Nanoemulsion of cinnamon oil to combat colistin-resistant Klebsiella pneumoniae and cancer cells.

This study aimed to investigate the potential of cinnamon oil nanoemulsion (CONE) as an antibacterial agent against clinical strains of colistin-resistant Klebsiella pneumoniae and its anticancer activity. The prepared and characterized CONE was found to have a spherical shape with an average size of 70.6 ± 28.3 nm under TEM and a PDI value of 0.076 and zeta potential value of 6.9 mV using DLS analysis. The antibacterial activity of CONE against Klebsiella pneumoniae strains was investigated, and it was found to have higher inhibitory activity (18.3 ± 1.2-30.3 ± 0.8 mm) against the tested bacteria compared to bulk cinnamon oil (14.6 ± 0.88-20.6 ± 1.2) with MIC values ranging from 0.077 to 0.31 % v/v which equivalent to 0.2-0.82 ng/ml of CONE. CONE inhibited the growth of bacteria in a dose and time-dependent manner based on the time-kill assay in which Klebsiella pneumoniae B-9 was used as a model among the bacterial strains under investigation. The study also investigated the expression of the mcr-1 gene in the Klebsiella pneumoniae strains and found that all strains were positive for the gene expression and subsequently its presence. The level of mcr-1 gene expression among the B-2, B-4, B-9, and B-11 control strains and that treated with colistin was similar, but it was different in both B-5 and B-2. However, all strains exhibited a significant downregulation in gene expression (ranging from 3.97 to 8.7-fold) after their treatment with CONE. Additionally, the CONE-treated bacterial cells appeared with a great deformation compared with control cells under TEM. Finally, CONE exhibited selective toxicity against different cancer cell lines depending on comparison with the normal cell lines.

A statistical approach to enhance the productivity of Streptomyces baarensis MH-133 for bioactive compounds.

The goal of this study was to use statistical optimization to change the nutritional and environmental conditions so that Streptomyces baarensis MH-133 could make more active metabolites. Twelve trials were used to screen for critical variables influencing productivity using the Placket-Burman Design method. S. baarensis MH-133 is significantly influenced by elicitation, yeast extract, inoculum size, and incubation period in terms of antibacterial activity. A total of 27 experimental trials with various combinations of these factors were used to carry out the response surface technique using the Box-Behnken design. The analyses revealed that the model was highly significant (p < 0.001), with a lack-of-fit of 0.212 and a coefficient determination (R2) of 0.9224. Additionally, the model predicted that the response as inhibition zone diameter would reach a value of 27 mm. Under optimal conditions, S. baarensis MH-133 produced 18.0 g of crude extract to each 35L and was purified with column chromatography. The active fraction exhibiting antibacterial activity was characterized using spectroscopic analysis. The MIC and MBC values varied between 37.5 and 300 µg/ml and 75 and 300 µg/ml, respectively. In conclusion, the biostatistical optimization of the active fraction critical variables, including environmental and nutritional conditions, enhances the production of bioactive molecules by Streptomyces species.

Antibacterial, Antibiofilm and Anti-Inflammatory Activities of Eugenol Clove Essential Oil against Resistant Helicobacter pylori.

