Ethno-medicinal study of Artemisia ordosica Krasch. (traditional Chinese/Mongolian medicine) extracts for the treatment of allergic rhinitis and nasosinusitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia ordosica Krasch. (AOK) has been used for rheumatic arthritis, cold headache, sore throat, etc. in traditional Chinese/Mongolian medicine and is used for nasosinusitis by local Mongolian "barefoot" doctors. Up to now, their mechanisms are still unclear. AIM: To evaluate the in vivo anti-inflammatory and allergic rhinitis (AR) alleviating effect as well as in vitro antimicrobial activities of AOK extracts to verify its ethno-medicinal claims. MATERIALS AND METHODS: Crude extracts (methanol/95%-ethanol/ethyl acetate) of AOK root/stem/leaf and fractions (petroleum ether/ethyl acetate/n-butanol/aqueous) of AOK root extract were prepared. Xylene-induced ear swelling model in mouse and ovalbumin (OVA)-induced AR model in guinea pig were established. Ear swelling degrees of mice were measured. The numbers of rubbing movement and sneezes of guinea pigs were counted to evaluate the symptoms of AR. The serum levels of histamine, INF-γ, IL-2/4/10, and VCAM-1 were measured by ELISA assay. The histological changes of nasal mucosa were investigated by light microscope after H&E staining. Antimicrobial activities of AOK extracts were also tested. LC-MS/MS analysis was performed to characterize the constituents of active extract and molecular docking was conducted to predict the biological mechanism. RESULTS: In ear-swelling model, extract (100.00 mg/kg) from the ethyl acetate layer of 95% ethanol (100.00 mg/kg) showed better swelling inhibition in mice than positive control (dexamethasone, 191.91 mg/kg). In AR model, extract from the ethyl acetate layer of 95% ethanol significantly alleviated the AR symptoms in guinea pigs, decreased the serum levels of histamine, INF-γ, IL-2/4/10, and VCAM-1, and reduced the infiltration of eosinophil in nasal mucosa. For Staphylococcus aureus, the ethyl acetate extract of AOK stem showed the highest inhibition (MIC=1.25 mg/mL), for Escherichia coli, n-butanol layer of 95% ethanol extract of AOK root showed the highest inhibition (MIC=15.00 mg/mL), for Candida glabrata, 95%-ethyl acetate extract of AOK leaf showed the best inhibition (MIC=0.064 mg/mL), while ethyl acetate and n-butanol layers showed similar inhibition on MRSA (MIC=7.50 mg/mL). LC-MS/MS characterization showed that dicaffeoylquinic acids account for more than 30% of ethyl acetate layer of AOK extract. Dicaffeoylquinic acids bind with histamine-1 receptor with high affinities and interesting modes. CONCLUSIONS: Extracts from AOK had interesting anti-inflammatory activity in mice, alleviating effect against OVA-induced AR in guinea pigs, and antimicrobial activities in vitro, which support the ethno-medicinal use of it. The main constituents in ethyl acetate layer of AOK root extract are dicaffeoylquinic acids and could bind with histamine-1 receptor well. These findings highlighted the importance of natural product chemistry study of AOK.

In Vitro Antimicrobial Activities of Commercially Available Tea Tree (Melaleuca alternifolia) Essential Oils.

Melaleuca alternifolia tea tree oil (TTO) is largely used in cutaneous infections. Clinical observations reported antibacterial, antifungal, and antiviral activities, whereas in vitro experiments ascribed most of biological properties to terpinen-4-ol. Since different plant chemotypes and storage conditions result in variations of chemical composition of commercially available TTO, in this study we investigated the antimicrobial activity and the chemical profile of ten commercially available TTO products. The antimicrobial activity was assessed against Candida glabrata, Herpes simplex virus type 1 (HSV-1), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa grown in planktonic mode or biofilms. Only five out of ten TTO batches reported significant antimicrobial activity. The identified TTO products reduced bacterial survival in biofilms, generated oxidative damage in C. glabrata, and diminished HSV-1 infectivity. GC-MS analysis revealed that all the analyzed TTO batches fitted into the terpinen-4-ol chemotype even if we reported great variability in composition of nine major ISO-specified TTO components. Overall, we were not able to ascribe the antimicrobial activity to the content in terpinen-4-ol. We therefore conclude that the antimicrobial activity of TTO results from complex interaction among different components.

