OPTIMIZATION OF A CULTURE MEDIUM FOR BIOMASS AND δ-ENDOTOXIN PRODUCTION BY A RECOMBINANT ESCHERICHIA COLI STRAIN

A recombinant strain of Escherichia coli harboring a plasmid containing the Bacillus thuringiensis δ-endotoxin synthesis gene, was tested for its efficacy to synthesize δ-endotoxin, in a complex medium containing sucrose and yeast extract. Also, the recombinant E. coli strain was tested for its efficacy against the 2nd instars of Spodoptera littoralis. The recombinant strain of E. coli showed a good activity against the 2nd instars of S. littoralis, the mortality was 70 % after 7 days at room temperature. A high cell biomass (8.8gL-1) and δ-endotoxin concentration (6.8 mg L-1), were obtained by the shake flask culture (100 ml medium/250 ml flask, at 200 rpm), of the recombinant E. coli in modified MR medium containing sucrose (20g/L), as carbon source and yeast extract as nitrogen source, in the presence of CaCO3, K2HPO4, MgSO4, FeSO4 and ZnSO4 as mineral salts. The best pH values for cell biomass production and endotoxin production were 7.0 and 7.5, respectively. The corresponding figures for the best temperature were 37oC and 30oC, respectively. The use of some byproducts such as black-strap molasses, corn-steep liquor and cheese whey, as an alternative for carbon and nitrogen sources of medium, were found to enhance the cell growth but showed no effect on endotoxin production

Novel approach to overcome the β-lactam resistant bacteria using an actinobacterial inhibitory protein

β-lactam resistance is a serious problem that the hospitals face worldwide; particularly in the developing countries. The widespread of this resistance is attributed to various mechanisms used by the nosocomial bacteria. The aims of this study were to monitor the spread of the β-lactam resistant bacteria in the different provinces of Egypt; to create a biocontrol strategy by producing the β-lactamase inhibitory protein from the Streptomyces bacteria, and to knowing its suitability for the human use. Seventy β-lactam resistant bacterial isolates were sampled randomly from several hospital laboratories across ten governorates of Egypt. The isolates were screened against six different antibiotics; mainly Amoxicillin; Amoxicillin-Clavulanate, Penicillin, Ampicillin-Sulbactam, Cefepime, and Piperacillin-Tazobactam at 250 µg/ ml, and their Minimum inhibitory concentration (MIC) was recorded. The Bn67 isolate was the most promising isolate, which was molecularly identified using 16SrRNA partial sequence as Pseudomonas aeruginosa (LC710315.1). So in order to overcome this bacterial resistance; eighty actinobacteria were isolated from several soil samples collected from Giza governorate, Egypt, and were screened for their effectiveness against Bn67. The actinobacterial isolate (Stn-01) showed the maximum inhibitory efficacy against Bn67 and was identified using 16SrRNA partial sequence as Streptomyces katsurahamanus (LC710314.1). The inhibitor protein (β-LIP-n) was isolated; precipitated, and purified to give 35 kDa with 17 amino acid sequences. The β-LIPn exhibited no cytotoxic potential against the Human Skin Fibroblast (HSF) cell line at 200 µg/ ml. This approach of using the bacterial soil-based inhibitor protein to biocontrol the β-lactam resistant bacteria is considered as novel and as a promising start-up to using the environmental bacteria to overcome this problem of β-lactam resistance

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Production of amylases from Bacillus amyloliquefaciens under submerged fermentation using some agro-industrial by-products

AbstractThirty-one bacterial isolates out of 133 isolates, were obtained from rhizosphere of Egyptian clover plants, and had variant capability for starch degradation on starch agar medium. The isolate E109 was the most potent being 72.5Uml−1 and 2.5 for amylase activity and starch hydrolysis ratio (SHR), respectively, at 50°C. The potent isolate E109 was identified based on phenotypic characteristics, phylogenetic positions based on 16S rRNA gene analysis and base sequences (submitted to NCBI Gen Bank). 16S rRNA gene analysis confirmed that this isolate belonged to the genus Bacillus and it was most closely related to B. amyloliquefaciens (95% similarity). For the production of amylases, nine agro-industrial residues were added as carbon sources to the basal medium. The medium supplemented with potato starchy waste as the sole carbon source enhanced the enzyme activity more than soluble starch as control for α, β and γ amylases activity, as it increased by B. amyloliquefaciens about 1.26 & 4 and 8-fold, respectively after 48h at 50°C using rotary shaker at 150rpm. B. amyloliquefaciens gave the maximum values of α, β and γ amylases activity on medium supplemented with 2% potato starchy waste after 30, 30 & 36h of fermentation periods at 50°C using shake flasks technique as a batch culture. These values were 155.2Uml−1 (R2=0.93), 1.0Uml−1 (R2=0.94) and 2.4Uml−1 (R2=0.95), respectively. It could be stated that productive medium supplemented with 2% potato starchy waste as a low price substrate could be more favorable than basal medium containing 1% starch for amylases production in submerged fermentation, as it increased α, β and γ amylase activity by 1.98, 7.69 and 12-fold than that produced in basal medium (control), respectively

