A Phenotypic and Genotypic Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits

Abstract

Moonmilk speleothems of limestone caves host a rich microbiome, among which Actinobacteria represent one of the most abundant phyla. Ancient medical texts reported that moonmilk had therapeutical properties, thereby suggesting that its filamentous endemic actinobacterial population might be a source of natural products useful in human treatment. In this work, a screening approach was undertaken in order to isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles in Belgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesis of antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusively affiliated to the genus Streptomyces and clustered into 31 distinct phylotypes displaying various pigmentation patterns and morphological features. Phylotype representatives were tested for antibacterial and antifungal activities and their genomes were mined for secondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases (NRPSs), and polyketide synthases (PKS). The moonmilk Streptomyces collection was found to display strong inhibitory activities against a wide range of reference organisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth of Gram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% of the cave strains induced strong growth suppression against the multi-drug resistant Rasamsonia argillacea, a causative agent of invasive mycosis in cystic fibrosis and chronic granulomatous diseases. No correlation was observed between the global antimicrobial activity of an individual strain and the number of NRPS and PKS genes predicted in its genome, suggesting that approaches for awakening cryptic metabolites biosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, our work supports the common belief that moonmilk might effectively treat various infectious diseases thanks to the presence of a highly diverse population of prolific antimicrobial producing Streptomyces, and thus may indeed constitute a promising reservoir of potentially novel active natural compounds.

Assessment of the Potential Role of Streptomyces in Cave Moonmilk Formation

“…The dominant role of bacteria in calcification is attributed to metabolic activities which increase the pH of the environment (above pH 8) and therefore favor a shift of the CO 2 – ${\text{HCO}}_3^{-}$ – ${\text{CO}}_3^{2-}$ equilibrium toward carbonate ions which precipitate with Ca 2+ ions. We screened 31 representative Streptomyces strains isolated from moonmilk (see Supplementary Figure 2 retrieved from Maciejewska et al, 2016) for their metabolic activities that could lead to a raise in pH, including ureolysis, peptides/amino acids ammonification, and dissimilatory nitrate/nitrite reduction to ammonia (Figure 1). Although these assays were performed under laboratory conditions, a qualitative assessment of these processes allowed ranking the tested phylotype representatives according to their metabolic performance, and therefore their potential to drive biomineralization through an increase in the extracellular pH.…”

Antimicrobial activities of culturable microorganisms (actinomycetes and fungi) isolated from Chaabe Cave, Algeria

“…Taking into consideration the numerous studies on cave microbial diversity, it is worth noting that each cave seems to be unique and diverse in its microbial compositions, however, there is a trend of finding dominating actinobacterial communities in many caves . The reason for the large recovery of Streptomyces in caves can lie in their spore dispersal, the ability to use a large variety of nutrient sources in synthetic rich media and their more rapid growth in comparison to other genera, recognized as rare Actinobacteria, which are isolated much less frequently (Subramani & Aalbersberg, 2013;Maciejewska et al, 2016).…”

“…However, we observed an absence of antimicrobial activity against P. aeruginosa. Maciejewska et al (2016) studied the cultivable Streptomyces isolated from cave moonmilk deposits which showed a much stronger antibacterial activity against Gram-positive bacteria (94% of the phylotypes) than against Gram-negative bacteria (71% of the phylotypes). In addition, the data showed that the antifungal activity was exhibited in 14.89% of all isolates.…”

“…From that time on, a number of papers reported the isolation of cave actinobacteria with production of bioactive compounds (Nakaew et al, 2009;Hodges et al, 2012;Cheeptham et al, 2013;Ghosh et al, 2016;Maciejewska et al, 2016). The most promising was the discovery of cervimycin A-D, a polyketide complex produced by a strain of Streptomyces tendae, isolated from Grotta dei Cervi, in Italy.…”

Bagremycin Antibiotics and Ferroverdin iron-chelators are synthetized by the Same Gene Cluster

“…Strains of Streptomyces lunaelactis, including the type-strain MM109 T , were previously isolated from cave moonmilk deposits [25][26][27]. The strain MM109 T produces a green pigment that was attributed to the biosynthesis of ferroverdin A [25], a homotrimer of p-vinylphenyl-3nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) complexed with one ferrous ion [18,19].…”