Frontiers in Cellular and Infection Microbiology
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2025
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IF 4.8
Yizhuo Zhang,Luqin Lv,Su Xu,Yijian Chen
Introduction: Clostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.Methods: Here, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.Results: The POLC platforms operated at 60 °C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/μL using a fluorescence reader and from 6 to 18 copies/μL via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.Discussion: Our research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).
研究领域:
医学诊断
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分子生物学
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DOI
PubMed
高影响因子
MOLECULES
·
2023
·Vol. 28
·期 16
·6152
·
IF 4.6
Lijun Xie,Li Chen,Yongbo Wei,Nannan Chen,Tong Wu,Jingming Zhou,Hong Jiang,Feng Lin
Rakicidin B1 was isolated and purified from the culture broth of a marineStreptomycessp. as a potent anti-cancer agent, and lately the compound and its derivatives have firstly been found to possess anti-Clostridium difficile(CD) activity but with high cytotoxicity. Herein, following our previous discovery on anti-CD activity of Rakicidin B1, structure modification was performed at the OH position of Rakicidin B1 and a new Rakicidin B1-PEG hybrids FIMP2 was facilely designed and synthesized by conjugating the PEG2000 with the scaffolds of Rakicidin B1 via the linkage of carbamate. The cytotoxicity of the FIMP2 was first evaluated against three different cancer cell lines, including HCT-8 cells, PANC-1, and Caco-2, with IC50values at 0.519 μM, 0.815 μM, and 0.586 μM, respectively. Obviously, as compared with a positive control group treated with Rakicidin B1, the IC50value of FIMP2 increased by nearly 91-fold, 50-fold, and 67-fold, suggesting that the PEGylation strategy significantly reduced the cytotoxicity of FIMP2. Thus, this preliminary result may be beneficial to increase its safety index (SI) value due to the decreased cytotoxicity of FIMP2. In addition, this decreased cytotoxicity of FIMP2 was further confirmed based on a zebrafish screening model in vivo. Thereafter, the anti-CD activity of FIMP2 was evaluated in vivo, and its efficacy to treat CDI was found to be better than that of vancomycin. The mortality and recurrence rate of FIMP2 is not as low compared with that of vancomycin; these results demonstrated that compound FIMP2 is a new, promising anti-CD agent with significant efficacy against CD recurrence with low cytotoxicity towards bodies.Keywords:Rakicidin B1;PEGylation strategy;anti-Clostridium difficile;low cytotoxicity
研究领域:
药物化学
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微生物学
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药理学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 91
·期 1
·
IF 3.9
Ji Zeng,Hao Wang,Yuxi Xu,Jianying Han,Yannan Li,Shu'an Wen,Changbu Wu,Dani Li,Zheng Liu,Xiaokang Zhang,Guo-Bao Tian,Min Dong
Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for in vitro expression. The prototyping capability of the C. difficile CFE system was assessed using a series of synthetic Clostridium promoters, demonstrating a good correlation between in vivo and in vitro expression. Additionally, we tested the expression of tcdB and tcdR from clinically relevant C. difficile strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for in vitro protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in Clostridioides difficile.
研究领域:
微生物学
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分子生物学
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生物技术
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DOI
高影响因子
Food Bioscience
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2021
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IF 4.24
Yujin Kim,Sejeong Kim,Heeyoung Lee,Nam-Su Oh,Min Suk Rhee,Yohan Yoon
Clostridium perfringens is an important bacterium among commensal bacteria in the gastrointestinal tract , which is known to possess potential pathogenicity during its inhabitation. In concordance with the concerns raised by its pathogenicity, this study aimed to elucidate if FMRP (fermented Maillard reaction products), the novel milk-derived material, can reduce the effect caused by the overabundance of C. perfringens in the gut. As a result, FMRP significantly reduced mRNA expression levels in the inflammatory cytokines (interleukin-10, interleukin-11, and interferon-γ) (p<0.05), and tumorigenic potential by activating the cell proliferation-suppressing factors (β-catenin, adenomatous polyposis coli , and E-cadherin) (p<0.05) and deactivation of the cell proliferation-inducing factor (cyclooxygenase-2) (p<0.05) in the colon. Moreover, the tight junction proteins (zonula occludens-1 and claudin-3) were recovered (p<0.05), and the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) were decreased by FMRP (p<0.05). However, biochemical analyses showed no significant difference. These results indicate that C. perfringens might cause inflammation, oxidative stress , disruption of tight junctions and abnormal cell proliferation to the colon, but FMRP may reduce inflammation and generation of reactive oxygen, and recover tight junction in the colon.
