Extracellular biosynthesis, OVAT/statistical optimization, and characterization of silver nanoparticles (AgNPs) using Leclercia adecarboxylata THHM and its antimicrobial activity.

BACKGROUND: The biosynthesis of silver nanoparticles (AgNPs) is an area of interest for researchers due to its eco-friendly approach. The use of biological approaches provides a clean and promising alternative process for the synthesis of AgNPs. We used for the first time the supernatant of Leclercia adecarboxylata THHM under optimal conditions to produce AgNPs with an acceptable antimicrobial activity against important clinical pathogens. RESULTS: In this study, soil bacteria from different locations were isolated and screened for their potential to form AgNPs. The selected isolate, which was found to have the ability to biosynthesize AgNPs, was identified by molecular methods as Leclercia adecarboxylata THHM and its 16S rRNA gene was deposited in GenBank under the accession number OK605882. Different conditions were screened for the maximum production of AgNPs by the selected bacteria. Five independent variables were investigated through optimizations using one variable at a time (OVAT) and the Plackett-Burman experimental design (PBD). The overall optimal parameters for enhancing the biosynthesis of AgNPs using the supernatant of Leclercia adecarboxylata THHM as a novel organism were at an incubation time of 72.0 h, a concentration of 1.5 mM silver nitrate, a temperature of 40.0 °C, a pH of 7.0, and a supernatant concentration of 30% (v/v) under illumination conditions. The biosynthesized AgNPs have been characterized by UV-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). The biosynthesized AgNPs showed an absorption peak at 423 nm, spherical shape, and an average particle size of 17.43 nm. FTIR shows the bands at 3321.50, 2160.15, and 1636.33 cm-1 corresponding to the binding vibrations of amine, alkyne nitrile, and primary amine bands, respectively. The biosynthesized AgNPs showed antimicrobial activity against a variety of microbial pathogens of medical importance. Using resazurin-based microtiter dilution, the minimum inhibitory concentration (MIC) values for AgNPs were 500 µg/mL for all microbial pathogens except for Klebsiella pneumoniae ATCC13883, which has a higher MIC value of 1000 µg/mL. CONCLUSIONS: The obtained data revealed the successful green production of AgNPs using the supernatant of Leclercia adecarboxylata THHM that can be effectively used as an antimicrobial agent against most human pathogenic microbes.

Investigation of the optimum conditions for electricity generation by haloalkaliphilic archaeon Natrialba sp. GHMN55 using the Plackett-Burman design: single and stacked MFCs.

The production of bioelectricity via the anaerobic oxidation of organic matter by microorganisms is recently receiving much interest and is considered one of the future alternative technologies. In this study, we aimed to produce electrical current by using facultative halophilic archaeon Natrialba sp. GHMN55 as a biocatalyst at the anode of a microbial fuel cell (MFC) to generate electrons from the anaerobic breakdown of organic matter to produce electrical current. Since the MFC's performance can be affected by many factors, the Plackett-Burman experimental design was applied to optimize the interaction between these factors when tested together and to identify the most significant factors that influence bioelectricity generation. We found that the factors that significantly affected electrical current generation were casein, inoculum age, magnet-bounded electrodes, NaCl, resistor value, and inoculum size; however, the existence of a mediator and the pH showed negative effects on bioelectricity production, where the maximum value of the 200 mV voltage was achieved after 48 h. The optimum medium formulation obtained using this design led to a decrease in the time required to produce bioelectricity from 20 days (in the basal medium) to 2 days (in the optimized medium). Also, the overall behavior of the cell could be enhanced by using multiple stacked MFCs with different electrical configurations (such as series or parallel chambers) to obtain higher voltages or power densities than the single chambers where the series chambers were recorded at 27.5 mV after 48 h of incubation compared with 12.6 mV and 1.1 mV for parallel and single chambers, respectively. These results indicate that the order of preferred MFC designs regarding total power densities would be series > parallel > single.

L-Glutaminase Synthesis by Marine Halomonas meridiana Isolated from the Red Sea and Its Efficiency against Colorectal Cancer Cell Lines.

