation, the World Health Organization released a report in 2014 with the aim of providing updated information on this critical scenario. Among the most worrying microorganisms, species from the genus Candida have exhibited a high rate of resistance to antifungal drugs. Therefore, the objective of this review is to show that the use of natural products (extracts or isolated biomolecules), along with conventional antifungal therapy, can be a very promising strategy to overcome microbial multiresistance. Some promising alternatives are essential oils of Melaleuca alternifolia (mainly composed of terpinen-4-ol, a type of monoterpene), lactoferrin (a peptide isolated from milk) and chitosan (a copolymer from chitin). Such products have great potential to increase antifungal therapy efficacy, mitigate side effects and provide a wide range of action in antifungal therapy.

In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

ABSTRACT Objective Halitosis can be caused by microorganisms that produce volatile sulphur compounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. Material and Methods The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. Results The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 mg/mL). M. alternifolia significantly reduced the growth and production of hydrogen sulfide (H2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). Conclusion M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.

Análisis multivariable de la composición química de aceite esencial dehojas de Melaleuca quinquenervia que crece en Cuba / Multivariate analysis of the chemical composition of the essential oil from leaves of Melaleuca quinquenervia growing in Cuba

El aceite esencial de Melaleuca quinquenervia de la Ciénaga de Zapata, Cuba, obtenido a escala piloto con un rendimiento promedio de 0,8%, presentó una densidad relativa y un índice de refracción promedios de 0,9253g/mL y 1,4827 respectivamente. La determinación de su composición química por cromatografía de gases acoplada a espectrometría de masas permitió identificar 42 compuestos, de los cuales 26 se sometieron al Análisis por Componentes Principales y al Análisis por Conglomerados. Dichos análisis permitieron la separación de las muestras en dos grupos con diferencias significativas (p<0,05) entre los contenidos promedios de sus principales constituyentes. A partir de dichos análisis se estableció la presencia de dos quimiotipos diferentes: uno con elevados contenidos de viridiflorol (33,8 por ciento), seguido por el 1,8-cineol (21,8 por ciento), y otro quimiotipo del tipo 1,8-cineol (30,0 por ciento) con un contenido relativamente elevado de viridiflorol (24,6 por ciento). Ambos quimiotipos presentaron concentraciones similares de los monoterpenos limoneno y αlfa-pineno.

The essential oil of Melaleuca quinquenervia from Ciénaga de Zapata, Cuba, obtained at pilot scale with average yield of 0.8%, showed average relative density and refraction index of 0.9253g/mL and 1.4827 respectively. It ́s chemical composition determination by gas chromatography coupled to Mass Spectrometry allow identifying 42 compounds, 26 of which were submitted to Principal Components Analysis and to Conglomerates Analysis. Such analyses allowed the separation of the samples into two groups with significant differences (p<0.05) between the average contents of its main components. From such analyses, it was established the presence of two different chemotypes: one with high contents of viridiflorol (33.8 percent), followed by 1,8-cineol (21.8 percent), and another chemotype rich in 1,8-cineol (30,0 percent) with a relatively high content in viridiflorol (24.6 percent). Both chemotypes showed similar concentrations of limonene and αlfa-pinene monoterpenes.

Antimicrobial activity of Melaleuca sp. oil against clinical isolates of antibiotics resistant Staphylococcus aureus

PURPOSE: To extract the Melaleuca sp. oil and to assess its in vitro inhibitory effect against Staphylococcus aureus isolates obtained from lower limb wounds and resistant to several antibiotics. METHODS: A total of 14 test-tubes containing Mueller-Hinton broth were used to determine the Minimum Inhibitory Concentration (MIC). The following concentrations of the Melaleuca sp. oil were added to the first 11 tubes: 8; 4; 2; 1; 0.5; 0.2; 0.1; 0.05; 0.025; 0,0125 and 0.00625%. The 12th and 13th tubes, with and without oil, were used as the positive and negative controls, respectively. The experimental study was carried out in triplicate at 37ºC for 18 hours. The Minimum Bactericidal Concentration (MBC), able of killing all the microorganisms, was also determined. Two S. aureus isolates were obtained from lower limb wounds of female patients and the identification of the microorganisms (Staphylococcus aureus) and the test for susceptibility to the antimicrobial agents were carried out by automation using the apparatus MicroScan(r). After identification, the isolates were preserved in liquid Trypticase Soy medium, and inoculated for determination of the MIC and MBC. RESULTS: The MIC was 0.2% and the MBC was 0.4%. CONCLUSION: The Melaleuca sp. oil showed antimicrobial properties in vitro against strains isolated from lower limb wounds which were resistant to multiple antibiotics. .

Atividade de nanoformulações de Melaleuca alternifolia e terpinen-4-ol em isolados de Rhodococcus equi / Melaleuca alternifolia activity in nanoformulations and terpinen-4-ol against Rhodococcus equi isolates

Rhodococcus equi é o agente etiológico da rodococose equina, importante doença respiratória de potros. Especialmente na última década, a emergência de cepas resistentes aos antimicrobianos empregados no tratamento da rodococose tem sido relatada. Nesse sentido, há a necessidade de estudos envolvendo terapias alternativas e novas tecnologias, incluindo o uso de plantas medicinais e nanotecnologia. Neste trabalho utilizou-se Melaleuca alternifolia nas seguintes formulações: óleo livre, nanocápsula, nanoemulsão e a combinação de óleo livre com nanocápsula e com nanoemulsão, além do seu composto majoritário, terpinen-4-ol, a fim de verificar a atividade antimicrobiana frente a isolados de R. equi de diferentes origens. Utilizou-se o método de microdiluição em caldo na determinação das concentrações inibitória mínima (CIM) e bactericida mínima (CBM) das diferentes formulações frente aos isolados (n=24). Verificou-se baixo potencial para atividade antibacteriana de M. alternifolia na formulação de óleo livre. Todavia, essa atividade foi potencializada quando se incorporou o óleo essencial às nanoformulações. O composto terpinen-4-ol demonstrou potencial atividade antibacteriana quando incorporado ao óleo essencial e quando utilizado isoladamente. Verificou-se que tanto M. alternifolia quanto terpinen-4-ol testados possuem atividade antimicrobiana contra isolados de R. equi, sugerindo seu emprego em estudos avaliando seu potencial para o tratamento da rodococose.

