Escherichia coli is a common cause of biofilm-associated urinary tract infections (UTIs). Biofilm formation in E. coli is responsible for various indwelling medical device-associated infections, including catheter-associated urinary tract infections (CAUTIs). This study aimed to reduce biofilm formation of E. coli ATCC 25922 by knocking out genes involved in quorum sensing (QS) (luxS) and adhesion (fimH and bolA) using the CRISPR/Cas9-HDR approach. Single-guide RNAs (sgRNAs) were designed to target luxS, fimH and bolA genes. Donor DNA for homologous recombination was constructed to provide accurate repairs of double-strand breaks (DSBs). A biofilm quantification assay (crystal violet assay) was performed to quantify the biofilm formation of mutant and wild-type strains. Morphological changes in biofilm architecture were confirmed by scanning electron microscopy (SEM). Further application of the biofilm formation of mutant and wild-type strains on urinary catheter was tested. Crystal violet assay showed that the biofilm formation of ΔfimH, ΔluxS, and ΔbolA strains was significantly reduced compared to the wild-type strain (P value<0.001). The percentage of biofilm reduction of mutant strains was as follows: ΔluxS1 77.51 %, ΔfimH1 78.37 %, ΔfimH2 84.17 %, ΔbolA1 78.24 %, and ΔbolA2 75.39 %. Microscopic analysis showed that all mutant strains lack extracellular polymeric substances (EPS) production compared to the wild-type strain, which was embedded in its EPS matrix. The adherence, cell aggregation, and biofilm formation of wild-type strain on urinary catheters were significantly higher compared to ΔfimH, ΔluxS and ΔbolA strains. Altogether, our results demonstrated that the knockout of luxS, fimH, and bolA genes reduced EPS matrix production, which is considered the main factor in the development, maturation, and maintenance of the integrity of biofilm. This pathway could be a potential strategy to disrupt E. coli biofilm-associated UTIs. This study suggests that CRISPR/Cas9-HDR system may provide an efficient and site-specific gene editing approach that exhibits a possible antibiofilm strategy through intervention with the QS mechanism and adhesion property to suppress biofilm formation associated with UTI catheter infections.

Curcumin (CUR) has impressive pharmacologic properties, including cardioprotective, neuroprotective, antimicrobial, and anticancer activity. However, the pharmaceutical application of CUR is limited due to its poor aqueous solubility and low bioavailability. The development of novel formulations has attracted considerable attention to the idea of applying nanobiotechnology to improve the therapeutic efficacy of these challenging compounds. In this study, CUR-loaded lecithin–chitosan nanoparticles (CUR/LCSNPs) were developed and optimized by the concentration of chitosan, lecithin, and stirring speed by a 3-factorial Box-Behnken statistical design, resulting in an optimal concentration of chitosan (A) and lecithin (B) with a 1200 rpm stirring speed (C), with applied constraints of minimal average particle size (Y₁), optimal zeta potential (Y₂), and maximum entrapment efficiency (%EE) (Y₃). The mean particle size of the checkpoint formulation ranged from 136.44 ± 1.74 nm to 267.94 ± 3.72, with a zeta potential of 18.5 ± 1.39 mV to 36.8 ± 3.24 mV and %EE of 69.84 ± 1.51% to 78.50 ± 2.11%. The mean particle size, zeta potential, %EE, and % cumulative drug release from the optimized formulation were 138.43 ± 2.09 nm, +18.98 ± 0.72 mV, 77.39 ± 1.70%, and 86.18 ± 1.5%, respectively. In vitro drug release followed the Korsmeyer–Peppas model with Fickian diffusion (n < 0.45). The optimized technique has proven successful, resulting in a nanoformulation that can be used for the high loading and controlled release of lipophilic drugs.

