Erythromycin Releasing PVA/sucrose and PVA/honey Hydrogels as Wound Dressings with Antibacterial Activity and Enhanced Bio-adhesion.

The present study deals with preparation and characterization of thermally crosslinked PVA-based hydrogels containing honey and sucrose for the purpose of erythromycin delivery. The hydrogels have been characterized and compared by scanning electron microscopy, Fourier transform infrared spectroscopy, and bio-adhesion tests. Swelling measurements showed that addition of sucrose and honey decreased the equilibrium swelling of the hydrogels. Results of release studies showed that the amount of erythromycin, released at the early hours was higher for PVA/sucrose and PVA/honey hydrogels compared to PVA hydrogel while the drug released at later times was highly reduced for PVA/honey hydrogel. Both Peppas-Sahlin and Korsmeyer-Peppas models fitted well to the release data. Fitting Peppas-Sahlin model to the release data showed that at the initial times, release of drug from the hydrogel network was mainly governed by Fickian mechanism; however, at later times the drug is dominantly released by relaxational mechanism due to swelling of the network,. Addition of honey improved the bio-adhesion of PVA/honey hydrogel as compared to PVA/sucrose and pure PVA hydrogel. Results of antibacterial tests showed growth inhibitory action of erythromycin-loaded PVA hydrogels against Pseudomonas aeruginosa and Staphylococcus aureus bacteria. This study indicates that these hybrid hydrogels are capable of being used as functional wound dressings aiming to control the rate of antibiotic delivery to the wound site and prevent the wounds from infection.

Pan drug-resistant Acinetobacter baumannii causing nosocomial infections among burnt children.

Background: Nosocomial infection caused by Acinetobacter baumannii has emerged as a world-wide serious problem in the emergence of multidrug-resistant (MDR). Infections caused by antibiotic-resistant strains of A. baumannii cannot be completely eliminated among the infected patients. This study aimed to monitor antibiotic resistance among A. baumannii strains isolated from burnt children. Methods: After performing biochemical identification tests on 115 isolates, 62 were detected as A. baumannii . Minimum inhibitory concentration (MIC) was used to test susceptibility to colistin, and disk agar diffusion was used for the susceptibility of the isolates to the antibiotics Ciprofloxacin, Amikacin, Gentamicin, Cefepime, Meropenem, Imipenem, Ceftazidime, Levofloxacin and Piperacillin/Tazobactam. Bacterial species were isolated and identified as multidrug-resistant (MDR), extensively drug-resistant (XDR) and pan drug-resistant (PDR), based on the susceptibility patterns to elected antibiotics, deputing different classes of antimicrobial. Results: The antibiotic susceptibility pattern out of a total of 62 bacterial strains used in this study. Thirty-six (58%) strains were categorized as MDR, 17 (27.5%) as XDR, and nine (14.5%) as PDR. Conclusion: To reduce the threat of antimicrobial resistance, MDR, XDR and PDR A. baumannii strains must be evaluated by all clinical microbiology laboratories.

Apoptotic Effects of Linum album Extracts on AGS Human Gastric Adenocarcinoma Cells and ZNF703 Oncogene Expression

Introduction: Linum album is a medicinal plant endemic in Iran that is very important pharmaceutically. The present study concerns the effect of different extracts of L. album on ZNF703 gene expression and apoptosis in human gastric carcinoma AGS cells. Method and material: Hydro alchoholic L. album extracts from various plant sources were produced by Maceration. AGS cells were treated with different concentrations (200, 400, 600, 800 and 1000 µg/ml) and the cytotoxicity potency was assessed after 24 h by MTT assay. Then, quantitative real time PCR was conducted for ZNF703 gene expression in AGS cells. Also, cell apoptosis/necrosis was assessed with the aid of Annexin V/PI staining and quantification by flow cytometry. Results: L. album extracts exerted dose-dependent toxicity in the AGS cell line. The mRNA levels of ZNF703 gene expression were significantly decreased with rhizome, fruit at fruiting, leaf and stem at anthesis (P<0.001), and leaf and stem at fruiting extracts as compared to the controls (P<0.01). Also, the number of apoptotic cells was increased from 2.70% (statistically significant; p<0.05) in untreated AGS cells to 44%, following treatment with the leaf and stem at anthesis example. Discussion: Our findings revealed that the L. album extracts can induce apoptosis and might modulate cytotoxicity by down regulating ZNF703 gene expression in AGS cells. Therefore, this extract could be a good candidate for inhibiting cancer cell growth, especially that of gastric cancer. In addition, ZNF703 may have potential as a therapeutic target.

