Helium Cold Atmospheric Plasma Causes Morphological and Biochemical Alterations in Candida albicans Cells.

(1) Background: Previous studies reported the promising inhibitory effect of cold atmospheric plasma (CAP) on Candida albicans. However, the exact mechanisms of CAP's action on the fungal cell are still poorly understood. This study aims to elucidate the CAP effect on C. albicans cell wall, by evaluating the alterations on its structure and biochemical composition; (2) Methods: C. albicans cells treated with Helium-CAP were analyzed by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) in order to detect morphological, topographic and biochemical changes in the fungal cell wall. Cells treated with caspofungin were also analyzed for comparative purposes; (3) Results: Expressive morphological and topographic changes, such as increased roughness and shape modification, were observed in the cells after CAP exposure. The alterations detected were similar to those observed after the treatment with caspofungin. The main biochemical changes occurred in polysaccharides content, and an overall decrease in glucans and an increase in chitin synthesis were detected; (4) Conclusions: Helium-CAP caused morphological and topographic alterations in C. albicans cells and affected the cell wall polysaccharide content.

Cold Atmospheric Plasma Jet as a Possible Adjuvant Therapy for Periodontal Disease.

Due to the limitations of traditional periodontal therapies, and reported cold atmospheric plasma anti-inflammatory/antimicrobial activities, plasma could be an adjuvant therapy to periodontitis. Porphyromonas gingivalis was grown in blood agar. Standardized suspensions were plated on blood agar and plasma-treated for planktonic growth. For biofilm, dual-species Streptococcus gordonii + P. gingivalis biofilm grew for 48 h and then was plasma-treated. XTT assay and CFU counting were performed. Cytotoxicity was accessed immediately or after 24 h. Plasma was applied for 1, 3, 5 or 7 min. In vivo: Thirty C57BI/6 mice were subject to experimental periodontitis for 11 days. Immediately after ligature removal, animals were plasma-treated for 5 min once-Group P1 (n = 10); twice (Day 11 and 13)-Group P2 (n = 10); or not treated-Group S (n = 10). Mice were euthanized on day 15. Histological and microtomography analyses were performed. Significance level was 5%. Halo diameter increased proportionally to time of exposure contrary to CFU/mL counting. Mean/SD of fibroblasts viability did not vary among the groups. Plasma was able to inhibit P. gingivalis in planktonic culture and biofilm in a cell-safe manner. Moreover, plasma treatment in vivo, for 5 min, tends to improve periodontal tissue recovery, proportionally to the number of plasma applications.

Cold Atmospheric Pressure Plasma Jet Reduces Trichophyton rubrum Adherence and Infection Capacity.

This study aimed to evaluate the effects of cold atmospheric pressure plasma (CAPP) jet on Trichophyton rubrum growth, germination and adherence to nail. The effects of plasma jet on T. rubrum conidia germination and on mycelial growth were evaluated by in vitro assays. An ex vivo nail infection model was used to evaluate the effects on conidia adherence and infection. Biochemical analyses of nail fragments exposed or not to CAPP were performed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Plasma jet exposure for 10 and 15 min completely inhibited mycelial growth after only one exposure. Fifteen minutes of exposure could reduce conidia germination in suspension. Fungal suspensions exposed to plasma jet for 10 and 15 min were not able to infect nail specimens. These results were corroborated by ATR-FTIR analyses of nail fragments. In conclusion, single exposure to CAPP for 15 min was able to inhibit fungal growth, adherence and infection capacity. The results suggest that cold atmospheric plasma jet can be a promising alternative for the treatment of onychomycoses caused by T. rubrum.

Amplitude-modulated cold atmospheric pressure plasma jet for treatment of oral candidiasis: In vivo study.

