In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153.

BACKGROUND: The emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues. OBJECTIVE: The objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153. METHODOLOGY: Enterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation. RESULTS: The Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 µg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 µg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA. CONCLUSION: The findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

Characterization and Statistical Optimization of Enterobatin Synthesized by Escherichia coli OQ866153.

Microorganisms produce siderophores, which are secondary metabolites with a high affinity for iron. Siderophores have received significant attention due to their diverse applications in ecological and clinical research. In this study, siderophores production by Escherichia coli OQ866153 was optimized using two-stage statistical approach involving Plackett-Burman design (PBD) and response surface methodology (RSM) using central composite design (CCD). Out of 23 variables, succinate, tryptophan, Na2HPO4, CaCl2, agitation, and KH2PO4 were found to have the most significant effect on siderophores production in the first optimization stage with the highest SU% of 43.67%. In the second stage, RSM using CCD was utilized, and the optimal conditions were determined to be 0.3 g/l succinate, 0 g/l tryptophan, 6 g/l Na2HPO4, 0.1 g/l CaCl2, 150 RPM agitation, and 0.6 g/l KH2PO4, resulting in a maximum siderophore units (SU%) of 89.13%. The model was significant, as indicated by the model f-value of 314.14 (p-value = 0.0004) and coefficient of determination R2 of 0.9950. During validation experiments, the obtained maximum SU% was increased up to 87.1472%, which was two times as the value obtained under ordinary conditions (46.62%). The produced siderophores were purified and characterized using 1H, 13C NMR, IR spectroscopy. The obtained results indicated that the compound was enterobactin and entABCDEF genes were further detected in Escherichia coli OQ866153 extracted DNA. To our knowledge, this is the first report of statistical optimization for enterobactin synthesis by an E. coli strain isolated from a clinical source in Egypt.

Antibiotic resistance and siderophores production by clinical Escherichia coli strains.

The phenomenon of antibiotic resistance has dramatically increased in the last few decades, especially in enterobacterial pathogens. Different strains of Escherichia coli have been reported to produce a variety of structurally different siderophores. In the present study, 32 E. coli strains were collected from different clinical settings in Cairo, Egypt and subjected to the antibiotic susceptibility test by using 19 antibiotics belonging to 7 classes of chemical groups. The results indicated that 31 strains could be considered as extensively drug-resistant and only one strain as pan drug-resistant. Siderophores production by all the tested E. coli strains was determined qualitatively and quantitatively. Two E. coli strains coded 21 and 49 were found to be the most potent siderophores producers, with 79.9 and 46.62%, respectively. Bacterial colonies with cured plasmids derived from strain 49 showed susceptibility to all the tested antibiotics. Furthermore, E. coli DH5α cells transformed with the plasmid isolated from E. coli strain 21 or E. coli strain 49 were found to be susceptible to ansamycins, quinolones, and sulfonamide groups of antibiotics. In contrast, both plasmid-cured and plasmid-transformed strains did not produce siderophores, indicating that the genes responsible for siderophores production were located on plasmids and regulated by genes located on the chromosome. On the basis of the obtained results, it could be concluded that there is a positive correlation between antibiotic resistance, especially to quinolones and sulfonamide groups, and siderophores production by E. coli strains used in this study.

Partial characterization of levan polymer from Pseudomonas fluorescens with significant cytotoxic and antioxidant activity.

Microbial levan has great potential as a functional biopolymer in different fields including foods, feeds, cosmetics, and the pharmaceutical and chemical industries. In this study, a good levan producer bacterial strain of Pseudomonas fluorescens strain ES, isolated from soil in Egypt in a previous study, was used. Levan production by this strain was optimized using Plackett-Burman experimental design (PBD) to screen the critical factors of several process variables and Centered Central Composite Design (CCD) was applied for further estimation of the relationship between the variables and the response as well as optimization of the levels. Plackett-Burman (P-B) design showed a p-value 0.0144 less than 0.05 indicated the significance of the model. Sucrose, potassium dihydrogen phosphate, yeast extract and pH value showed the most significant effect on levan concentration at the values of 89.17, 65.83, 24.17, and 15.83, respectively. The purified levan polymer was characterized using different Physico-chemical methods such as Fourier Transform Infrared Spectrometer (FTIR), Nuclear magnetic resonance (NMR), and High-Performance Liquid Chromatography (HPLC) to determine the main composition and functional groups in the obtained polymer. HPLC results indicated that the polymer purification increased the percentage of fructose residue from 75 up to 89. Furthermore, 1H and 13C NMR spectroscopy analysis showed great matching between the obtained signal for our polymer with that reported in other people's work. The obtained levan polymer exhibited cytotoxic activity against Human epidermoid Skin carcinoma and Hepatocellular carcinoma with IC50 of 469 and 222.7 µg/ml, respectively. Antioxidant activity was determined using DPPH assay and the percentage of inhibition at 1000 µg/ml was found to be <50 (13.89 ± 1.07) with IC50 of (24.42 ± 0.87).

Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis.

Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight were obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment.