In Vitro Antifungal Activity of Selected Essential Oils against Drug-Resistant Clinical Aspergillus spp. Strains.

BACKGROUND: Treatment options for aspergillosis include amphotericin B (AMB) and azole compounds, such as itraconazole (ITZ). However, serious side effects related to these antifungal agents are increasingly evident, and resistance continues to increase. Currently, a new trend in drug discovery to overcome this problem is represented by natural products from plants, or their extracts. Particularly, there is a great interest in essential oils (EOs) recognized for their antimicrobial role towards bacteria, fungi and viruses. METHODS: In this study, we evaluated the antifungal activity of eleven commercial EOs-clove, eucalyptus, geranium, hybrid lavender, lavender, lemon, lemongrass, neroli, oregano, tea tree and red red thyme-in comparison with AMB and ITZ against Aspergillus flavus, A. fumigatus and A. niger clinical isolates. Antifungal activity was determined by broth microdilution method, agar diffusion technique, fungistatic and fungicidal activities and vapor contact assay. RESULTS: Gas chromatography-mass spectrometry analysis displayed two groups of distinct biosynthetical origin: monoterpenes dominated the chemical composition of the most oils. Only two aromatic compounds (eugenol 78.91% and eugenyl acetate 11.64%) have been identified as major components in clove EO. Lemongrass EO exhibits the strongest antimicrobial activity with a minimum inhibitory concentration of 0.56 mg/mL and a minimum fungicidal concentration of 2.25-4.5 mg/mL against Aspergillus spp. strains. Clove and geranium EOs were fairly effective in inhibiting Aspergillus spp. growth. CONCLUSIONS: These results demonstrate the antimicrobial potential of some EOs and support the research of new alternatives or complementary therapies based on EOs.

Design, Characterization, and Biological Activities of Erythromycin-Loaded Nanodroplets to Counteract Infected Chronic Wounds Due to Streptococcus pyogenes.

Streptococcus pyogenes causes a wide spectrum of diseases varying from mild to life threatening, despite antibiotic treatment. Nanoparticle application could facilitate the foreign pathogen fight by increasing the antimicrobial effectiveness and reducing their adverse effects. Here, we designed and produced erythromycin-loaded chitosan nanodroplets (Ery-NDs), both oxygen-free and oxygen-loaded. All ND formulations were characterized for physico-chemical parameters, drug release kinetics, and tested for biocompatibility with human keratinocytes and for their antibacterial properties or interactions with S. pyogenes. All tested NDs possessed spherical shape, small average diameter, and positive Z potential. A prolonged Ery release kinetic from Ery-NDs was demonstrated, as well as a favorable biocompatibility on human keratinocytes. Confocal microscopy images showed ND uptake and internalization by S. pyogenes starting from 3 h of incubation up to 24 h. According to cell counts, NDs displayed long-term antimicrobial efficacy against streptococci significantly counteracting their proliferation up to 24 h, thanks to the known chitosan antimicrobial properties. Intriguingly, Ery-NDs were generally more effective (104-103 log10 CFU/mL), than free-erythromycin (105 log10 CFU/mL), in the direct killing of streptococci, probably due to Ery-NDs adsorption by bacteria and prolonged release kinetics of erythromycin inside S. pyogenes cells. Based on these findings, NDs and proper Ery-NDs appear to be the most promising and skin-friendly approaches for the topical treatment of streptococcal skin infections allowing wound healing during hypoxia.

Immune Defences: A View from the Side of the Essential Oils.