L-glutaminase is an important anticancer agent that is used extensively worldwide by depriving cancer cells of L-glutamine. The marine bacterium, Halomonas meridian was isolated from the Red Sea and selected as the more active L-glutaminase-producing bacteria. L-glutaminase fermentation was optimized at 36 h, pH 8.0, 37 °C, and 3.0% NaCl, using glucose at 1.5% and soybean meal at 2%. The purified enzyme showed a specific activity of 36.08 U/mg, and the molecular weight was found to be 57 kDa by the SDS-PAGE analysis. The enzyme was highly active at pH 8.0 and 37 °C. The kinetics' parameters of Km and Vmax were 12.2 × 10-6 M and 121.95 µmol/mL/min, respectively, which reflects a higher affinity for its substrate. The anticancer efficiency of the enzyme showed significant toxic activity toward colorectal adenocarcinoma cells; LS 174 T (IC50 7.0 µg/mL) and HCT 116 (IC50 13.2 µg/mL). A higher incidence of cell death was observed with early apoptosis in HCT 116 than in LS 174 T, whereas late apoptosis was observed in LS 174 T more than in HCT 116. Also, the L-glutaminase induction nuclear fragmentation in HCT 116 was more than that in the LS 174T cells. This is the first report on Halomonas meridiana as an L-glutaminase producer that is used as an anti-colorectal cancer agent.

Novel oxygen-generation from electrospun nanofibrous scaffolds with anticancer properties: synthesis of PMMA-conjugate PVP-H2O2 nanofibers, characterization, and in vitro bio-evaluation tests.

Released oxygen plays a critical role in reducing destructive tumor behavior. This study aims to utilize decomposed hydrogen peroxide as an oxygen source by conjugating it with polyvinylpyrrolidone (PVP). PVP-hydrogen peroxide complex (PHP) composed of different ratios of (PVP : H2O2) (0.5 : 1, 1 : 1, 1 : 1.5, 1 : 5, and 1 : 10) were successfully synthesized. PHP complex with a ratio of 1 : 1.5 was chosen as the optimized ratio, and it was incorporated into the polymethyl methacrylate (PMMA) nanofibrous scaffold via the electrospinning technique. Results have revealed that the PMMA-10% PHP complex provided a significant morphological structure of nanofibrous scaffolds. The mechanical properties of PMMA-10% PHP nanofibers showed the most suitable mechanical features such as Young's modulus, elongation-at-break (%), and maximum strength, in addition to the highest degree of swelling. All PHP complex scaffolds released oxygen in a sustained manner. However, the PMMA-10% PHP complex gave the highest concentration of released-oxygen with (∼8.9 mg L-1, after 2.5 h). PMMA-10% PHP nanofibers provided an ideal model for released-oxygen scaffold with anti-cancer effect and high selectivity for cancer cells, especially for breast cancer cells. Nanofibrous scaffolds with different composition revealed high cell viability for normal cells. Such outcomes support the suitability of using synthesized nanofibrous scaffolds as released-oxygen biomaterials to enhance cancer cells' sensitivity and maximize the treatment effect.

Electrospun PVA/hyaluronic acid/L-arginine nanofibers for wound healing applications: Nanofibers optimization and in vitro bioevaluation.

Hyaluronic acid (HA) based nanofibers (NFs) represented a novel class of bioactive wound dressings that have a vital role in wound management due to their unique properties as an extracellular-matrix and accelerating wound healing. Novel L-arginine-loaded citric acid crosslinked PVA-HA NFs were fabricated by electrospinning and proposed for potential wound healing purposes. However, poor mechanical properties of HA NFs might limit its biological usage, thus this study aims to develop reinforced PVA/HA NFs by incorporation of cellulose nanocrystals (CNCs) as nanofiller and loading L-arginine as wound healing accelerator. Results revealed that incorporation of CNCs into PVA/HA significantly improved mechanical and swelling properties of NFs, compared to CNC-free NFs. Biological performance of NFs was evaluated on normal human skin melanocyte (HFB-4) and lung fibroblast (WI38) cell-lines. PVA/HA/CNC/L-arginine NFs exhibited excellent hemocompatibility, high protein adsorption, outstanding proliferative and adhesive potential on HFB-4 cells expressed by high wound gap-closure 99.9% after 48 h of exposure. Released arginine from PVA/HA/CNC NFs showed sustained release about 46.5% and ~90% after 24 h and 48 h, respectively. Briefly, PVA/HA/CNCs/L-arginine showed adequate antibacterial activity especially against Klebsiella pneumonia, as an acute popular pathogen causing a skin infection. These results indicate that PVA/HA/CNC/L-arginine could act as promising and multifunctional wound dressings.

Biochemical characterisation and application of keratinase from Bacillus thuringiensis MT1 to enable valorisation of hair wastes through biosynthesis of vitamin B-complex.

