Investigation of a novel bilayered PCL/PVA electrospun nanofiber incorporated Chitosan-LL37 and Chitosan-VEGF nanoparticles as an advanced antibacterial cell growth-promoting wound dressing.

Chronic wounds have become a growing concern as they can have a profound impact on individuals, potentially resulting in mortality. It is crucial to prevent and manage bacterial infections, particularly drug-resistant ones. Antimicrobial peptides, such as LL-37, can firmly eliminate pathogens. Additionally, the process of angiogenesis, facilitated by growth factors like VEGF, is essential for tissue repair and wound healing. To enhance the stability and bioavailability of therapeutic agents, targeted delivery strategies utilizing Chitosan-based carriers have been employed. Electrospun biopolymers in advanced wound dressings have revolutionized wound care by providing a more effective and efficient solution for promoting tissue regeneration and speeding up the healing process. The present investigation utilized Chitosan nanoparticles to encapsulate the recombinant LL37 peptide and VEGF. An in-depth investigation was carried out to analyze the biophysical and morphological traits of the LL37-CSNPs and VEGF-CSNPs. The first support layer consisted of PCL electrospun nanofiber, followed by the electrospinning of PVA/CsLL37, PVA/CsVEGF, and PVA/CsLL37/CsVEGF onto the PCL layer. An in vitro examination assessed the fabricated nanofibers' morphological, mechanical, and biological characteristics. The antimicrobial effects were tested on methicillin-resistant Staphylococcus aureus (MRSA). The in vivo experiments assessed the antibacterial and wound-healing capabilities of the nanofibers. The findings validated the continuous release of LL37 and VEGF. The composite material PCL/PVA/CsLL37/CsVEGF demonstrated potent bactericidal and antioxidant characteristics. The cytotoxic assay demonstrated the biocompatibility of the fabricated nano mats and their potential to accelerate fibroblast cell proliferation. The efficacy of PVA/CsLL37/CsVEGF in promoting wound healing was confirmed through an in vivo wound healing assay. Furthermore, the histological analysis provided evidence of faster epidermal formation and improved antibacterial activity in wounds covered with PVA/CsLL37/CsVEGF. Adding LL37 and VEGF to the composite material improves the immune response and promotes blood vessel formation, accelerating wound healing and decreasing inflammation.

Diabetic wound healing function of PCL/cellulose acetate nanofiber engineered with chitosan/cerium oxide nanoparticles.

The use of cerium oxide nanoparticles (CeO2NPs) in diabetic wound repair substances has shown promising results. Therefore, the study was conducted to introduce a novel nano-based wound dressing containing chitosan nanoparticles encapsulated with green synthesized cerium oxide nanoparticles using Thymus vulgaris extract (CeO2-CSNPs). The physical properties and structure of the nanoparticles were analyzed using XRD, DLS, FESEM and FTIR techniques. The electrospun PCL/cellulose acetate-based nanofiber was prepared and CeO2-CSNPs were integrated on the PCL/CA membrane by electrospraying. The physicochemical properties, morphology and biological characteristics of the electrospun nanocomposite were evaluated. The results showed that the nanocomposite with 0.1 % CeO2-CSNPs exhibited high antibacterial performance against S. aureus (<58.59 µg/mL). The PCL/CA/CeO2-CSNPs nanofiber showed significant antioxidant activity up to 89.59 %, cell viability improvement, and cell migration promotion up to 90.3 % after 48 h. The in vivo diabetic wound healing experiment revealed that PCL/CA/CeO2-CSNPs nanofibers can significantly increase the repair rate of diabetic wounds by up to 95.47 % after 15 days. The results of this research suggest that PCL/CA nanofiber mats functionalized with CeO2-CSNPs have the potential to be highly effective in treating diabetes-related wounds.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD.

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

In vivo efficiency of the produced recombinant lysostaphin antimicrobial peptide in treatment of methicillin-resistant Staphylococcus aureus (MRSA) skin infection in a mouse model.

Background and Objectives: Staphylococcus simulans secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell wall. Therefore, this unique property indicates the high ability of lysostaphin in the treatment of staphylococcal infections and is considered as an anti-staphylococcal agent. Materials and Methods: Escherichia coli BL21 (DE3) competent cells were transformed with pET32a-lysostaphin clone and induced by isopropyl-ß-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant lysostaphin -A-based ointment was used for external wound healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: Our results showed the recombinant protein was produced exactly. The results of checkerboard tests showed MIC, MBC and antibacterial activity test an acute reduction of cell viability during the use of lysostaphin, and SEM results approved the intense wrecking effects of lysostaphin in combination on bacterial cells. Macroscopic findings and microscopic data showed that the recombinant lysostaphin ointment was effective on excisional wound healing. Conclusion: Our findings proved that the recombinant lysostaphin ointment was effective on wound healing due to Staphylococcus aureus infection.

Production of mutant streptavidin protein and investigation of its effect on the performance of streptavidin.

Objectives: Streptavidin is a versatile protein in cell science. The tetramer structure of streptavidin plays a key role in this binding, but this form interferes with some assays. If monomer streptavidin is still capable of binding to biotin, it can overcome the limitations of the streptavidin application. So, we examined the elimination of tryptophan 120 and its effect on the function of streptavidin. Materials and Methods: Mutant streptavidin gene was synthesized in a pBSK vector. Then it was ligated to the pET32α vector. This vector is expressed in Escherichia coli BL21 (DE3) pLysS host. After purification and refolding of the recombinant protein, its structure was analyzed on the SDS_PAGE gel. Recombinant streptavidin binding affinity to biotin was evaluated by spectrophotometric and HABA color compound. Results: Mutant streptavidin gene was successfully expressed in E. coli BL21 (DE3) pLysS host and the purified protein was observed as a single band in the 36 kDa area. The best condition for dialysis was PBS buffer+arginine. The molar ratio of biotin/protein of mutant streptavidin was not only near but also more than standard protein. Mutant streptavidin remained in the monomeric state in the presence or absence of biotin. Conclusion: Results of this study showed that 120 tryptophan is one of the most important factors in tetramer streptavidin formation and its deletion produces the monomer form that has a high binding affinity to biotin. This mutant form of streptavidin can therefore be used in studies requiring monovalent binding as well as in studies facing limitations due to the size of streptavidin tetramer.

Wound healing and antibacterial capability of electrospun polyurethane nanofibers incorporating Calendula officinalis and Propolis extracts.

In order to produce a healing accelerator antibacterial wound dressing, different electrospun polyurethane (PU)-based nanofibers inclusive Calendula officinalis and Propolis ethanolic extracts were fabricated. The measurement of minimum inhibitory concentrations (MIC) against Methicillin-resistant Staphylococcus aureus (MRSA) determined the concentrations of incorporating extracts. Then the morphological properties of the produced polyurethane (PU), polyurethane/C. officinalis (PU/CO), polyurethane/Propolis (PU/PR), polyurethane/C. officinalis/Propolis (PU/CO/PR) were analyzed by scanning electron microscopy (SEM). The physicochemical features and biological characteristics of the fabricated nanofibers were evaluated. Subsequently, the antibacterial and wound-healing efficiency of electrospun wound dressings were tested under in vivo situation. The electrospun PU/CO/PR nanofiber illustrated the most degree of antibacterial, antioxidant, and cell proliferation efficiencies. In vivo examination and histological analysis confirmed significant improvement in the complete, well-organized wound-healing process in MRSA-infected wounds treated with PU/CO/PR. These outcomes described PU/CO/PR electrospun nanofibers as a wound dressing that can significantly facilitate wound healing with notable antibacterial, antioxidant, and cell proliferation properties.

The LL-37 Antimicrobial Peptide as a Treatment for Systematic Infection of Acinetobacter baumannii in a Mouse Model.