The use of essential oils is increasingly being investigated among new therapeutic approaches based on medicinal plants and their extracts. With the wide use of synthetic and semi-synthetic antimicrobial drugs, the spread of drug-resistant clinical isolates has increased, and research is directed towards natural products, such as essential oils, as useful antimicrobial resources. In the context of a prospective infection, we compared the impact of essential oils and common antimicrobial agents on the microbicidal activity of human phagocytes. Here, we present the results of our decades-long investigation into the effectiveness of thyme red oil (26.52% thymol chemotype), tea tree oil (TTO), and Mentha of Pancalieri [(Mentha x piperita (Huds) var. officinalis (Sole), form rubescens (Camus) (Lamiaceae)] essential oils on human polymorphonuclear leukocytes (PMNs) capacity to kill clinical strains of Candida albicans and C. krusei when compared to three antifungal drugs used to treat candidiasis (fluconazole, anidulafungin, and caspofungin) These essential oils demonstrate antifungal drug-like and/or superior efficacy in enhancing intracellular killing by PMNs, even at subinhibitory concentrations. Our results are compared with data in the literature on essential oils and immune system interactions. This comparison would aid in identifying therapeutic solutions to the increasingly prevalent antibiotic resistance as well as filling in any remaining knowledge gaps on the bioactivity of essential oils.

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species.

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

Smart nano-in-microparticles to tackle bacterial infections in skin tissue engineering.

Chronic wounds (resulting from underlying disease, metabolic disorders, infections, trauma, and even tumours) pose significant health problems. In this work, microparticles, based on polysaccharides (maltodextrin or dextran) and amino acids, and doped with antibacterial nanoparticles (CuO or ZnO NPs) are designed. Smart nano-in-microparticles with a hierarchical 3D structure are developed. The ultimate goal aims at an innovative platform to achieve skin repair and to manage skin colonization by avoiding infection that could delay and even impair the healing process. The microparticles are prepared by spray-drying and cross-linked by heating, to obtain insoluble scaffolds able to facilitate cell proliferation in the wound bed. The nano-in-microparticles are characterized using a multidisciplinary approach: chemico-physical properties (SEM, SEM-EDX, size distribution, swelling and degradation properties, structural characterization - FTIR, XRPD, SAXS - mechanical properties, surface zeta potential) and preclinical properties (in vitro biocompatibility and whole-blood clotting properties, release studies and antimicrobial properties, and in vivo safety and efficacy on murine burn/excisional wound model) were assessed. The hierarchical 3D nano-in microparticles demonstrate to promote skin tissue repair in a preclinical study, indicating that this platform deserves particular attention and further investigation will promote the prototypes translation to clinics.

Disinfection and Biocompatibility of Titanium Surfaces Treated with Glycine Powder Airflow and Triple Antibiotic Mixture: An In Vitro Study.

The aim of this in vitro study was to compare three disinfection protocols of biofilm-coated machined (MAC) and acid etched (SLA) commercial pure Grade 4 Titanium disks. Samples were infected with a vial of polymicrobial biofilm to simulate peri-implantitis in vitro. Seventeen MAC and twenty SLA titanium disks were randomly assigned to: (1) glycine powder air-flow (GYPAP) for 1 min; (2) a local delivered triple paste antibiotic composed by a gel mixture with ciprofloxacin, metronidazole, and clarithromycin (3MIX) for 1 h; and (3) a combination of both (GYPAP + 3MIX). Biocompatibility of the titanium disks after each treatment protocol was assessed by measurement of adhesion and growth of adipose-derived mesenchymal stem cells (ASCs) after 24 and 72 h. A confocal laser scanning microscope (CLSM) assessed the antibacterial effect of each treatment. Data of the antibacterial efficacy and cell viability were presented as mean with standard deviation and calculated by one-way ANOVA with multiple comparisons via Bonferroni tests. Results were considered significant with p < 0.05. The higher cell viability was achieved by the 3MIX and GYPAP combination on the SLA surfaces after 72 h. CLSM analysis showed a mean ratio of dead bacteria statistically higher in the 3MIX + GYPAP group compared with the GYPAP and 3MIX subgroups (p < 0.05). In conclusion, data showed that the combination of GYPAP and 3MIX could be preferred to the other protocols, especially in presence of SLA titanium surface.

Antibacterial and Antifungal Efficacy of Medium and Low Weight Chitosan-Shelled Nanodroplets for the Treatment of Infected Chronic Wounds.

Purpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) - methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata - and for their physical interaction with cell walls/membranes. Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy. Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h. Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.

Confocal Laser Scanner Evaluation of Bactericidal Effect of Chitosan Nanodroplets Loaded with Benzalkonium Chloride.

