First report of Aquitalea spp. From cave aquatic environments of Roraima Tepui, Guayana highlands

Aquitalea has mainly been identified from freshwater environments, including lakes, springs, waterfalls, and wetland-associated habitats. Notwithstanding the importance of wetland bacteria in mineral processes, knowledge about this genus from cave aquatic ecosystems is limited. Here, we investigated the presence of Aquitalea spp. in freshwaters from Roraima Sur Cave (Guayana Highlands), the world's longest and deepest quartzite karst system, using culture, microscopy and 16S rRNA gene sequencing. Although initially we attempted Helicobacter isolation using HP selective medium, this approach unexpectedly revealed four clones with nearly complete 16S rRNA sequences showing 99.34-99.49% identity to Aquitalea aquatica strain HSC-21Su07. Phylogenetic analysis positioned these clones within the Aquitalea genus, forming a clade with A. aquatica, A. aquatilis, and A. palustris. Furthermore, fluorescent in situ hybridization (FISH) with a eubacterial probe detected coccoid red-fluorescent cells in HP enrichments. Such morphology suggests the typical size and volume reduction experienced by rod gram-negative bacteria in the viable but non culturable state, not previously described in this genus. Given the limited microbial exploration in subterranean silica ecosystems, targeted cultivation of Aquitalea spp. is crucial to uncover novel species and elucidate their physiological adaptations to oligotrophic conditions. Our findings represent the first report of these bacteria within a quartzite cave, extending their known habitats and laying the groundwork for investigating their possible ecological role, as well as putative associations with geochemical processes within silica-rich, energy-deprived environments of evolutionary importance on Earth.