Malva sylvestris inhibits Candida albicans biofilm formation

Introduction: Candidiasis-associated biofilm formed by Candida species complicates treatment and contributes to unacceptable high mortality rates. We performed the aqueous and ethanol extracts of the different parts of Malva sylvestris, Dorema aucheri, Ferulago angulata and Citrullus colocynthis plants to identify best plant extract that inhibits growth of Candida albicans or Candida krusei, and conducted a series of follow-up studies to examine the inhibitors of C. albicans biofilm formation of the identified plant extract.Methods: The antifungal activities of the aqueous and ethanol extracts of the different parts of M. sylvestris, D. aucheri, F. angulata and C. colocynthis plants were evaluated in vitro using disk diffusion test and broth microdilution test against C. albicans and C. krusei. The crystal violet assay, morphological response and expression pattern of hyphal wall protein 1 (HWP1) gene were carried out to investigate the biofilm-inhibitory properties of the best plant extract tested in C. albicans.Results: The screen identified ethanol extract of M. sylvestris root that largely represented antifungal activity among the tested extracts. M. sylvestris root inhibited C. albicans biofilm formation. Ethanol extract of M. sylvestris root demonstrated significant reduction in C. albicans biofilm formation (P &lt; 0.005). Moreover, morphological observation of ethanol extract of M. sylvestris root treated cells confirmed a decrease in biofilm thickness and cellular density. Finally, ethanol extract of M. sylvestris root displayed significant down-regulation of HWP1.Conclusion: These results provide proof of concept for the implementation of ethanol extract of M. sylvestris root as inhibitor of C. albicans biofilm formation.</p

Mycosynthesis of Silver Nanoparticles from Candida albicans and its Antibacterial Activity against Escherichia coli and Staphylococcus aureus

Purpose: To produce and characterize silver nanoparticles using Candida albicans and evaluate its antibacterial properties.Methods: Extracellular silver nanoparticles were biosynthesized using C. albicans. The biomass obtained from cultures of C. albicans was used to synthesize silver nanoparticles in 1.5 mM silver nitrate solution. Characterization of the biosynthesized nanoparticles was carried out using ultraviolet (UV)- visible spectrometry and scanning electron microscopy (SEM). The antibacterial properties of the nanoparticles were determined by agar disc diffusion method against Escherichia coli and Staphylococcus aureus.Results: Incubation of C. albicans with silver nitrate solution produced silver nanoparticles after 2 – 4 h as evidenced by change in the color of the reaction mixture. The formation of silver nanoparticles was confirmed by UV-visible spectroscopy which showed absorption peak between 300 – 800 nm, being the characteristic wavelength range of silver nanoparticles. SEM revealed the varying morphology of the nanoparticles which had a size range of 20 – 80 nm. Furthermore, the nanoparticles showed significant antimicrobial activity (p &lt; 0.05).Conclusion: The biosynthesized silver nanoparticles hold some promise for various industrial applications, including drug delivery.Keywords: Antibacterial activity, Candida albicans, Escherichia coli, Mycosynthesis, Silver nanoparticles, Staphylococcus aureu

Inhibitory effect of menthol on expression of aspartyl proteinase 1 in fluconazole-resistant Candida albicans

Introduction: Fluconazole-resistant Candida albicans is one of the biggest problems seen in clinical practices. One of the most common ways to resolve this problem is the use of natural pure compounds such as menthol. The aims of this study were to investigate the hyphal formation and gene expression profiling of fluconazole-resistant C. albicans treated by menthol. Methods: Colonization of vaginal isolates of C. albicans was recognized and fluconazole-resistant yeasts detected by WHONET software. The relative minimum inhibitory concentrations (MICs) of menthol were determined by broth microdilution for fluconazole-resistant isolates. The potency of menthol to inhibit hyphal formation was exploited using a light microscope. A quantitative real-time RT- PCR was used to measure the expression of SAP1 . Results: Almost 100% of colonized vaginal isolates of C. albicans was found to be fluconazole-resistant. MIC90 for menthol in fluconazole-resistant isolates was 1.6 to 25 μg/mL. Furthemore, all isolates treated with menthol showed a significant reduction in hyphae and number of planktonic cells. In the fluconazole-resistance C. albicans cells treated with fluconazole, the expression levels of SAP1 increased by 1.53- (2×MIC conc.) and 1.43-fold (1×MIC conc.). However, treatment with menthol down regulated the SAP1 expression by 2.02- and 1.85-fold at concentrations of 2 × MIC and 1 × MIC, respectively (P ≤ 0.05). Conclusion: This study suggests that menthol might have potential applications in treatment of infections, due to fluconazole-resistance C. albicans. In addition, SAP1 could be probable molecular target of menthol in C. albicans