The aim was to evaluate the antibacterial efficacy and penetration depth into dentinal tubules of a solution of chitosan nanodroplets (NDs) loaded with Benzalkonium Chloride (BAK). Seventy-two human single-root teeth with fully formed apex were used. Cylindrical root dentin blocks were longitudinally sectioned and enlarged to a size of a Gates Glidden drill #4. After sterilization, root canals were infected with Enterococcus faecalis ATCC 29212 and further incubated for three weeks. Specimens were assigned to three experimental groups (n = 20), plus positive (n = 6) and negative (n = 6) controls. In the first group, irrigation was achieved with 2 mL of NDs solution loaded with BAK (NDs-BAK), in the second with 2 mL of 5% sodium hypochlorite (NaOCl) and in the last with 2 mL of 2% chlorhexidine (CHX). Specimens were rinsed and vertically fractured. Confocal laser scanning microscopy (CLSM) and viability staining were used to analyze the proportions of dead and live bacteria quantitatively. The volume ratio of red fluorescence (dead) was calculated in 3D reconstructions. Data were analyzed by one-way ANOVA and post hoc Bonferroni tests (p < 0.05). The ratio of red fluorescence over the whole green/red fluorescence resulted in a significant comparison of NDs-BAK with NaOCl (p < 0.01) and NaOCl with CHX (p < 0.01). No differences were found between NDs-BAK and CHX (p > 0.05). The mean depth of efficacy was, respectively: NDs-BAK 325.25 µm, NaOCl 273.36 µm and CHX 246.78 µm with no statistical differences between groups. The NaOCl solution showed the highest antimicrobial efficacy, but nanodroplets with BAK seemed to have the same effect as CHX with a high depth of efficacy.

Exploitation of the Antibacterial Properties of Photoactivated Curcumin as 'Green' Tool for Food Preservation.

In the search for non-chemical and green methods to counteract the bacterial contamination of foods, the use of natural substances with antimicrobial properties and light irradiation at proper light waves has been extensively investigated. In particular, the combination of both techniques, called photodynamic inactivation (PDI), is based on the fact that some natural substances act as photosensitizers, i.e., produce bioactive effects under irradiation. Notably, curcumin is a potent natural antibacterial and effective photosensitizer that is able to induce photodynamic activation in the visible light range (specifically for blue light). Some practical applications have been investigated with particular reference to food preservation from bacterial contaminants.

Antimicrobial oxygen-loaded nanobubbles as promising tools to promote wound healing in hypoxic human keratinocytes.

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Additionally, CWs are often worsened by microbial infections. Oxygen-loaded nanobubbles (OLNBs), displaying a peculiar structure based on oxygen-solving perfluorocarbons such as perfluoropentane in the inner core and polysaccharydes including chitosan in the outer shell, have proven effective in delivering oxygen to hypoxic tissues. Antimicrobial properties have been largely reported for chitosan. In the present work chitosan/perfluoropentane OLNBs were challenged for biocompatibility with human skin cells and ability to promote wound healing processes, as well as for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. After cellular internalization, OLNBs were not toxic to human keratinocytes (HaCaT), whereas oxygen-free NBs (OFNBs) slightly affected their viability. Hypoxia-dependent inhibition of keratinocyte migratory ability after scratch was fully reversed by OLNBs, but not OFNBs. Both OLNBs and OFNBs exerted chitosan-induced short-term bacteriostatic activity against MRSA (up to 6 h) and long-term fungistatic activity against C. albicans (up to 24 h). Short-term antibacterial activity associated with NB prolonged adhesion to MRSA cell wall (up to 24 h) while long-term antifungal activity followed NB early internalization by C. albicans (already after 3 h of incubation). Taken altogether, these data support chitosan-shelled and perfluoropentane-cored OLNB potential as innovative, promising, non-toxic, and cost-effective antimicrobial devices promoting repair processes to be used for treatment of MRSA- and C. albicans-infected CWs.

The Inhibition of Non-albicans Candida Species and Uncommon Yeast Pathogens by Selected Essential Oils and Their Major Compounds.

