Anti-biofilm activity of a sophorolipid-amphotericin B niosomal formulation against Candida albicans.

Sophorolipids (SLs) have gained interest in the pharmaceutical industries due to their anti-microbial, anti-adhesive and anti-biofilm properties. In the present study, the production of SL was increased by using low-cost media components. The potential of a SL-based niosomal formulation of amphotericin B (AmB) was determined against biofilm of the opportunistic fungal pathogen Candida albicans. In-house prepared SL-AmB niosomes were characterized by different microscopic techniques. The mean entrapment efficiency of AmB within SL-AmB niosome was 63.20% ± 3.86. The cytotoxicity of SL-AmB on mature C. albicans biofilm was compared with an expensive, marketed drug, viz. phosome (a liposomal formulation of AmB). Fewer hyphae were observed in C. albicans biofilm treated with SL-AmB niosome whereas more budding cells were found in phosome treated biofilm. The present study has established the affordable production of SL and the suitability of this approach for delivery of poorly soluble drugs such as AmB against candidiasis infections.

Biogenic selenium nanoparticles induce ROS-mediated necroptosis in PC-3 cancer cells through TNF activation.

BACKGROUND: Selenium is well documented to inhibit cancer at higher doses; however, the mechanism behind this inhibition varies widely depending on the cell type and selenium species. Previously, we have demonstrated that Bacillus licheniformis JS2 derived biogenic selenium nanoparticles (SeNPs) induce non-apoptotic cell death in prostate adenocarcinoma cell line, PC-3, at a minimal concentration of 2 µg Se/ml, without causing toxicity to the primary cells. However, the mechanism behind its anticancer activity was elusive. RESULTS: Our results have shown that these SeNPs at a concentration of 2 µg Se/ml were able to induce reactive oxygen species (ROS) mediated necroptosis in PC-3 cells by gaining cellular internalization. Real-time qPCR analysis showed increased expression of necroptosis associated tumor necrotic factor (TNF) and interferon regulatory factor 1 (IRF1). An increased expression of RIP1 protein was also observed at the translational level upon SeNP treatment. Moreover, the cell viability was significantly increased in the presence of necroptosis inhibitor, Necrostatin-1. CONCLUSION: Data suggest that our biogenic SeNPs induce cell death in PC-3 cells by the ROS-mediated activation of necroptosis, independent to RIP3 and MLKL, regulated by a RIP1 kinase.

Crataeva nurvala nanoparticles inhibit virulence factors and biofilm formation in clinical isolates of Pseudomonas aeruginosa.

Green synthesized nanoparticles have gained great attention due to their non-toxic and non-hazardous nature. In the present study, bark extract of the medicinal plant in Ayurveda Crataeva nurvala (Buch-Ham) (CN) was chosen for the biosynthesis of silver nanoparticles (AgNPs). These NPs were characterized by Ultra violet visible spectroscopy, Fourier Transform Infra Red, Atomic Force Microscopy, and Transmission Electron Microscopy (TEM). The average particle size of green synthesized CN-AgNPs was 15.2 ± 1.01 nm. Gas chromatography- mass spectrometry analysis of methanolic bark extract involved in the formation of CN-AgNPs revealed lupeol as a major active component. In this study, CN-AgNPs (15 µg ml-1 ) efficiently suppressed the production of quorum sensing mediated virulence factors viz. pyocyanin, protease, hemolysin, and biofilm formation in Pseudomonas aeruginosa. The pyocyanin production was strongly inhibited (74.64%) followed by proteolysis (47.3%) and hemolysin production (47.7%). However, the biofilm forming ability was maximally reduced up to 79.70%. Moreover, the Confocal Laser Scanning Microscopic Analysis showed that CN-AgNPs inhibit colonization of P. aeruginosa on to the surface. Furthermore, TEM analysis revealed internalization of CN-AgNPs inside the bacterial cell. It is concluded that green synthesized AgNPs have great potential to inhibit virulence factors and biofilm forming ability of drug-resistant clinical isolates of P. aeruginosa.

Biosurfactant/s from Lactobacilli species: Properties, challenges and potential biomedical applications.