Evaluation the cytotoxic effect of cytotoxin-producing Klebsiella oxytoca isolates on the HEp-2 cell line by MTT assay.

BACKGROUND: The cytotoxic effects on epithelial cells of the human are not observed in other strains of Klebsiella spp and are only observed in K. oxytoca strains. MTT assay was used to evaluate cytotoxic activity. In this study, colorimetric method was used to evaluate the cytotoxic effect of cytotoxin-producing isolates on Hep-2 cell line and determines the percentage of surviving cells. MATERIALS AND METHODS: In this study, we collected a total of 75 K. oxytoca strains isolate and we detected the production of toxins and their cytotoxic effects on HEp-2 cells. Colorimetric method such as MTT assay was used to evaluate the cytotoxic effect of cytotoxin-producing isolates on Hep-2 cell line and determines the percentage of surviving cells. RESULTS: Nine isolates had cytotoxic effects on HEp-2 cells. The results of MTT assay showed that the isolated strains were different from the control stain in terms of toxinogenicity and cytotoxic effects on HEp-2 cells at the studied dilutions (1:3, 1:6, 1:12, 1:24, 1:48, and 1:96). CONCLUSIONS: In the current study, Percentage of Hep-2 surviving cells exposed to 1:3, 1:6, 1:12, 1:24, 1:48, and 1:96 supernatant dilutions of cytotoxin-producing Klebsiella oxytoca isolates was different.

Identification of cytotoxin-producing Klebsiella oxytoca strains isolated from clinical samples with cell culture assays.

BACKGROUND: Klebsiella oxytoca is an opportunistic pathogen which damages intestinal epithelium through producing cytotoxin tilivalline. This toxin plays a role in the pathogenesis of bacteria and is the main virulence factor which leads to antibiotic-associated hemorrhagic colitis progress. MATERIALS AND METHODS: In this study, we collected a total of 75 K. oxytoca strains isolated from the stool, urine, blood, wounds, and sputum and evaluated them in terms of the production of toxins; we detected their cytotoxic effects on HEp-2 cells. RESULTS: Of all the isolates, five K. oxytoca strains isolated from the stool cultures, two strains isolated from the blood cultures, one strains isolated from the wound cultures, and one strains isolated from the urine cultures had cytotoxic effects on HEp-2 cells. The strains isolated from sputum cultures had no cytotoxic effects on HEp-2 cells. CONCLUSIONS: In the current study, the majority of strains isolated from the stool of the patients included cytopathic effects on HEp-2 cells.

Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines.

BACKGROUND: About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. METHODS: For the construction of pET-21a/ entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/ entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/µL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively. RESULTS: Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/µL and 1000 ng/µL ( P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon γ and tumor necrosis factor α compared to control. CONCLUSION: According to our results, the biological activity of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A is identical; therefore, these procedures may be approved as an efficient method to express and purify this protein in a large scale.

Identification of Klebsiella pneumoniae Carbapenemase-producing Klebsiella oxytoca in Clinical Isolates in Tehran Hospitals, Iran by Chromogenic Medium and Molecular Methods.

