Expression and purification of recombinant Shiga toxin 2B from Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli are important human food-borne pathogens. Recently, Shiga toxinproducing E. coli (STEC) causes life-threatening hemolytic-uremic syndrome (HUS). In this study, Stx2B gene, a subunit of Shiga toxin, was amplified via polymerase chain reaction (PCR) from the chromosomal DNA of clinical fecal sample using appropriate primers. The PCR product was cloned to commercially available plasmid pH6HTN His6HaloTag® T7 containing two purification tags, namely, six histadine tag and Halo tag. The integrity of the constructed plasmid was confirmed using restriction enzyme mapping and sequencing. Then, Stx2B protein expressed after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli JM109 (DE3) under the control of the T7 promotor. The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at 37°C. Protein identity was confirmed by a band at ~11.4 kDa using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and StxB2 yield was 450 μg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology.Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Biosynthesis, Characterization, Antibacterial and Synergistic Effect of Silver Nanoparticles using Fusarium oxysporum

Biosynthesis of silver nanoparticles was achieved using cell filtrate from submerged fermentation of Fusarium oxysporum. It was found that AgNO3 reduced to Ag nanoparticles when exposed to the cell filtrate and the colour of solution was dark brown with absorbance peak at 430 nm wavelength. TEM micrograph showed spherical AgNPs with range 10-25 nm in dimension and was well dispersed. AgNPs show high stability in solution due to biological stabilizing and capping agents released from fungus, and have negative charge -25mv.Biosynthesized AgNPs have high potential antibacterial and antifungal activity, highest inhibitory zone was (27) mm against Candida albicans. The synergistic effect of AgNPs gave highest fold increase (10) against E. coli, followed by (5) fold against Staphylococcus auras using Azithromycin and levofloxacin as standard antibiotics respectively

Direct Detection of Burkholderia cepacia in Susceptible Pharmaceutical Products Using Semi-Nested PCR

Burkholderia cepaciahas recently received a considerable attention as one of the major risks in susceptible pharmaceutical products. This microorganism can easily propagate and cause vast and severe contamination, especially to the water supplies for pharmaceutical companies. Moreover, it proliferates within the products and can cause severe infections for humans. Therefore, fast and sensitive detection of these bacteria is of a great demand. The present study introduces improved application of a polymerase chain reaction assay with relatively high sensitivity and specificity for the direct detection ofB. cepaciafrom the aqueous pharmaceutical products. A semi-nested polymerase chain reaction approach using the primer set BCR1/BCR2 followed by BCR1/Mr yielding a 465 bp fragment of the recA gene was applied and tested using both crude lysate from isolated colonies and DNA directly extracted from artificially prepared and spiked reference syrup. The polymerase chain reaction assay showed no interference with other bacterial reference and environmental strains tested, includingStaphylococcus aureusATCC® 6538,Pseudomonas aeruginosaATCC® 9027,Escherichia coliATCC® 8739,Salmonella abonyNCTC® 6017,Bacillus subtilisATCC® 6633,Micrococcus luteus, Staphylococcus warneri, Pseudomonas fluorescens, Pseudomonas putida, andRalstonia pickettii Moreover, this semi-nested assay showed a detection limit of around 10 colony-forming units per sample and could detectB. cepaciastrains isolated from a municipal pre-treated potable water tank. Comparing the results for detection ofB. cepaciain 100 randomly collected commercial syrup preparations using both conventional standard method and polymerase chain reaction assay revealed thatB. cepaciawas detected in two samples using polymerase chain reaction assay while all samples showed negative results by conventional culturing and biochemical methods. These results highlight the advantage of using this polymerase chain reaction assay to detectB. cepaciain contaminated pharmaceutical products and even water for pharmaceutical purposes, without the need of culturing or pre-enrichment, where it may give false-negative results and may be misidentified when biochemically tested

Biological treatment of industrial wastes in a photobioreactor

An algal-bacterial consortium was tested for the treatment from a coke factory. A Chlorella vulgaris strain and a phenol-degrading Alcaligenes sp. were first isolated from the wastewater treatment plant to serve as inocula in the subsequent biodegradation tests. Batch tests were then conducted with samples from the real wastewater or using a synthetic wastewater containing 325 mg phenol/l and 500 mg NH4+/l as target pollutants. Direct biological treatment of-the real wastewater was not possible due to the toxicity of organic compounds. Activated carbon adsorption and UV(A-B)-irradiation were efficient in detoxifying the effluent for subsequent biological treatment as inoculation of pretreated samples with the algal-bacterial consortium was followed by complete phenol removal and NH4+ removal of 45%. Complete phenol removal and 33% NH4+ removal were achieved during the fed-batch treatment of artificial wastewater. at 6 d hydraulic retention time (HRT). Under continuous feeding at 3.6 d HRT, phenol and NH4+ removal dropped to 58 and 18%, respectively. However, complete phenol removal and 29% NH4+ removal were achieved when 8 g NaHCO3/1 was added to the artificial wastewater to enhance algal growth. This study confirms the potential of solar-based industrial wastewater treatment based on solar-based UV pretreatment followed by algal-bacterial biodegradation

Detection, Characterization, and Molecular Typing of Human Mycoplasma spp. from Major Hospitals in Cairo, Egypt

