Gut microbiome changes in mouse, Mongolian gerbil, and hamster models following Clostridioides difficile challenge.

Introduction: Clostridioides difficile infection (CDI), as well as its etiology and pathogenesis, have been extensively investigated. However, the absence of suitable CDI animal models that reflect CDI symptoms and the associated gut microbiome changes in humans has limited research progress in this field. Thus, we aimed to investigate whether Mongolian gerbils, which present a range of human pathological conditions, can been used in studies on CDI. Methods: In this study, we infected Mongolian gerbils and two existing CDI model animals, mice and hamsters, with the hypervirulent ribotype 027 C. difficile strain, and comparatively analyzed changes in their gut microbiome composition via 16S rRNA gene sequencing. Methods: In this study, we infected Mongolian gerbils and two existing CDI model animals, mice and hamsters, with the hypervirulent ribotype 027 C. difficile strain, and comparatively analyzed changes in their gut microbiome composition via 16S rRNA gene sequencing. Results: The results obtained showed that C. difficile colonized the gastrointestinal tracts of the three rodents, and after the C. difficile challenge, C57BL/6J mice did not manifest CDI symptoms and their intestines showed no significant pathological changes. However, the hamsters showed explosive intestinal bleeding and inflammation and the Mongolian gerbils presented diarrhea as well as increased infiltration of inflammatory cells, mucus secretion, and epithelial cell shedding in their intestinal tissue. Further, intestinal microbiome analysis revealed significant differences with respect to intestinal flora abundance and diversity. Specifically, after C. difficile challenge, the Firmicutes/Bacteroidetes ratio decreased for C57BL/6J mice, but increased significantly for Mongolian gerbils and hamsters. Furthermore, the abundance of Proteobacteria increased in all three models, especially in hamsters, while that of Verrucomicrobia only increased significantly in C57BL/6J mice and Mongolian gerbils. Our results also indicated that differences in the relative abundances of Lactobacillaceae and Akkermansia were primarily responsible for the observed differences in response to C. difficile challenge. Conclusion: Based on the observed responses to C. difficile challenge, we concluded for the first time that the Mongolian gerbil could be used as an animal model for CDI. Additionally, the taxa identified in this study may be used as biomarkers for further studies on CDI and to improve understanding regarding changes in gut microbiome in CDI-related diseases.

Rapid discrimination between clinical Clostridioides difficile infection and colonization by quantitative detection of TcdB toxin using a real-time cell analysis system.

Objectives: It is important to accurately discriminate between clinical Clostridioides difficile infection (CDI) and colonization (CDC) for effective antimicrobial treatment. Methods: In this study, 37 stool samples were collected from 17 CDC and 20 CDI cases, and each sample were tested in parallel through the real-time cell analysis (RTCA) system, real-time PCR assay (PCR), and enzyme-linked immunosorbent assay (ELISA). Results: RTCA-measured functional and toxical C. difficile toxin B (TcdB) concentrations in the CDI group (302.58 ± 119.15 ng/mL) were significantly higher than those in the CDC group (18.15 ± 11.81 ng/mL) (p = 0.0008). Conversely, ELISA results revealed no significant disparities in TcdB concentrations between the CDC (26.21 ± 3.57 ng/mL) and the CDI group (17.07 ± 3.10 ng/mL) (p = 0.064). PCR results indicated no significant differences in tcdB gene copies between the CDC (774.54 ± 357.89 copies/µL) and the CDI group (4,667.69 ± 3,069.87 copies/µL) (p = 0.407). Additionally, the functional and toxical TcdB concentrations secreted from C. difficile isolates were measured by the RTCA. The results from the CDC (490.00 ± 133.29 ng/mL) and the CDI group (439.82 ± 114.66 ng/mL) showed no significant difference (p = 0.448). Notably, RTCA-measured functional and toxical TcdB concentration was significantly decreased when mixed with pooled CDC samples supernatant (p = 0.030). Conclusion: This study explored the novel application of the RTCA assay in effectively discerning clinical CDI from CDC cases.

Preparation of PLGA microspheres loaded with niclosamide via microfluidic technology and their inhibition of Caco-2 cell activity in vitro.