Rhodococcus equi causes rodococose in horses, characterized by bronchopneumonia in foals. Due to reports of antimicrobial resistance, it is important to develop studies involving alternative therapies and new technologies, including the use of medicinal plants and nanotechnology. In this work, the plant Melaleuca alternifolia in oil free formulations, nanocapsule, nanoemulsion and the combination of free and nanocapsule oil nanoemulsion, besides its major compound, terpinen-4-ol, were used in order to verify antimicrobial activity against isolates of R. equi. The broth microdilution method was employed to determine the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of different formulations against 24 isolates. There was low antibacterial activity of M. alternifolia in oil free formulation; however, it was observed that the activity was enhanced when incorporated as essential oil the nanoformulations. The major compound, terpinen-4-ol, showed bactericidal and bacteriostatic activity when used alone. It is suggested that M. alternifolia, in association with nanocarriers systems, as well as terpinen -4-ol, presents potential for future studies concerning the equine rodococosis therapy.

Cumulative mortality of Aedes aegypti larvae treated with compounds / Mortalidade acumulativa de larvas de Aedes aegypti tratadas com compostos

OBJECTIVE To evaluate the larvicidal activity of Azadirachta indica, Melaleuca alternifolia, carapa guianensis essential oils and fermented extract of Carica papaya against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae). METHODS The larvicide test was performed in triplicate with 300 larvae for each experimental group using the third larval stage, which were exposed for 24h. The groups were: positive control with industrial larvicide (BTI) in concentrations of 0.37 ppm (PC1) and 0.06 ppm (PC2); treated with compounds of essential oils and fermented extract, 50.0% concentration (G1); treated with compounds of essential oils and fermented extract, 25.0% concentration (G2); treated with compounds of essential oils and fermented extract, 12.5% concentration (G3); and negative control group using water (NC1) and using dimethyl (NC2). The larvae were monitored every 60 min using direct visualization. RESULTS No mortality occurred in experimental groups NC1 and NC2 in the 24h exposure period, whereas there was 100% mortality in the PC1 and PC2 groups compared to NC1 and NC2. Mortality rates of 65.0%, 50.0% and 78.0% were observed in the groups G1, G2 and G3 respectively, compared with NC1 and NC2. CONCLUSIONS The association between three essential oils from Azadirachta indica, Melaleuca alternifolia, Carapa guianensis and fermented extract of Carica papaya was efficient at all concentrations. Therefore, it can be used in Aedes aegypti Liverpool third larvae stage control programs. .

OBJETIVO Avaliar a eficácia de composto de óleos essenciais de Azadirachta indica, Melaleuca alternifolia, Carapa guianensis e extrato fermentado de Carica papaya sobre larvas de Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae). MÉTODOS O ensaio larvicida foi realizado em triplicata com 300 larvas para cada grupo experimental utilizando larvas de terceiro estádio, as quais foram expostas por 24h, em 2013. Os grupos foram: controles positivos com larvicida industrial (BTI) nas concentrações de 0,37 ppm (CP1) e 0,06 ppm (CP2); tratado com composto de óleos essenciais e extrato fermentado na concentração de 50,0% (G1); tratado composto e óleos essenciais e extrato fermentado na concentração de 25,0% (G2); tratado com composto de óleos essenciais e um extrato fermentado na concentração de 12,5% (G3); controle negativo com água (CN1) e controle dimetil sulfóxido (CN2). As larvas foram monitoradas a cada 60 min através de visualização direta. RESULTADOS Larvas dos grupos CN1 e CN2 não tiveram mortalidade durante o período de 24h de exposição, mas os grupos CP1 e CP2 apresentaram taxa de mortalidade de 100% em relação a CN1 e CN2. Os tratamentos G1, G2 e G3 exerceram atividade larvicida de 65,0%, 50,0% e 78,0%, respectivamente, quando comparados a CN1 e CN2. CONCLUSÕES A associação entre os três óleos essenciais de Azadirachta indica, Melaleuca alternifolia, Carapa guianensis e extrato fermentado de Carica papaya foi eficiente em todas as concentrações testadas, podendo ser utilizado no controle de larvas de terceiro estádio de A. aegypti linhagem Liverpool. .

In vitro anti-microbial activity of the Cuban medicinal plants Simarouba glauca DC, Melaleuca leucadendron L and Artemisia absinthium L

In the present study, an extensive in vitro antimicrobial profiling was performed for three medicinal plants grown in Cuba, namely Simarouba glauca, Melaleuca leucadendron and Artemisia absinthium. Ethanol extracts were tested for their antiprotozoal potential against Trypanosoma b. brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum. Antifungal activities were evaluated against Microsporum canis and Candida albicans whereas Escherichia coli and Staphylococcus aureus were used as test organisms for antibacterial activity. Cytotoxicity was assessed against human MRC-5 cells. Only M. leucadendron extract showed selective activity against microorganisms tested. Although S. glauca exhibited strong activity against all protozoa, it must be considered non-specific. The value of integrated evaluation of extracts with particular reference to selectivity is discussed.