Eugenol essential oil (EEO) is the major component in aromatic extracts of Syzygium aromaticum (clove) and has several biological properties, such as antibacterial, antioxidant, and anti-inflammatory activities, as well as controlling vomiting, coughing, nausea, flatulence, diarrhea, dyspepsia, stomach distension, and gastrointestinal spasm pain. It also stimulates the nerves. Therefore, the aim of this study was to extract and purify EEO from clove buds and assess its ability to combat resistant Helicobacter pylori. Additionally, EEO's anti-inflammatory activity and its ability to suppress H. pylori biofilm formation, which is responsible for antibiotic resistance, was also investigated. Syzygium aromaticum buds were purchased from a local market, ground, and the EEO was extracted by using hydro-distillation and then purified and chemically characterized using gas chromatography-mass spectrometry (GC-MS). A disk-diffusion assay showed that Helicobacter pylori is sensitive to EEO, with an inhibition zone ranging from 10 ± 06 to 22 ± 04 mm. The minimum inhibition concentration (MIC) of EEO ranged from 23.0 to 51.0 µg/mL against both Helicobacter pylori clinical isolates and standard strains. In addition, EEO showed antibiofilm activity at 25 µg/mL and 50 µg/mL against various Helicobacter pylori strains, with suppression percentages of 49.32% and 73.21%, respectively. The results obtained from the anti-inflammatory assay revealed that EEO possesses strong anti-inflammatory activity, with human erythrocyte hemolysis inhibition percentages of 53.04, 58.74, 61.07, and 63.64% at concentrations of 4, 8, 16, and 32 µg/L, respectively. GC-MS analysis revealed that EEO is a major component of Syzygium aromaticum when extracted with a hydro-distillation technique, which was confirmed by its purification using a chemical separation process. EEO exhibited antibacterial action against resistant Helicobacter pylori strains, as well as antibiofilm and anti-inflammatory activities, and is a promising natural alternative in clinical therapy.

Biogenic synthesis of CuO-NPs as nanotherapeutics approaches to overcome multidrug-resistant Staphylococcus aureus (MDRSA).

Due to the misuse of antibiotics, the multidrug-resistant Staphylococcus aureus (MDRSA) has caused serious infections and become more difficult to deal with. Here we propose to synthesise copper oxide nanoparticles (CuO-NPs) using a cell-free filter of Streptomyces rochei to enhance antibiotics activity against (MDRSA) and kill them. Characterisation of CuO-NPs using ultraviolet, dynamic light scattering, zeta potential, transmission electron microscopic (TEM), and X-ray diffraction, were investigated. The antibacterial action of the CuO-NPs was tested against standard strain and clinical isolates using the agar well diffusion method and the microdilution assay. The results showed the monodispersed spherical shape CuO-NPs with a mean diameter of 10.7 nm and were found to be active against (MDRSA). By a combination of CuO-NPs with different antibiotics, the highest synergistic effect was observed with cefoxitin, the minimum inhibitory concentration (MIC) was reduced to 6.5 for CuO-NPs, and 19.5 for cefoxitin. Time-kill assay showed the highest reduction in log10 colony-forming unit (CFU)/ml of initial inoculum of MRSA after 24 h. The HFB-4 cells cultured in the presence of CuO-NPs showed normal morphology with 100% viability at 8 µg/ml. TEM showed that combination (1/4 MIC cefoxitin +1/16 MIC CuO-NPs) highly damages bacterial cells' shape. The biosynthesis CuO-NPs showed antibacterial activity against S. aureus suggesting a promising alternative in clinical.

Antibacterial activity of essential oils for combating colistin-resistant bacteria.

OBJECTIVES: Colistin (polymyxin E) is a bactericidal antibiotic used to treat severe infections caused by multidrug-resistant Gram-negative bacteria. The product of the mcr1 gene generates transferable plasmid-mediated colistin resistance, which has arisen as a worldwide health-care problem. This study aimed to isolate and identify colistin-resistant bacteria, and evaluate the ability of essential oils in its fights. METHODS: Twenty-seven bacterial isolates were collected from patients who were admitted to the National Cancer Institute, Cairo, Egypt, and processed using standard microbiological methods. Essential oils were purchased from AB Chem Company, Egypt, screened for antibacterial, cytotoxic activity, and (GC-MS) analysis. RESULTS: A total of 5 bacterial isolates were resistant to colistin with minimum inhibitory concentration (MIC) ranging from 6.25->200 µg/ml. Cinnamon oil exhibited the highest activity against colistin-resistant strains followed by thyme and eucalyptus oil. The (MIC) of cinnamon oils against resistant strains ranged from 4.88 to 312.5 µg/ml. Moreover, mcr-1 gene expression was extremely down-regulated after the treatment of bacterial strains with cinnamon oil and decreased to 20-35-fold. Examination of treated bacterial cells with sub-inhibitory concentrations under transmission electron microscopy showed various abnormalities occurred in most of these cells. CONCLUSIONS: Cinnamon oil exhibits antibacterial activity against colistin-resistant strains, showing it as a promising natural alternative in clinical therapy.