Phenotypic and Genotypic Identification of Metallobetalactamase Genes in Resistant Enterobacteriaceae Isolated from Medical Centers in Isfahan

Background: The emergence of resistant Enterobacteriaceae and the abundance of antibiotic-resistant genes is one of the major problems of the global health system. The present study aimed to determine the phenotypic and genotypic expression levels of metallobetalactamase coding genes (blaVIM, blaNDM, blaIMP, blaSIM, blaSPM, and blaGIM) in Enterobacteriaceae isolates (Escherichia, Enterobacter, Citrobacter, Klebsiella, and Serratia) from patients referred to the clinical centers in Isfahan city and typing of these isolates.Materials and Methods: Enterobacteriaceae isolates were identified and isolated after sample collection. Antibiotic sensitivity pattern was investigated by disk diffusion method. MIC was performed in carbapenem-resistant isolates by the E-test method, and the frequency of strains with multidrug resistance was determined. The presence of metallobetalactamase genes was investigated phenotypically using a combined disk test and modified Hodge test. The genotypic identification of the above genes was done by PCR and sequencing techniques. Finally, PCR based on the sequence of repetitive elements was performed for molecular typing of metallobetalactamase-producing Enterobacteriaceae.Results: In the present study, 580 isolates of Enterobacteriaceae were isolated by examining 3500 samples. Klebsiella and Escherichia were the most common isolates, and the frequency of MDR was 60% in Klebsiella and 59.53% in Escherichia. Moreover, MIC results showed that 33.7% Klebsiella, 4.1% Escherichia, 5.7% Enterobacter, 3.5% Citrobacter, and 5.5% Serratia were resistant to carbapenems. Frequency of isolates with multidrug resistance in Escherichia (MDR 59.53% and XDR 1.5%), Klebsiella (MDR 60%, XDR% 3 and PDR 0.8%), Enterobacter (MDR 44%), Citrobacter (MDR 53.5%) and Serratia (MDR 55.5%) were reported. Metallobetalactamase production was confirmed by phenotypic analysis in Escherichia (1.8%) and Klebsiella (10.4%). Genotypic tests showed that blaSIM, blaSPM, and blaGIM genes were absent in any Enterobacteriaceae isolates. The presence of blaVIM, blaIMP, and blaNDM genes was confirmed in 6.2% of Klebsiella isolates and 1.3% of Escherichia isolates. The frequency of detected metallobetalactamase genes in Klebsiella and Escherichia isolates was 4.58% and 1.39% for blaVIM, 0.83% and 1.39% for blaIMP and 0.83% and 1.39% for blaNDM. The rep-PCR results showed that 11 metallobetalactamase-producing Klebsiella isolates are in 4 main groups, and 9 Escherichia isolates and 4 Enterobacter isolates are classified in two main clusters.Conclusion: The present study shows the prevalence of Klebsiella and Escherichia isolates and their resistance to metallobetalactamase-producing Enterobacteriaceae. These genes in the horizontal transfer of antibiotic resistance identification of metallobetalactamase-producing isolates in clinical environments are essential to reduce the spread of antibiotic resistance. The high homology of resistant isolates of Enterobacteriaceae inclinical samples indicates the high power of these genotypes in causing infection in hospitalized patients, which can play an important role in increasing antibiotic resistance

Analysis of ergosterol and gene expression profiles of sterol ∆5,6-desaturase (ERG3) and lanosterol 14α-demethylase (ERG11) in Candida albicans treated with carvacrol

Introduction: Usually, for treatment of fungal infections, antifungals such as azoles are used, but one of the biggest problems faced in clinical practice is the emergence of resistance for most of these drugs. Antifungal drugs derived from plants may alleviate this problem. The aims of this study were to analyse the ergosterol and gene expression profiles of ERG genes in Candida albicans treated with carvacrol. Methods:We used carvacrol and conducted a series of follow-up studies to examine the inhibitors of Candida species isolated from immunocompromised patients. Antifungal susceptibility test, time-kill study, ergosterol binding assay and ergosterol content were investigated. Eventually, the expression of ERG3 and ERG11genes was carried out to investigate the inhibitory properties of antifungal activity against Candida albicans using quantitative real time RT-PCR. Results: Carvacrol was able to inhibit Candida species and reduce time-kill kinetic in C. albicans. This phytoconstituent acted by binding to ergosterol in the fungal membrane and caused a reduction of 52% of the ergosterol content compared to the untreated growth control. Finally, carvacrol displayed significant down-regulation of ERG3 and ERG11genes in C. albicans. Conclusion: These results provide proof of concept for the implementation of carvacrol inhibitors of Candida species. In addition, ERG3 and ERG11 genes could be probable target of carvacrol against C. albicans

In vitro investigation of antifungal activity of allicin alone and in combination with azoles against Candida species.