The epidemiology of yeast infections and resistance to available antifungal drugs are rapidly increasing, and non-albicans Candida species and rare yeast species are increasingly emerging as major opportunistic pathogens. In order to identify new strategies to counter the threat of antimicrobial resistant microorganisms, essential oils (EOs) have become an important potential in the treatment of fungal infections. EOs and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities. We investigated the in vitro antifungal activity of nine commercial EOs such as Thymus vulgaris (thyme red), Origanum vulgare (oregano), Lavandula vera (lavender), Pinus sylvestris (pine), Foeniculum vulgare (fennel), Melissa officinalis (lemon balm), Salvia officinalis (sage), Eugenia caryophyllata (clove) and Pelargonium asperum (geranium), and some of their main components (α-pinene, carvacrol, citronellal, eugenol, γ-terpinene, linalool, linalylacetate, terpinen-4-ol, thymol) against non-albicans Candida strains and uncommon yeasts. The EOs were analyzed by GC-MS, and their antifungal properties were evaluated by minimum inhibitory concentration and minimum fungicidal concentration parameters, in accordance with CLSI guidelines, with some modifications for EOs. Pine exhibited strong antifungal activity against the selected non-albicans Candida isolates and uncommon yeasts. In addition, lemon balm EOs and α-pinene exhibited strong antifungal activity against the selected non-albicans Candida yeasts. Thymol inhibited the growth of all uncommon yeasts. These data showed a promising potential application of EOs as natural adjuvant for management of infections by emerging non-albicans Candida species and uncommon pathogenic yeasts.

Antifungal Activity of Melaleuca alternifolia Essential Oil (TTO) and Its Synergy with Itraconazole or Ketoconazole against Trichophyton rubrum.

Over the past 20-30 years, Trichophyton rubrum represented the most widespread dermatophyte with a prevalence accounting for 70% of dermatophytosis. The treatment for cutaneous infections caused by Trichophyton spp. are imidazoles (ketoconazole (KTZ)) and triazoles (itraconazole (ITZ)). T. rubrum can develop resistance to azoles after prolonged exposure to subinhibitory concentrations resulting in therapeutic failures and chronic infections. These problems have stimulated the search for therapeutic alternatives, including essential oils, and their potential use in combination with conventional antifungals. The purpose of this study was to evaluate the antifungal activity of tea tree oil (TTO) (Melaleuca alternifolia essential oil) and the main components against T. rubrum and to assess whether TTO in association with KTZ/ITZ as reference drugs improves the antifungal activity of these drugs. We used a terpinen-4-ol chemotype (35.88%) TTO, and its antifungal properties were evaluated by minimum inhibitory and minimum fungicidal concentrations in accordance with the CLSI guidelines. The interaction between TTO and azoles was evaluated through the checkerboard and isobologram methods. The results demonstrated both the fungicide activity of TTO on T. rubrum and the synergism when it was used in combination with azoles. Therefore, this mixture may reduce the minimum effective dose of azole required and minimize the side effects of the therapy. Synergy activity offered a promise for combination topical treatment for superficial mycoses.

Influence of Photon-Induced Photoacoustic Streaming (PIPS) on Root Canal Disinfection and Post-Operative Pain: A Randomized Clinical Trial.

The aim of this study was to evaluate the ability of a PIPS (photon-induced photoacoustic streaming) Er:YAG laser to reduce the root canal system bacterial count in vivo in comparison to the traditional irrigation technique. The post-operative patients' quality of life (QoL) after endodontic therapy was evaluated through a questionnaire. Fifty-four patients affected by pulp necrosis with or without apical periodontitis biofilm disease were selected for endodontic treatment and randomly assigned to Group A (n = 27) with traditional irrigation and Group B (n = 27), with PIPS irrigation applied according to the protocol. Shaping was performed with ProGlider and ProTaper Next, and irrigation was performed with 5% NaOCl and 10% EDTA. Intracanal samples for culture tests were collected before and after irrigation. The microbiological analysis was evaluated by the Kolmogorov-Smirnov normality and Mann-Whitney tests (p < 0.05). A self-assessment questionnaire was used to evaluate the QoL during the 7 days after treatment; differences were analysed with Student's t-test. Irrigation with the PIPS device was significantly effective in reducing bacterial counts, which were higher for facultative than obligate anaerobic strains, particularly for Gram-negative bacteria, without statistical significance (p > 0.05). There were no significant differences among the QoL indicators, except for the maximum pain (p = 0.02), eating difficulty (p = 0.03) and difficulty performing daily functions (p = 0.02) in the first few days post-treatment. PIPS may represent an aid to root canal disinfection not affecting the patients' QoL, particularly for the first day after treatment.