Boosting the Anticancer Activity of Aspergillus flavus "endophyte of Jojoba" Taxol via Conjugation with Gold Nanoparticles Mediated by γ-Irradiation.

Taxol production by fungi is one of the promising alternative approaches, regarding to the natural and semisynthetic sources; however, the lower yield and rapid loss of Taxol productivity by fungi are the major challenges that halt their further industrial implementation. Thus, searching for fungal isolates with affordable Taxol-production stability, in addition to enhance its anticancer activity via conjugation with gold nanoparticles, is the main objectives of this study. Twenty-four endophytic fungal isolates were recovered from the barks, twigs, and leaves of jojoba plant, among these fungi, Aspergillus flavus MW485934.1 was the most potent Taxol producer (88.6 µg/l). The chemical identity of the extracted Taxol of A. flavus was verified by the TLC, HPLC, HNMR, and FTIR analyses. The yield of Taxol produced by A. flavus was optimized by the response surface methodology (RSM) using Plackett-Burman (PBD) and faced central composite designs (FCCD). The yield of Taxol by A. flavus was increased by about 3.2 folds comparing to the control cultures (from 96.5 into 302.7 µg/l). The highest Taxol yield by was obtained growing A. flavus on a modified malt extract medium (g/l) (malt extract 20.0, peptone 2.0, sucrose 20.0, soytone 2.0, cysteine 0.5, glutamine 0.5, and beef extract 1.0 adjusted to pH 6.0) and incubated at 30 °C for 16 days. From the FCCD design, the significant variables affecting Taxol production by A. flavus were cysteine, pH, and incubation time. Upon A. flavus γ-irradiation at 1.0 kGy, the Taxol yield was increased by about 1.25 fold (375.9 µg/l). To boost its anticancer activity, the purified Taxol was conjugated with gold nanoparticles (AuNPs) mediated by γ-rays irradiation (0.5 kGy), and the physicochemical properties of Taxol-AuNPs composite were evaluated by UV-Vis, DLS, XRD, and TEM analyses. The IC50 values of the native-Taxol and Taxol-AuNPs conjugates towards HEPG-2 cells were 4.06 and 2.1 µg/ml, while the IC50 values against MCF-7 were 6.07 and 3.3 µg/ml, respectively. Thus, the anticancer activity of Taxol-AuNPs composite was increased by 2 folds comparing to the native Taxol towards HEPG-2 and MCF-7 cell lines. Also, the antimicrobial activity of Taxol against the multidrug resistant bacteria was dramatically increased upon conjugation with AuNPs comparing to authentic AuNPs and Taxol, ensuring the higher solubility, targetability, and efficiency of Taxol upon AuNPs conjugation.

Solar radiation-induced synthesis of bacterial cellulose/silver nanoparticles (BC/AgNPs) composite using BC as reducing and capping agent.

In the present work, a simple, novel, and ecofriendly method for synthesis of silver nanoparticles (AgNPs) and BC/AgNP composite using bacterial cellulose (BC) nanofibers soaked in AgNO3 solution under induction action of solar radiation. The photochemical reduction of silver Ag + ions into silver nanoparticles (Ago) was confirmed using UV visible spectra; the surface plasmon resonance of synthesized AgNPs was localized around 425 nm. The mean diameter of AgNPs obtained by DLS analysis was 52.0 nm with a zeta potential value of - 9.98 mV. TEM images showed a spherical shape of AgNPs. The formation of BC/AgNP composite was confirmed by FESEM, EDX, FTIR, and XRD analysis. FESEM images for BC showed the 3D structures of BC nanofibers and the deposited AgNPs in the BC crystalline nanofibers. XRD measurements revealed the high crystallinity of BC and BC/AgNP composite with crystal sizes of 5.13 and 5.6 nm, respectively. BC/AgNP composite and AgNPs exhibited strong antibacterial activity against both Gram-positive and Gram-negative bacteria. The present work introduces a facile green approach for BC/AgNP composite synthesis and its utility as potential food packaging and wound dressings, as well as sunlight indicator application.