BACKGROUND: The antimicrobial peptides (AMPs) played a critical role in the innate immunity of the host and are considered natural sources illustrating a broad-spectrum antimicrobial activity with high specificity and low cytotoxicity. AMPs generally possess a net positive charge and have amphipathic structures. Thus, AMPs can bind and interact with negatively charged bacterial cell membranes, leading to destructive defects in biomembranes and ending in cell death. LL37 is the only human cathelicidin-derived antimicrobial peptide that shows a broad spectrum of antimicrobial activity. MATERIALS AND METHODS: To determine the antibacterial efficiency of LL37 in a mouse model of systemic A. baumannii infection, LL37 corresponding gene was expressed in E. coli, purification and refolding situations were optimized. The antimicrobial performance of produced LL-37 against A. baumannii was evaluated in vitro via MIC and Time Kill assays, and its destructive effects on the bacterial cell were confirmed by SEM image. RESULTS: The recombinant LL37 showed strong antibacterial function against A. baumannii at 1.5 µg/mL concentration. Time kill assay showed a sharp reduction of cell viability during the first period of exposure, and complete cell death was recorded after 40 min exposure. CONCLUSION: Furthermore, in vivo results represented a significant ability of LL37 in the treatment of systematic infected mouse models, and all infected mice receiving LL37 protein survived without no trace of bacteria in their blood samples.

Wound healing performance of PVA/PCL based electrospun nanofiber incorporated green synthetized CuNPs and Quercus infectoria extracts.

In this study, copper nanoparticles (CuNPs) were synthetized through green chemistry approach using C. officinalis flowers extract. The biosynthetized nanoparticles were characterized by FESEM, XRD, DLS and FTIR analysis. Subsequently, PCL nanofiber was fabricated as first supportive layer by electrospinning method. Afterward, PVA/Quercus infectoria galls (QLG) extracts/biosynthetized CuNPs blending solution was electrospinned as second bioactive topical layer. The morphology, physicochemical properties and biological characteristics of the produced PCL, PCL/PVA, PCL/PVA/CuNPs, PCL/PVA/QLG and PCL/PVA/QLG/CuNPs were investigated. Eventually, in vivo wound healing effectiveness was examined. Histologic investigation was carried out for visualization of the healing wounds architecture in different treated groups. FESEM, XRD and DLS assays confirmed the successful synthesis of CuNPs in range of 40-70 nm and FTIR spectrum approve the presence of functional constituents of C. officinalis extract on synthesized CuNPs. The incorporation of CuNPs and QLG extract into PCL/PVA based nanofibers improved their biological capabilities and physicochemical properties. Furthermore, PCL/PVA/QLG/CuNPs illustrated significant wound healing potentials and excellent antibacterial function against at wounds infected with MRSA. Histological assay demonstrated complete wound healing and less inflammation on day 10th. These outcomes recommended the utilization of PCL/PVA/QLG/CuNPs as a novel promising wound dressings with considerable antibacterial features.

Ib-AMP4 antimicrobial peptide as a treatment for skin and systematic infection of methicillin-resistant Staphylococcus aureus (MRSA).

Objectives: Antimicrobial peptide compounds (AMPs) play important roles in the immune system. They also exhibit significant anti-tumor and antibacterial properties. Most AMPs are cationic and are able to bind bacterial cell membranes through electrostatic affinity. Ib-AMP4 is a plant-derived AMP that exerts rapid bactericidal functions. In the present study, the antibacterial efficiency of the produced recombinant Ib-AMP4 in elimination of Methicillin-resistant Staphylococcus aureus (MRSA) bacterial infection, was investigated under in vitro and in vivo situations. Materials and Methods: The synthesized Escherichia coli codon-optimized gene sequences of the Ib-AMP4 were expressed in E. coli BL21 (DE3) pLysS. The recombinant Ib-AMP4 was purified and refolding conditions were optimized. The antibacterial efficiency of the refolded peptide against MRSA was tested under in vivo and in vitro situations for treatment of skin and systematic infection of MRSA in a mouse model. Results: Antibacterial assays confirmed the antibacterial function of Ib-AMP4 against MRSA. SEM results proved the destructive effects of applying Ib-AMP4 on MRSA biomembrane. Time-kill curve and growth kinetic assay illustrated rapid antibacterial activity of the produced Ib-AMP4. Moreover, Ib-AMP4 showed significant infection treatment ability in a mouse model and all infected mice receiving Ib-AMP4 protein survived and there was no trace of bacteria in their blood samples. Conclusion: The results confirmed the rapid antibacterial potential of the produced recombinant Ib-AMP4 to be used for efficient treatment of MRSA infection.

Cloning, expression and purification of antigenic fragments of the Ureaplasma urealyticum UreD protein and its value in serology.

