Green Synthesis of Silver Nanoparticles Using Cirsium Congestum Extract Modified by Chitosan/Alginate: Bactericidal Activity against Pathogenic Bacteria and Cytotoxicity Analysis in Normal Cell Line.

AIM: The study aimed to determine in vitro pharmacological effects of modified Ag nanoparticles (AgNPs). BACKGROUND: AgNPs are considered antimicrobial agents. However, the cytotoxicity of chemically synthesized AgNPs (cAgNPs) has raised challenges that limit their use. OBJECTIVE: The purpose of the study was to examine the antimicrobial and cytotoxicity effects of AgNPs synthesized using Cirsium congestum extract modified by chitosan/alginate AgNPS (Ch/ALG-gAgNPs). METHODS: Nanoparticles were characterized using TEM, DLS, XRD, and FTIR. Resistant strains of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used for the antimicrobial analysis of Ch/ALG-gAgNPs using disc diffusion and microdilution methods. The effects of NPs on cell viability and apoptosis in L929 normal cells were determined using MTT assay and annexin/PI staining, respectively. RESULTS: Physicochemical characterizations confirmed Ch/ALG-gAgNPs to be spherical and uniformly dispersed, and their size ranged from 50 to 500 nm. Ch/ALG-gAgNPs inhibited the growth of microbial strains in a dose-dependent manner. The antibacterial effect of Ch/ALG-gAgNPs was significantly higher than cAgNPs. The Ch/ALG-gAgNPs showed little cytotoxicity against normal cells at concentrations less than 50 µg/ml. Cytotoxicity effects of Ch/ALG-gAgNP were less than cAgNPs. Flow cytometry and real-time PCR results showed a decrease in apoptosis percentage and BAX marker in the presence of Ch/ALG-gAgNPs relative to when the cell was treated with cAgNPs. CONCLUSION: Current findings introduce novel gAgNPs modified with chitosan/alginate for use in medicine.

Anticancer Potential of Doxorubicin in Combination with Green-Synthesized Silver Nanoparticle and its Cytotoxicity Effects on Cardio-Myoblast Normal Cells.

BACKGROUND: The use of DOX as an anticancer agent is associated with serious side effects on normal cells especially in cardiovascular tissue. OBJECTIVE: Here, it is proposed that the combination of a low dose of DOX with AgNPs provides ideal cytotoxicity against cancer cells and decreases side effects on normal human cells. This study evaluates the cytotoxic effects of green-synthetized AgNPs (GS-AgNPs) in combination with DOX in cancerous cells (MCF7) and investigates its influences on cell growth and apoptosis in a normal cell line of the heart (H9c2). METHODS: We used coffee extracts, as a reducing and stabilizing agent for the green-synthesis of AgNPs. GSAgNPs were characterized by using various analytical methods. MTT assay was used for cell toxicity analysis in cancerous and normal cells. Moreover, Annexin-V /PI staining and mRNA expression of Bax, Bcl2 and p53 were performed for apoptosis measurement in heart normal cell line. RESULTS: GS-AgNPs showed more biocompatibility for normal cells and higher cytotoxicity for cancerous cells compared to that reported for chemically synthesized nanoparticles. Our results also demonstrated that a selected combination of DOX and AgNPs, 20 µM AgNPs / 0.3 µM DOX, had a suitable cytotoxic effect against cancerous cells with a minimum toxic effect on normal cells. So, no significant alteration was observed in cell migration capacity, apoptosis and gene expression of BAX, Bcl-2 and P53 when H9c2 cells were treated with 20 µM AgNPs / 0.3 µM DOX relative to the non-treated control. CONCLUSION: Finally, it seems that the combination of GS-AgNPs and DOX could be a potent strategy to combat cancer.

The Efficacy of Imipenem Conjugated with Synthesized Silver Nanoparticles Against Acinetobacter baumannii Clinical Isolates, Iran.