Control of extensively drug-resistant Pseudomonas aeruginosa co-harboring metallo-ß-lactamase enzymes with oprD gene downregulation.

PURPOSE: to study control and treatment of infection with extensive drug-resistant carbapenem-resistant Pseudomonas aeruginosa (XDR-CRPA). METHODS: Eleven Pseudomonas aeruginosa (XDR-CRPA) strains used in this study were isolated from a clinical sample, identified, and antibiotics susceptibility recorded in a previous study. Real-time PCR (RT-PCR) was performed to determine the expression level of the OprD gene. Besides, a checkerboard technique was performed to assess the effect of polymyxin-B (POX), colistin (COL), rifampicin (RIF), imipenem (IPM), and meropenem (MEM) during 2 and 3- dimensional antibiotic combinations. Further, the time-kill study was determined for the most potent combination against four representative strains, log10 changes of viable cell counts were expressed as their mean value (±SD) values. RESULTS: Molecular analysis by Real-time PCR revealed that the diminished expression level of OprD mRNA was overwhelming to various degrees. The checkerboard method demonstrated that the relevant synergism was achieved in 90.9% of strains for both carbapenem antibiotics during the triple combinations. While an additive effect was noted for all the dual regimen assays. Regarding time-kill experiments, a remarkable bactericidal effect with [99.9% killing rate] was observed toward only one strain whilst a bacteriostatic attitude was proven with ≥95% bacterial eradication against the three remaining strains. CONCLUSIONS: These findings underscore the promising implications of these combinations for treatment against XDR-Pseudomonas aeruginosa even they are resistant to carbapenems due to multiple mechanisms of action.

Correlation between phenotypic virulence traits and antibiotic resistance in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is a ubiquitous pathogen capable of infecting virtually all tissues and its one of the standout amongst the most hazardous microorganisms of high morbidity and mortality rates especially in debilitated patients with few successful antibiotic choices available. This pathogen regulating most virulence traits by that so-called quorum sensing (QS), a cell to cell communication system. the present study was intended to phenotypically evaluate the activity of specific virulence traits (including swarming and swimming motility, protease, pyocyanin, and biofilm production) in Pseudomonas aeruginosa clinical isolates and assess the statistical correlation between these traits and antibiotic resistance. One hundred and thirteen bacterial isolates were obtained from different clinical samples and identified as P. aeruginosa, among them, 73.4% have the ability to forming biofilm with different degrees; 59.2% were able to produce pyocyanin pigment while all isolates having the ability to make swarming and swimming motility and able to produce protease enzyme with different degrees. The isolates that produce the higher levels of the virulence traits were identified by both biochemical using Vitek2 automated system and genetically via 16s rRNA gene analysis. The statistical analysis results indicate that a positive significant correlation was found between biofilm formation and other studied virulence traits except for protease (r = 0.584: 0.324, P < 0.05) while a non-significant correlation was found between biofilm formation and protease activity (r = 0.105, P ˃ 0.05). Swimming and swarming motility have a positive significant correlation with other studied virulence traits (r = 0.613: 0.297, P < 0.05) except for protease. Pyocyanin pigment production have a positive significant correlation with other studied virulence traits (r = 0.33: 0.297, P < 0.05) except for protease. on the other hand, negative significant correlations were found between biofilm formation, swimming; and swarming motility, Pyocyanin pigment production, and the susceptibility of antibiotics (r = -0.512: -0.281, P < 0.05). Detection of such correlations in P. aeruginosa is useful for study the behavior of this pathogen and may be provide a new target for the treatment of MDR infections.

Enhanced removal of fifteen pesticide mixture by a single bacterial strain using response surface methodology and its application in raw milk.