This study reports on the exploitation of keratinous hydrolysate by keratinase enzymes to produce vitamin B-complex. Toward this end, keratinase enzyme was produced by Bacillus thuringiensis strain MT1, newly isolated from cattle-yard utilising donkey hairs. Scanning electron microscope (SEM) and Fourier transform infrared spectrophotometer (FTIR) analyses demonstrated hairs disintegration and the disruption of the disulphide bonds of the keratin structure, respectively. The biochemical characterisation of the produced enzyme exhibited optimal activity of 422 U/ml at 50 °C and pH 9 with a molecular mass of 80 kDa. The enzyme activity was entirely deactivated by Ethylenediaminetetraacetic acid (EDTA), implying the existence of a metallokeratinase group. Donkey hairs were thus treated with metallokeratinase, emancipating eight essential and eight more non-essential amino acids, which were identified employing amino acid analyser. These amino acids were subsequently utilised by Saccharomyces cerevisiae strain ATCC 64712, at different concentrations, to produce vitamin B-complex. High-performance liquid chromatography (HPLC) analysis revealed the synthesis of vitamins B1, B2, and B12 at various levels associated with concentrations of supplemented amino acids. This report thus highlights the feasible application of keratinase enzyme as an eco-friendly approach to managing hair waste, and concurrently promotes the implementation of hair-based hydrolysate in vitamin B-complex biosynthesis.

The green exfoliation of graphite waste and its suitability for biosensor applications.

This work is concerned with the bio-exfoliation of graphite using a soil bacterium. The isolated bacterium showed a detectable ability to oxidize and change its physical appearance and chemical structure. Multiple characterization procedures were used to study the physical and chemical changes. Raman and FTIR spectroscopy proved that the isolate G3 partially exfoliated the graphite into multi-layer sp2 graphitic layers. Scanning electron microscopy (SEM) proved that there was a change in morphology between untreated graphite waste and that manipulated by bacteria. Cyclic voltammetry results proved that the green exfoliated graphite (GEG) was suitable for use in biosensor applications and showed a noticeable ability to detect methanol, even at lower concentrations. These findings are considered as promising for the biological manipulation of graphite waste for environmental purposes. In addition, it is proved that the bacterial transformation of graphite into other GEG structures occurs without needing the chemically hazardous methods that are currently applied.

Tested functionalization of alginate-immobilized ureolytic bacteria for improvement of soil biocementation and maximizing water retention.

The future expected water scarcity in the world invites the scientists to figure out sustainable solutions for agriculture needs. One of suggested solutions could be the improvement of soil stability and increasing its water retention ability. The current proposal is concerning by the improvement of soil stability through biocementation process. While, water retention ability was enhanced through the amendment of tested soils with alginate hydrogel. An ureolytic bacterial isolate showed a detectable ability to dissociate urea and act as a nucleation site for calcium carbonate precipitation. The bacterium was identified as Bacillus sp. after comparing with other strains in GenBank. The mechanical properties of three tested soil types (sand, calcareous, and clay) were improved after the biocementation by calcium carbonate from 119.8, 45.9, and 5 (N) to 187.5, 423.9, and 337.2, respectively. The Energy-dispersive X-ray spectroscopy (EDX) analysis confirmed the appearance of carbon element in samples containing bacterial-immobilized beads and free bacterial cells indicating calcium carbonate formation. The water uptake measurements investigated the ability of alginate beads to retain water with a percentage of 55%. The overall results prove the capability of Bacillus sp. strain combined with alginate hydrogel to improve the soil stability and water retention ability.

Thermo-and pH-sensitive hydrogel membranes composed of poly(N-isopropylacrylamide)-hyaluronan for biomedical applications: Influence of hyaluronan incorporation on the membrane properties.

