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Several highly antibiotic-resistant infections are caused by methicillin-resistant Staphylococcus aureus (MRSA) due to its ability to form biofilms. Antibiofilm strategies cannot be developed without the use of comprehensive microscopic techniques. This study aimed to examine the antibiofilm activity of linoleic acid, ferulic acid, vanillic acid, and iso-vanillic acid against the MRSA and its biofilm. The microdilution method was used to study the antibacterial activity of selected natural products against MRSA in vitro. SEM was used to examine the effect of selected natural products on the degradation of extracellular polymeric substances of MRSA biofilms. Metabolic activity was monitored using the tetrazolium (MTT) assay. SEM images revealed that sub-inhibitory concentrations of linoleic acid, vanillic acid, ferulic acid, and isovanillic acid significantly prevented MRSA cell adhesion, and altered bacterial morphology. Whereas, treatment of mature biofilms with higher concentrations of vanillic acid, linoleic acid, ferulic acid, and isovanillic acid resulted in significant destruction of biofilms and their extracellular polymeric substances. Linoleic acid, ferulic acid, vanillic acid, and iso-vanillic acid, therefore, could help in the prevention of MRSA biofilm formation, and the removal of preformed biofilms, which will lead to the development of combinatorial strategies against biofilm-related MRSA infections.

Dental caries arises from dysbiosis of the oral microbiome, wherein acidogenic pathogens such as Streptococcus mutans dominate over protective commensals. Sodium fluoride (NaF) remains a cornerstone in caries prevention; however, its limited efficacy in high-risk individuals and the emergence of fluoride-resistant strains highlight the need for enhanced therapeutic strategies. The present study investigates the synergistic antibacterial potential of NaF combined with ricinoleic acid (RA), a bioactive fatty acid with established antimicrobial activity. Checkerboard and time-kill assays revealed a strong synergistic interaction between RA and NaF, with a combinatorial minimum inhibitory concentration (CMIC) of 64 + 128 μg/mL, respectively. Complete eradication of S. mutans was achieved within 120 min at CMIC (64 + 128 μg/mL). The combination significantly disrupted mature biofilms, resulting in an 82% reduction in total biomass as confirmed by confocal microscopy. Mechanistic analyses indicated that RA triggered reactive oxygen species (ROS) generation and membrane perturbation, which facilitated enhanced NaF uptake and intracellular antibacterial action. Moreover, the RA-NaF combination markedly inhibited key virulence attributes of S. mutans, including acidogenesis and aciduricity, by approximately 76% and 71%, respectively. The treatment also exhibited a sustained post-antimicrobial effect lasting up to 12 h and did not induce bacterial resistance upon repeated exposure. Collectively, these findings highlight that RA potentiates the anticariogenic efficacy of NaF through a multi-targeted cellular mechanism involving oxidative stress induction, membrane disruption, and metabolic suppression. This synergistic combination represents a promising and fluoride-efficient strategy for the prevention and management of dental caries.

To investigate, through immunohistochemistry, the interaction between the epithelial-mesenchymal transition (EMT) status and the tumor microenvironment (TME) in oral tongue squamous cell carcinomas (OTSCCs). A total of 61 cases of OTSCC were selected. In all cases, the EMT status was determined through the analysis of E-cadherin and N-cadherin immunoexpression, while the TME was characterized using TGF-β and α-SMA for cancer-associated fibroblasts, CD163 for M2 macrophages, and Foxp3 for regulatory T cells. Associations between the expression patterns of these proteins and clinicopathological features, as well as overall survival (OS) and disease-free survival (DFS) parameters, were investigated. High α-SMA expression in OTSCC cases was significantly associated with a positive EMT status (p = 0.011). This marker also showed a significant positive correlation with TGF-β immunostaining (p = 0.028). Foxp3 and CD163 also demonstrated a significant positive correlation (p = 0.010). Multivariate analysis identified high Foxp3 (p = 0.011) and TGF-β (p = 0.002) expression as independent prognostic factors for OS, and high TGF-β expression (p = 0.031) as an independent prognostic factor for DFS. The results suggest an interaction among TME biomarkers, with implications for EMT development and progression, as well as for the prognosis of OTSCC.

Biofilms are microbial communities enclosed in an extracellular polymeric substance (EPS), significantly contributing to antimicrobial resistance (AMR) in medical, industrial, and environmental settings. Their matrix enhances microbial survival, inhibits antibiotic penetration, and facilitates horizontal gene transfer, worsening the AMR crisis. Conventional antimicrobial treatments often fail against biofilms, necessitating novel therapeutic strategies. Emerging biofilm-targeted interventions, such as nanotechnology-based antimicrobials, bacteriophage therapy, and CRISPR-Cas9 gene editing, offer promising solutions. Nanoparticles improve drug delivery, bacteriophages selectively lyse resistant bacterial populations, and CRISPR-Cas9 disrupts AMR-related genes and biofilm virulence factors. Additionally, AI and ML are advancing biofilm prediction models and antimicrobial optimization, paving the way for precision-targeted interventions. This review explores biofilm biology and next-generation biofilm control strategies, with a focus on AI-driven bioinformatics. Future research should focus on clinical translation, regulatory standardization, and scalable implementation in healthcare and industrial settings to combat biofilm-associated AMR.

