Actinobacteria from Cave Moonmilk Deposits: Isolation of Rare Taxa and Assessment of Antimicrobial Activities

During the golden age of the discovery of natural products of microbial origin, numerous antimicrobial agents were identified, and their exploitation led to a marked reduction in mortality from infectious diseases. Today, the increasing resistance of pathogenic microorganisms to most antibiotics available on the market represents a major threat to global health. This situation, combined with a growing interest in natural products in the agri-food sector, has revived the search for new bioactive compounds. In this context, the exploration of extreme or still poorly studied ecological niches appears to be a promising strategy for accessing new microorganisms and new metabolites. Caves and their calcareous deposits known as ‘moonmilk’ are among such environments, where an unexpectedly rich microbial diversity has been identified despite their nutrient-poor conditions. To explore this potential, this work focused on the following objectives: (i) improving the isolation of rare or difficult-to-culture actinobacteria from moonmilk, (ii) characterizing their phylogenetic diversity, and (iii) investigating the bioactive potential of the isolated strains, with a particular focus on the production of antibacterial and antifungal compounds.
The first part focused on optimizing isolation methods. By combining the rehydration-centrifugation method with other strategies, it was possible to obtain forty isolates belonging to eight different genera, including rare taxa such as Kocuria, Micrococcus and Micromonospora, isolated for the first time from moonmilk. Bioactivity assays revealed that several strains exhibited strong antimicrobial activity, and genomic analysis highlighted a significant potential for the production of natural compounds.
The second part focused on the antifungal activity observed in many strains isolated from moonmilk against Rasamsonia argillacea, an emerging pathogen notably associated with cystic fibrosis. Bipartite Petri dish assays revealed that this inhibition occurred only under certain conditions and was mediated by volatile compounds rather than diffusible metabolites. The effect, reproduced with phylogenetically diverse bacteria and against several fungi, suggested the involvement of ubiquitous volatiles. A comparative volatilomics approach led to the identification of two compounds, dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS), whose production was specifically increased under conditions inducing antifungal activity. Exposure tests with the pure compounds confirmed their ability to inhibit R. argillacea.
Taken together, these results show that moonmilk deposits represent a still under-exploited source of actinobacterial diversity and natural metabolites. The optimisation of isolation methods enabled access to previously inaccessible microorganisms, while the study of their bioactive potential revealed antibacterial and antifungal activities of particular interest. This work highlights the importance of further exploring understudied environments to address the challenges associated with antimicrobial resistance.