It is well known that extensive osteoclast formation plays a key role in osteoporosis in post‑menopausal women and the elderly. The suppression of extensive osteoclastogenesis and bone resorption may be an effective preventive strategy for osteoporosis. Zoledronic acid (ZOL) has been indicated to play an essential role in regulating bone mineral density and has already been used in large clinical trials. However, the effects of ZOL on osteoclastogenesis remain to be fully elucidated. Therefore, the present study aimed to determine the effects of ZOL on osteoclastogenesis, and to explore the corresponding signalling pathways. By using a cell viability assay, as well as in vitro osteoclastogenesis, immunofluorescence and resorption pit assays, we demonstrated that ZOL (0.1‑5 µM) suppressed receptor activator of nuclear factor‑κB ligand (RANKL)‑induced osteoclast differentiation and bone resorptive activity. Furthermore, western blot analysis and reverse transcription‑quantitative PCR indicated that ZOL inhibited the RANKL‑induced activation of NF‑κB and the phosphorylation of JNK in RAW264.7 cells, and subsequently decreased the expression of osteoclastogenesis‑associated genes, including calcitonin receptor, tartrate‑resistant acid phosphatase and dendritic cell‑specific transmembrane protein. ZOL inhibited osteoclast formation and resorption in vitro by specifically suppressing NF‑κB and JNK signalling. On the whole, the findings of this study indicate that ZOL may serve as a potential agent for the treatment of osteoclast‑associated diseases, including osteoporosis.

Clostridioides difficile, a major cause of antibiotic-associated diarrhea and pseudomembranous colitis, is increasingly resistant to antibiotics and poses a significant threat due to its regulated virulence. The alternative sigma factor σ⁵⁴ (rpoN) is known to regulate gene expression broadly, affecting microbial adaptation. Our study investigates how rpoN influences gene expression, physiology, and virulence in C. difficile. We used a modified CRISPR-Cpf1 system to create a rpoN deletion strain (∆rpoN) and a complemented strain (::rpoN) in the CD630 background, comparing their phenotypes and transcriptomes with the wild type. The ∆rpoN strain showed reduced motility and increased susceptibility to seven antibiotics, including β-lactams (amoxicillin, ampicillin, cefoxitin), nitroimidazoles (metronidazole), glycopeptides (vancomycin), fluoroquinolones (norfloxacin), and aminoglycosides (kanamycin). It also exhibited increased toxin gene expression, higher autolysis rates, and enhanced cytotoxicity and virulence in animal models. Additionally, rpoN deletion led to a decrease in glucose metabolic rate, which we attribute to the downregulation of glycolytic enzymes. Transcriptomic analysis indicated that reduced motility in ∆rpoN is due to downregulation of flagellar biosynthesis genes, while increased autolysis is linked to upregulation of autolysin genes like cwp19 and acd. The enhanced release of toxins due to higher autolysis rates contributes to the increased virulence of ∆rpoN. Our findings establish rpoN as a global regulator critical for antibiotic resistance, motility, metabolism, toxin production, and pathogenicity in C. difficile, suggesting its potential as a therapeutic target to mitigate virulence and resistance.

Background: Clostridium difficileinfection (CDI) is the leading cause of nosocomial diarrhea. Co-colonization of key bacterial taxa may prevent the transition from asymptomaticC. difficilecolonization to CDI. However, little is known about the composition of key bacterial taxa in asymptomatic patients.Methods: In the present study, the culture method was used to examine the composition of stool microbiota in two asymptomatic patients from Guizhou, China.Results: A total of 111 strains were isolated and phylogenetic relationships were determined by 16S ribosomal gene sequencing and Molecular Evolutionary Genetics Analysis version 7. The results demonstrated thatEscherichia(33.3%, 37/111),Clostridium(24.3%, 27/111) andEnterococcus(11.7%, 13/111) exhibited a high ratio in asymptomatic patients. These isolates derived from two phyla:Firmicutes(51.3%, 57/111) andProteobacteria(44.1%, 49/111). In addition, co-colonization of human pathogensFusobacterium nucleatum,Ralstonia pickettii,Klebsiella pneumoniae,Klebsiella quasipneumoniaeandClostridium tertiumwithC. difficilewas identified. To the best of our knowledge, these pathogens have not been co-isolated withC. difficilepreviously.Conclusions: In summary, the present study identified the composition of fecal microbiota in two asymptomatic patients in Guizhou, China. These results suggested that co-infection with human pathogens may be ubiquitous during CDI progression.