Fusarium Keratitis is a corneal infection that causes blindness if left untreated. Immune profiling of Fusarium-infected corneas may facilitate the development of an effective immunotherapy. The ex vivo caprine (goat) cornea model examines immune cell and cytokine activity in response to Fusarium keratitis. Dissected caprine corneas maintained in ex vivo culture conditions were infected with the clinical Fusarium isolate (CSH_1) to study local immune responses. Immuno-phenotyping was performed using immune cell markers CD45, CD11b, and HLA-DR/DQ, followed by quantification of IL-1ß, IL-6, IFN-γ, TGF-ß, IL-4, IL-10, and MCP-1 through ELISA. The efficacy of antifungals was evaluated by sectioning drug-treated infected corneas by H&E staining and cytokine profiling to assess the immunomodulatory effect of antifungal treatment. Results show that CD45⁺ lymphocytes, recognized as a universal marker for immune cells, were abundant in the cornea. Activated myeloid cells (CD 11b⁺HLA DR/DQ⁺) and cytokines IL-1ß, IL-6, IFN-γ, TGF-ß, IL-4, IL-10, and MCP-1 were significantly elevated in infected corneas relative to uninfected samples. Among the drugs tested, posaconazole showed complete inhibition of Fusarium, while natamycin and amphotericin B showed the least inhibition and moderate inhibition was shown by voriconazole. The ex vivo model offers a valuable framework for evaluating pre-clinical efficacy of different treatment protocols for fungal keratitis on local tissue.

Pseudomonas aeruginosa is a prominent uropathogen associated with biofilm-related urinary tract infections. It can coexist in a biofilm environment with other pathogens, including Staphylococcus aureus, and the availability of metabolites in urine may influence interspecies interactions that can be cooperative or competitive. Here, a dual-species biofilm model consisting of uropathogenic and reference strains of P. aeruginosa and S. aureus was used to understand their coexistence under different nutritional conditions using synthetic urine supplemented with creatinine, glucose, albumin, and haem. A dual-species biofilm was developed using equal initial cell densities of pathogens. Biofilm intensity was quantified by crystal violet staining. Biofilm biomass, growth, ureolysis were estimated and compared with mono-species cultures. P. aeruginosa formed higher biofilm than S. aureus (p < 0.01) under mono-species culture. Overall, dual-species biofilm intensity was significantly higher than mono-species biofilm (p < 0.01), except in albumin, where S. aureus mono-species biofilm was higher. Biofilm biomass enumeration indicates near-equilibrium coexistence of species within the biofilm matrix. The ureolytic activity correlated with growth and biofilm observations. Our study highlights the complex interactions in dual-species biofilms and emphasizes the need for further studies involving diverse uropathogenic strains to assess metabolite influences on biofilm structural dynamics and its effect on antibiotic responses for targeted therapeutics.

Biofilms are structured communities of microorganisms embedded in extracellular polymeric substances (EPSs) that adhere to surfaces or interfaces. These biofilms are resistant to antimicrobial agents and are responsible for persistent infections. An effective strategy is needed to inhibit the biogenesis of polymeric substances by Enterococcus faecalis. This study explored a synergistic approach using an indole terpenoid molecule, rhodethrin, combined with chloramphenicol to disrupt EPS production by E. faecalis. TGA revealed three stages of decomposition: moisture content (15% at 35°C–200°C), pyrolysis temperature (40%–60% at 200°C–400°C), and polysaccharide crystals (< 10% at < 600°C). X-ray diffraction analysis indicated that the EPS consisted of 40%–60% crystalline and 60%–75% amorphous domains. FTIR analysis also identified various functional groups, like aliphatic CH₂, hydroxyl groups, aliphatic methyl groups, asymmetrical C=H stretching, and amine groups. LCMS analysis provided insights into the molecular mass of EPS constituents. SEM analysis revealed a condensed matrix characterized by polymeric carbohydrates and filamentous structures. The results showed a significant therapeutic importance of their combined effects on the biosynthesis of EPSs in E. faecalis. Further research on their molecular mechanisms and clinical applications is essential to harness their benefits and develop effective treatment against biofilm-associated infections caused by E. faecalis.

Intraoperative margin evaluation is very important in the management of breast cancer patients, specifically in breast conserving therapy in which attaining negative margins is a prerequisite. Microscopic involvement of margins shows two to three times increase in local recurrence. Frozen section is mostly employed for intraoperative assessment of margins. It has, however, the inherent limitations of being expensive, more time-consuming, with difficult serial sectioning, associated technical issues regarding adipose tissue freezing and tissue consumption during the procedure that could leave limited tissue for the permanent sections, thus compromising the final diagnosis, in addition to its intrinsic procedural standardization deficits. A variety of other techniques and methodologies have been developed that can complement frozen section or be employed independently for increased diagnostic efficacy and accuracy like intraoperative touch preparation cytology, assessment by margin probes, flow cytometry, optical coherence tomography (OCT), integrated OCT with microscopy or with dye-enhanced field polarization, polarization-sensitive multimodal imaging, and nonlinear microscopy. This review article states what defines free margins, highlights the different histological options for margin evaluation, and reflects on the evolving methodologies to diagnose the margins intraoperatively.