Microbial Polysaccharides Extracted from Different Mature Muds of the Euganean Thermal District Show Similar Anti-Inflammatory Activity In Vivo.

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.

Thermal mud maturation: organic matter and biological activity.

OBJECTIVE: Many of the therapeutic and cosmetic treatments offered in spas are centred on mud therapy, to moisturize the skin and prevent skin ageing and rheumatic diseases. Thermal mud is a complex matrix composed of organic and inorganic elements which contribute to its functions. It is a natural product derived from the long mixing of clay and thermal water. During its maturation, organic substances are provided by the microalgae, which develop characteristic of the composition of thermal water. METHODS: The aim of this study was to identify methods for introducing objective parameters as a basis for characterizing thermal mud and assessing its efficacy. Samples of thermal mud were collected at the Saturnia spa, where there are several sulphureous pools. The maturation of the mud was evaluated by organic component determination using extractive methods and chromatographic analysis (HPLC, GC-MS, SPME). We also studied the radical scavenging activity of mud samples at different stages of maturation, in a homogeneous phase, using several tests (DPPH, ORAC, ABTS). RESULTS: We identified several classes of compounds: saturated and unsaturated fatty acids, hydroxyl acids, dicarboxylic acids, ketoacids, alcohols and others. SPME analysis showed the presence of various hydrocarbons compounds (C(11) -C(17)) and long-chain alcohols (C(12) -C(16)). Six or seven months seemed appropriate to complete the process of maturation, and the main effect of maturation time was the increase of lipids. Six-month mud showed the highest activity. The hydrophilic extract was more active than the lipophilic extract. CONCLUSION: The results indicate that maturation of thermal mud can be followed on the basis of the changes in its organic composition and antioxidant properties along the time. They also highlight the need to develop reference standards for thermal muds in relation to assess their use for therapeutic and cosmetic purposes.

Diversity of cyanobacteria from thermal muds (Balaruc-Les-Bains, France) with the description of Pseudochroococcus coutei gen. nov., sp. nov.

Cyanobacteria are able to synthesize a high diversity of natural compounds that account for their success in the colonization of a variety of ecological niches. Many of them have beneficial properties. The mud from the thermal baths of Balaruc-Les-Bains, one of the oldest thermal baths in France, has long been recognized as a healing treatment for arthro-rheumatic diseases. To characterize the cyanobacteria living in these muds, several strains were isolated from the water column and biofilms of the retention basin and analyzed using a polyphasic approach. Morphological, ultrastructural and molecular (16S rRNA gene and 16S-23S ITS region sequencing) methods were employed to identify nine cyanobacterial strains belonging to the orders Chroococcales, Synechococcales, Oscillatoriales and Nostocales. The combination of morphological and genetic characteristics supported the description of a new genus and species with the type species as Pseudochroococcus coutei. The taxonomic diversity in the muds from Thermes de Balaruc-Les-Bains appears higher than previously documented, providing new candidate taxa for their observed therapeutic properties.

Microbiological Hygiene Quality of Thermal Muds: A Pilot Study in Pelotherapy Facilities of the Euganean Thermal District (NE Italy).

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.

Anti-Inflammatory, Antioxidant, and Wound-Healing Properties of Cyanobacteria from Thermal Mud of Balaruc-Les-Bains, France: A Multi-Approach Study.

Background: The Balaruc-les-Bains' thermal mud was found to be colonized predominantly by microorganisms, with cyanobacteria constituting the primary organism in the microbial biofilm observed on the mud surface. The success of cyanobacteria in colonizing this specific ecological niche can be explained in part by their taxa-specific adaptation capacities, and also the diversity of bioactive natural products that they synthesize. This array of components has physiological and ecological properties that may be exploited for various applications. Methods: Nine cyanobacterial strains were isolated from Balaruc thermal mud and maintained in the Paris Museum Collection (PMC). Full genome sequencing was performed coupled with targeted and untargeted metabolomic analyses (HPLC-DAD and LC-MS/MS). Bioassays were performed to determine antioxidant, anti-inflammatory, and wound-healing properties. Results: Biosynthetic pathways for phycobiliproteins, scytonemin, and carotenoid pigments and 124 metabolite biosynthetic gene clusters (BGCs) were characterized. Several compounds with known antioxidant or anti-inflammatory properties, such as carotenoids, phycobilins, mycosporine-like amino acids, and aeruginosins, and other bioactive metabolites like microginins, microviridins, and anabaenolysins were identified. Secretion of the proinflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8 appeared to be inhibited by crude extracts of Planktothricoides raciborskii PMC 877.14, Nostoc sp. PMC 881.14, and Pseudo-chroococcus couteii PMC 885.14. The extract of the Aliinostoc sp. PMC 882.14 strain was able to slightly enhance migration of HaCat cells that may be helpful in wound healing. Several antioxidant compounds were detected, but no significant effects on nitric oxide secretion were observed. There was no cytotoxicity on the three cell types tested, indicating that cyanobacterial extracts may have anti-inflammatory therapeutic potential without harming body cells. These data open up promising uses for these extracts and their respective molecules in drugs or thermal therapies.