Candidiasis is a term describing infections by yeasts from the genus Candida, and the type of infection encompassed by candidiasis ranges from superficial to systemic. Treatment of such infections often requires antifungals such as the azoles, but increased use of these drugs has led to selection of yeasts with increased resistance to these drugs. In this study, we used allicin, an allyl sulfur derivative of garlic, to demonstrate both its intrinsic antifungal activity and its synergy with the azoles, in the treatment of these yeasts in vitro. In this study, the MIC50 and MIC90 of allicin alone against six Candida spp. ranged from 0.05 to 25 μg/ml. However, when allicin was used in combination with fluconazole or ketoconazole, the MICs were decreased in some isolates. Our results demonstrated the existing synergistic effect between allicin and azoles in some of the Candida spp. such as C. albicans, C. glabrata and C. tropicalis, but synergy was not demonstrated in the majority of Candida spp. tested. Nonetheless, In vivo testing needs to be performed to support these findings

Differential expression of oil palm pathology genes during interactions with Ganoderma boninense and Trichoderma harzianum.

The expression profiles of Δ9 stearoyl–acyl carrier protein desaturase (SAD1 and SAD2) and type 3 metallothionein (MT3-A and MT3-B) were investigated in seedlings of oil palm (Elaeis guineensis) artificially inoculated with the pathogenic fungus Ganoderma boninense and the symbiotic fungus Trichoderma harzianum. Expression of SAD1 and MT3-A in roots and SAD2 in leaves were significantly up-regulated in G. boninense inoculated seedlings at 21 d after treatment when physical symptoms had not yet appeared and thereafter decreased to basal levels when symptoms became visible. Our finding demonstrated that the SAD1 expression in leaves was significantly down-regulated to negligible levels at 42 and 63 d after treatment. The transcripts of MT3 genes were synthesized in G. boninense inoculated leaves at 42 d after treatment, and the analyses did not show detectable expression of these genes before 42 d after treatment. In T. harzianum inoculated seedlings, the expression levels of SAD1 and SAD2 increased gradually and were stronger in roots than leaves, while for MT3-A and MT3-B, the expression levels were induced in leaves at 3 d after treatment and subsequently maintained at same levels until 63 d after treatment. The MT3-A expression was significantly up-regulated in roots at 3 d after treatment and thereafter were maintained at this level. Both SAD and MT3 expression were maintained at maximum levels or at levels higher than basal. This study demonstrates that oil palm was able to distinguish between pathogenic and symbiotic fungal interactions, thus resulting in different transcriptional activation profiles of SAD and MT3 genes. Increases in expression levels of SAD and MT3 would lead to enhanced resistance against G. boninense and down-regulation of genes confer potential for invasive growth of the pathogen. Differences in expression profiles of SAD and MT3 relate to plant resistance mechanisms while supporting growth enhancing effects of symbiotic T. harzianum

The anti-obesity effects of Lactobacillus casei strain shirota versus orlistat on high fat diet-induced obese rats

BACKGROUND:Obesity and overweight are major public health problems. Various factors, such as daily nutritional habits, physical inactivity, and genetic, are related to the prevalence of obesity. Recently, it was revealed that the gut microflora may also play an important role in weight management. Thus, this study aimed to determine the anti-obesity effects of Lactobacillus casei strain Shirota (LcS) compared with those of orlistat in an animal model fed a high-fat diet (HFD). DESIGN:Thirty-two male Sprague-Dawley rats were assigned to four groups fed various diets as follows: a standard diet group, HFD group, HFD supplemented with LcS (108109 colony-forming units (HFD-LcS) group, and HFD group treated with Orlistat (10 mg/kg body weight)). After 15 weeks, the weights of organs, body weight, body fat mass and serological biomarkers were measured. In addition, histological analysis of the liver and adipose tissue was performed. RESULTS:Body weight, body mass index, fat mass, leptin and glucose levels were lower, and high-density lipoprotein and adiponectin levels were higher in the HFD-LcS and HFD-orlistat groups than in the HFD group. In addition a significant difference in body fat mass was observed between HFD-LcS group with HFD-orlistat group (19.19±5.76 g vs. 30.19±7.98 g). Although the interleukin-6 level was significantly decreased in the HFD-LcS and HFD-orlistat groups compared with the HFD group, no significant change was observed in other inflammatory biomarkers. CONCLUSION:The results of the present study show that LcS supplementation improves body weight management and the levels of some related biomarkers. In addition, LcS supplementation showed a better result in fat mass and alanine aminotransferase reduction than Orlistat. Further studies are needed to elucidate the anti-obesity effects of LcS, with a longer period of supplementation