Lactic acid bacteria are generally believed to have positive roles in maintaining good health and immune system in humans. A number of Lactobacilli spp. are known to produce important metabolites, among which biosurfactants in particular have shown antimicrobial activity against several pathogens in the intestinal tract and female urogenital tract partly through interfering with biofilm formation and adhesion to the epithelial cells surfaces. Around 46 reports are documented on biosurfactant production from Lactobacillus spp. of which six can be broadly classified as cell free biosurfactant and 40 as cell associated biosurfactants and only approximately 50% of those have reported on the structural composition which, in order of occurrence were mainly proteinaceous, glycolipidic, glycoproteins, or glycolipopeptides in nature. Due to the proteinaceous nature, most biosurfactant produced by strains of Lactobacillus are generally believed to be surlactin type with high potential toward impeding pathogens adherence. Researchers have recently focused on the anti-adhesive and antibiofilm properties of Lactobacilli-derived biosurfactants. This review briefly discusses the significance of Lactobacilli-derived biosurfactants and their potential applications in various fields. In addition, we highlight the exceptional prospects and challenges in fermentation economics of Lactobacillus spp.-derived biosurfactants' production processes.

Biosurfactant as a Promoter of Methane Hydrate Formation: Thermodynamic and Kinetic Studies.

Natural gas hydrates (NGHs) are solid non-stoichiometric compounds often regarded as a next generation energy source. Successful commercialization of NGH is curtailed by lack of efficient and safe technology for generation, dissociation, storage and transportation. The present work studied the influence of environment compatible biosurfactant on gas hydrate formation. Biosurfactant was produced by Pseudomonas aeruginosa strain A11 and was characterized as rhamnolipids. Purified rhamnolipids reduced the surface tension of water from 72 mN/m to 36 mN/m with Critical Micelle Concentration (CMC) of 70 mg/l. Use of 1000 ppm rhamnolipids solution in C type silica gel bed system increased methane hydrate formation rate by 42.97% and reduced the induction time of hydrate formation by 22.63% as compared to water saturated C type silica gel. Presence of rhamnolipids also shifted methane hydrate formation temperature to higher values relative to the system without biosurfactant. Results from thermodynamic and kinetic studies suggest that rhamnolipids can be applied as environment friendly methane hydrate promoter.

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5).

An extracellular uricase producing bacterium (VITPCB5) was isolated from soil of the duck farm near Chidambaram, Tamilnadu, India and it was identified based on its 16S rRNA as Sphingobacterium thalpophilum. Uric acid was used as an effective inducer. The enzyme kinetics was studied using uric acid as a substrate. The Km and Vmax for the enzyme was found to be 0.28mM and 0.92µM/minml, respectively. Maximum uricase production was observed when lactose was used as a carbon source. Among the nitrogen sources tested, urea gave the maximum uricase production. The enzyme was successfully purified using a weak cation exchange convective interaction media carboxy methyl (CIM-CM) monolith column with a recovery of 79.7%±0.1 and 14.2±1.8-fold purification. The optimal reaction temperature of the enzyme was observed between 25 and 45°C. The pH optimum of the enzyme was 8.0. The enzyme activity was enhanced by copper and partially inhibited by calcium, iron, zinc and nickel ions. Treatment with ethylene diamine tetraacetic acid completely inhibited the enzyme activity. The in-gel trypsin digested peptides of 48-kDa uricase when analyzed using mass spectrometry, gave 32% sequence coverage with the uricase (30-kDa) from Cyberlindnera jadinii.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp.

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Development of novel membrane active lipidated peptidomimetics active against drug resistant clinical isolates.

A new series of small cationic lipidated peptidomimetics have been synthesized and found to be highly active against several susceptible as well as drug resistant clinical isolates of bacteria and fungi. All lipidated peptidomimetics do not cause significant lysis of human erythrocytes (HC50>200µg/mL). Calcein dye leakage experiment revealed membranolytic effect of LPEP08 which was further confirmed by scanning electron microscopy (SEM). The involvement of intracellular targets as an alternate mode of action was precluded by DNA retardation assay. Additionally, LPEP08 exhibit high proteolytic stability and dose not elicit resistance against drug resistant clinical isolate of Staphylococcusaureus, even after 16 rounds of passaging. These results demonstrate the potential of lipidated peptidomimetics as biocompatible anti-infective therapeutics.

Synthesis, in vitro evaluation, and molecular docking studies of azetidinones and thiazolidinones of 2-amino-5-cyclopropyl-1,3,4-thiadiazole as antibacterial agents.

In an attempt to find a new class of antimicrobial agents, a series of novel azetidin-2-ones 3a-e and thiazolidin-4-ones 4a-e of 2-amino-5-cyclopropyl-1,3,4-thiadiazole were synthesized. The synthesized compounds were confirmed by melting point, IR, (1) H NMR, (13) C NMR, and mass spectroscopy. The ß-lactam derivative (3e) was found to be the most potent compound of the series displaying excellent antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa with MIC values of 15.60, 31.50, 62.50, and 125 µg/mL, respectively, as compared to the positive control drug ampicillin. Molecular docking studies and determination of the leakage of UV260 - and UV280 -absorbing material (nucleic acid material and protein) confirmed that the synthesized compounds inhibit cell wall synthesis by inhibiting PTB (transpeptidase enzyme). Lipinski's rule and in silico ADME pharmacokinetic parameters are within the acceptable range defined for human use, thereby indicating their potential as a drug-like molecules.