研究领域:
微生物学
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消化系统疾病
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营养学
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产品关联
DOI
高影响因子
Frontiers in Microbiology
·
2025
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IF 4.5
Yulei Tai,Meng Zhang,Yuning Han,Hui Hu,Shan Lin,Fangya Zhai,Menglun Tian,Xiaojun Song,Shuangshuang Wan,Yu Chen,Dazhi Jin
Introduction: Niclosamide (NIC) has significant potential as a clinical therapeutic agent for Clostridioides difficile infection (CDI); however, its strong hydrophobicity hampers its oral bioavailability, and its active effects against C. difficile remain unclear.Methods: Niclosamide-loaded controlled-release hyaluronic acid-modified poly (lactic-co-glycolic acid) naosphernes (NIC@PLGA-HAs) were synthesized using an oil-in-water emulsion technique and their effects on C. difficile cell growth, spore germination, biofilm formation, and NIC interaction sites with C. difficile toxin B (TcdB) were analyzed.Results: NIC@PLGA-HAs exhibited enhanced solubility and stability, with a water contact angle on a hydrophilic surface of 65.1° and a zeta potential of 31.57 ± 2.08 mV, and pH-responsive (pH 7.4) controlled-release characteristics compared to free NIC. The NIC@PLGA-HAs killed C. difficile vegetative cells at a minimum inhibitory concentration (MIC) of 4 μg/mL. When C. difficile cells were treated with NIC@PLGA-HAs at the 1/4 MIC, spore germination and biofilm formation were significantly inhibited compared to those in untreated cells (P < 0.01). NIC was found to interact with the receptor-binding domain of TcdB at 24 amino acid sites via an enthalpy-driven reaction (enthalpy change, 36.21 kJ/mol and entropy change, 212.9 J⋅mol/K). In vivo experimental findings in Mongolian gerbils indicated that NIC@PLGA-HAs outperformed free NIC in reducing pathological damage, diarrhea severity, weight loss, and TcdB production and enhanced the survival rate.Conclusion: These findings presented the therapeutic potential of NIC@PLGA-HAs with high solubility and stability, which simultaneously exerted multiple biological activities against C. difficile.
研究领域:
药剂学
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微生物学
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纳米技术
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药物递送
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传染病学
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产品关联
DOI
PubMed
高影响因子
Frontiers in Cellular and Infection Microbiology
·
2019
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IF 3.518
Yang Jingpeng,Yang Hong
Bifidobacterium breve (YH68) is widely used in the fields of food fermentation and biomedicine. In this study, we explored the antibacterial activity of the cell free culture supernatant (CFCS) of YH68 against Clostridioides difficile ATCC 9689 (CD) by measuring multiple indexes, including the growth, spores production, toxin A/B production, and the expression levels of the tcdA and tcdB genes of CD. In addition, we examined the changes in major cellular functional groups, structures, permeability, integrity, and the proton motive force (PMF) of the cytoplasmic membrane. The results showed that double-dilution ratio of YH68-CFCS (3 × 109 CFU/mL) was the MIC value. The cell density, spores production, and the toxin production of CD treated with YH68-CFCS were lower than that of the control (p < 0.05). In addition, the gene expression levels of tcdA and tcdB in CD treated with YH68-CFCS were significant downregulated (p < 0.05). Marked differences were observed in the cell membrane and cell wall by a FT-IR spectroscopy and SEM. Analysis of the cell membrane permeability and integrity of the CD cells revealed that YH68-CFCS induced the leakage of a large amount of intracellular K+, inorganic phosphate, ATP, nucleic acids and proteinaceous substances. Furthermore, PMF analysis indicated that there was a significant change in Δψ and ΔpH. These findings demonstrated that the antibacterial activity of YH68-CFCS against CD involved the inhibition of growth, spore production, toxin production, and virulence genes expression; a consumption of PMF in the cytoplasmic membrane, the formation of pore in the cell membrane, together with the enhanced cell membrane permeability; and, eventually, cell completely disintegration.