Background and Objectives: The detection of Ureaplasma urealyticum is usually done through culture. With the change of the smallest effective factor in culture, we face the lack of growth of these bacteria, which is one of the important reasons to find a suitable alternative for the diagnosis of this bacterium. UreD is a protected gene in this bacterium. The aim of this was to evaluate the ability of antigenic regions of UreD protein to bind to patients' serum antibodies. Materials and Methods: Antigenic regions of UreD protein were predicted using IEDB software with five different methods: Emini Surface Accessibility Prediction, Kolaskar and Tongaonkar Antigenicity, Chou and Fasman beta turn prediction, Karplus and Schulz flexibility scale, Ellipro-Epitope prediction based upon structural protrusion. Antigenic regions of UreD gene was clonned, expressed and purified. The antigenicity of this recombinant protein against the antibodies in the serum of people infected with U. urealyticum infections was checked in western blotting. Results: The results showed that the antigenic regions of the UreD protein was producted and its antigenicity was demonstrated in western blotting. Moreover, all sera from patients infected with U. urealyticum reacted to the recombinant antigen. Conclusion: Specimens from people infected with U. urealyticum infection was positive in Western blotting suggesting that UreD protein has antigenic properties. Therefore, it can be used as a suitable candidate for the design of diagnostic kits and U. urealyticum vaccine.

Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Lactococcus-based vaccine against brucellosis: IgG immune response in mice with rOmp16-IL2 fusion protein.

This study was designed to introduce the recombinant Lactococcus lactis MG1363 as a cell factory candidate for production of recombinant Brucella melitensis Omp16-Human IL2 (r-Omp16-IL2) and to suggest it as a promising safe, non-pathogenic mucosal live vaccine against brucellosis. Three groups of BALB/c mice (10 mice per group) were intragastrically administrated with phosphate-buffered saline (PBS), L. lactis harboring the empty pAMJ2008 plasmid and with L. lactis expressing rOmp-IL2. The first two groups were classified as control groups and the third one is indicated as treatment group. Another group was injected by the intraperitoneal (i.p.) route with purified rOmp16-IL2 protein. The total serum IgG of each group was assessed with indirect ELISAs at two days before immunization and also two weeks after the last immunization. Results showed that BALB/c mice intragastrically administrated with L. lactis expressing rOmp-IL2 had dominant IgG response compared to the control (PBS administrated) group (P < 0.05). The level of IgG was significantly increased by intraperitoneally injection of recombinant Omp-IL2 in adjuvant compared to the intragastrically administration of PBS and L. lactis/pAMJ2008 as control groups, and also compared to L. lactis/pAMJ2008-rOmp-IL2 (P < 0.05). Our findings provide the use of L. lactis rOmp16-IL2 as a new promising alternative safe strategy than presently live attenuated vaccines toward developing an oral vaccine or subunit-based vaccine against brucellosis.

Contamination of obsterics and gynecology hospital air by bacterial and fungal aerosols associated with nosocomial infections.

Bacterial and fungal bioaerosols are a global concern due to nosocomial infections, especially in developing countries. Our study aimed to detect fungal and bacterial bioaerosols in different wards of an obstetrics and gynecology hospital air samples. 240 bioaerosol samples were collected by performing impaction method from different wards of a hospital in the central part of Iran, during two seasons. Fungi genera and bacteria species are recognized by cultivation. Concentrations of bacteria and fungi were ranged from 44 to 75 CFU/m3 and 8 to 22 CFU/m3, respectively. Labor Delivery and Recovery (LDR) and Emergency room had first and second most contaminated air among all the hospital wards. No significant difference between microbial load of wards which used natural ventilation and heating, ventilating, and air conditioning (HVAC) system was observed. The microbial load was not affected significantly by temperature, working shift, and Inpatient Bed Occupancy Rate (IBOR). Fungal load related significantly with relative humidity. Staphylococcus aureus (detected in 48.3% of samples) and Penicillium (27%) were the most predominant isolated bacteria and fungi, respectively. The results revealed that the level of bacteria and fungi responsible for nosocomial infections in the air of this hospital is very low. Although levels of microbial contamination are relatively low, it is important to investigate the effect of bioaerosols on nosocomial infections, especially in neonates.

