Balneotherapeutic water springs, such as those with thermal, saline, sulfur, or any other characteristics, have recently been the subject of phylogenetic studies with a closer focus on the description and/or isolation of phylogenetically novel or biotechnologically interesting microorganisms. Generally, however, most such microorganisms are rarely obtained in pure culture or are even, for now, unculturable under laboratory conditions. In this culture-dependent study of radioactive water springs of Jáchymov (Joachimstahl), Czech Republic, we investigated a combination of classical cultivation approaches with those imitating sampling source conditions. Using these environmentally relevant cultivation approaches, over 1,000 pure cultures were successfully isolated from 4 radioactive springs. Subsequent dereplication yielded 121 unique taxonomic units spanning 44 genera and 9 taxonomic classes, ~10% of which were identified as hitherto undescribed taxa. Genomes of the latter were sequenced and analyzed, with a special focus on endogenous defense systems to withstand oxidative stress and aid in radiotolerance. Due to their origin from radioactive waters, we determined the resistance of the isolates to oxidative stress. Most of the isolates were more resistant to menadione than the model strain Deinococcus radiodurans DSM 20539T. Moreover, isolates of the Deinococcacecae, Micrococcaceae, Bacillaceae, Moraxellaceae, and Pseudomonadaceae families even exhibited higher resistance in the presence of hydrogen peroxide. In summary, our culturomic analysis shows that subsurface water springs contain diverse bacterial populations, including as-yet-undescribed taxa and strains with promising biotechnological potential. Furthermore, this study suggests that environmentally relevant cultivation techniques increase the efficiency of cultivation, thus enhancing the chance of isolating hitherto uncultured microorganisms. IMPORTANCE The mine Svornost in Jáchymov (Joachimstahl), Czech Republic is a former silver-uranium mine and the world's first and for a long time only radium mine, nowadays the deepest mine devoted to the extraction of water which is saturated with radon and has therapeutic benefits given its chemical properties. This healing water, which is approximately 13 thousand years old, is used under medical supervision for the treatment of patients with neurological and rheumatic disorders. Our culturomic approach using low concentrations of growth substrates or the environmental matrix itself (i.e., water filtrate) in culturing media combined with prolonged cultivation time resulted in the isolation of a broad spectrum of microorganisms from 4 radioactive springs of Jáchymov which are phylogenetically novel and/or bear various adaptive or coping mechanisms to thrive under selective pressure and can thus provide a wide spectrum of capabilities potentially exploitable in diverse scientific, biotechnological, or medical disciplines.

• Klíčová slova
• environmentally relevant cultivation approaches, extremophiles, novel taxa, oxidative stress response, radioactive water springs, subsurface microbiology,
• MeSH
• Bacteria MeSH
• fylogeneze MeSH
• lidé MeSH
• mladiství MeSH
• peroxid vodíku MeSH
• radium * MeSH
• radon * MeSH
• síra MeSH
• stříbro MeSH
• uran * MeSH
• vitamin K 3 MeSH
• voda MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peroxid vodíku MeSH
• radium * MeSH
• radon * MeSH
• síra MeSH
• stříbro MeSH
• uran * MeSH
• vitamin K 3 MeSH
• voda MeSH

BACKGROUND: The extreme conditions of thermal springs constitute a unique aquatic habitat characterized by low nutrient contents and the absence of human impacts on the microbial community composition. Thus, these springs may host phylogenetically novel microorganisms with potential use in biotechnology. With this hypothesis in mind, we examined the microbial composition of four thermal springs of the world-renowned spa town of Karlovy Vary (Carlsbad), Czechia, which differ in their temperature and chemical composition. RESULTS: Microbial profiling using 16S rRNA gene sequencing revealed the presence of phylogenetically novel taxa at various taxonomic levels, spanning from genera to phyla. Many sequences belonged to novel classes within the phyla Hydrothermae, Altiarchaeota, Verrucomicrobia, and TA06. Cultivation-based methods employing oligotrophic media resulted in the isolation of 44 unique bacterial isolates. These include strains that withstand concentrations of up to 12% NaClw/v in cultivation media or survive a temperature of 100 °C, as well as hitherto uncultured bacterial species belonging to the genera Thermomonas, Paenibacillus, and Cellulomonas. These isolates harbored stress response genes that allow them to thrive in the extreme environment of thermal springs. CONCLUSIONS: Our study is the first to analyze the overall microbial community composition of the renowned Karlovy Vary thermal springs. We provide insight into yet another level of uniqueness of these springs. In addition to their unique health benefits and cultural significance, we demonstrate that these springs harbor phylogenetically distinct microorganisms with unusual life strategies. Our findings open up avenues for future research with the promise of a deeper understanding of the metabolic potential of these microorganisms.

• Klíčová slova
• Amplicon sequencing analysis, Cultivation analysis, Phylogenetic novelty, Thermal water springs,
• Publikační typ
• časopisecké články MeSH