Characterization of Three Pleiotropic Drug Resistance Transporter Genes and Their Participation in the Azole Resistance of Mucor circinelloides.

Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.

Silver nanopaticles synthesized using the seed extract of Trigonella foenum-graecum L. and their antimicrobial mechanism and anticancer properties.

BACKGROUND: Synthesis of silver nanoparticles (AgNPs) through biological route plays an important role in their applications in the medical field, especially in the prevention of disease causing microbial pathogens and arresting the propagation of cancer cells. The stable, green synthesis of AgNPs is very much welcomed in the medical field because of their low toxicity. Therefore, the demands of AgNPs synthesised biologically is on the rise. The present study aimed to investigate the antimicrobial mechanisms and anticancer properties of the AgNPs synthesized using the seed extract of Trigonella foenum-graecum L. The AgNPs were characterized by UV-vis, SEM, XRD, FTIR and EDAX analysis. The minimum inhibitory concentrations (MIC) of the AgNPs were determined by the broth micro dilution method. RESULTS: The formation of brownish red color indicated the formation NPs with the absorption maximum at 420 nm. The average size was found to be 33.93 nm and sphere shaped. The FTIR spectrum revealed the absorption bands at 3340 cm-1 and 1635 cm-1 indicated the presence of -OH or -COOH and amide group stretching in the AgNPs. The X-ray diffraction report confirmed the presence of strong peak values of 2θ within the angle of 37.1°. The lowest MIC of the AgNPs against Staphylococcus aureus was 62.5 µg mL-1. MIC values against Escherichia coli and Klebsiella pneumonia, were 125 and 250 µg mL-1 respectively. The MIC of the AgNPs against Aspergillus flavus, Trichophyton rubrum and Trichoderma viridiae were each 250 µg mL-1, respectively. The extracellular protein concentration, levels of lactate dehydrogenase and alkaline phosphtase enzyme in the AgNPs treated bacterial pathogens demonstrated greater antimicrobial mechanism. Additionally, the AgNPs exhibited significant anticancer activity against the MCF7 and Vero cell lines. CONCLUSION: The synthesized AgNPs could be further evaluated in large scale as a botanical antimicrobial agent.

In vitro investigation on probiotic, anti-Candida, and antibiofilm properties of Lactobacillus pentosus strain LAP1.

OBJECTIVE: To investigate the probiotic characteristics, anti-Candida activity, and antibiofilm attributes of Hentak derived Lactobacillus pentosus strain LAP1. DESIGN: The probiotic properties of strain LAP1 were depicted by adapting standard protocols. The anti-Candida and antibiofilm properties of isolate were determined using agar well diffusion assay and ELISA reader test, respectively. The time-kill assay was performed using viable colony count assay. Further, the co-aggregation property of strain LAP1 was determined based on standard methodology. RESULTS: Strain LAP1 exhibited not only tolerance to acidic pH but also showed resistivity (P ≤ 0.05) to simulated gastric juice exposure. Similarly, the strain was able to tolerate bile salt, showed hyperproteolytic activity, and also depicted susceptibility to most of the antibiotics tested. Auto-aggregation phenomenon (37.5-60%), hydrophobicity nature (42.85%), and survival potentiality of strain LAP1 under freeze-dried condition (9.0 ±â€¯0.01 log CFU/ml) made the isolate a promising probiotic candidate. Cell-free neutralized supernatant (CFNS) of strain LAP1 exhibited potent antifungal activities against C. albicans, C. tropicalis, and C. krusei with arbitrary unit of 150 ±â€¯4.34, 200 ±â€¯5.21, and 130 ±â€¯5.13 AU/ml, respectively and depicted remarkable reduction in the biofilm formation of respective Candida sp. in a concentration dependent manner. Moreover, time-kill assay data provided the growth inhibition of all Candida sp. in a time dependent manner. Additionally, strain LAP1 revealed significant co-aggregate percentage with C. albicans, C. tropicalis, and C. krusei. CONCLUSIONS: L. pentosus strain LAP1 exhibited a good probiotic characteristics, potent anti-Candida activity, and significant antibiofilm property that could be undoubtedly recommended for its vast applications not only in food industries but also as biotherapeutic agent against Candida infections in pharmaceutical industries.

In-vitro antioxidative, antiinflammatory properties of Aurea helianthus leaf extract a Korean traditional medicinal plant.

The leaf of Aurea helianthus (A. helianthus Jinhuakui) is popularly used in China traditional medicine, however, scientific evidence on its antioxidant properties rarely studied. In this study, biological activities of A. helianthus leave's 80% ethanol extract (AHL) were investigated. The measured total polyphenol and flavonoid content of AHL was 184.24 ±â€¯5.01 mg GAE/g and 102.53 ±â€¯0.98 mg NAR/g. AHL showed the highest α, α-diphenyl-ß-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical scavenging activities of 98.30 ±â€¯0.18% at 1000 µg/mL. DPPH and ABTS radical scavenging activities significantly increased in a AHL concentration-dependent manner. AHL treatment significantly suppressed the generation of pro-inflammatory mediators, including nitric oxide (NO), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. AHL demonstrated strong anti-inflammatory activity that reduced NO production in LPS-stimulated RAW 264.7 cells. To test the potential protective effect of AHL, the antioxidant capacity, on the cell growth, viability of a human hepatoma cell (HepG2) and Raw 264.7 cell were investigated. AHL also enhanced cytotoxicity on the proliferation of HepG2 cells and was capable of inhibiting 56% against LPS at 400 µg/mL. The results of this study the potential of AHL as an excellent antioxidant substance for inhibiting inflammatory mediators. Therefore, AHL may be used as a therapeutic approach to various inflammatory diseases.