Toxicity assessment of green synthesized Cu nanoparticles by cell-free extract of Pseudomonas silesiensis as antitumor cancer and antimicrobial

Spherical homogeneous 32 nm, protein coated Pseudomonas silesiensis strain A3 CuNPs was investigated for their cytotoxicity effect as well as antimicrobial and antitumor activity. CuNPs cytotoxicity was estimated using human normal lung cell lines (Wi38) against CuNPs with concentrations ranging from 25 to 150 μg/mL using neutral red uptake assay. The cytotoxicity study revealed that the bacterial CuNPs had an impact on Wi38 cell viability at concentrations of 25, 50, 100 and 150 μg/mL CuNPs were 95.8, 91.1, 89.2 and 82.3%, respectively, with a strong correlation coefficient (r = 0.94) and a CuNPs IC50 value of 1057.0 μg/mL. CuNPs exhibit a broad-spectrum antimicrobial activity against various microorganism species, including fungi and Gram positive and negative bacteria using the agar-well diffusion method. The findings revealed that the most sensitive pathogens were Staphylococcus aureus ATCC5638 and Aspergillus flavus ATCC 9643 which tended to have a high inhibition zone diameter (50 and 47 mm, respectively). The minimum inhibitory concentration (MIC) of CuNPs was 50 μg/mL. The minimum lethal concentration (MLC) values were 50 and 75 μg/mL for S. aureus ATCC 5638 and A. flavus ATCC 9643, respectively. MLC/MIC ratio was ≤2, suggesting the CuNPs had a bactericidal or fungicidal effect on both pathogenic strains. Results also indicated that bacterial CuNPs at varying concentrations of 25, 50, 100 and 150 μg/mL were such a good antitumor agent against A549 lung carcinoma cell lines with an IC50 value of 137.5 μg/mL and a cell viability of 89.3, 79.6, 64.9 and 44.1%, respectively. The results also suggested that the biosynthesized-CuNPs were an antimicrobial and anticancer agent that could be used in future in food preservation, biomedicine and pharmaceutical fields

Genetic characterisation of multidrug-resistant <i>Salmonella enterica</i> serotypes isolated from poultry in Cairo, Egypt

Background: Food-borne diseases pose serious health problems, affecting public health and economic development worldwide. Methods: Salmonella was isolated from samples of chicken parts, skin samples of whole chicken carcasses, raw egg yolks, eggshells and chicken faeces. Resulting isolates were characterised by serogrouping, serotyping, antimicrobial susceptibility testing and detection of extended-spectrum β-lactamase (ESBL) production. Antibiotic resistance genes and integrons were identified by polymerase chain reaction (PCR). Results: The detection rates of Salmonella were 60%, 64% and 62% in chicken parts, skin, and faeces, respectively, whereas the egg yolks and eggshells were uniformly negative. Salmonella Kentucky and S. Enteritidis serotypes comprised 43.6% and 2.6% of the isolates, respectively, whilst S. Typhimurium was absent. Variable resistance rates were observed against 16 antibiotics; 97% were resistant to sulfamethoxazole, 96% to nalidixic acid and tetracycline and 76% to ampicillin. Multidrug resistance was detected in 82% (64/78) of the isolates and ESBL production was detected in 8% (6/78). The β-lactamase blaTEM-1 gene was detected in 57.6% and blaSHV-1 in 6.8% of the isolates, whilst the blaOXA gene was absent. The sul1gene was detected in 97.3% and the sul2 gene in 5.3% of the isolates. Sixty-four of the 78 isolates (82%) were positive for the integrase gene (int I) from class 1 integrons, whilst int II was absent. Conclusion: This study reveals the presence of an alarming number of multidrug-resistant Salmonella isolates in the local poultry markets in Cairo. The high levels of drug resistance suggest an emerging problem that could impact negatively on efforts to prevent and treat poultry and poultry-transmitted human diseases in Egypt

Biochemical and biotechnological studies on a novel purified <i>bacillus</i> cholesterol oxidase tolerant to solvent and thermal stress

<p>A novel bacterial strain was isolated and identified as <i>Bacillus pumilus,</i> with the capability to produce cholesterol oxidase enzyme (55 kDa). The production of the enzyme was optimized via two-step statistical approach. Out of eight factors screened in Plackett–Burman, only four had significant effects on enzyme activity. The optimization process of these four variables by Box–Behnken revealed that the maximum enzyme activity (90 U/mL) was significantly obtained after 6 days of fermentation with 0.3%, 1% and 0.2% of NH₄NO₃, yeast extract and Tween 80, respectively. The purified enzyme showed optimum activity at pH 7.5 and temperature of 40 °C. The enzyme retained 100% of its activity after storage at 40 °C for 60 min. The enzyme also exhibited enhanced stability in the presence of Tween 80, methanol and isopropanol. This solvent and thermal stress tolerant enzyme, produced by <i>B. pumilus</i>, may provide a practical option for industrial and analytical applications.</p