Niclosamide (NIC) is a multifunctional drug that regulates various signaling pathways and biological processes. It is widely used for the treatment of cancer, viral infections, and metabolic disorders. However, its low water solubility limits its efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) and hyaluronic acid (HA), which exhibit good biocompatibility, biodegradability, and non-immunogenicity, were conjugated with niclosamide to prepare PLGA-HA-niclosamide polymeric nanoparticles (NIC@PLGA-HA) using microfluidic technology. The obtained microspheres had a uniform size distribution, with an average mean size of 442.0 ± 18.8 nm and zeta potential of -25.4 ± 0.41 mV, indicating their stable dispersion in water. The drug-loading efficiency was 8.70%. The drug-loaded microspheres showed sustained release behavior at pH 7.4 and 5.0, but not at pH 2.0, and the drug release kinetics were described by a quasi-first-order kinetic equation. The effect of the drug-loaded microspheres on the proliferation of Caco-2 cells was detected using the MTT assay. Hydrophilic HA-modified NIC@PLGA-HA microspheres prepared via microfluidic technology increased the cellular uptake by Caco-2 cells. Compared to the same concentration of NIC, the NIC@PLGA-HA microspheres demonstrated a stronger inhibitory effect on Caco-2 cells owing to the combined effect of PLGA, HA, and NIC. Therefore, the pH-responsive NIC@PLGA-HA microspheres synthesized using microfluid technology increased the solubility of NIC and improved its biological activity, thus contributing to the demand for intestinal drug carriers.

Design and evaluation of lidocaine- and prilocaine-coloaded nanoparticulate drug delivery systems for topical anesthetic analgesic therapy: a comparison between solid lipid nanoparticles and nanostructured lipid carriers.

PURPOSE: Topical anesthesia analgesic therapy has diverse applicability in solving the barrier properties of skin and unfavorable physicochemical properties of drugs. Lidocaine (LID) combined with prilocaine (PRI) has been used as a topical preparation for dermal anesthesia for treatment of conditions such as paresthesia. MATERIALS AND METHODS: In this study, for combination anesthesia and overcoming the drawbacks of LID and PRI, respectively, LID- and PRI-loaded solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were prepared and characterized by determination of their particle size, drug loading capacity, stability, in vitro drug release behavior and in vitro cellular viability. Ex vivo skin permeation and in vivo anesthesia analgesic efficiency of these two systems were also evaluated and compared. RESULTS: Results revealed that combination delivery of the dual drugs exhibited more remarkable efficiency than signal drug-loaded systems. SLN systems have better ex vivo skin permeation ability than NLCs. NLC systems revealed a stronger in vivo anesthesia analgesic effect than SLN systems. CONCLUSION: It can be concluded that SLNs and NLCs have different advantages, and that both carriers are promising dual drug delivery systems for topical anesthetic analgesic therapy.

A novel local anesthetic system: transcriptional transactivator peptide-decorated nanocarriers for skin delivery of ropivacaine.

PURPOSE: Barrier properties of the skin and physicochemical properties of drugs are the main factors for the delivery of local anesthetic molecules. The present work evaluates the anesthetic efficacy of drug-loaded nanocarrier (NC) systems for the delivery of local anesthetic drug, ropivacaine (RVC). METHODS: In this study, transcriptional transactivator peptide (TAT)-decorated RVC-loaded NCs (TAT-RVC/NCs) were successfully fabricated. Physicochemical properties of NCs were determined in terms of particle size, zeta potential, drug encapsulation efficiency, drug-loading capacity, stability, and in vitro drug release. The skin permeation of NCs was examined using a Franz diffusion cell mounted with depilated mouse skin in vitro, and in vivo anesthetic effect was evaluated in mice. RESULTS: The results showed that TAT-RVC/NCs have a mean diameter of 133.2 nm and high drug-loading capacity of 81.7%. From the in vitro skin permeation results, it was observed that transdermal flux of TAT-RVC/NCs was higher than that of RVC-loaded NCs (RVC/NCs) and RVC injection. The evaluation of in vivo anesthetic effect illustrated that TAT-RVC/NCs can enhance the transdermal delivery of RVC by reducing the pain threshold in mice. CONCLUSION: These results indicate that TAT-decorated NCs systems are useful for overcoming the barrier function of the skin, decreasing the dosage of RVC and enhancing the anesthetic effect. Therefore, TAT-decorated NCs can be used as an effective transdermal delivery system for local anesthesia.