OBJECTIVES: Production of carbapenemase, especially Klebsiella pneumoniae carbapenemases (KPC), is one of the antibiotic resistance mechanisms of Enterobacteriaceae such as Klebsiella oxytoca. This study aimed to investigate and identify KPC-producing K. oxytoca isolates using molecular and phenotypic methods. METHODS: A total of 75 isolates of K. oxytoca were isolated from various clinical samples, and were verified as K. oxytoca after performing standard microbiological tests and using a polymerase chain reaction (PCR) method. An antibiotic susceptibility test was performed using a disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. CHROMagar KPC chromogenic culture media was used to examine and confirm the production of the carbapenemase enzyme in K. oxytoca isolates; in addition, PCR was used to evaluate the presence of blaKPC gene in K. oxytoca strains. RESULTS: Of a total of 75 K. oxytoca isolates, one multidrug resistant strain was isolated from the urine of a hospitalized woman. This strain was examined to assess its ability to produce carbapenemase enzyme; it produced a colony with a blue metallic color on the CHROMagar KPC chromogenic culture media. In addition, the blaKPC gene was confirmed by PCR. After sequencing, it was confirmed and deposited in GenBank. CONCLUSION: To date, many cases of KPC-producing Enterobacteriaceae, in particular K. pneumoniae, have been reported in different countries; there are also some reports on the identification of KPC-producing K. oxytoca. Therefore, to prevent the outbreak of nosocomial infections, the early detection, control, and prevention of the spread of these strains are of great importance.

Rising Cellular Immune Response after Injection of pVax/iutA: A Genetic DNA Cassette as Candidate Vaccine against Urinary Tract Infection.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are major bacterial agent of Urinary Tract Infection (UTI). This infection is more prevalent among women because approximately half of all women will experience a UTI in their life-time and near a quarter of them will have a recurrent infection within 6-12 months. IutA protein has a major role during UPEC pathogenesis and consequently infection. Therefore, the aim of current study was assessment of IutA protein roles as a potential candidate antigen based for vaccine designing. METHODS: This survey was conducted during 2014-2015 at the University of Tehran, Iran. Chromosomal DNA extracted from E. coli 35218 and iutA gene amplified by PCR. The amplicon cloned to pVax.1 eukaryotic expression vector and recombinant vector confirmed by sequencing. The iutA gene expression in genetic cassette of pVax/iutA was evaluated in COS7 cell line by RT-PCR. Then, injected to mouse model, which divided to three groups: injected with pVax vector, PBS and pVax/iutA cassette respectively in two stages (d 1 and 14). One week after the second injection, bleeding from immunized mouse was performed and IFN-gamma was measured. RESULTS: The mice immunized with pVax/iutA showed increased interferon-γ responses significantly higher than two non-immunized groups (P<0.05). CONCLUSION: Cellular immune response has a main protective role against UTI. Raising this kind of immune response is important to preventing of recurrent infection. Moreover, the current DNA cassette will be valuable for more trying to prepare a new vaccine against UTI.

A systems biology approach to reconcile metabolic network models with application to Synechocystis sp. PCC 6803 for biofuel production.

Production of biofuels has been one of the promising efforts in biotechnology in the past few decades. The perspective of these efforts can be reduction of increasing demands for fossil fuels and consequently reducing environmental pollution. Nonetheless, most previous approaches did not succeed in obviating many big challenges in this way. In recent years systems biology with the help of microorganisms has been trying to overcome these challenges. Unicellular cyanobacteria are widespread phototrophic microorganisms that have capabilities such as consuming solar energy and atmospheric carbon dioxide for growth and thus can be a suitable chassis for the production of valuable organic materials such as biofuels. For the ultimate use of metabolic potential of cyanobacteria, it is necessary to understand the reactions that are taking place inside the metabolic network of these microorganisms. In this study, we developed a Java tool to reconstruct an integrated metabolic network of a cyanobacterium (Synechocystis sp. PCC 6803). We merged three existing reconstructed metabolic networks of this microorganism. Then, after modeling for biofuel production, the results from flux balance analysis (FBA) disclosed an increased yield in biofuel production for ethanol, isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and propanol. The numbers of blocked reactions were also decreased for 2-methyl-1-butanol production. In addition, coverage of the metabolic network in terms of the number of metabolites and reactions was increased in the new obtained model.