研究领域:
微生物学
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生物医学
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食品科学
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分子生物学
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DOI
PubMed
高影响因子
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
·
2023
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IF 4.9
Xiaojun Hu,Renhan Dong,Sheng Huang,Yisheng Zeng,Wei Zhan,Xiaofang Gao,Dong Tian,Jian Peng,Jiewei Xu,Ting Wang,Yaying Zhang,Xiaohui Wang,Xiaoxia Zhang,Jin Liu,Bing Guang,Tai Yang
Clostridioides difficile infection (CDI) causes severe diarrhea and colitis, leading to significant morbidity, mortality, and high medical costs worldwide. Oral vancomycin, a first-line treatment for CDI, is associated with a high risk of recurrence, necessitating novel therapies for primary and recurrent CDI. A novel small-molecule compound, CDBN-YGXZ, was synthesized by modifying the benzene ring of nitazoxanide with lauric acid. The mechanism of action of CDBN-YGXZ was validated using a pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR) inhibition assay. The efficacy of CDBN-YGXZ was evaluated using the MIC test and CDI infection model in mice and hamsters. Furthermore, metagenomics was used to reveal the underlying reasons for the effective reduction or prevention of CDI after CDBN-YGXZ treatment. The inhibitory activity against PFOR induced by CDBN-YGXZ. MIC tests showed that the in vitro activity of CDBN-YGXZ against C. difficile ranging from 0.1 to 1.5 μg/mL. In the mouse and hamster CDI models, CDBN-YGXZ provided protection during both treatment and relapse, while vancomycin treatment resulted in severe relapse and significant clinical scores. Compared with global effects on the indigenous gut microbiota induced by vancomycin, CDBN-YGXZ treatment had a mild influence on gut microbes, thus resulting in the disappearance or reduction of CDI recurrence. CDBN-YGXZ displayed potent activity against C. difficile in vitro and in vivo, reducing or preventing relapse in infected animals, which could merit further development as a potential drug candidate for treating CDI.
研究领域:
药理学
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传染病学
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微生物学
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药物开发
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DOI
PubMed
高影响因子
ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH
·
2023
·Vol. 30
·期 49
·108162-108175
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IF 5.8
Lu Yuanyuan,Pang Lina,Chatzisymeon Efthalia,Liu Xuna,Xu Kailin,Yang Ping,Gou Min
The metagenomics-based behavior and risk of antibiotic resistance genes (ARGs) were investigated during cattle manure thermophilic anaerobic digestion with tetracycline and copper, namely, bulk-copper oxide, nano-copper oxide, and copper sulfate, which are common feed additives. Although bulk-copper oxide reduced ARGs’ diversity, it enriched high-risk ARGs the most than the other two copper species, while copper sulfate could strongly mitigate the ARG risk by decreasing their abundances. Compared to corresponding individual effects, copper and tetracycline combinations may decrease ARGs’ co-occurrence potential by 22.0%, and particularly, tetracycline combined separately with copper sulfate and nano-copper oxide reduces the ARGs’ risk in abundance (by 7.2%) and human health (by 4.0%). These were mainly driven by bioavailable copper, volatile fatty acids, and pH, as well as the main potential hosts in phyla Firmicutes, Coprothermobacterota, and Euryarchaeota. Notably, the twin risks of pathogenicity and ARGs should be emphasized due to the ARGs’ positive correlation with human pathogens of Clostridioides difficile and Arcobacter peruensis . These findings are important for understanding the potential ARGs’ risk in treatments of livestock wastes containing feed additives of different sizes and speciation.
研究领域:
环境科学
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微生物学
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宏基因组学
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生物技术
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动物废物管理
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）