The Euganean Thermal District (Italy) represents the oldest and largest thermal center in Europe, and its therapeutic mud is considered a unique product whose beneficial effects have been documented since Ancient Roman times. Mud properties depend on the heat and electrolytes of the thermal water, as well as on the bioactive molecules produced by its biotic component, mainly represented by cyanobacteria. The investigation of the healing effects of compounds produced by the Euganean cyanobacteria represents an important goal for scientific validation of Euganean mud therapies and for the discovering of new health beneficial biomolecules. In this work, we evaluated the therapeutic potential of exopolysaccharides (EPS) produced by Phormidium sp. ETS05, the most abundant cyanobacterium of the Euganean mud. Specifically, Phormidium EPS resulted in exerting anti-inflammatory and pro-resolution activities in chemical and injury-induced zebrafish inflammation models as demonstrated using specific transgenic zebrafish lines and morphometric and expression analyses. Moreover, in vivo and in vitro tests showed no toxicity at all for the EPS concentrations tested. The results suggest that these EPS, with their combined anti-inflammatory and pro-resolution activities, could be one of the most important therapeutic molecules present in the Euganean mud and confirm the potential of these treatments for chronic inflammatory disease recovery.

The Euganean Thermal District has been known since Roman times for the therapeutic properties of peloids, obtained from natural clays that have undergone a traditional maturation process. This leads to the growth of a green microbial biofilm with Cyanobacteria and the target species Phormidium sp. ETS-05 as fundamental components for their ability to synthetize anti-inflammatory molecules. Currently, in-depth studies on the microbiota colonizing Euganean peloids, as in general on peloids utilized worldwide, are missing. This is the first characterization of the microbial community of Euganean thermal muds, also investigating the effects of environmental factors on its composition. We analysed 53 muds from 29 sites (Spas) using a polyphasic approach, finding a stable microbiota peculiar to the area. Differences among mud samples mainly depended on two parameters: water temperature and shading of mud maturation plants. In the range 37–47 °C and in the case of irradiance attenuation due to the presence of protective roofs, a statistically significant higher mud Chl a content was detected. Moreover, in these conditions, a characteristic microbial and Cyanobacteria population composition dominated by Phormidium sp. ETS-05 was observed. We also obtained the complete genome sequence of this target species using a mixed sequencing approach based on Illumina and Nanopore sequencing.

Evaluation of hygienic aspects of thermal mud microbiology is still neglected. This study evaluates the microbiological hygiene quality of thermal muds, providing a comprehensive assessment of the whole mud cultivation chain. Maturing mud, peloid and used mud samples were collected twice in a year from 30 SPAs of the Euganean Thermal District, NE Italy. Samples were processed with an ad hoc laboratory method. The following indicator parameters were assessed: Total Count at 22, 37 and 55 °C; total coliforms; Escherichia coli; enterococci; Staphylococcus aureus; Pseudomonas aeruginosa; sulfite-reducing clostridia; dermatophytes. Statistical significance of differences between the two sampling campaigns and correlation between temperature and indicator parameters were evaluated. One-hundred eighty samples were analyzed. Widespread presence of environmental species was found, as well as hints of possible microorganism transfer from the patient’s skin to the mud. Proper setting of thermal water temperature resulted critical, in terms of hygienic quality. Although optimal maturation should be granted (thermal water at 30–42 °C), a pasteurization step at 60–65 °C is strongly recommended to sanitize peloids before pelotherapy. Facilities re-using thermal muds should also implement a regeneration step at ≥65 °C. Core evaluation of thermal mud hygienic quality could encompass the following guidelines: absence (i.e., 0 colony forming units (CFU)/g) of E. coli, P. aeruginosa, S. aureus and dermatophytes.

The Euganean Thermal District, situated in North-East Italy, is one of Europe’s largest and oldest thermal centres. The topical application of its therapeutic thermal muds is recognised by the Italian Health System as a beneficial treatment for patients suffering from arthro-rheumatic diseases. Polysaccharides produced by the mud microbiota have been recently identified as anti-inflammatory bioactive molecules. In this paper we analysed the efficacy of Microbial-Polysaccharides (M-PS) derived from mature muds obtained at different maturation temperatures, both within and outside the codified traditional mud maturation range. M-PSs were extracted from six mature muds produced by five spas of the Euganean Thermal District and investigated for their chemical properties, monosaccharide composition and in vivo anti-inflammatory potential, using the zebrafish model organism. Additionally, mature muds were characterized for their microbiota composition using Next-Generation Sequencing. The results showed that all M-PSs exhibit similar anti-inflammatory potential, referable to their comparable chemical composition. This consistency was observed despite changes in cyanobacteria populations, suggesting a possible role of the entire microbial community in shaping the properties of these biomolecules. These findings highlight the importance of scientific research in untangling the origins of the therapeutic efficacy of Euganean Thermal muds in the treatment of chronic inflammatory conditions.

Background: Although treatments using thermal water have yielded beneficial effects in respiratory tract infections, the effects of thermal water under experimental conditions similar to those triggered by SARS-CoV-2 have yet to be evaluated. This study aimed to assess whether thermal water could interfere with the interaction between SARS-CoV-2 and host cells and influence inflammatory factors. Methods: Human nasal epithelial primary cells (HNEpCs) were stimulated with SARS-CoV-2 spike protein in the presence or absence of thermal water or tap water. Cell viability, cytokine concentration, ACE2 and TMPRSS2 levels, and ACE2 activity were determined in the cell cultures. Results: Exposure of HNEpCs to spike protein increased IL-6, IL-8, and IL-1β production, with decreased production observed in the presence of thermal water at an optimal dose. Treatment of cells with tap water did not affect cytokine release in unstimulated or spike-stimulated cells. Spike-protein-stimulated HNEpCs showed reduced levels of ACE2, which were partially restored only in the presence of thermal water. Spike protein did not affect the TMPRSS2 levels of the cell lysates. Stimulation with spike protein induced an increase in the concentration of both receptors in the supernatants, while treatment with thermal water reduced TMPRSS2 levels in both the cells and supernatants. Stimulation with spike protein increased ACE2 activity, which was reduced with thermal water. Conclusions: This study shows the regulatory effects of mineral-rich thermal water on spike-protein-induced pro-inflammatory cytokine production and the amount and activity of receptors mainly involved in viral entry, suggesting a potential use of this treatment as a support therapy for SARS-CoV-2 infection of the upper respiratory tract.

Thermal mud produced by spas of the Euganean Thermal District (Italy) has been used since ancient times for therapeutic purposes. Recently, the anti-inflammatory activity of microbial polysaccharides (M-PS), extracted from traditionally maturated muds, was demonstrated using the zebrafish model organism. However, the downstream signalling pathways regulated by M-PS remain largely unknown. In this study, to investigate the underlying mechanisms of inflammation resolution, we performed a transcriptome analysis on zebrafish larvae inflamed with copper sulphate and treated with M-PS. Our findings revealed that M-PS treatment down-regulated the expression of key genes involved in several inflammatory pathways. Gene Set Enrichment Analysis identified eleven up-regulated pathways (e.g., TNF-α signalling via NFκB, IL6–JAK–STAT signalling, p53 pathway, apoptosis, and interferon response) with components reduced in number and expression level in M-PS-treated larvae compared to the inflamed ones. Additionally, seven down-regulated pathways were identified (e.g., transcription factors E2F, MYC, and the G2M checkpoint). DEG-pseudotime analysis further confirmed the association of these genes with the pathways identified by GSEA. These results provide valuable insights into the anti-inflammatory properties of M-PS and the therapeutic potential of Euganean thermal muds for inflammatory diseases.