Proanthocyanidin polymeric tannins from Stryphnodendron adstringens are effective against Candida spp. isolates and for vaginal candidiasis treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem bark of Stryphnodendron adstringens (Mart.) Coville is popularly used as anti-inflammatory, astringent and in the treatment of wounds and vaginal infections. Several pharmacological activities have been scientifically proven by in vitro and in vivo experimental assays for antibacterial, antiviral, antiprotozoan, anti-inflammatory and antioxidant. AIM OF THE STUDY: We investigated whether proanthocyanidin polymeric tannins from the Stryphnodendron adstringens stem bark with antifungal activity against Candida albicans in vitro are also active against planktonic and biofilm cells of Candida non-albicans (CNA, including fluconazole-resistant isolates) and are capable of controlling Candida vaginitis in vivo. MATERIALS AND METHODS: A total of 46 clinical isolates and 5 reference Candida spp. strains were used in this study. The antifungal effects in vitro of tannins (F2 and sub-fraction F2.4) from S. adstringens stem bark were evaluated using a broth microdilution assay (for planktonic yeasts and biofilm dispersion cells) or by XTT assay (for biofilm sessile cells). For in vivo antifungal activity analysis, mice with vaginal infection by C. albicans or C. glabrata were treated with a topical gel containing F2 (alone or combined with oral fluconazole), and the vaginal histopathology and fungal burden (by CFU counts from vaginal homogenates) were analyzed. RESULTS: F2 and F2.4 inhibited the proliferation of planktonic cells of Candida spp., especially that of fluconazole- and/or amphotericin B-resistant isolates. F2 and F2.4 also inhibited the proliferation of Candida biofilm dispersion cells. Moreover, a gel containing F2 efficiently controlled vaginal infection by C. albicans and C. glabrata in mice, with no noticeable toxicity to vaginal tissue. CONCLUSIONS: Our data show that proanthocyanidin polymeric tannins obtained from S. adstringens have antifungal activity in vitro against C. albicans and CNA (including fluconazole-resistant isolates) and presented efficacy in the control of candidiasis in murine model. Therefore, these tannins have potential use in the treatment of vaginal candidiasis, representing interesting alternatives to current antifungals.

Boric Acid and Commercial Organoboron Products as Inhibitors of Drug-Resistant Candida albicans.

Clinical use of boric acid as a topical antifungal in women who have failed standard antifungal therapy with azole drugs has been used sporadically for decades. Our previous in vitro work showing inhibition of Candida albicans growth was conducted on clinical isolates without antifungal drug susceptibility profiling. Here, we report that boric acid restricts growth of drug-resistant Candida albicans and inhibits hyphal growth and diminishes cell volume. The availability of over-the-counter organoboron compounds intended for use as oral nutritional supplements led us to determine if these also were inhibitory toward resistant Candida and show here that they also possess antifungal activity. Candida glabrata was also found to be inhibited by boric acid and organoboron compounds. Further development of organoboron compounds as topical therapeutics is of potential value.

In vitro activity of essential oils extracted from condiments against fluconazole-resistant and -sensitive Candida glabrata.

In the present study, the antifungal activity of essential oils obtained from Origanum vulgare (oregano), Cinnamomum zeylanicum (cinnamon), Lippia graveolens (Mexican oregano), Thymus vulgaris (thyme), Salvia officinalis (sage), Rosmarinus officinalis (rosemary), Ocimum basilicum (basil) and Zingiber officinale (ginger) were assessed against Candida glabrata isolates. One group contained 30 fluconazole-susceptible C. glabrata isolates, and the second group contained fluconazole-resistant isolates derived from the first group after the in vitro induction of fluconazole-resistance, for a total of 60 tested isolates. The broth microdilution methodology was used. Concentrations of 50µg/mL, 100µg/mL, 200µg/mL, 400µg/mL, 800µg/mL, 1600µg/mL and 3200µg/mL of the essential oils were used, and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined. Thyme, sage, rosemary, basil and ginger essential oils showed no antifungal activity at the tested concentrations. Antimicrobial activity less than or equal to 3200µg/mL was observed for oregano, Mexican oregano and cinnamon essential oils. Both the oregano and Mexican oregano essential oils showed high levels of antifungal activity against the fluconazole-susceptible C. glabrata group, whereas the cinnamon essential oil showed the best antifungal activity against the fluconazole-resistant C. glabrata isolates.

Comparative effects of hypoxia and hypoxia mimetic cobalt chloride on in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata.