Evaluation of antimicrobial activity of the hydrolate of Coridothymus capitatus (L.) Reichenb. fil. (Lamiaceae) alone and in combination with antimicrobial agents.

BACKGROUND: Hydrolates, complex mixtures containing traces of essential oils (EOs), are inexpensive, easy to make and less toxic than their corresponding EOs. The antibacterial and antifungal activity of the hydrolate of Coridothymus capitatus (L.) Reichenb. fil. (Lamiaceae) alone and in combination with antimicrobial drugs, such as tetracycline and itraconazole, were evaluated. METHODS: The chemical composition was analysed by gas-chromatography-mass spectrometry (GC-MS). Standard methods were performed to evaluate the susceptibility of some Gram-positive and Gram-negative bacteria, and Candida spp. to the hydrolate, in comparison with its EO. The hydrolate mechanism of action was assayed by propidium iodide and MitoTracker staining. Checkerboard tests were carried out for combinations studies. RESULTS: GC-MS identified 0.14% (v/v) of total EO content into hydrolate and carvacrol as a dominant component. The hydrolate showed a good antimicrobial activity against bacteria and yeasts. It exhibited a synergistic effect with itraconazole against Candida krusei, and an additive effect with tetracycline against methicillin-resistant Staphylococcus aureus strains. Hydrolate changed the membranes permeability of bacteria and yeasts and altered mitochondrial function of yeasts. CONCLUSIONS: Our study extends the knowledge by exploiting non-conventional antimicrobial agents to fight the emergence of antibiotic resistance.

Enhanced Killing of Candida krusei by Polymorphonuclear Leucocytes in the Presence of Subinhibitory Concentrations of Melaleuca alternifolia and "Mentha of Pancalieri" Essential Oils.

The aim of this study was to evaluate the influence of tea tree oil (TTO) and "Mentha of Pancalieri" essential oil (MPP) on intracellular killing of Candida krusei, often resistant to conventional drugs, by polymorphonuclear leucocytes (PMNs). Intracellular killing was investigated by incubating yeasts and PMNs with essential oils (EOs) at 1/4 and 1/8 × MIC (Minimal Inhibitory Concentration), in comparison with anidulafungin, used as a reference drug. Killing values were expressed as Survival Index (SI) values. The cytotoxicity of EOs was evaluated by 3-[4,-5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Both EOs were more efficaceous at 1/8 × MIC than 1/4 × MIC, with killing values higher than observed in EO-free systems and in presence of anidulafungin, indicating that the decreasing concentrations did not cause lower candidacidal activity. This better activity at 1/8 × MIC is probably due to the EOs' toxicity at 1/4 × MIC, suggesting that at higher concentrations EOs might interfere with PMNs functionality. TTO and MPP at 1/8 × MIC significantly increased intracellular killing by PMNs through their direct action on the yeasts (both EOs) or on phagocytic cells (MPP), suggesting a positive interaction between EOs and PMNs to eradicate intracellular C. krusei. These data showed a promising potential application of TTO and "Mentha of Pancalieri" EO as natural adjuvants in C. krusei infection management.

Evaluation of the Antifungal Activity of Mentha x piperita (Lamiaceae) of Pancalieri (Turin, Italy) Essential Oil and Its Synergistic Interaction with Azoles.