Wound healing performance of PCL/chitosan based electrospun nanofiber electrosprayed with curcumin loaded chitosan nanoparticles.

In this study, the electrospun poly(ε-caprolactone) (PCL)/Chitosan (CS)/curcumin (CUR) nanofiber was fabricated successfully with curcumin loaded chitosan nano-encapsulated particles (CURCSNPs). The morphology of the produced CURCSNPs, PCL, PCL/CS, PCL/CS/CUR, and PCL/CS/CUR electrosprayed with CURCSNPs were analyzed by scanning electron microscopy (SEM). The physicochemical properties and biological characteristics of fabricated nanofibers such as antibacterial, antioxidant, cell viability, and in vivo wound healing efficiency and histological assay were tested. The electrospraying of CURCSNPs on surface PCL/CS/CUR nanofiber resulted in the enhanced antibacterial, antioxidant, cell proliferation efficiencies and higher swelling and water vapor transition rates. In vivo examination and Histological analysis showed PCL/CS/CUR electrosprayed with CURCSNPs led to significant improvement of complete well-organized wound healing process in MRSA infected wounds. These results suggest that the application of PCL/CS/CUR electrosprayed with CURCSNPs as a wound dressing significantly facilitates wound healing with notable antibacterial, antioxidant, and cell proliferation properties.

Effect of competition between petroleum-degrading bacteria and indigenous compost microorganisms on the efficiency of petroleum sludge bioremediation: Field application of mineral-based culture in the composting process.

The effect of competition between isolated petroleum-degrading bacteria (PDB) and indigenous compost microorganisms (ICM) on the efficiency of composting process in bioremediation of petroleum waste sludge (PWS) was investigated. After isolating two native PDB (Acinetobacter radioresistens strain KA5 and Enterobacter hormaechei strain KA6) from PWS, their ability for growth and crude oil degradation was examined in the mineral-based culture (MBC). Then, the PDB isolate were inoculated into the composting experiments and operated for 12 weeks. The results showed that the PDB degraded 21.65-68.73% of crude oil (1-5%) in the MBC after 7 days. The PDB removed 84.30% of total petroleum hydrocarbon (TPHs) in the composting bioreactor containing the initial TPH level of 20 g kg-1. Removal of petroleum hydrocarbons (PHCs) in the composting experiments proceeded according to the first-order kinetics. The computed values of degradation rate constants and half-lives showed a better performance of the PDB than ICM for TPHs removal. This finding suggests that simultaneous application of the PDB and ICM in the composting reactors resulted in a decline in the effectiveness of the PDB which is due to competition between them. The study also verified that the capability of PDB in degrading PHCs can be successfully scaled-up from MBC to composting process.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis.

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Biodegradation of high concentrations of petroleum compounds by using indigenous bacteria isolated from petroleum hydrocarbons-rich sludge: Effective scale-up from liquid medium to composting process.

The scale-up of petroleum hydrocarbons-rich sludge (PHRS) bioremediation from liquid medium to a composting method bioaugmentated with two indigenous bacteria, capable of degrading high levels of crude oil, was surveyed. After isolating the strains (Sphingomonas olei strain KA1 and Acinetobacter radioresistens strain KA2) and determining their biomass production, emulsification index (E24), bacterial adhesion to hydrocarbon (BATH), and crude oil degradation in liquid medium, they were inoculated into the composting experiments. In liquid medium, the removal rate of crude oil were 67.25, 70.86, 61.77, 42.13, and 27.92%, respectively for the initial oil levels of 1, 2, 3, 4, and 5% after 7 days. Degradation of 10, 20, 30, 40 and 50 g kg-1 concentrations of total petroleum hydrocarbons (TPH) were also calculated to be 91.24, 87.23, 84.69, 74.08, and 60.14%, respectively after a composting duration of 12 weeks. The values of the rate constants (k) and half-lives (t1/2) of petroleum hydrocarbons degradation were 0.083-0.212 day-1 and 3.27-8.35 days for the first-order and 0.003-0.089 g kg-1day-1 and 1.12-6.67 days for the second-order model, respectively. This study verified the suitability of the isolated strains for PHRS bioremediation. Successful scale-up of PHRS bioremediation from a liquid medium to a composting process for degrading high amounts of TPH was also confirmed.