OBJECTIVES: Candida glabrata has emerged as potent pathogen in nosocomial infections. The objective of this study is to investigate the effects of hypoxia (an important host factor) and hypoxia mimetic cobalt chloride upon growth, in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata. MATERIALS AND METHODS: Growth was checked by spotting assays. Expression of TDH3, a gene of glycolytic pathway was analyzed as intracellular hypoxia marker by reverse transcription PCR. In vitro adhesion, biofilm development and susceptibility of biofilm to amphotericin B were performed on polystyrene plates and quantified by XTT assay in RPMI 1640 and YNB media. Experiments were performed in triplicates and Student's t-test was used for statistical analysis. RESULTS: Hypoxia did not compromise the growth of C. glabrata unlike CoCl2. Hypoxia and CoCl2 upregulated TDH3 expression. Adhesion was reduced upon exposure to hypoxia and CoCl2. Biofilm activity remained unchanged in the presence of CoCl2 in both media. In comparison to normoxia control, hypoxia increased biofilm activity to 259.33 ± 22.05% in RPMI 1640, while hypoxia reduced it to 70.99 ± 2.99% in YNB. Biofilm susceptibility to amphotericin B was significantly decreased in RPMI 1640 and remained unaffected in YNB in hypoxia. CONCLUSIONS: C. glabrata grows well even under hypoxia but not upon CoCl2 exposures. CoCl2 mimics hypoxia like expression of TDH3 but affects the virulence properties unlike hypoxia. Both, hypoxia and CoCl2 affects adhesion adversely. Hypoxia increases biofilm development and reduces the susceptibility of biofilm to amp B in RPMI 1640 but not in YNB.

Abdominal candidiasis is a hidden reservoir of echinocandin resistance.

FKS mutant Candida isolates were recovered from 24% (6/25) of abdominal candidiasis patients exposed to echinocandin. Candida glabrata (29%) and Candida albicans (14%) mutants were identified. Multidrug-resistant bacteria were recovered from 83% of FKS mutant infections. Mutations were associated with prolonged echinocandin exposure (P = 0.01), breakthrough infections (P = 0.03), and therapeutic failures despite source control interventions (100%). Abdominal candidiasis is a hidden reservoir for the emergence of echinocandin-resistant Candida.

Growth inhibitory response and ultrastructural modification of oral-associated candidal reference strains (ATCC) by Piper betle L. extract.

Candida species have been associated with the emergence of strains resistant to selected antifungal agents. Plant products have been used traditionally as alternative medicine to ease mucosal fungal infections. This study aimed to investigate the effects of Piper betle extract on the growth profile and the ultrastructure of commonly isolated oral candidal cells. The major component of P. betle was identified using liquid chromatography-mass spectrophotometry (LC-MS/MS). Seven ATCC control strains of Candida species were cultured in yeast peptone dextrose broth under four different growth environments: (i) in the absence of P. betle extract; and in the presence of P. betle extract at respective concentrations of (ii) 1 mg⋅mL(-1); (iii) 3 mg⋅mL(-1); and (iv) 6 mg⋅mL(-1). The growth inhibitory responses of the candidal cells were determined based on changes in the specific growth rates (µ). Scanning electron microscopy (SEM) was used to observe any ultrastructural alterations in the candida colonies. LC-MS/MS was performed to validate the presence of bioactive compounds in the extract. Following treatment, it was observed that the µ-values of the treated cells were significantly different than those of the untreated cells (P<0.05), indicating the fungistatic properties of the P. betle extract. The candidal population was also reduced from an average of 13.44×10(6) to 1.78×10(6) viable cell counts (CFU)⋅mL(-1). SEM examination exhibited physical damage and considerable morphological alterations of the treated cells. The compound profile from LC-MS/MS indicated the presence of hydroxybenzoic acid, chavibetol and hydroxychavicol in P. betle extract. The effects of P. betle on candida cells could potentiate its antifungal activity.

The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Effect of pH on in vitro susceptibility of Candida glabrata and Candida albicans to 11 antifungal agents and implications for clinical use.

The treatment of vulvovaginal candidiasis (VVC) due to Candida glabrata is challenging, with limited therapeutic options. Unexplained disappointing clinical efficacy has been reported with systemic and topical azole antifungal agents in spite of in vitro susceptibility. Given that the vaginal pH of patients with VVC is unchanged at 4 to 4.5, we studied the effect of pH on the in vitro activity of 11 antifungal agents against 40 C. glabrata isolates and compared activity against 15 fluconazole-sensitive and 10 reduced-fluconazole-susceptibility C. albicans strains. In vitro susceptibility to flucytosine, fluconazole, voriconazole, posaconazole, itraconazole, ketoconazole, clotrimazole, miconazole, ciclopirox olamine, amphotericin B, and caspofungin was determined using the CLSI method for yeast susceptibility testing. Test media were buffered to pHs of 7, 6, 5, and 4. Under conditions of reduced pH, C. glabrata isolates remained susceptible to caspofungin and flucytosine; however, there was a dramatic increase in the MIC(90) for amphotericin B and every azole drug tested. Although susceptible to other azole drugs tested at pH 7, C. albicans strains with reduced fluconazole susceptibility also demonstrated reduced susceptibility to amphotericin B and all azoles at pH 4. In contrast, fluconazole-sensitive C. albicans isolates remained susceptible at low pH to azoles, in keeping with clinical observations. In selecting agents for treatment of recurrent C. glabrata vaginitis, clinicians should recognize the limitations of in vitro susceptibility testing utilizing pH 7.0.

