霍乱是一种腹泻病，被认为是由产生毒素的霍乱弧菌 (V.) 引起的。本研究旨在评估封装在壳聚糖纳米颗粒（CFSb-CsNPs 和 CFSa-CsNPs）中的两歧双歧杆菌 (B.) 和嗜酸乳杆菌 (L.) 衍生的无细胞上清液 (CFS) 的杀弧菌和免疫调节特性。霍乱弧菌 O1 El Tor 的耐药 (MDR) 分离株。我们使用离子凝胶技术合成了 CFSb-CsNPs 和 CFSa-CsNPs。对新纳米结构的尺寸、表面 zeta 电位、形态、封装功效 (EE)、不同 pH 值和温度下的稳定性、释放曲线和体外细胞毒性进行了表征。分别通过微肉汤稀释法和结晶紫染色研究了所获得的纳米复合材料对霍乱弧菌 E1 Tor O1 临床 MDR 分离株（N = 5）的抗菌和抗生物膜作用。我们进行了定量实时 PCR (qRT-PCR) 来分析 Bap、Rbmc、CTXAB 和 TCP 对 CFSb-CsNP 和 CFSa-CsNP 的相对基因表达。此外，还研究了配制结构对人结直肠腺癌细胞（Caco-2）中 TLR2 和 TLR4 基因表达的免疫调节作用。纳米表征分析表明，CFSb-CsNPs 和 CFSa-CsNPs 在扫描电子显微镜下呈现球形形状（ SEM）成像，平均直径分别为 98.16±0.763 nm 和 83.90±0.854 nm。两种类型的纳米颗粒都具有正表面电荷。 CFSb-CsNPs 的 EE% 为 77±4.28%，而 CFSa-CsNPs 的 EE% 为 62.5±7.33%。壳聚糖 (Cs) 封装可提高 CFS 在模拟胃肠道 pH 条件下的稳定性，其中在 pH = 7.4 时，CFSb-CsNPs 和 CFSa-CsNPs 的释放率分别达到 58.00±1.24% 和 55.01±1.73% 。从 CFSb-CsNP 和 CFSa-CsNP 共同给药观察到的协同杀弧菌效应，抑制浓度分数 (FIC) 指数为 0.57，导致 MIC 显着降低，为 2.5 ± 0.05 mg/mL (p < 0.0001) 与单独给药相比。 CFSb-CsNPs 和 CFSa-CsNPs 对 Bap (0.14±0.05)、Rbmc (0.24±0.01)、CTXAB (0.30±0.09) 和 TCP (0.38±0.01) 基因表达的联合抗菌作用显着 (p<0.00) 1） 。此外，CFSb-CsNPs 和 CFSa-CsNPs 的共同给药还证明了抑制 TLR 2/4（分别为 0.20±0.01 和 0.12±0.02）基因表达（p=±0.0019）并减少 Caco-2 细胞附着细菌的效力。 526,000 ± 51,46 菌落形成单位/mL (11.19%) (p < 0.0001)。我们的研究表明，将 CFS 封装在 CsNP 内可通过提高稳定性并在宿主之间的相互作用位点启用受控释放机制来增强其杀弧菌活性和细菌。此外，同时使用 CFSb-CsNP 和 CFSa-CsNP 对 MDR 霍乱弧菌 O1 El Tor 菌株表现出优异的杀弧菌功效，表明这些组合是对抗 MDR 细菌的潜在新方法。© 2024。作者。

Cholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor.We synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied.Nano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001).Our study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria.© 2024. The Author(s).