Association of the Exotoxin A and Exoenzyme S with Antimicrobial Resistance in Pseudomonas Aeruginosa Strains.

INTRODUCTION: Pseudomonas aeruginosa is a gram-negative and opportunistic bacterium that causes opportunistic infections in immunosuppressed patients. The main purpose of this study was to investigate the association between exotoxin A and exoenzyme S production with antibiotic resistance patterns. METHODS: The toxA and exoS genes were detected in 102 clinical isolates by PCR. Antibiotic susceptibility tests were performed by disk diffusion method (Kirby Bauer). The Chi-square and Fisher's test were used for evaluation of the association between "toxins and infections source" and "toxins and antibiotic resistance" respectively. RESULTS: Frequency of toxA+ and exoS+ strains was 81% and 61%, respectively. The association between drug resistance and toxA+ genotype was significant for all antibiotics tested (P < 0.05) except aztreonam; however, no significant association was observed between drug resistance and exoS gene (P > 0.05). CONCLUSIONS: High frequency of toxA+ resistant strains isolated from inpatients and significant association between the toxin and drug resistance in more antibiotics, reinforces possible role of exotoxin A as an extracellular protein in the regulation of drug resistance genes. The results may be further verified by Southern blot analysis of toxA and exoS gene expression and elucidation of the mechanism of antibiotic resistance.

Antibiotic Resistance Patterns and a Survey of Metallo-ß-Lactamase Genes Including bla-IMP and bla-VIM Types in Acinetobacter baumannii Isolated from Hospital Patients in Tehran.

BACKGROUND: Metallo-ß-lactamases (MBLs) producing strains of Acinetobacter baumannii are serious etiological agents of hospital infections worldwide. Among the ß- lactams, carbapenems are the most effective antibiotics used against A. baumannii. However, resistance to these drugs among clinical strains of A. baumannii has been increasing in recent years. In this study, the antimicrobial sensitivity patterns of A. baumannii strains isolated from eleven different hospitals in Tehran, Iran, and the prevalence of MBL genes (bla-VIM and bla-IMP) were determined. METHOD: During a period of 5 months, 176 isolates of A. baumannii were collected from different clinical specimens from hospitalized patients in Tehran. All isolates were confirmed by biochemical methods. The isolates were tested for antibiotic sensitivity by the Kirby-Bauer disk diffusion method. Following minimum inhibitory concentration determination, imipenem-resistant isolates were further tested for MBL production by the double disk synergy test (DDST) method. PCR assays were performed for the detection of the MBL genes bla-IMP and bla-VIM. RESULTS: The DDST phenotypic method indicated that among the 169 imipenem-resistant isolates, 165 strains were MBL positive. The PCR assays revealed that 63 of the overall isolates (36%) carried the bla-VIM gene and 70 strains (40%) harbored bla-IMP. CONCLUSIONS: It is obvious that nosocomial infections associated with multidrug-resistant Acinetobacter spp. are on the rise. Therefore, the determination of antibiotic sensitivity patterns and screening for MBL production among A. baumannii isolates is important for controlling clinical Acinetobacter infections.