Substrates and modulators of the multidrug transporter Cdr1p of Candida albicans in antifungal extracts of medicinal plants.

The effective treatment of infections caused by the most frequent human fungal pathogens Candida albicans and Candida glabrata is hindered by a limited number of available antifungals and development of resistance. In this study, we identified new extracts of medicinal plants inhibiting the growth of C. glabrata, a species generally showing low sensitivity to azoles. The methanolic extract of Anacardium occidentalis with an MIC of 80 microg ml(-1) proved to be the most active. In contrast to higher azole sensitivity, C. albicans showed increased resistance to several extracts. Investigation of the possible contribution of the multidrug transporter of the ATP-binding cassette superfamily Cdr1p of C. albicans to extract tolerance revealed a differential response upon overproduction of this protein in Saccharaomyces cerevisiae. Whereas the growth inhibitory activity of many extracts was not affected by CDR1 overexpression, increased sensitivity to some of them was observed. In contrast, extracts showing no detectable anticandidal activity including the ethyl acetate extract of Trichilia emetica were detoxified by Cdr1p. The presence of a non-toxic Cdr1p-mediated ketoconazole resistance modulator accompanying growth-inhibitory Cdr1p substrates in this extract was revealed by further fractionation experiments.

Posaconazole against Candida glabrata isolates with various susceptibilities to fluconazole.

We investigated the in vitro activities of posaconazole (POS), fluconazole (FLC), amphotericin B (AMB), and caspofungin (CAS) against four clinical isolates of Candida glabrata with various susceptibilities to FLC (FLC MICs ranging from 1.0 to >64 microg/ml). POS MICs ranged from < or =0.03 to 0.5 microg/ml; AMB MICs ranged from 0.25 to 2.0 microg/ml, while CAS MICs ranged from 0.03 to 0.25 microg/ml. When FLC MICs increased, so did POS MICs, although we did not observe any isolate with a POS MIC greater than 0.5 mug/ml. Time-kill experiments showed that POS, FLC, and CAS were fungistatic against all isolates, while AMB at eight times the MIC was fungicidal against three out of four isolates of C. glabrata tested. Then, we investigated the activity of POS in an experimental model of disseminated candidiasis using three different isolates of C. glabrata: one susceptible to FLC (S; FLC MICs ranging from 1.0 to 4.0 microg/ml; POS MIC of < or =0.03 microg/ml), one susceptible in a dose-dependent manner (SDD; FLC MICs ranging from 32 to 64 microg/ml; POS MICs ranging from 0.125 to 0.25 microg/ml), and another one resistant to FLC (R; FLC MIC of >64 microg/ml; POS MIC of 0.5 microg/ml). FLC significantly reduced the kidney burden of mice infected with the S strain (P = 0.0070) but not of those infected with the S-DD and R strains. POS was significantly effective against all three isolates at reducing the kidney fungal burden with respect to the controls (P ranging from 0.0003 to 0.029). In conclusion, our data suggest that POS may be a useful option in the management of systemic infections caused by C. glabrata. Additionally, the new triazole may be a therapeutic option in those cases where an FLC-resistant isolate is found to retain a relatively low POS MIC.

Caspofungin in combination with amphotericin B against Candida glabrata.

The effects of caspofungin combined with amphotericin B were investigated with Candida glabrata. Although in vitro experiments showed an indifferent interaction, the combination regimen was the only therapeutic approach yielding organ sterilization in a murine candidemia model.

[Addition of TCA cycle intermediates enhances pyruvate production].

The capability of utilizing the intermediates of TCA-cycle as the sole carbon source by the multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019 under the conditions of vitamins limitation was demonstrated. Furthermore, the colony numbers grown on medium supplemented with glucose, acetate and one of the intermediates of TCA-cycle was higher than that of medium used glucose and acetate or medium used one of the intermediates of TCA-cycle carbon source. Among the intermediates of TCA-cycle used in this study, oxaloacetate was the best carbon source for the yeast and it was found that its presence stimulated the conversion of acetate to acetyl-CoA. In batch fermentation with glucose medium, the addition of 10 g/L of oxaloacetate improved the dry cell weight from 11.8 g/L to 13.6 g/L, and the productivity of pyruvate from 0.96 g x L(-1) x h(-1) to 1.19 g x L(-1) x h(-1), a 24% increase after 56 h growth. The yield of pyruvate on glucose was also improved as well, from 0.63 g/g to 0.66 g/g.