Development of antifungal fibrous ocular insert using freeze-drying technique.

Candida species is one of the pathogenic fungi of the eye responsible for keratitis that frequently causes vision impairment and blindness. Effective treatment requires long-term use of antifungal drugs, which is opposed by the defensive mechanisms of the eye and inadequate corneal penetration. The objective of this study was to develop a carrier for prolonged ocular application of fluconazole (FLZ) to treat keratitis. FLZ was encapsulated into chitosan fibrous matrices (F1-F4) using different chitosan concentrations (0.02, 0.1, 0.5, and 1%w/v, respectively) by freeze-drying as a single-step technique. Studying the morphology and surface properties of the inserts revealed a porous matrix with fibrous features with a large surface area. Thermal stability and chemical compatibility were confirmed by DSC/TGA/DTA and FT-IR, respectively. Loading capacity (LC) and entrapment efficiency (EE) were determined. According to the in vitro release study, F4 (0.11 mg mg-1 LC and 87.53% EE) was selected as the optimum insert because it had the most sustained release, with 15.85% burst release followed by 75.62% release within 12 h. Ex vivo corneal permeation study revealed a 1.2-fold increase in FLZ permeation from F4 compared to FLZ aqueous solution. Also, in the in vivo pharmacokinetic study in rabbits, F4 increased the AUC0-8 of FLZ by 9.3-fold and its concentration in aqueous humor was maintained above the MIC through the experimentation time. Studies on cytotoxicity (MTT assay) provide evidence for the safety and biocompatibility of F4. Therefore, the freeze-dried FLZ-loaded chitosan fibrous insert could be a promising candidate for treating ocular keratitis.

Comparative genomic analysis of strong biofilm-forming Klebsiella pneumoniae isolates uncovers novel ISEcp1-mediated chromosomal integration of a full plasmid-like sequence.

BACKGROUND: The goal of the current study was to elucidate the genomic background of biofilm formation in Klebsiella pneumoniae. METHODS: Clinical isolates were screened for biofilm formation using the crystal violet assay. Antimicrobial resistance (AMR) profiles were assessed by disk diffusion and broth microdilution tests. Biofilm formation was correlated to virulence and resistance genes screened by PCR. Draft genomes of three isolates that form strong biofilm were generated by Illumina sequencing. RESULTS: Only the siderophore-coding gene iutA was significantly associated with more pronounced biofilm formation. ST1399-KL43-O1/O2v1 and ST11-KL15-O4 were assigned to the multidrug-resistant strain K21 and the extensively drug-resistant strain K237, respectively. ST1999-KL38-O12 was assigned to K57. Correlated with CRISPR/Cas distribution, more plasmid replicons and prophage sequences were identified in K21 and K237 compared to K57. The acquired AMR genes (blaOXA-48, rmtF, aac(6')-Ib and qnrB) and (blaNDM-1, blaCTX-M, aph(3')-VI, qnrS, and aac(6')-Ib-cr) were found in K237 and K21, respectively. The latter showed a novel ISEcp1-mediated chromosomal integration of replicon type IncM1 plasmid-like structure harboring blaCTX-M-14 and aph(3')-VI that uniquely interrupted rcsC. The plasmid-mediated heavy metal resistance genes merACDEPRT and arsABCDR were spotted in K21, which also exclusively carried the acquired virulence genes mrkABCDF and the hypervirulence-associated genes iucABCD-iutA, and rmpA/A2. Pangenome analysis revealed NTUH-K2044 accessory genes most frequently shared with K21. CONCLUSIONS: While less virulent to Galleria mellonella than ST1999 (K57), the strong biofilm former, multidrug-resistant, NDM-producer K. pneumoniae K21 (ST1399-KL43-O1/O2v1) carries a novel chromosomally integrated plasmid-like structure and hypervirulence-associated genes and represents a serious threat to countries in the area.

Green Biosynthesis of Silver Nanoparticles Using Annona glabra and Annona squamosa Extracts with Antimicrobial, Anticancer, Apoptosis Potentials, Assisted by In Silico Modeling, and Metabolic Profiling.

Annona glabra L. (AngTE) and Annona squamosa L. (AnsTE) fruits have been widely used in cancer treatment. Accordingly, their extracts were used to synthesize silver nanoparticles via a biogenic route (Ang-AgNPs) and (Ans-AgNPs), respectively. Chemical profiling was established using UPLC-QTOF-MS/MS. All species were tested for anticancer activity against human cervical cancer cells (HeLa), prostate adenocarcinoma metastatic (PC3), and ovary adenocarcinoma (SKOV3) using sulphorhodamine B assay. Apoptosis was determined using Annexin flow cytometry along with cell cycle analysis and supported by a molecular docking. The antibacterial and synergistic effect when combined with gentamicin were evaluated. A total of 114 compounds were tentatively identified, mainly acetogenins and ent-kaurane diterpenes. AnsTE and Ans-AgNPs had the most potent cytotoxicity on HeLa and SKOV3 cells, inducing a significant apoptotic effect against all tumor cells. The AnsTE and Ans-AgNPs significantly arrested PC3, SKOV3, and HeLa cells in the S phase. The nanoparticles demonstrated greater antibacterial and antifungal activities, as well as a synergistic effect with gentamicin against P. aeruginosa and E. coli. Finally, a molecular docking was attempted to investigate the binding mode of the identified compounds in Bcl-2 proteins' receptor, implying that the fruits and their nanoparticles are excellent candidates for treating skin infections in patients with ovarian or prostatic cancer.