Interpenetrating hydrogel membranes consisting of pH-sensitive hyaluronan (HA) and thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAM) were synthesized using redox polymerization, followed by N,N-methylenebisacrylamide (BIS) and epichlorohydrin (EPI) were added as chemical crosslinkers. The interaction between membrane compositions has been characterized by FTIR spectroscopy and discussed intensively. The result indicates that HA incorporation in membranes increase the gel fraction, swelling uptake, and the flexibility/elasticity of crosslinked membranes, however it reduced oppositely the mechanical elongation of membranes. PNIPAAm-HA hydrogels responded to both temperature and pH changes and the stimuli-responsiveness was reversible. However, in vitro bioevaluation results revealed that the released ampicillin during the burst release time was sharply influenced and increased with increasing HA contents in membranes; afterwards it became sustainable. Whereas, high HA contents in hydrogels unexpectedly impacted negatively on the cells viability, owing to the viscosity of cell culture media changed. A big resistance was observed against microbial growth of Staphylococcus aureus, Salmonella typhi, and Candida albicans in case of pure PNIPAAm hydrogel membranes without HA or ampicillin. However, HA incorporation or the loaded ampicillin in membranes showed unexpected easily microbial growth. The fast release performance with dual pH-thermo-sensitive hydrogels were suggested as promising materials for quick drug carrier in the biomedical field.

Hydrolytic Enzyme Production by Thermophilic Bacteria Isolated from Saudi Hot Springs.

Hydrolytic enzyme production by thermophilic bacteria isolated from hot springs in the southern region of Saudi Arabia was investigated. The physical and chemical properties of the hot springs prove to be an important environment for hydrolytic-enzyme-producing thermophilic bacteria. Eighty-four bacterial isolates were obtained from three hot springs: Al-Majardah, Al-Khubah and Al-Ardah. Screening of the isolates for enzyme production indicated that 78 isolates showed activity for one or more enzymes. Molecular identification and phylogenic analysis of selected promising isolates confirmed the identity of the isolates as Bacillus aerius, Bacillus licheniformis and Bacillus sonorensis, which have potential to produce the target enzymes α-amylase, protease and lipase, respectively. Optimization of hydrolytic enzyme production by bacterial strains was investigated using kitchen waste as a cheap carbon energy source. Maximum enzyme production was achieved after 72 hours of incubation at the beginning of the stationary phase of growth. Enzyme production was dependent on the initial pH value in the range of pH 7.5-8.5 and an optimal incubation temperature of between 55-60°C. Enzyme production increased gradually in proportion to the kitchen waste concentration; whereas maximum lipase production was gained at 5.0% (w/v) kitchen waste, 7.0% (w/v) of waste was optimal for both α-amylase and protease productivity. The results indicated that hot springs in Saudi Arabia are a substantial source of thermophilic bacteria producing industrially important enzymes using cheap and unexploited waste.

Polyvinyl alcohol/Sodium alginate integrated silver nanoparticles as probable solution for decontamination of microbes contaminated water.

It is well known that the pathogenic multidrug resistant microbes are highly distributed and transferred to human through contaminated food and water. Advanced technologies have been developed for controlling these microbes using synthesized nanoparticles. In this study, biosynthesized silver nanoparticles were prepared, characterized and integrated with two synthetic and natural polymers. The polymers-silver nanoparticles were characterized using SEM, FTIR and mechanical properties of the membrane synthesized from either polymers with/without nanosilver. Both of pathogenic bacteria and yeast were tested for their resistance against 10 different antibiotics. The pathogens showed high resistance against 9 antibiotics and only one was recorded as potent. The cytotoxicity of nanosilver integrated polymers were tested against Hamster kidney cells and Human skin fibroblast cells, and the non-cytotoxic dose was checked for its antimicrobial activity against the selected pathogens. The obtained results in this study confirm that the using of the nanomaterials in safe doses could be a good substitution for biogenic antibiotics and chemicals used in water treatment. Moreover, the beads which were used in this study could be lasts for long period in water treatment station with high antibacterial capacity, in addition, it can be gathered easily at the end of the run.

Supplementation of Cerelac baby food with yeast-probiotic cocktail strains induces high potential for aflatoxin detoxification both in vitro and in vivo in mother and baby albino rats.

BACKGROUND: Aflatoxins (AFs) are a group of toxic, nephrotoxic, hepatotoxic and carcinogenic fungal metabolites. Heat- and acid-treated yeasts, probiotic bacteria and their combination were used to remove AFB1, AFB2, AFG1 and AFG2 from human and animal food. RESULTS: The in vitro study revealed that the highest removal percentage of AFs in phosphate-buffered saline was recorded after 72 h with the yeast-probiotic coctile, reaching 95.59%. Therefore, this coctile was added to Cerelac contaminated with AFB1, AFB2, AFG1 and AFG2, and the removal percentages were 8.17%, 36.12%, 44.75%, 64.72% and 93.21% after 6, 12, 24, 48 and 72 h of treatment, respectively. Cerelac yeast-probiotic coctile was administered to female rats and the results showed that all AFs (AFB1, AFB2, AFG1 and AFG2) were detected in the serum of mother rats for both AF groups III and IV. On the other hand, AFM1 and AFM2 metabolites were not observed in mothers' sera but were detected in all infants of groups III and IV. Meanwhile, AFB1, AFB2, AFG1 and AFG2 were not observed in infants' sera. CONCLUSION: A mixture of yeast-probiotic coctile was successful in reducing the level of AF in rat sera and diminished the deleterious effect of AFs on animal health. © 2017 Society of Chemical Industry.

