Prevalence of Bacillus species in the lytic cultural heritage of Santa Lucia alle Malve Rupestrian Church

Santa Lucia alle Malve (SLM) is a unique rupestrian heritage site, entirely carved into limestone. This monument, which was a church in the ancient settlement of Benedictine nuns over a millennium ago in southern Italy, holds exceptional value not only from an architectural and cultural perspective but also in terms of its microbial ecology. Until now, the specific microbiota of this site had remained unexplored. In this study, the bacterial community inhabiting the interior walls of Santa Lucia alle Malve was investigated using a metagenomic approach, alongside the isolation and comprehensive characterization of cultivable strains from various sampling sites. Both methodologies consistently revealed a dominance of sporeforming bacteria from the phylum Bacillota, particularly the genus Bacillus. Notably, most of the cultivable strains belonged to the Bacillus cereus sensu lato group and the Bacillus. licheniformis clade. Despite the high genetic similarity among these microorganisms, each strain exhibited a unique set of phenotypic traits, highlighting the potential complexity of the SLM metabolome. Additionally, two isolates were identified as Bacillus thuringiensis, entomopathogenic bacteria with possible applications in biological pest management. Finally Staphylococcus warneri, a human skin commensal found in the church, suggests human influence on the microbial landscape.