Omneya Mohamed Helmy

Background: Treating infected wounds is challenging, and conventional dressings delay healing. Resistance to the last-resort antibiotics is on the rise. Antimicrobial peptides (AMPs) offer advantages over traditional antibiotics against multidrug-resistant (MDR) bacterial strains. We aimed to use a computer-aided approach to design an anti-Gram-negative AMP with reduced mammalian cell toxicity and to functionalize an electrospun nanofibrous dressing with the AMP for in vitro and vivo evaluation. Methods: Colistin and polymyxin sequences were modified to generate analogs using a template-based approach. The membrane activity was predicted using the collection of antimicrobial peptide database. Three shortlisted sequences were synthesized, and their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against MDR Gram-negative bacterial isolates were determined. The time-kill kinetics of the potent candidate against Pseudomonas aeruginosa SM016 were analyzed, and toxicity to human skin fibroblasts (HSF) was determined by the MTT assay. A sodium alginate (SA)/polyvinyl alcohol (PVA) nanofibrous wound dressing was synthesized by electrospinning. The produced scaffolds were gamma-irradiated and examined using scanning electron microscopy (SEM). The AMP was loaded onto the scaffold by physical adsorption, followed by in vitro and in vivo testing against Pseudomonas aeruginosa SM016 in open infected wounds in BALB/c mice. Results: EM86 exhibited bactericidal activity against all tested isolates. EM86 killed Pseudomonas aeruginosa SM016 within 60 min, with morphological changes indicative of cell death confirmed by SEM after 30 min of treatment. EM86 was safe to HSF(IC50 > 300 μg/mL). The polymeric mixture of 1:12.3 SA/PVA, crosslinked with 1.25% glutaraldehyde, electrospun at 30 kV, 0.045 mm/min, and 12.5 cm, produced nanofibrous dressings with a mean diameter of 238 ± 78 nm and a swelling capacity of 510%. EM86-loading was confirmed by Fourier-transform infrared spectroscopy. Fibers loaded with 35 ± 18 μg EM86 significantly reduced P. aeruginosa SM016 counts (P < 0.001) in open-wound infected BALB/c mice, compared to the untreated mice, after 4 days of a once-daily treatment. Conclusion: Using computer-aided approaches, we designed EM86 with a bactericidal activity against MDR Gram-negative microorganisms and reduced toxicity to HSF compared to colistin. EM86-functionalized SA/PVA nanofibrous dressings offer a better treatment alternative for colistin-resistant infections in open wounds.

Burkholderia cenocepacia causes life-threatening infections in immunocompromised patients. Treatment is challenging due to intrinsic antibiotic multiresistance, so vaccination provides an alternative approach. We aimed to identify vaccine candidates using reverse vaccinology and evaluate their efficacy as protein-loaded chitosan: pectin nanoparticles (C:P NPs) in a vaccine model. Applying strict subtractive channels, three proteins were shortlisted: WP_006481710.1 (LY), WP_012493605.1 (KT), and WP_006492970.1 (BD). Proteins were cloned, purified as His-tagged proteins, and loaded onto C:P NPs. Vaccinated mice had significantly higher systemic IgG and mucosal IgA antibody responses and induced IL-6 and IL-17A. 6x-His-LY-CS:P NPs and 6x-His-KT-CS:P NPs vaccines induced TNF-α. Vaccines conferred significant protection against B. cenocepacia intranasal infections. In conclusion, cyclic-di-AMP phosphodiesterase (WP_012493605.1) is a promising vaccine candidate that elicited IgG and IgA antibodies, Th1, Th2, and Th17 cellular immunity in BALB/c mice and protected against B. cenocepacia infection. This provides hope for saving lives of people at high risk of infection.

To reduce high mortality and morbidity rates, timely and proper treatment of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is required. A multiplex polymerase reaction (mPCR)-based DNA lateral flow assay (MBDLFA) was developed as a point-of-care diagnostic for simultaneous identification of S. aureus, methicillin resistance, and vancomycin resistance directly from blood or blood cultures. A mPCR was developed to detect nuc, mecA, and vanA/B; its sensitivity, specificity, and limit of detection (LOD) were determined. The developed reaction was further modified for use in MBDLFA and its sensitivity for detection of target genes from artificially inoculated blood samples was checked. The optimized mPCR successfully detected nuc, mecA, and vanA/B from genomic DNA of bacterial colonies with LODs of 10⁷, 10⁷, and 10⁵ CFU/mL, respectively. The reaction was sensitive and specific. The optimized mPCR was used in MBDLFA that detected nuc, mecA, and vanA/B with LODs of 10⁷, 10⁸, and 10⁴ CFU/mL, respectively, directly from artificially inoculated blood. The developed MBDLFA can be used as a rapid, cheap point-of-care diagnostic for detecting S. aureus, MRSA, and vancomycin resistance directly from blood and blood cultures in ~2 h with the naked eye. This will reduce morbidity, mortality, and treatment cost in S. aureus bacteremia.