Multidrug-resistant Shigella infection in pediatric patients with diarrhea from central Iran.

Background: Shigella spp. are primary pathogens of diarrhea in children worldwide. Emergence of resistance to fluoroquinolones and third-generation cephalosporins is crucial in the management of pediatric shigellosis. We determined the prevalence and the antibiotic resistance patterns of Shigella species isolated from pediatric patients in central Iran. Materials and methods: Pediatric diarrhea samples (n=230) were cultured on MacConkey and XLD agar media and in GN broth. Genus-specific PCR for ipaH was also used for detection directly from fecal specimens. Antibiotic resistance and the frequency of ESBL and AmpC genes were determined. Results: Out of the 230 samples, 19 (8.2%) cases of Shigella spp. were identified using culture. Twenty-six samples were positive by PCR (11.3%), S. flexneri (4/19; 21%) and S. sonnei (15/19; 78.9%) being the most detected. The highest antibiotic resistance rates were found for cotrimoxazole (19/19; 100%), ampicillin (16/19; 84.2%), cefixime (13/19; 68.4%) and ceftriaxone (12/19; 63.1%). Ten cases showed phenotypic ESBL presence and all these strains were positive for bla TEM, bla CTX-M-1, and bla CTX-M-15. Three strains were AmpC positive, all of which harbored bla CMY-2 and two contained bla CIT. Of the 19 Shigella isolates 5 (26.3%), 2 (10.5%), and 1 (5.2%) were phenotypically resistant to nalidixic acid, ciprofloxacin, and norfloxacin, respectively. Class 1 integron was found in 18 (94.7%) isolates whereas class 2 integron was found in 19 (100%) strains. Conclusion: We found a considerable presence of Shigella species with elevated antibiotic resistance levels. In particular, the resistance to third-generation cephalosporins (ESBL) and ciprofloxacin must be taken seriously.

Prediction of the Omp16 Epitopes for the Development of an Epitope-based Vaccine Against Brucellosis.

BACKGROUND: Brucellosis is an infectious disease caused by Brucella bacteria that cause disease in animals and humans. Brucellosis is one of the most common zoonotic diseases transmitted from animals-to-human through direct contact with infected animals and also consumption of unpasteurized dairy products. Due to the wide incidence of brucellosis in Iran and economical costs in industrial animal husbandry, Vaccination is the best way to prevent this disease. All of the available commercial vaccines against brucellosis are derived from live attenuated strains of Brucella but because of the disadvantage of live attenuated vaccines, protective subunit vaccine against Brucella may be a good candidate for the production of new recombinant vaccines based on Brucella Outer Membrane Protein (OMP) antigens. In the present study, comprehensive bioinformatics analysis has been conducted on prediction software to predict T and B cell epitopes, the secondary and tertiary structures and antigenicity of Omp16 antigen and the validation of used software confirmed by experimental results. CONCLUSION: The final epitope prediction results have proposed that the three epitopes were predicted for the Omp16 protein with antigenicity ability. We hypothesized that these epitopes likely have the protective capacity to stimulate both the B-cell and T-cell mediated immune responses and so may be effective as an immunogenic candidate for the development of an epitope-based vaccine against brucellosis.

Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity.

BACKGROUND: Streptavidin is a protein produced by Streptomyces avidinii with strong biotin-binding ability. The non-covalent, yet strong bond between these two molecules has made it a preferable option in biological detection systems. Due to its extensive use, considerable attention is focused on streptavidin production by recombinant methods. METHODS: In this study, streptavidin was expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS cells and purified by affinity chromatography. Various dialysis methods were employed to enable the protein to refold to its natural form and create a strong bond with biotin. RESULTS: Streptavidin was efficiently expressed in E. coli. Streptavidin attained its natural form during the dialysis phase and the refolded protein bound biotin. The addition of proline or arginine to the dialysis buffer resulted in a refolded streptavidin with greater affinity for biotin than refolding in dialysis buffer with no added amino acids. CONCLUSION: Dialysis of recombinant streptavidin in the presence of arginine or proline resulted in proper refolding of the protein. The recombinant dialyzed streptavidin bound biotin with affinity as great as that of a commercial streptavidin.