Carbapenem-resistant Acinetobacter baumannii (CRAB) remains as a serious cause of infectious diseases and septic mortality in hospitalized patients worldwide. This study was conducted to evaluate the antimicrobial effect of imipenem conjugated silver nanoparticles (AgNPs) on resistant isolated A. baumannii from nosocomial infections.The antimicrobial susceptibility test of 100 A. baumannii clinical isolates against different antibiotics was performed. PCR was used to confirm bacterial resistance and to identify different genes encoding Ambler class ß-lactamases. The chemically synthesized AgNPs were characterized using UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier-transform infrared (FTIR). The stability, drug release kinetic, and cytotoxicity (MTT assay) of AgNPs were also investigated. The imipenem were conjugated with AgNPs, and conjugants were characterized as discussed above. Minimum inhibitory concentration (MIC) of the AgNPs and conjugants were tested against A. baumannii isolates and compared with imipenem alone.The results revealed that among all isolated A. baumannii, 76% showed resistant to imipenem (MIC ≥ 64 µg/mL to ≥ 256 µg/mL). The blaOXA-23, blaPER, blaOXA-40, and blaIMP genes were the most prevalent genes. UV-vis spectroscopy, XRD, TEM, and FTIR analysis confirmed synthesis of AgNPs (average size of 10-40 nm) and conjugation with imipenem. The release of imipenem from AgNPs can be defined as Fickian diffusion model. The MIC values of AgNPs conjugated with imipenem against resistant A. baumannii were decreased in a dose dependent manner and were based on existence of resistant genes. The AgNPs also showed low cytotoxic effects.The results suggest that imipenem-AgNPs has a strong potency as a powerful antibacterial agent against multi-resistant A. baumannii.

Antifungal effects of Ziziphora tenuior, Lavandula angustifolia, Cuminum cyminum essential oils against clinical isolates of Candida albicans from women suffering from vulvovaginal candidiasis / Efecto antifungico de los aceites esenciales de Ziziphora tenuior, Lavandula angustifolia y Cuminum cyminum contra aislados clínicos de Candida albicans de mujeres que sufren de candidiasis vulvovaginal

Candida sp. especially C. albicans is the main cause of candidiasis in women in reproductive ages. The prevalence of drug resistant C. albicans along with adverse effects of current treatments have encouraged the scientists to research on medicinal plant's essential oils. The aim of this study was to evaluate the potential effects of Ziziphora tenuior, Lavandula angustifolia, Cuminum cyminum essential oils against clinical isolates of C. albicans, which were isolated from women with vulvovaginal candidiasis. The anti-candidal effects of these commercial essential oils were screened against these clinical isolates (n=80) by disc diffusion and micro-broth dilution assays. The means of inhibition zone diameters, MIC and MFCs were reported for each essential oil. Also, the capability of fungal strain for biofilm formation in comparison with C. albicans ATCC 10231 were determined. The anti-biofilm effects of essential oils against 11 biofilm producing strains of C. albicans were determined. The results showed C. cyminum and Z. tenuior essential oils had the best anti-candidal effects, while the best biofilm killing effects was for L. angustifolia essential oil. C. cyminum and Z. tenuior essential oils can be formulated for more evaluations in preclinical and clinical studies.

Candida sp., especialmente C. albicans, es la principal causa de candidiasis en mujeres en edad reproductiva. La aparición de resistencia a los antifungicos de C. albicans, junto con el riesgo de efectos adversos de los tratamientos actuales, ha llevado a los científicos a buscar alternativas en los aceites esenciales derivados de plantas. Los objetivos del estudio fueron evaluar los efectos potenciales de los aceites esenciales de Ziziphora tenuior, Lavandula angustifolia, Cuminum cyminum, contra aislados clínicos de C. albicans, obtenidos de mujeres con candidiasis vulvovaginal. Los efectos anti-Candida de estos aceites esenciales comerciales fueron probados contra estos aislados clínicos (n=80) por difusión en disco y ensayos de microdilución. Se obtuvo el promedio de diámetro de inhibición, MIC y MFCs para cada aceite esencial. También se comparó la capacidad de formación de biopelículas de 11 cepas de C. albicans de cada aislado frente a la cepa de referencia C. albicans ATCC 10231 y la capacidad de cada aceite esencial para evitar la formación de biopelículas. Los resultados muestran que los aceites esenciales de C. cyminum y Z. tenuior tuvieron la mejor actividad anti-Candida, mientras que los mejores efectos para destruir biopelículas se obtuvieron con los aceites esenciales de L. angustifolia. Los aceites esenciales de C. cyminum y Z. tenuior ameritan tener mayores evaluaciones preclínicas y clínicas.