Helicobacter pylori (H. pylori) has been identified as a group-1 definite carcinogen. As of yet, there is no available vaccine for this microorganism. Our study aimed to identify antigenic peptides in H. pylori using an in silico proteomic approach, and to evaluate their effectiveness as potential vaccine candidates. Four different peptide sequences were prioritized using the reverse vaccinology, namely, CagA¹, CagA², VacA, and SabA. Peptides emulsified with Freunde’s adjuvant were used to immunize BALB/C mice. Subcutaneously immunized mice were challenged by oral administration of H. pylori. IgG, IgA, IL4, and IL17 were detected in mice sera. Histopathology of the dissected stomach of vaccinated and control mice were assessed using H&E stain. IgG was significantly higher in mice vaccinated with SabA. IL-4 was significantly increased in CagA¹, CagA², VacA, and SabA vaccinated mice compared to the adjuvant group. Additionally, histopathological examination of gastric tissue showed a protective effect in the vaccinated groups compared to adjuvant and PBS groups. Our findings indicate a promising effect of the tested epitopes, particularly the SabA antigen, to induce an immune response against H. pylori.

We set out to isolate alpha-amylase producers from soil samples, optimize the production, and immobilize the enzyme on chitosan-loaded barium ferrite nanoparticles (CLBFNPs). Alpha-amylase producers were isolated on starch agar plates and confirmed by dinitrosalicylic acid assay. The potent isolate was identified by phenotypic methods, 16S-rRNA sequencing, and phylogenetic mapping. Sequential optimization of α-amylase production involved the use of Plackett–Burman (P–BD) and central composite designs (CCD), in addition to exposing the culture to different doses of gamma irradiation. Alpha-amylase was immobilized on CLBFNPs, and the nanocomposite was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy, with energy-dispersive analysis of X-ray analysis. Forty-five α-amylase producers were isolated from 100 soil samples. The highest activity (177.12 ± 6.12 U/mg) was detected in the MS009 isolate, which was identified as Bacillus paramycoides. The activity increased to 222.3 ± 5.07 U/mg when using the optimal culture conditions identified by P–BD and CCD, and to 319.45 ± 4.91 U/mg after exposing the culture to 6 kGy. Immobilization of α-amylase on CLBFNPs resulted in higher activity (246.85 ± 6.76 U/mg) compared to free α-amylase (222.254 ± 4.89 U/mg), in addition to retaining activity for up to five cycles of usage. Gamma irradiation improved α-amylase production, while immobilization on CLBFNPs enhanced activity, facilitated enzyme recovery, and enabled its repetitive use.

Neonatal sepsis is a life-threatening condition and Staphylococcus aureus is one of its major causes. However, to date, no rapid and sensitive diagnostic tool has been developed for its direct detection. Bioinformatics analyses identified a surface-exposed 112-amino acid polypeptide of the cell wall protein NWMN_1649, a surface protein involved in cell aggregation and biofilm formation, as being a species-specific and highly conserved moiety. The polypeptide was cloned, purified, and used to immunize mice to raise specific immunoglobulins. The purified antibodies were conjugated to gold nano-particles and used to assemble an immunochromatographic strip (ICS). The developed prototype ICS detected as low as 5 µg purified polypeptide and 10² CFU/mL S. aureus within 15 min. The strip showed superior ability to directly detect S. aureus in neonatal sepsis blood specimens without prior sample processing. Moreover, it showed no cross-reaction in specimens infected with two other major causes of neonatal sepsis; coagulase-negative staphylococci and Klebsiella pneumoniae. The selected NWMN_1649-derived polypeptide demonstrates success as a promising biomolecule upon which a prototype ICS has been developed. This ICS provides a rapid, direct, sensitive, and specific option for the detection of S. aureus causing neonatal sepsis. Such a tool is urgently needed especially in resources-limited countries.