Expression and Purification of the Uropathogenic Escherichia coli PapG Protein and its Surface Absorption on Lactobacillus reuteri: Implications for Surface Display System Vaccines.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is one of the most common bacteria that can cause urinary tract infections (UTIs). Unfortunately, no human vaccine against UTIs has been developed. Therefore, it is necessary to develop an efficient and safe vaccine that is able to induce mucosal and systemic immune responses. The use of lactic acid bacteria as a delivery system is a promising method to induce the immune system. OBJECTIVES: The aim of this study was to establish Lactobacillus reuteri harboring the E. coli PapG antigen on its surface. MATERIALS AND METHODS: In this study, the gene encoding PapG was fused to the AcmA gene (which encodes an anchor protein in Lactobacillus) and cloned into the pEX A vector. The PapG.AcmA fusion gene was digested with BamHI and NdeI and sub-cloned into the pET21a expression vector at the digestion sites. Subsequently, the recombinant plasmids (pET21a-PapG.AcmA and pET21a-PapG) were transformed into the E. coli Origami strain using the calcium chloride method and the fusion protein was expressed under 1 mM IPTG induction. The expression of the fusion protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Purification of the PapG and PapG.AcmA proteins was carried out using a Ni-NTA column, and surface adsorption was estimated on Lactobacillus. Finally, surface localization of the fusion protein was verified by an enzyme-linked immunosorbent assay (ELISA). RESULTS: The PapG.AcmA fusion was successfully sub-cloned in the pET21a expression vector. The expression of PapG and PapG.AcmA proteins in the E. coli Origami strain was indicated as protein bands in SDS-PAGE and confirmed by western blotting. In addition, the fusion protein was displayed on the surface of L. reuteri. CONCLUSIONS: In conclusion, we developed a method to express the PapG.AcmA protein on the surface of Lactobacillus. This is the first report on the successful application of lactic acid bacteria displaying the PapG.AcmA fusion protein. It will be interesting to determine the immune responses against the PapG protein in near future using this surface display strategy.

Genome-wide analysis of alternative splicing events in Hordeum vulgare: Highlighting retention of intron-based splicing and its possible function through network analysis.

In this study, using homology mapping of assembled expressed sequence tags against the genomic data, we identified alternative splicing events in barley. Results demonstrated that intron retention is frequently associated with specific abiotic stresses. Network analysis resulted in discovery of some specific sub-networks between miRNAs and transcription factors in genes with high number of alternative splicing, such as cross talk between SPL2, SPL10 and SPL11 regulated by miR156 and miR157 families. To confirm the alternative splicing events, elongation factor protein (MLOC_3412) was selected followed by experimental verification of the predicted splice variants by Semi quantitative Reverse Transcription PCR (qRT-PCR). Our novel integrative approach opens a new avenue for functional annotation of alternative splicing through regulatory-based network discovery.

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine.

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-γ and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

Antibiotic Resistance Pattern and Evaluation of Metallo-Beta Lactamase Genes Including bla- IMP and bla- VIM Types in Pseudomonas aeruginosa Isolated from Patients in Tehran Hospitals.

Beta-lactamase producing strains of Pseudomonas aeruginosa are important etiological agents of hospital infections. Carbapenems are among the most effective antibiotics used against Pseudomonas infections, but they can be rendered infective by group B ß -lactamase, commonly called metallo-beta lactamase. In this study, the antimicrobial sensitivity patterns of P. aeruginosa strains isolated from 9 different hospitals in Tehran, Iran, as well as the prevalence of MBLs genes (bla- VIM and bla- IMP ) were determined. A total of 212 strains of P. aeruginosa recovered from patients in hospitals in Tehran were confirmed by both biochemical methods and PCR. Their antimicrobial sensitivity patterns were determined by Kirby-Bauer disk diffusion method. Following MIC determination, imipenem resistant strains were selected by DDST method which was followed by PCR tests for determination of MBLs genes: bla- IMP and bla- VIM . The results indicated that, in the DDST phenotypic method, among the 100 imipenem resistant isolates, 75 strains were MBLs positive. The PCR test indicated that 70 strains (33%) carried bla- VIM gene and 20 strains (9%) harbored bla- IMP . The results indicated that the extent of antibiotic resistance among Pseudomonas aeruginosa is on the rise. This may be due to production of MBLs enzymes. Therefore, determination of antibiotic sensitivity patterns and MBLs production by these bacteria, can be important in control of clinical Pseudomonas infection.