Mycoplasmas are fastidious slow growing organisms lacking a cell wall and mostly isolated from the mucosal surfaces of the respiratory and genitourinary tracts. There is a dearth of information regarding clinical Mycoplasma spp. isolates among Egyptian patients. A total of 170 samples were collected from patients and apparently healthy personnel in local public hospitals in Cairo, Egypt. Isolation of Mycoplasma spp. was carried out using appropriate culture media and further identification was carried out by biochemical tests followed by serotyping using specific antisera. Confirmation was done by PCR for detection of different Mycoplasma spp. using genus-specific primers targeting 16S ribosomal RNA gene. Characterization of the antibiotic resistance and sensitivity pattern against different antimicrobials was carried out using disc diffusion test. The results indicated the presence of six Mycoplasma spp. in 22.94% of the samples. Mycoplasmas were detected more frequently in throat swabs than sputum. Mycoplasma pneumoniae was highly sensitive to macrolides and quinolones but less sensitive to aminoglycosides and tetracyclines. Molecular techniques were found to be of more rapid, highly sensitive, able to detect nonviable organisms, and cost effective. These results shed light on difficulties of Mycoplasma detection and the superiority of molecular techniques over culture

Optimization and enhancement of textile reactive Remazol black B decolorization and detoxification by environmentally isolated pH tolerant Pseudomonas aeruginosa KY284155

Abstract Azo dyes are complex derivatives of diazene used in food and textile manufacture. They are highly recalcitrant compounds, and account for severe environmental and health problems. Different strains of Pseudomonas species were isolated from textile wastewater effluents. The bioconversion of Remazol black B (a commonly used water soluble dye) by Pseudomonas aeruginosa was observed in static conditions. The bio-decolorization process was optimized by a multi factorial Plackett–Burman experimental design. Decolorization of 200 mg L−1 reached 100% in 32 h. Interestingly, the presence of yeast extract, magnesium and iron in the culture media, highly accelerated the rate of decolorization. Moreover, one of our isolates, P. aeruginosa KY284155, was kept high degradation rates at high pH (pH = 9), which represents the pH of most textile wastewater effluents, and was able to tolerate high concentration of dye up to 500 mg L−1. In bacteria, azo-dye degradation is often initiated by reductive azo compound cleavage catalyzed by azo-reductases. Three genes encoding azo-reductases, paazoR1, paazoR2 and paazoR3, could be identified in the genome of the isolated P. aeruginosa stain (B1). Bioinformatics analyses of the paazoR1, paazoR2 and paazoR3 genes reveal their prevalence and conservation in other P. aeruginosa strains. Chemical oxygen demand dramatically decreased and phyto-detoxification of the azo dye was accomplished by photocatalytic post treatment of the biodegradation products. We suggest applying combined biological photocatalytic post treatment for azo dyes on large scale, for effective, cheap decolorization and detoxification of azo-dyes, rendering them safe enough to be discharged in the environment

Studying the influence of formulation and process variables on Vancomycin-loaded polymeric nanoparticles as potential carrier for enhanced ophthalmic delivery

Ocular topically applied Vancomycin (VCM) suffers poor bioavailability due to its high molecular weight and hydrophilicity. In the Present investigation, VCM-loaded polymeric nanoparticles (PNPs) were developed aiming to enhance its ocular bioavailability through prolonging its release pattern and ophthalmic residence. PNPs were prepared utilizing double emulsion (W/O/O), solvent evaporation technique. 2 3X4 1 full factorial design was applied to evaluate individual and combined influences of polymer type, Eudragit® RS100, sonication time, and Span® 80 concentration on PNPs particle size, encapsulation efficiency, and zeta potential. Further, the optimized formulae were incorporated in 1% Carbopol® - based gel. In-vivo evaluation of the optimized formulae was performed via Draize test followed by microbiological susceptibility testing on albino rabbits. Results revealed successful formulation of VCM- loaded PNPs was achieved with particle sizes reaching 155 nm and up to 88% encapsulation. Draize test confirmed the optimized formulae as non-irritating and safe for ophthalmic administration. Microbiological susceptibility testing confirmed prolonged residence, higher Cmax. with more than two folds increment in the AUC(0.25- 24) of VCM-PNPs over control groups. Thus, VCMloaded PNPs represent promising carriers with superior achievements for enhanced Vancomycin ophthalmic delivery over the traditional use of commercially available VCM parenteral powder after constitution into a solution by the ophthalmologists

Nanoparticles as tool for enhanced ophthalmic delivery of vancomycin: a multidistrict-based microbiological study, solid lipid nanoparticles formulation and evaluation

Context: A microbiological multidistrict-based survey from different Egyptian governorates was conducted to determine the most prevalent causative agents of ocular infections in the Egyptian population. Antibiotic sensitivity testing was then performed to identify the most potent antimicrobial agent. Vancomycin (VCM) proved the highest activity against gram-positive Staphylococcus bacteria, which are the most commonly isolated causative agents of ocular infection. However, topically applied VCM suffers from poor ocular bioavailability because of its high molecular weight and hydrophilicity. Objective: the aim of the present study was to develop VCM-loaded solid lipid nanoparticles (SLNs) using water-in-oil-in-water (W/O/W) double emulsion, solvent evaporation technique to enhance ocular penetration and prolong ophthalmic residence of VCM. Method: Two consecutive full factorial designs (24 followed by 32 ) were adopted to study the effect of different formulation and process parameters on SLN formulation. The lipid type and structure, polyvinyl alcohol (PVA) molecular weight and concentration, sonication time, as well as lipid:drug ratio were studied as independent variables. The formulated SLN formulae were evaluated for encapsulation efficiency, particle size, and zeta potential as dependent variables. Results: The statistically-optimized SLN formula (1:1 ratio of glyceryltripalmitate:vancomycin with 1% low molecular weight PVA and 1 min sonication time) had average particle size of 277.25 nm, zeta potential of -20.45, and 19.99% drug encapsulation. Scanning and transmission electron micrographs showed well-defined, spherical, homogenously distributed particles. Conclusion: The present study suggests that VCM incorporation into SLNs is successfully achievable; however, further studies with different nanoencapsulation materials and techniques would be valuable for improving VCM encapsulation