Simplex and triplex polymerase chain reaction (PCR) for identification of three medically important Candida species

Candida species are a major cause of invasive infections in both critically ill and immunocompromised patients. Hence, rapid identification of these pathogens may facilitate specific therapy and patient management. The development of rapid and specific diagnostic methods remains a challenge. Herein, we developed the simplex and triplex polymerase chain reaction (PCR) for the identification of three medically important Candida species namely C. albicans, C. parapsilosis and C. tropicalis. The developed methods target the phospholipase B gene (PLB). The primers designed achieved highly specific identification of the selected species using both the simplex PCR and the triplex PCR formats, which were confirmed by DNA sequencing. The primers did not show any non-specific amplification when tested with DNA from other Candida species and other fungal species such as Aspergillus and Cryptococcus. These results showed that the PLB gene provides a novel target that could be used for the detection of medically important Candida species from clinical specimens.Key words: Candida species, primers, phospholipase B gene (PLB), polymerase chain reaction (PCR)

Characterisation of Allicin Activity in Candida Albicans

Candidiasis is a term describing infections by yeasts from the genus Candida, and the type of infection encompassed by candidiasis ranges from superficial to systemic.Treatment of such infections often requires antifungals such as the azoles, but increased use of these drugs has led to selection of yeasts with increased resistance to these drugs. Essential oils such as allicin are an example of natural compounds which when used in synergy with antimicrobial agents may increase the efficacies of these therapeutic agents. In the present study, it is hypothesized that allicin has antifungal effect against different genera and species of fungi including Candida spp. and allicin acts via suppressing some of the virulence attributes of Candida. For determination of minimal inhibitory concentration (MIC) of allicin against Candida spp., a standard broth dilution method (NCCLS-M27 A2) was used. The determination of fractional inhibitory concentration (FIC) for combination of allicin with fluconazole and ketoconazole was performed. The results showed a significant synergistic effect between allicin combined with azoles in some of the Candida spp. This demonstrates that allicin decreased the growth of C. albicans almost as efficiently as fluconazole (p < 0.05). For the murine model of candidiasis, female BALB/c mice, 4-6 weeks old, were inoculated intravenously via the lateral tail vein with C. albicans. Infection was followed up for 4 weeks and evaluated in terms of mortality, the latter being assessed by determination of fungal colonization of viscera. Fluconazole was also tested as a comparative standard antifungal drug. It has been shown that the allicin treated group (5mg/kg/day) was compatible to fluconazole treated group in terms of the fungal load reduction in kidney and spleen (p < 0.001) as well as improvement in the survival time (50% reduction in mortality) during 28 days period. For quantification of biofilm of Candida (treated and untreated), Crystal Violet and XTT assays have been performed. Results showed a significant reduction of biofilm in C. albicans treated with allicin. On the other hand, the morphological changes of biofilms treated with allicin at different concentrations have been observed with scanning electron microscopy (SEM) and compared to normal biofilms. The presence of pits on the cell surface and cellular collapse with high concentrations of allicin indicates that the cell membrane could be one of the targets of allicin in Candida. In order to observe the effect of allicin on hyphae production and biofilm formation, measurement of expression of SAP1-4, SIR2 (hyphae) and HWP1, INT1 (biofilm) genes after treatment with allicin has been conducted through semi quantitative RT-PCR and then relative real time RT-PCR. RNA was extracted and converted to cDNA, which was used as a template in RT-PCR.These significant results have demonstrated the strong down-regulated expression of SIR2 and HWP1 genes at 5.54 and 138.889 fold changes respectively (p < 0.0001), after treatment with allicin along with observation of suppression of hyphae and biofilm via SEM. For evaluation of ERG3, ERG 11 (Ergosterol) and CDR1 (ABC Transporter) expression after treatment by allicin, semi quantitative RT-PCR was used for investigating the effect of allicin on expression of membrane-related genes. These results were significant for fluconazole (down-regulated expression) but allicin-treated samples did not show any significant change in gene expression for ERG3, ERG 11 and CDR1. Taken together, it can be concluded from the results in this study that allicin displays significant efficacy when applied in synergy with fluconazole and ketoconazole. Results from the in vivo mouse model of systemic candidiasis also indicated that allicin is able to reduce fungal load and prolong the survival time. In conclusion, HWP1 and SIR2 genes are suggested as the targets of allicin in Candida cells