Background: Clostridioides difficile infection (CDI) represents a significant global public health concern. The Phosphatidylinositol Glycan Class L ( pig-L ) gene in C. difficile encodes an enzyme critical for the biosynthesis of Glycosylphosphatidylinositol (GPI) anchor, which play a vital role in bacterial surface protein localization and function. Methods: To investigate the role of pig-L in C. difficile pathogenesis, we utilized CRISPR-Cas9 gene editing to generate a pig-L knockout strain and a complementation strain in the wild-type (WT) background. Phenotypic characterization of these strains was performed through a suite of assays, including virulence assays, biofilm formation assays, oxidative stress sensitivity testing, and antimicrobial susceptibility testing. Proteomics analysis was conducted to identify differentially expressed proteins in the knockout strain. Results: Deletion of the pig-L gene resulted in a significant reduction in C. difficile virulence, decreased biofilm formation, and increased susceptibility to oxidative stress. Proteomic analysis revealed significant alterations in protein expression, with 170 proteins exhibiting upregulation and 101 proteins demonstrating downregulation in the knockout strain. Complementation of the pig-L gene partially restored the phenotypes observed in the deletion strain. Conclusion: These findings demonstrate that the pig-L gene functions as a crucial regulator of C. difficile virulence, biofilm formation, and peroxide resistance. Targeting the pig-L gene or its downstream effectors represents a promising avenue for the development of novel therapeutic strategies to effectively control C. difficile infection.

Excessive dieting (ED), a common weight-control strategy, often causes neurological and emotional disturbances, yet its gut-brain interaction mechanisms remain unclear. Employing a short-term dietary (SDR) adult male rabbit model, we found that SDR can induced cerebral cortex up-regulation of the immune-related genes (e.g., C1QC, SAA3) enriched in NF-kappa B signaling pathways, contrasted with down-regulation of sex hormone-related genes (e.g., PRLR, SPA17) implicated in metabolic homeostasis. Furthermore, dysregulated expression of metabolic genes (e.g., PPM1J, GALNT18) in the cecum of the SDR group interacted to impair the immune protection pathways related to intestinal mucosa. Then, SDR significantly increased the cecal Firmicutes/Bacteroidetes ratio (from 3.38 to 5.57) and reduced microbial diversity. Specifically, beneficial bacteria involved in tryptophan metabolism and neurotransmitter synthesis (e.g., Bacteroidales_bacterium, Alistipes_indistinctus) decreased, whereas bile acid-metabolizing bacteria (e.g., Clostridium_sp._CAG:710, Ruminococcus_sp._Marseille-P6503) linked to increase energy metabolism. The top 20 genes from the brain-gut axis analysis (e.g., ITPR1, CAMK4, CDK5R1) were enriched in critical neural pathways like axon guidance, GABAergic synapse, and long-term potentiation. Notably, key neurodevelopmental genes (e.g., GPR37, GPX3) correlated with these microbial shifts, implicating oxidative stress, synaptic plasticity, and mitochondrial function in microbiota-host crosstalk. This study highlights a "microbial-metabolism-neural" axis in SDR, providing novel targets for future obesity intervention strategies.

Periodontitis is a chronic inflammatory disease driven by bacterial biofilms and host immune responses. Conventional treatments, such as mechanical debridement and antibiotics, have inherent limitations. These include antibiotic resistance and incomplete pathogen elimination. Consequently, there is a growing need for novel therapeutic strategies. Probiotics represent a promising adjunct therapy due to their potential to modulate the oral microbiota and host immunity. Probiotic bacteria were isolated from infant fecal samples, and Guiyang fermented cabbage using spread plating on Man, Rogosa, and Sharpe (MRS) agar. Following isolation, candidate strains were screened in vitro for anti-inflammatory activity using an inflammatory cell model, assessing the levels of IL-1β, IL-6, and TNF-α. Subsequently, a ligature-induced rat periodontal disease model was used to evaluate the in vivo therapeutic efficacy. Clinical periodontal parameters, including probing depth and gingival bleeding, were systematically evaluated, along with assessment of inflammatory markers. In vitro screening of the anti-inflammatory capacity of six isolated probiotic strains and their combinations revealed that Lacticaseibacillus rhamnosus LHJY (LGG), Weissella confusa LHJY (W. c), and the LGG + W. c combination exhibited superior anti-inflammatory activity compared to other strains. In vitro experiments demonstrated that the probiotic combination effectively inhibited the growth of key periodontal pathogens. Furthermore, the combination exhibited potential for modulating inflammatory responses. In a ligature-induced rat periodontitis model, the combined probiotic treatment significantly improved clinical parameters, including probing depth and gingival bleeding, compared to single-strain treatments, while concurrently suppressing host inflammatory responses. These results suggest that the combined intervention of LGG and W. c, exerts a significant effect in regulating local immunity, inhibiting pathogenic bacteria, and alleviating periodontal tissue destruction. This study provides novel experimental evidence for the efficacy of a specific probiotic combination. It offers a theoretical rationale for developing targeted therapies to treat periodontitis. The online version contains supplementary material available at 10.1186/s12903-026-08015-0.