PURPOSE: Environmental contamination with various pesticides accompanied by uncontrolled use contributes to severe ecological and health problems. Although extensive research was conducted on pesticides degradation, very few reports have demonstrated the degradation of mixed pesticides. Consequently, this study aimed to evaluate the removal efficacy of highly potent bacterial isolate for pesticide mixture under optimal culture conditions, followed by their application in milk. METHODS: Isolation and selection of bacterial isolates were performed from 40 milk samples by enrichment culture technique and were screened to obtain highly potent bacterial strain identified by 16 S rDNA analysis. The statistics-based experimental designs were applied to optimize the culture conditions towards the best degradation of pesticides mixture, followed by subsequent utilization in milk. The degradation ratio of pesticides was analyzed using gas chromatography-mass spectrometry. RESULTS: In this study, a bacterial strain S6A identified as Bacillus subtilis-mw1 efficiently eliminated environmental contaminants from different groups of pesticide residues. The statistical optimization showcased optimum settings that accomplished the highest pesticide mixture degradation (61.59 %). The application experiment manifested that degradation of pesticide mixtures of sterile milk (STM) was relatively faster than non-sterile milk (NSTM). CONCLUSIONS: The obtained results assist in eliminating environmental contamination with various groups of pesticide residues. Furthermore, it can be employed in reducing pesticide residues that cause milk contamination to increase safety and quality.Graphical abstract. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40201-021-00683-0.

Production, bioprocess optimization and γ-irradiation of Penicillium polonicum, as a new Taxol producing endophyte from Ginko biloba.

Twenty-eight fungal endophytes were recovered from the different parts of Ginkgo biloba and screened for their Taxol producing potency. Among these isolates, Penicillium polonicum AUMC14487 was reported as the potent Taxol producer (90.53 µg/l). The chemical identity of the extracted Taxol was verified from the TLC, HPLC, NMR, EDX, and FTIR analyses. The extracted Taxol displayed a strong antiproliferative activity against HEPG2 (IC50 4.06 µM) and MCF7 (IC50 6.07 µM). The yield of Taxol by P. polonicum was optimized by nutritional optimization with the Response Surface Methodology (RSM) using Plackett-Burman and Central Composite Designs. In addition to nutritional optimization, the effect of γ-irradiation of the spores of P. polonicum on its Taxol producing potency was evaluated. The yield of Taxol by P. polonicum was increased via nutritional optimization by response surface methodology with Plackett-Burman and FCCD designs, and γ-irradiation by about 4.5 folds, comparing to the control culture. The yield of Taxol was increased by about 1.2 folds (401.2 µg/l) by γ -irradiation of the isolates at 0.5-0.75 kGy, comparing to the control cultures (332.2 µg/l). The highest Taxol yield was obtained by growing P. polonicum on modified Czapek's- Dox medium (sucrose 40.0 g/l, malt extract 20.0 g/l, peptone 2.0 g/l, K2PO4 2.0 g/l, KCl 1.0 g/l, NaNO3 2.0 g/l, MgSO4. 5H2O 1.0 g/l) of pH 7.0 at 30.0 °C for 7.0 days. From the FCCD design, sucrose, malt extract and incubation time being the highest significant variables medium components affecting the Taxol production by P. polonicum.

New combination approaches to combat methicillin-resistant Staphylococcus aureus (MRSA).