Simulation and Stability Assessment of Anti-EpCAM Immunotoxin for Cancer Therapy.

Purpose: Epithelial cell adhesion molecule (EpCAM) is a dominant antigen in human colon carcinoma tissue. Topology features of this antigen are different in normal and malignant conditions; for instance, EpCAM is much less accessible to antibodies in normal cells than in cancerous tissues. Hence, EpCAM has been considered as a suitable candidate for cancer target therapy via immunotoxins (ITs) development. In this study, attention was focused on the stability assessment of anti-EpCAM-IT (anti-Ep-IT) to design a novel IT. Methods: The 3D structures of the antibody template and the toxin segment of anti-Ep-IT were retrieved from PDB. Discovery Studio3.0 was used to separate the ligands and water molecules. The antibody (Ab) fragment of anti-Ep-IT was aligned using protein blast (BLAST-p), and SWISS-MODEL database was used for Ab modeling. IT modeling was accomplished using MODELLER 9.15. Also, GROMACS 5.07 was used for molecular dynamic (MD) simulation step. Moreover, ERRAT and RAMPAGE databases were used for quality assessment of the structures. Results: BLAST-p results indicated that antibody moiety of IT has the highest E-value and query coverage scores to the monoclonal antibody (mAb) 4D5MOC-B. Modeling by SWISS-MODEL provided a reasonable template for Ab portion compared to MODELLER. The best modeled full-length IT with the lowest RMSD values was selected. Finally, RMSD plot for MD stage demonstrated constant values from 7000ps to 20000ps. Conclusion: In general, both modeling results and their quality evaluations were satisfactory for designing IT. Moreover, RMSD plot revealed that IT stability was preserved during the simulation. Overall, our findings led to modeling and simulation of the anti-Ep-IT with more structural stability.

Evaluating the anti-biofilm and antibacterial effects of Juglans regia L. extracts against clinical isolates of Pseudomonas aeruginosa.

OBJECTIVE: Pseudomonas aeruginosa, an opportunistic pathogen, can cause serious health problems and produces several virulence factors. The most important of these factors is biofilm. Many studies suggest administration of new generation of antibiotics, as P. aeruginosa biofilm has developed high resistance to antimicrobial drugs. Emergence of multidrug resistant (MDR) strains has resulted in screening biofilm inhibitors from natural products or modified from natural compounds. To test this hypothesis, we evaluated the inhibitory effects (antibacterial and antibiofilm) of Juglans regia L. extract on biofilm formation by clinical isolates of P. aeruginosa. METHODS: Samples collected from burn, tracheal and urine infections of hospitalized patients (Shahid Motahari Hospital, Tehran, Iran) were identified as P. aeruginosa using traditional biochemical tests. Antibiotic susceptibility testing of isolates was performed using disk diffusion method. The microtiter plate method was used to evaluate the ability of pathogenic strains in producing biofilm. Antibacterial and antibiofilm effects of aqueous and methanol Juglans regia L. leaf extracts were determined by microtiter plate method. RESULTS: 46.7% of P. aeruginosa isolates (n = 50) were resistant to gentamicin and 100% of them could form a biofilm. All isolates (100%) exhibited MDR phenotype. Various concentrations of Juglans regia L. extracts exhibited significant effects on the growth and biofilm inhibition of the isolates. In addition, aqueous Juglans regia L. leaf extract had better inhibition activity on planktonic growth, and methanol extract was more effective on inhibiting biofilm of P. aeruginosa. CONCLUSIONS: The results of this study indicate that antibiotic-resistant strains were significantly associated with biofilm formation. The J. regia L. extract, at various concentrations, may provide an alternative to control biofilm-related infections caused by P. aeruginosa. Further analyses are needed to validate the antibiofilm activity of these medicinal plants.

Expression and purification of recombinant HTLV-I/-II linear epitopes antigen and its application for screening of suspected patients.