Persistence and biodegradation of quinalphos using soil microbes.

The present study reports the degradation of the persistent and toxic organophosphate, quinalphos, by employing microorganisms that were already members of the natural soil community for degradation. Bacillus and Pseudomonas spp., both of which are capable of degrading quinalphos from aqueous streams, were isolated from different contaminated soils. Batch experiments were performed to determine the natural and induced biodegradation of quinalphos in the aqueous medium. The rate of degradation was analyzed through determination of residual concentration using UV-Vis spectrophotometer and high-performance liquid chromatography. A single peak of a metabolite was observed on the 160th day, and identified as dihydroxy quinalphos oxon by mass spectrometry. The presence of quinalphos and its metabolite in water over an extended period prompted the authors to investigate its induced biodegradation using indigenous microbes extracted from soil. For biodegradation studies, the isolated microbes were inoculated into minimal media with quinalphos for 17 days. The results revealed that > 80% of quinalphos was degraded in 17 days in the presence of isolated microbes, and no metabolite was observed during the biodegradation process.

In vitro and in vivo antibacterial evaluation and mechanistic study of ornithine based small cationic lipopeptides against antibiotic resistant clinical isolates.

We investigated the in vitro activities of short lipopeptides against a large panel of clinical isolates of antibiotic resistant bacteria. In the animal model, LP16 (5 mg/kg) significantly decreased the burden of viable colony forming unit (CFU) of bacteria. MTT assay results revealed the high selectivity of lipopeptides toward microbial cells. Calcein dye leakage experiments and flow cytometric analysis suggests the membranolytic effect of lipopeptides, which was further confirmed by visualizing bacterial damage via electron microscopy tool (SEM & TEM). Moreover, stability in human blood plasma and no sign of resistance development against clinical isolates of Escherichia coli and Staphylococcus aureus were observed for lead lipopeptides. These results demonstrate the potential of short lipopeptides as a novel class of anti-infectives.

Antibacterial evaluation of structurally amphipathic, membrane active small cationic peptidomimetics: synthesized by incorporating 3-amino benzoic acid as peptidomimetic element.

A new series of small cationic peptidomimetics were synthesized by incorporating 3-amino benzoic acid (3-ABA) in a small structural framework with the objective to mimic essential properties of natural antimicrobial peptides (AMPs). The new design approach resulted into improvement of activity and selectivity in comparison to linear peptides and allowed us to better understand the influence of structural amphipathicity on biological activity. Lead peptidomimetics displayed antibacterial activities against resistant pathogens (MRSA & MRSE). A calcein dye leakage experiment revealed a membranolytic effect of 4g and 4l which was further confirmed by fluorescence microscopy. In addition, proteolytic stability and no sign of resistance development against Staphylococcus aureus and MRSA demonstrate their potential for further development as novel antimicrobial therapeutics.

Substrate dependent in vitro antifungal activity of Bacillus sp strain AR2.

BACKGROUND: Biosurfactants are a structurally diverse group of secondary metabolites with lots of potential to serve mankind. Depending upon the structure and composition they may exhibit properties that make them suitable for a particular application. Structural and compositional diversity of biosurfactant is unambiguously substrate dependent. The present study investigates the qualitative and quantitative effect of different water soluble carbon source on the biosurfactant produced by Bacillus amylofaciens strain AR2. RESULTS: Strain AR2 produced lipopeptide type biosurfactant while growing on water soluble carbon sources. Maximum biosurfactant production was observed in the sucrose supplemented minimal salt medium (MSM). Strain AR2 exhibited carbon source dependent surface tension reduction in the range of 30-37 mN/m, critical micelle concentration (CMC) in the range 80-110 mg/l and emulsification index (EI24 kerosene) in the range of 32-66%. In dextrose, sucrose and glycerol supplemented MSM, strain AR2 produced lipopeptides as a mixture of surfactin, iturin and fengycin. However, in the presence of maltose, lactose and sorbitol only iturin was produced. This substrate dependent compositional variation in the lipopeptides significantly influenced antifungal activity. Lipopeptides produced by strain AR2 while growing on sucrose and dextrose based MSM was observed to be most efficient as an antifungal agent. CONCLUSIONS: These results suggest that carbon source provided for the growth and biosurfactant production not only influences the yield but also the type of biosurfactant. Sucrose is the most suitable carbon source for production of lipopeptide biosurfactant with antifungal activity.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Lipopeptides from Bacillus strain AR2 inhibits biofilm formation by Candida albicans.