The promising antimicrobial activity of essential oils (EOs) has led researchers to use them in combination with antimicrobial drugs in order to reduce drug toxicity, side effects, and resistance to single agents. Mentha x piperita, known worldwide as "Mentha of Pancalieri", is produced locally at Pancalieri (Turin, Italy). The EO from this Mentha species is considered as one of the best mint EOs in the world. In our research, we assessed the antifungal activity of "Mentha of Pancalieri" EO, either alone or in combination with azole drugs (fluconazole, itraconazole, ketoconazole) against a wide panel of yeast and dermatophyte clinical isolates. The EO was analyzed by GC-MS, and its antifungal properties were evaluated by minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) parameters, in accordance with the CLSI guidelines, with some modifications. The interaction of EO with azoles was evaluated through the chequerboard and isobologram methods. The results suggest that this EO exerts a fungicidal activity against yeasts and a fungistatic activity against dermatophytes. Interaction studies with azoles indicated mainly synergistic profiles between itraconazole and EO vs. Candida spp., Cryptococcus neoformans, and Trichophyton mentagrophytes. Thus, the "Mentha of Pancalieri" EO may act as a potential antifungal agent and could serve as a natural adjuvant for fungal infection treatment.

A case report of tinea capitis in infant in first year of life.

BACKGROUND: Tinea capitis is a cutaneous fungal infection common among 3 to 7 year old children but it is rare in the first year of life. CASE PRESENTATION: We present a case of a 12-month-old infant with erythematous scalp lesions combined with hair loss. He was suspected of dermatophytosis and mycological analysis of all suspected lesions was performed. Clinical features and culture results confirmed tinea capitis caused by Microsporum canis. The infant patient was treated with griseofulvin for 2 months. However, 15 days later at the end of treatment he presented with a single vesicle positive for M. canis. Griseofulvin therapy continued for another month. After 3 months of follow-up, no recurrence was observed. CONCLUSIONS: In infant, sometimes tinea capitis is misdiagnosed and underreported because it is similar to other scalp pathologies. Therefore, if erythematous scalp lesions are present, they must be examined from a mycological point of view to inform the differential diagnosis. Once diagnosed, treatment of tinea capitis can pose a dilemma because different factors may influence the choice between equally effective therapies (i.e. safety, age, formulation, cost). This case report suggests that it is important to establish an accurate diagnosis and  treatment for this dermatophytosis to avoid recurrences or therapeutic failures, especially in infants.

Use of Pinus sylvestris L. (Pinaceae), Origanum vulgare L. (Lamiaceae), and Thymus vulgaris L. (Lamiaceae) essential oils and their main components to enhance itraconazole activity against azole susceptible/not-susceptible Cryptococcus neoformans strains.

BACKGROUND: Cryptococcal infections, besides being a problem for immunocompromised patients, are occasionally being a problem for immunocompetent patients. In addition, the lower susceptibility of this yeast to azoles is a growing problem in health care. To date, there are very few molecules with any activity towards Cryptococcus neoformans, leading to heightened interest in finding new alternatives or adjuvants to conventional drugs for the treatment of mycosis caused by this yeast. Since the essential oils (EOs) are considered as a potential rich source of bioactive antimicrobial compounds, we evaluated the antifungal activity of Origanum vulgare (oregano), Pinus sylvestris (pine), and Thymus vulgaris (thyme red) EOs, and their components (α-pinene, carvacrol, thymol) compared with fluconazole, itraconazole, and voriconazole, against C.neoformans clinical strains. Then, we investigated the effect of EOs and components in combination with itraconazole. METHODS: EO composition was analysed by Gas chromatography-mass spectrometry (GC-MS). A broth microdilution method was used to evaluate the susceptibility of C.neoformans to azoles, EOs and components. Checkerboard tests, isobolograms and time-kill assays were carried out for combination studies. RESULTS: Six C.neoformans isolates were susceptible to azoles, while one C.neoformans exhibited a reduced susceptibility to all tested azole drugs. All EOs exerted a good inhibitory activity against all C.neoformans strains. Pine EO was the most effective. Among components, thymol exerted the most remarkable activity. By checkerboard testing and isobolographic analysis, combinations of itraconazole with oregano, pine, or thyme EOs, and carvacrol were found to be synergistic (FICI≤0.5) against azole susceptible C.neoformans. Regarding the azole not susceptible C.neoformans strain, the synergistic effect with itraconazole was observed with thyme EO (chemotype: thymol 26.52%; carvacrol 7.85%), and carvacrol. Time-kill assays confirmed the synergistic effects of itraconazole and oregano or thyme EO against azole susceptible C.neoformans. Binary mixtures of itraconazole/thyme EO or carvacrol yielded additive effects on the azole not susceptible C.neoformans. CONCLUSIONS: Our findings highlight the potential effectiveness of thyme, oregano EOs, and carvacrol as natural and cost-effective adjuvants when used in combination with itraconazole. Identification of EOs exerting these effects could be one of the feasible ways to overcome drug resistance, reducing drug concentration and side effects.