Background: Obesity and its related diseases are increasing worldwide. One of the best therapeutic strategies for obesity management is through the inhibition of pancreatic lipase (PL) enzyme. So far orlistat is the only FDA approved PL inhibitor, but with unpleasant side effects. New efficacious anti-obesity drugs are needed to achieve a successful reduction in the incidence and prevalence of obesity. Many microbial metabolites have PL inhibitory activity. Screening soil inhabitants for PL inhibitors could help in increasing the available anti-obesity drugs. We aimed to isolate and identify alternative PL inhibitors from soil flora.Results: We screened the crude mycelial methanolic extracts of 39 soil samples for PL inhibitory activity by the quantitative lipase colorimetric assay, using the substratep-nitrophenyl palmitate and orlistat as positive control. AspsarO, a PL inhibitor producer, was isolated from an agricultural field soil in Giza, Egypt. It was identified asAspergillus oryzaeusing colony morphology, microscopical characteristics,18SrDNA sequencing, and molecular phylogeny. Increasing the PL inhibitor activity, in AspsarO cultures, from 25.9 ± 2% to 61.4 ± 1.8% was achieved by optimizing the fermentation process using a Placket–Burman design. The dried 100% methanolic fraction of the AspsarO culture had an IC₅₀of 7.48 μg/ml compared to 3.72 μg/ml for orlistat. It decreased the percent weight gain, significantly reduced the food intake and serum triglycerides levels in high-fat diet-fed Sprague–Dawley rats. Kojic acid, the active metabolite, was identified using several biological guided chromatographic and¹H and¹³C NMR techniques and had an IC₅₀of 6.62 μg/ml. Docking pattern attributed this effect to the interaction of kojic acid with the key amino acids (Lys80, Trp252, and Asn84) in PL enzyme binding site.Conclusion: Combining the results of the induced obesity animal model, in silico molecular docking and the lipase inhibitory assay, suggests that kojic acid can be a new therapeutic option for obesity management. Besides, it can lower serum triglycerides in obese patients.

The class 1 carcinogen, Helicobacter pylori, is one of the World Health Organization’s high priority pathogens for antimicrobial development. We used three subtractive proteomics approaches using protein pools retrieved from: chokepoint reactions in the BIOCYC database, the Kyoto Encyclopedia of Genes and Genomes, and the database of essential genes (DEG), to find putative drug targets and their inhibition by drug repurposing. The subtractive channels included non-homology to human proteome, essentiality analysis, sub-cellular localization prediction, conservation, lack of similarity to gut flora, druggability, and broad-spectrum activity. The minimum inhibitory concentration (MIC) of three selected ligands was determined to confirm anti-helicobacter activity. Seventeen protein targets were retrieved. They are involved in motility, cell wall biosynthesis, processing of environmental and genetic information, and synthesis and metabolism of secondary metabolites, amino acids, vitamins, and cofactors. The DEG protein pool approach was superior, as it retrieved all drug targets identified by the other two approaches. Binding ligands (n = 42) were mostly small non-antibiotic compounds. Citric, dipicolinic, and pyrophosphoric acid inhibited H. pylori at an MIC of 1.5–2.5 mg/mL. In conclusion, we identified potential drug targets in H. pylori, and repurposed their binding ligands as possible anti-helicobacter agents, saving time and effort required for the development of new antimicrobial compounds.

In the era of antibiotic resistance, antimicrobial polymers represent state of the art innovation evolved to fight biofilm-associated infections. In the present study, novel self-disinfecting polyurethane (PU) catheter materials were developed. Gamma radiation-induced graft copolymerization was used to functionalize PU using acrylic acid-co-glycidyl methacrylate (AAc/GMA) binary comonomer. The grafted PU, PU-g-(AAc-co-GMA), was subsequently modified by covalent immobilization of cefepime and/or wet in-situ intermatrix synthesis of ZnO (NPs) to produce PU-g-(AAc-co-GMA)/cefepime, PU-g-(AA-co-GMA-cefepime/ZnO and PU-g-(AAc-co-GMA)/ZnO nanocomposites, respectively. Modified polymers were characterized by Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and scanning electron microscope (SEM). Finally, the antibacterial and antibiofilm characteristics were evaluated against five multidrug-resistant (MDR) clinical bacterial isolates; including Gram-positive and Gram-negative microorganisms. FTIR confirmed the successful grafting and subsequent immobilization of cefepime. Formation of ZnO was verified by EDX analysis, while XRD analysis revealed the crystalline nature of ZnO NPs with a size range of 43–62 nm. Moreover, SEM showed the morphology, particle size and distribution of ZnO NPs within the prepared nanocomposites. The modified PU catheter nancomposites with or without cefepime showed excellent antibacterial and anti-biofilm characteristics. The prepared polymeric biocides could be potential candidates in medical care to combat biofilm formation on biomaterials.