The inadequate eradication of pulmonary infections and chronic inflammation are significant complications in cystic fibrosis (CF) patients, who usually suffer from persistent and frequent lung infections caused by several pathogens, particularly Pseudomonas aeruginosa (P. aeruginosa). The ability of pathogenic microbes to protect themselves from biofilms leads to the development of an innate immune response and antibiotic resistance. In the present work, a reference bacterial strain of P. aeruginosa (PA01) and a multidrug-resistant isolate (MDR 7067) were used to explore the microbial susceptibility to three antibiotics (ceftazidime, imipenem, and tobramycin) and an anti-biofilm peptide (IDR-1018 peptide) using the minimum inhibition concentration (MIC). The most effective antibiotic was then encapsulated into liposomal nanoparticles and the IDR-1018 peptide with antibacterial activity, and the ability to disrupt the produced biofilm against PA01 and MDR 7067 was assessed. The MIC evaluation of the tobramycin antibacterial activity showed an insignificant effect on the liposomes loaded with tobramycin and liposomes encapsulating tobramycin and IDR-1018 against both P. aeruginosa strains to free tobramycin. Nevertheless, the biofilm formation was significantly reduced (p < 0.05) at concentrations of ≥4 μg/mL and ≤32 μg/mL for PA01 and ≤32 μg/mL for MDR 7067 when loading tobramycin into liposomes, with or without the anti-biofilm peptide compared to the free antibiotic, empty liposomes, and IDR-1018-loaded liposomes. A tobramycin concentration of ≤256 µg/mL was safe when exposed to a lung carcinoma cell line upon its encapsulation into the liposomal formulation. Tobramycin-loaded liposomes could be a potential candidate for treating lung-infected animal models owing to the high therapeutic efficacy and safety profile of this system compared to the free administration of the antibiotic.

Skin infection compromises the body’s natural defenses. Several antibiotics are no longer effective owing to the evolution of antimicrobial-resistant (AMR) bacteria, hence, the constant development of novel antibacterial agents. Naturally occurring antibacterial agents may be potential candidates for AMR bacterial infection treatments; however, caution should be taken when administering such agents due to the high incidence of toxicity. A fibrous material system from a biocompatible polymer that could be used as a skin patch for skin infections treatment caused by AMR bacteria is proposed in this study. Bee venom’s active ingredient, melittin, was fabricated using electrospinning technology. Scanning electron microscopy showed that melittin-loaded fibers had smooth surfaces with no signs of beads or pores. The average diameter of this fibrous system was measured to be 1030 ± 160 nm, indicating its successful preparation. The melittin fibers’ drug loading and entrapment efficiency (EE%) were 49 ± 3 µg/mg and 84 ± 5%, respectively. This high EE% can be another successful preparatory criterion. An in vitro release study demonstrated that 40% of melittin was released after 5 min and achieved complete release after 120 min owing to the hydrophilic nature of the PVP polymer. A concentration of ≤10 µg/mL was shown to be safe for use on human dermal fibroblasts HFF-1 after 24-h exposure, while an antibacterial MIC study found that 5 μg/mL was the effective antimicrobial concentration for S. aureus, A. baumannii, E. coli and Candida albicans yeast. A melittin-loaded fibrous system demonstrated an antibacterial zone of inhibition equivalent to the control (melittin discs), suggesting its potential use as a wound dressing patch for skin infections.

One of the key challenges in developing a dry powder inhaler (DPI) of an inhalable potent fixed-dose combination (FDC) is the ability of the formulation to generate an effective and reproducible aerosol able to reach the lower parts of the lungs. Herein, a one-step approach is presented to expedite the synthesis of nanoaggregates made from a biocompatible and biodegradable polyamide based on L-lysine amino acid employing market-leading active pharmaceutical ingredients (fluticasone propionate (FP) and salmeterol xinafoate (SAL)) for the management of asthma. The nanoaggregates were synthesized using interfacial polycondensation that produced nanocapsules with an average particle size of 226.7 ± 35.3 nm and zeta potential of −30.6 ± 4.2 mV. Differential scanning calorimetric analysis and x-ray diffraction, as well as scanning electron microscopy of the produced FDC, revealed the ability of the produced nanocapsules to encapsulate the two actives and display the best aerodynamic performance. The FDC nanocapsules displayed 88.5% and 98.5% of the emitted dose for FP and SAL, respectively. The fine particle fraction of the nominated dose was superior to the marketed product (Seretide Diskus^®, Brentford, United Kingdom). The in-vitro release study showed an extended drug release profile. Our findings suggest that nanoaggregates using polyamides based on L-lysine and interfacial polycondensation can serve as a good platform for pulmonary drug delivery of FDC systems.