The Microbiological@mind project: a public engagement initiative of Turin University bringing microbiology and health education into primary schools.

Despite ongoing global efforts, antimicrobial resistance continues to threaten the treatment of an ever-increasing range of bacterial infections. There is substantial evidence that public education programs that foster microbial literacy amongst young school audiences may improve correct knowledge of specific health issues, such as prevention of microbial infections and responsible use of antibiotics. The aim of the Microbiological@mind project was to engage primary school students with the subject of microbiology, to promote both scientific interest and awareness towards correct behaviors that may ensure a safer lifestyle. Interactive workshops based on a full ''hands-on'' approach were carried out by an expert team from the University of Turin to over 1200 children aged 9-11 years at primary schools in Turin. A questionnaire (pre- and post-activity test) on the main topic (i.e. antibiotics) was used to assess project effectiveness. The workshops provided a useful means to strengthen the understanding of basic microbiology concepts amongst students. Students' baseline knowledge of antibiotics was quite low, as low percentages of correct answers on antibiotic action and use (5.0% and 12.1%, respectively) were found in the pre-activity tests. A significant increase (P <0.0001) in correct knowledge was observed in the post-activity tests, after implementation of the teaching activity. Our findings support the idea that microbial literacy in early childhood through hands-on educational programs is of great importance to foster children's interest in science learning, and to provide young people with information about general and specific health-related issues, such as prudent antibiotic use, for a more responsible citizenship.

Liquid and vapour-phase antifungal activities of essential oils against Candida albicans and non-albicans Candida.

BACKGROUND: The management of Candida infections faces many problems, such as a limited number of antifungal drugs, toxicity, resistance of Candida to commonly antifungal drugs, relapse of Candida infections, and the high cost of antifungal drugs. Though azole antifungal agents and derivatives continue to dominate as drugs of choice against Candida infections, there are many available data referring to the anticandidal activity of essential oils. Since we have previous observed a good antimicrobial activity of some essential oils against filamentous fungi, the aim of this study was to extend the research to evaluate the activity of the same oils on Candida albicans, C.glabrata and C.tropicalis clinical strains, as well as the effects of related components. Essential oils selection was based both on ethnomedicinal use and on proved antibacterial and/or antifungal activity of some of these oils. Fluconazole and voriconazole were used as reference drugs. METHODS: The minimum inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) of essential oils (thyme red, fennel, clove, pine, sage, lemon balm, and lavender) and their major components were investigated by the broth microdilution method (BM) and the vapour contact assay (VC). RESULTS: Using BM, pine oil showed the best activity against all strains tested, though C.albicans was more susceptible than C.glabrata and C.tropicalis (MIC50-MIC90 = 0.06 %, v/v). On the contrary, sage oil displayed a weak activity (MIC50-MIC90 = 1 %, v/v). Thyme red oil (MIC50-MIC90 ≤ 0.0038 %, v/v for C.albicans and C.tropicalis, and 0.0078- < 0.015 %, v/v for C.glabrata), followed by lemon balm, lavender and sage were the most effective by VC. Carvacrol and thymol showed the highest activity, whereas linalyl acetate showed the lowest activity both by two methods. α-pinene displayed a better activity by BM than VC. CONCLUSION: Results show a good activity of essential oils, mainly thymus red and pine oils, and their components carvacrol, thymol and α-pinene against Candida spp., including fluconazole/voriconazole resistant strains. These data encourage adequately controlled and randomized clinical investigations. The use in vapour phase could have additional advantages without requiring direct contact, resulting in easy of environmental application such as in hospital, and/or in school.