The herbal products proved to be more promising antimicrobials even though their antimicrobial activity is milder than commercially available antibiotics. Moreover, herbal drugs may act synergistically with antibiotics to kill microbes. In this study, we aimed to enhance the activity of penicillin against MRSA through combination with the active saponin fraction isolated from the Zygophyllum album plant. Three different types of metabolites (saponins, sterols, and phenolics) have been extracted from Zygophyllum album with ethanol and purified using different chromatographic techniques. The antibacterial activity of crude extract and the separated metabolites were checked against MRSA isolates, Saponin fraction (ZA-S) was only the active one followed by the crude extract. Therefore, the compounds in this fraction were identified using ultra-high-performance liquid chromatography connected to quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) operated in positive and negative ionization modes. UHPLC/QTOF-MS revealed the presence of major six ursane-type tritepenoidal saponins (Quinovic acid, Quinovic acid 3ß-O-ß-D-quinovopyranoside, Zygophylloside C, Zygophylloside G, Zygophylloside K and Ursolic acid), in addition to Oleanolic acid. Interaction studies between saponin fraction and penicillin against MRSA were performed through the checkerboard method and time-kill assay. According to checkerboard results, only three combinations showed a fractional inhibitory concentration index less than 0.5 at concentrations of (62.5 + 312.5, 62.5 + 156.25, and 62.5 + 78.125 of penicillin and ZA-S, respectively). Time kill assay results showed that the highest reduction in log10 colony-forming unit (CFU)/ml of initial inoculum of MRSA after 24 h occurred by 3.7 at concentrations of 62.5 + 312.5 (µg/µg)/ml of penicillin and ZA-S, respectively. Thus, the combination between saponin fraction of Zygophyllum album and penicillin with these concentrations could be a potential agent against MRSA that can serve as possible model for new antibacterial drug.

Corrigendum to "Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 α-amylase on Chitosan-magnetic nanoparticles" [Biotechnol. Rep. 26 (2020) e00443].

[This corrects the article DOI: 10.1016/j.btre.2020.e00443.].

Catalytic, kinetic and thermal properties of free andimmobilized Bacillus subtilis -MK1 α-amylase on Chitosan-magnetic nanoparticles.

Bacillus subtilis strain-MK1 α-amylase was successfully immobilized on Chitosan-magnetic nanoparticles (Ch-MNP) that had been modified with polyethyleneimine (PEI) and glutaraldehyde (GA). Optimization of Ch-MNP/PEI/GA beads modification by Central Composite design enhanced the immobilization yield (IY %) by 1.5-fold. Ch-MNP/PEI/GA was characterized before and after modification and immobilization by FTIR and SEM. Ch-MNP/PEI/GA/Enzyme showed the same pH optima of free enzyme, while an elevation 10 °C in temperature optima was observed after its immobilization. Ch-MNP/PEI/GA/Enzyme displayed higher Km and Vmax values (2.1 and 1.2-fold) and lower Vmax/Km ratio (1.7-fold), respectively than the free enzyme. Compared to the free enzyme, Ch-MNP/PEI/GA/Enzyme exhibited lower activation energy, lower deactivation constant rate, higher D-values, higher half-life, and higher energy for denaturation. Immobilization of α-amylase increased enthalpy (4.2-fold), free energy (1.1-fold) and decreased entropy (4.6-fold) of thermal inactivation. A significant increase in pH stability of Ch-MNP/PEI/GA/Enzyme was observed especially at alkaline pH values. In addition, Ch-MNP/PEI/GA/Enzyme preserved 83.2 % of its initial activity after 15 consecutive cycles. When storing Ch-MNP/PEI/GA/Enzyme at 4 °C the residual activity was 100 and 86 %, respectively after 21 and 40 days. Finally, immobilization process improved the catalytic properties and stabilities, thus raising the suitability for industrial processes with lower cost and time.

Nematicidal activity of thermostable alkaline protease produced by Saccharomonospora viridis strain Hw G550.

Application of thermostable alkaline protease to control the harmful nematodes was investigated in the current study. A total of 14 proteolytic actinomycetes were isolated from Egyptian harsh environments. Out of them, isolate G550 exhibited the highest proteolytic activity (528.9 U/ml). Protease from isolate G550 exhibited high nematicidal activity against M. incognita under laboratory conditions and caused hydrolysis of J2S cuticle. This isolate was identified using molecular techniques and deposited in GenBank under name of Saccharomonospora viridis strain Hw G550 with accession number: MF152631. The G550 protease was extracted, characterized and applied under greenhouse conditions as nematicidal agent. This enzyme exhibited maximum activity and stability at alkaline pH (8) and thermal conditions (50-60 °C). Also, the results showed that, all treatments using protease caused a significant decrease in nematode reproduction and increasing in the plant properties. Finally, the thermo alkaliphilic protease could be used as bio-control agent against RKN.