Differential-display reverse transcription-PCR (DDRT-PCR): a new technology for molecular detection and studying one of the antagonistic factors of Bacillus endophyticus strain SA against Staphylococcus aureus (MRSA).

Differential Display (DDRT-PCR) is a powerful technique for analyzing differences in gene expression. In-vivo expression technologies and differential display RT-PCR are providing new approaches to further examine a microbe's response to experimental conditions which more closely resemble natural microbial associations and habitats. In this study, Bacillus endophyticus strain SA isolated from the inner tissue of the stem of the cultivated plant (Salvadora persica, Asir, Kingdom of Saudi Arabia) produces an antagonistic factor. This factor has a broad spectrum of activity against Gram-positive and specifically against Staphylococcus aureus (MRSA). The antagonistic factor was isolated from the bacterial culture medium and purified by thin layer chromatography technique, then analyzed by GC-MS analysis. Identification of the producer strain was performed using the partial nucleotide sequence of 16S rRNA gene, which indicated that this strain is identical to B. endophyticus with 99 % similarity. The sequence of this strain was deposited at NCBI GenBank under accession number KF011545. Application of differential display RT-PCR revealed that the isolate was able to up-regulate a gene with serine protease like protein. The protein is well known as antimicrobial agent and was reported to be produced by plants, animals and insects. Serine protease is also known to be produced by bacteria for purposes oth er than bacterial-bacterial antagonistic effect, which has been confirmed by this study.

Advanced trends in controlling Helicobacter pylori infections using functional and therapeutically supplements in baby milk.

Helicobacter pylori is a common human pathogen infecting about 30 % of children and 60 % of adults worldwide. It is responsible for diseases such as gastritis, peptic ulcer and gastric cancer. H. pylori treatment based on antibiotics with proton pump inhibitor, but therapy failure is shown to be higher than 20 % and is essentially due to an increasing in prevalence of antibiotic-resistant bacteria, which has led to the search for alternative therapies. In this study, we discuss the usage of natural extracts mixture as alternative or complementary agents in controlling H. pylori infection so here, we focused on the plant extracts of (Cloves, Pepper, Cumin, Sage, Pomegranate peel, Ginger, Myrrh and Licorice). To that end, Phytochemical constituents detection like Tannins, Glycosides, Alkaloids, Flavonoids, Terpenoids, Saponins, Phenolic compounds, Reducing sugars, Volatile oils, Amino acids and Proteins was demonstrated. Each plant extract was examined individually or in combination for its antimicrobial activity against H. pylori. Out of the used extracts, four mixes were prepared and tested against H. pylori. The antibacterial activities of the four mixes, represented by the diameter of inhibition clear zone, recorded 21, 39, 23 and 28 mm. The most potent mix (mix2) was chosen and mixed with baby milk as a new combination for H. pylori infections treatment in babies.

Utilization of dental pulp DNA as diagnostic molecular marker for fertility detection in men.

UNLABELLED: Recent advances in DNA technology have revolutionized forensic identification procedures. Teeth dentin and pulp are rich sources of DNA material, which can be successfully extracted and it provides us with valuable information on individuals, systemic health including fertility status. AIM OF STUDY: The aim of this study was to use DNA material extracted from human teeth pulp for detection of fertility status of men. MATERIALS AND METHODS: Twenty extracted premolar teeth of systemic disease free male Saudi individuals (45 years average age) were collected; eight of them were infertile while others were fertile and were used as control group. This information was concealed until the PCR analysis was performed. The results of recorded patient information was matched with the results of the DNA analysis. RESULTS: Results showed that the gene (sY83) an important gene of AZFa region in Y chromosome is important for male fertility. It was later evident that the infertile patients suffered from azoospermia, and that information is completely matched with our results. CONCLUSION: Using DNA extracted from dental pulp can be used successfully in determining fertility status of human which may help in an accurate personal identification specially in extreme circumstances.