BACKGROUND AND OBJECTIVES: The linear epitopes of gp46-I, gp46-II, gp21 and p19 are used in diagnosis of HTLV-I/-II infections. The aims of this study was to obtain high-level expression and purification of recombinant antigen (RA) containing these epitopes. Large-scale preparation of such antigen probably worths for diagnostic purpose. MATERIALS AND METHODS: The synthetic DNA encoding RA was synthesized and over-expressed as soluble in Escherichia coli BL21 (DE3) cells. Expression and distribution of the His-GST-RA protein were evaluated using SDS-PAGE. The soluble RA was purified utilizing Ni-NTA agarose beads under native conditions and was concentrated by ultra filtration. Using 20 sera specimens from HTLV infected patients, the antigenicity of the purified protein was confirmed in ELISA and western blotting analysis. RESULTS: SDS-PAGE revealed that the purified protein was more than 90% pure. The final yield was approximately 25 mg per liter of culture medium. ELISA results showed that RA could specifically bind to anti-HTLV-I/-II antibodies in infected sera. CONCLUSION: RA could be a candidate for HTLV-I/-II screening and the strategy presented in this study could be used for easy production of this diagnostic protein.

Elevated prevalence of multidrug-resistant Acinetobacter baumannii with extensive genetic diversity in the largest burn centre of northeast Iran.

OBJECTIVES: The emergence and spread of multidrug-resistant (MDR) Acinetobacter baumannii isolates is now frequently associated with nosocomial infections. The aim of this study was to evaluate the genetic relatedness and patterns of antimicrobial resistance amongst A. baumannii isolated from a burn centre at a teaching hospital in Iran. METHODS: A total of 54 A. baumannii isolates were collected from burn wound infections of hospitalised patients. Antimicrobial susceptibility of the isolates was determined, and genotyping analysis was performed by repetitive extragenic palindromic PCR (rep-PCR). PCR assay was performed to investigate the distribution of ß-lactamase, aminoglycoside-modifying enzyme and efflux pump genes. RESULTS: Etest results revealed that the most active antimicrobial agent was colistin (100% susceptibility), followed by tigecycline (96.3%). The blaOXA-51 and blaADC genes were detected in all of the isolates, but blaOXA-58-like was not detected. The prevalence of blaTEM, blaIMP, blaVIM, blaOXA-23-like and blaOXA-24-like genes was 64.8%, 70.4%, 70.4%, 66.7% and 68.5%, respectively. ISAba1 was detected upstream of blaOXA-23-like and blaADC in 66.7% and 77.8% of isolates, respectively. This study showed a high level of distribution of adeB (72.2%), aphA6 (81.5%), aacC1 (85.2%), aadA1 (59.3%), aadB (31.5%), tetB (70.4%) and aphA1 (29.6%) in A. baumannii strains. Based on rep-PCR analysis, four clusters (I-IV) were defined. CONCLUSIONS: The elevated prevalence of MDR A. baumannii strains in this burn centre suggests that local antibiotic prescription policies should be precisely revised. Moreover, strict infection control procedures to prevent further dissemination need to be prioritised immediately.

Isolation and drug-resistant patterns of Campylobacter strains cultured from diarrheic children in Tehran.

To detect campylobacteriosis and determine the drug susceptibility of causative organisms, we acquired 500 diarrheic samples in Cary-Blair transfer medium from two pediatric hospitals in Tehran between October 2004 and October 2005. The samples were also enriched in Preston broth (with supplements) and defibrinated sheep blood (7%). They were plated from both media on Brucella agar containing antibiotics and blood. Isolates were identified through biochemical tests and by the polymerase chain reaction method. Drug susceptibility testing was performed using the disk diffusion method. In total, 40 Campylobacter strains were isolated (8%). C. jejuni was the dominant species (85.8%) followed by C. coli (14.2%). The rates of resistance to antimicrobial agents were as follows: ciprofloxacin (61.7%), ceftazidime (47%), carbenicillin (35%), tetracycline (20.5%), cefotaxime (14.7%), ampicillin (11.7%), neomycin erythromycin and chloramphenicol (2.9%), gentamicin, streptomycin, imipenem and colistin (0.0%). Campylobacter is an important cause of diarrhea among Iranian children. The detection of Campylobacter increases by 25% if samples are treated in enrichment broth prior to plating. The high rate of resistance to ciprofloxacin is alarming, and further investigation into the possible reasons for this is imperative.