The ability of the human fungal pathogen Candida albicans to reversibly switch between different morphological forms and establish biofilms is crucial for establishing infection. Targeting phenotypic plasticity and biofilm formation in C. albicans represents a new concept for antifungal drug discovery. The present study evaluated the influence of cyclic lipopeptide biosurfactant produced by Bacillus amyloliquefaciens strain AR2 on C. albicans biofilms. The biosurfactant was characterized as a mixture of iturin and fengycin by MALDI-TOF and amino acid analysis. The biosurfactant exhibited concentration dependent growth inhibition and fungicidal activity. The biosurfactant at sub-minimum growth inhibition concentration decreased cell surface hydrophobicity, hindered germ tube formation and reduced the mRNA expression of hyphae-specific gene HWP1 and ALS3 without exhibiting significant growth inhibition. The biosurfactants inhibited biofilm formation in the range of 46-100 % depending upon the concentration and Candida strains. The biosurfactant treatment dislodged 25-100 % of preformed biofilm from polystyrene plates. The biosurfactant retained its antifungal and antibiofilm activity even after exposure to extreme temperature. By virtue of the ability to inhibit germ tube and biofilm formation, two important traits of C. albicans involved in establishing infection, lipopeptides from strain AR2 may represent a potential candidate for developing heat stable anti-Candida drugs.

Gum arabic capped-silver nanoparticles inhibit biofilm formation by multi-drug resistant strains of Pseudomonas aeruginosa.

Clinical isolates (n = 55) of Pseudomonas aeruginosa were screened for the extended spectrum ß-lactamases and metallo-ß-lactamases activities and biofilm forming capability. The aim of the study was to demonstrate the antibiofilm efficacy of gum arabic capped-silver nanoparticles (GA-AgNPs) against the multi-drug resistant (MDR) biofilm forming P. aeruginosa. The GA-AgNPs were characterized by UV-spectroscopy, X-ray diffraction, and high resolution-transmission electron microscopy analysis. The isolates were screened for their biofilm forming ability, using the Congo red agar, tube method and tissue culture plate assays. The biofilm forming ability was further validated and its inhibition by GA-AgNPs was demonstrated by performing the scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM analysis of GA-AgNPs treated bacteria revealed severely deformed and damaged cells. Double fluorescent staining with propidium iodide and concanavalin A-fluorescein isothiocyanate concurrently detected the bacterial cells and exopolysaccharides (EPS) matrix. The CLSM results exhibited the GA-AgNPs concentration dependent inhibition of bacterial growth and EPS matrix of the biofilm colonizers on the surface of plastic catheters. Treatment of catheters with GA-AgNPs at 50 µg ml(-1) has resulted in 95% inhibition of bacterial colonization. This study elucidated the significance of GA-AgNPs, as the next generation antimicrobials, in protection against the biofilm mediated infections caused by MDR P. aeruginosa. It is suggested that application of GA-AgNPs, as a surface coating material for dispensing antibacterial attributes to surgical implants and implements, could be a viable approach for controlling MDR pathogens after adequate validations in clinical settings.

Surfactin restores and enhances swarming motility under heavy metal stress.

The present work reports the importance of lipopeptide biosurfactant on swarming motility of multi-metal resistant (MMR) bacterium under heavy metal stress. The MMR bacteria strain CM100B, identified as Bacillus cereus, was isolated from the coal mine sample. The strain was able to grow and reduce several metals namely Cd(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Pb(2+) ions which are common environmental pollutants. Presence of toxic heavy metal ions in the swarming medium significantly altered the motility of CM100B. Presence of Cd(2+) and Pb(2+) ions inhibited development of peritrichous flagella, thus inhibiting swarming motility. However, the addition of anionic biosurfactant surfactin restored (in case of Cd(2+) and Pb(2+) ions) or enhanced (in case of Co(2+), Cu(2+), Ni(2+) and Mn(2+)) the swarming ability of CM100B. Zeta potential studies for determining bacterial cell surface charge indicated that surfactin provided a suitable swarming environment to bacteria even under metal stress by chelating to cationic metal ions. Non-ionic surfactant Triton X-100 was unable to restore swarming under Cd(2+) and Pb(2+) ion stress. Thus, suggesting that surfactin can aid in motility not only by reducing the surface tension of swarming medium but also by binding to metal ions in the presence of metal ions stress.