The aim of this study was to establish an effective and safe protocol for in vivo oral candidiasis treatment with atmospheric plasma jets. A novel amplitude-modulated cold atmospheric pressure plasma jet (AM-CAPPJ) device, operating with Helium, was tested. In vitro assays with Candida albicans biofilms and Vero cells were performed in order to determine the effective parameters with low cytotoxicity. After the determination of such parameters, the protocol was evaluated in experimentally induced oral candidiasis in mice. AM-CAPPJ could significantly reduce the viability of C. albicans biofilms after 5 minutes of plasma exposure when compared to the non-exposed group (p = 0.0033). After this period of exposure, high viability of Vero cells was maintained (86.33 ± 10.45%). Also, no late effects on these cells were observed after 24 and 48 hours (83.24±15.23% and 88.96±18.65%, respectively). Histological analyses revealed significantly lower occurrence of inflammatory alterations in the AM-CAPPJ group when compared to non-treated and nystatin-treated groups (p < 0.0001). Although no significant differences among the values of CFU/tongue were observed among the non-treated group and the groups treated with AM-CAPPJ or nystatin (p = 0.3201), histological analyses revealed marked reduction in candidal tissue invasion. In conclusion, these results point out to a clinical applicability of this protocol, due to the simultaneous anti-inflammatory and inhibitory effects of AM-CAPPJ with low cytotoxicity.

Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity.

The objective of this study was to evaluate the effects of Cymbopogon citratus essential oil and its main compound (citral) against primary dental colonizers and caries-related species. Chemical characterization of the essential oil was performed by gas chromatography/mass spectroscopy (GC/MS), and the main compound was determined. Antimicrobial activity was tested against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus. Minimum inhibitory and bactericide concentrations were determined by broth microdilution assay for streptococci and lactobacilli reference, and for clinical strains. The effect of the essential oil on bacterial adhesion and biofilm formation/disruption was investigated. Negative (without treatment) and positive controls (chlorhexidine) were used. The effect of citral on preformed biofilm was also tested using the same methodology. Monospecies and microcosm biofilms were tested. ANOVA or Kruskal-Wallis tests were used (α=0.05). Cytotoxicity of the essential oil to human keratinocytes was performed by MTT assay. GC/MS demonstrated one major component (citral). The essential oil showed an inhibitory effect on all tested bacterial species, including S. mutans and L. acidophilus. Essential oil of C. citratus (10X MIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p < 0.05). The essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p < 0.01). Citral significantly reduced the number of viable cells of streptococci biofilm (p < 0.001). The essential oil showed low cytotoxicity to human keratinocytes. Based on these findings, this study can contribute to the development of new formulations for products like mouthwash, against dental biofilms.

Gallic acid/hydroxypropyl-ß-cyclodextrin complex: Improving solubility for application on in vitro/ in vivo Candida albicans biofilms.

The aim of this study was to increase the solubility of gallic acid (GA) for the treatment of Candida albicans biofilm, which is very difficult to treat and requires high drug concentrations. Cyclodextrins (CDs) were used for this purpose. Complexes were evaluated by phase-solubility studies, prepared by spray drying and characterized by drug loading, scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The complexes were tested on C. albicans biofilm using in vitro and in vivo models. HPßCD formed soluble inclusion complexes with GA. The percentage of GA in GA/HPßCD was 10.8 ± 0.01%. The SEM and DSC analyses confirmed the formation of inclusion complexes. GA/HPßCD maintained the antimicrobial activity of the pure GA. GA/HPßCD was effective on C. albicans biofilms of 24 and 48h. The in vivo results showed an anti-inflammatory activity of GA/HPßCD with no difference in invading hypha counting among the groups. This study encourages the development of new antifungal agents.

Screening of plants with antimicrobial activity against enterobacteria, Pseudomonas spp. and Staphylococcus spp.

AIM: This study screened plants for antibacterial properties against bacteria of medical importance. MATERIALS & METHODS: 60 extracts were obtained from the leaves of ten plants (Jatropha weddelliana, Attalea phalerata, Buchenavia tomentosa, Croton doctoris, Mouriri elliptica, Mascagnia benthamiana, Senna aculeata, Unonopis guatterioides, Allagoptera leucocalyx and Bactris glaucescens) using different extraction methods: A) Ethanol 70°C/72 h; B) Water/5 min/100°C; C) Water/1 h/55°C; D) Water/72 h; E) Hexane/72 h and F) Ethanol 99°C/72 h. Enterobacteria/Pseudomonas and staphylococci reference strains and 201 clinical isolates were used. Primary screening was done using agar well-diffusion assay. MIC/minimum bactericidal concentration and chemical characterization were determined. RESULTS: Extracts 5F and 3A showed the best MIC/minimum bactericidal concentration against clinical isolates and showed the presence of phenols. CONCLUSION: The present study demonstrated that Mouriri elliptica and Buchenavia tomentosa were the most active plants against the studied bacteria.

Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity

Abstract The objective of this study was to evaluate the effects of Cymbopogon citratus essential oil and its main compound (citral) against primary dental colonizers and caries-related species. Chemical characterization of the essential oil was performed by gas chromatography/mass spectroscopy (GC/MS), and the main compound was determined. Antimicrobial activity was tested against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus. Minimum inhibitory and bactericide concentrations were determined by broth microdilution assay for streptococci and lactobacilli reference, and for clinical strains. The effect of the essential oil on bacterial adhesion and biofilm formation/disruption was investigated. Negative (without treatment) and positive controls (chlorhexidine) were used. The effect of citral on preformed biofilm was also tested using the same methodology. Monospecies and microcosm biofilms were tested. ANOVA or Kruskal-Wallis tests were used (α=0.05). Cytotoxicity of the essential oil to human keratinocytes was performed by MTT assay. GC/MS demonstrated one major component (citral). The essential oil showed an inhibitory effect on all tested bacterial species, including S. mutans and L. acidophilus. Essential oil of C. citratus (10X MIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p < 0.05). The essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p < 0.01). Citral significantly reduced the number of viable cells of streptococci biofilm (p < 0.001). The essential oil showed low cytotoxicity to human keratinocytes. Based on these findings, this study can contribute to the development of new formulations for products like mouthwash, against dental biofilms.

Atividade antifúngica e citotoxicidade do jato de plasma frio sob pressão atmosférica / Antifungal activity and cytotoxicity of atmospheric pressure nonthermal plasma jet

As doenças fúngicas representam grande desafio para a área médica e odontológica devido à crescente prevalência e aumento da resistência aos antifúngicos. O plasma frio sob pressão atmosférica (PFPA) é uma mistura gasosa contendo partículas carregadas, radicais livres e radiação. Sua potencial aplicação em doenças infecciosas foi relatada, contudo a literatura carece de estudo sistemático sobre o efeito antifúngico, mecanismos de ação e potencial citotóxico. Neste trabalho foi avaliado o efeito antifúngico do PFPA sobre Candida albicans através de ensaios em células planctônicas e biofilmes, efeitos sobre a integridade de parede celular e membrana plasmática, morfogênese, produção de exoenzimas, aderência às células epiteliais e efeito no tratamento in vivo de lesões de candidose oral induzida em modelo murino. Ainda, avaliou-se o efeito do PFPA sobre culturas de Trichophyton rubrum, além de efeitos sobre capacidade de adesão de conídios. Ainda, o potencial citotóxico foi investigado usando células epiteliais. PFPA em modo de tensão contínuo (MC) foi capaz de reduzir a aderência de C. albicans às células epiteliais, modular a transição levedura-hifa na cepa SC 5314 e comprometer a viabilidade de biofilmes. PFPA-MC se mostrou citotóxico em parâmetros efetivos frente a biofilmes de C. albicans. Porém, não foi observado efeito citotóxico quando o PFPA foi utilizado em modo de tensão pulsada (MP). A exposição ao PFPA-MP reduziu a invasão de C. albicans no epitélio in vivo. O PFPA-MP foi capaz de afetar o crescimento de T. rubrum a partir de 10 minutos e de afetar a sua capacidade de aderência. Assim, conclui-se que PFPA apresenta efeito antifúngico contra C. albicans e T. rubrum e é capaz de interferir em fatores de virulência de ambos os micro-organismos(AU)

Fungal diseases represent a great challenge to the medical and dental areas, due to the increasing prevalence and antifungal resistance. Atmospheric pressure plasma jet (APPJ) is a gaseous mixture containing charged particles, free radicals and radiation. Its potential application in infectious disease has been reported, however there is still a lack of a systematic study on the antifungal effect, mechanism of action and citotoxicc potential. The general aim of this project was to evaluate the antifungal effect of APPJ on Candida albicans in planktonic and biofilm cultures, effects on cell wall and cell membrane integrity, morphogenesis, exoenzymes production, adherence to epithelial cells and in vivo effect in the treatment of oral candidosis in murine model will be performed. The effect of APPJ on Trichophyton rubrum cultures and on adherence capability were also evaluated. The cytotoxic potential was evaluated in vitro. APPJ in continuous tension mode (CM) was able to reduce the adherence and yeast-hyphae transition in C. albicans SC 5314 and to decrease biofilm viability. APPJ-CM showed cytotoxic effect in the parameters effective to C. albicans biofilm. Conversely, no cytotoxic effect on epithelial cells were observed when pulsed (PM) plasma jet was used. In vivo tests showed that APPJ-PM was able to prevent C. albicans invasion to the epithelium. T. rubrum cultures were affectd by APPJ-PM after 15 minutes of exposure and conidia adherence was impaired by 10 minutes exposure. In conclusion, APPJ showed antifungal effect against C. albicans and T. rubrum and can also impair virulence factors in both microorganisms(AU)

Investigação das propriedades antimicrobianas de frações obtidas a partir dos extratos hidroalcoólico e etanólico de Croton doctoris S. Moore / Evaluation of antimicrobial properties of fractions obtained from hydroalcoholic and ethanolic extracts from Croton doctoris S. Moore

Na última década, tratamentos com plantas medicinais se expandiram pelo mundo e ganharam popularidade. O objetivo desse trabalho foi avaliar o efeito antimicrobiano de frações de extratos de Croton doctoris S. Moore sobre micro-organismos envolvidos na etiologia cárie dentária e identificar substâncias com efeito antimicrobiano dentro das frações mais ativas. Extratos foram obtidos por maceração do pó das folhas em etanol 70º ou etanol 99,5º (1 g / 20 ml de solvente; 72h). Em seguida, frações foram obtidas por extração líquido-líquido dos extratos com diferentes solventes. As frações mais ativas foram selecionadas através do teste de difusão em ágar utilizando cepas de referência (Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mitis ATCC 9811, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478). As concentrações inibitórias e bactericidas mínimas (CIM e CBM) das frações mais ativas foram determinadas para as cepas de referência e isolados clínicos de dentina cariada de estreptococos do grupo mutans e lactobacilos. O efeito sobre biofilme em modelo de microcosmo a partir da saliva foi avaliado para as frações mais ativas em concentrações 2, 4 e 10 vezes o valor de CIM. As mesmas frações, em concentração referente a ½ CIM, foram analisadas quanto a capacidade de inibir a adesão de micro-organismos ao esmalte dentário em modelo de microcosmo. A caracterização química das frações ativas foi realizada através de espectometria de massas. A triagem mostrou que as frações diclorometânicas dos extratos hidroalcoólico (HD) e etanólico (ED) e a fração hexânica do extrato hidroalcoólico (HH) apresentaram atividade antimicrobiana. Os valores de CIM variaram de 0,1 a 3,1 mg/ml e CBM de 0,1 a >50 mg/ml para as cepas de referências, sendo que os melhores resultados foram encontrados nas frações HD e ED. Os valores de CIM e CBM para isolados clínicos ...

In the past decade, treatment with medicinal plants expanded worldwide and gained popularity. The aim of this study was to evaluate the antimicrobial effect of fractions from extracts of Croton doctoris S. Moore against microorganisms of cariogenic relevance and to identify substances with antimicrobial action in the most active fractions. Crude extracts was obtained by maceration of powdered leaves in 70% ethanol or 99.5% ethanol (1 g / 20 ml, 72 h).The fractions were obtained by liquid-liquid extraction with different solvents. The most active fractions were selected by agar well diffusion test against reference strains (Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mitis ATCC 9811, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478). Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of most active fractions were evaluated against reference strains and clinical isolates of lactobacilus and mutans streptococci. The activity against microcosm biofilm obtained from human saliva was evaluated for the most active fractions at 2, 4 and 10 times MIC. These fractions (½ MIC) were also tested for inhibition of microorganism adhesion to enamel. Chemical characterization of most active fractions was made using mass spectrometry. The screening showed that dichoromethanic fractions of hydroalcoholic and ethanolic extracts (HD and ED) and hexanic fration of hydroalcoholic extract (HH) had antimicrobial activity. The MIC and MBC values for reference strains ranged from 0.1 to 3.1 mg/ml and 0.1 to >50 mg/ml. HD and ED fractions showed the best values. For lactobacilli clinical isolates MIC and MBC ranged from 0.1 to 25 mg/ml and 0.2 to >50 mg/ml. MIC and MBC for mutans streptococci clinical isolates ranged from 0.8 to 25 mg/ml and 3.1 a >50 mg/ml. Activity against biofilms showed that HD fraction...