Isolation, Characterization, and Antibacterial Activity of Hard-to-Culture Actinobacteria from Cave Moonmilk Deposits

Abstract

Cave moonmilk deposits host an abundant and diverse actinobacterial population that has a great potential for producing novel natural bioactive compounds. In our previous attempt to isolate culturable moonmilk-dwelling Actinobacteria, only Streptomyces species were recovered, whereas a metagenetic study of the same deposits revealed a complex actinobacterial community including 46 actinobacterial genera in addition to streptomycetes. In this work, we applied the rehydration-centrifugation method to lessen the occurrence of filamentous species and tested a series of strategies to achieve the isolation of hard-to-culture and rare Actinobacteria from the moonmilk deposits of the cave "Grotte des Collemboles". From the "tips and tricks" that were tested, separate autoclaving of the components of the International Streptomyces Project (ISP) medium number 5 (ISP5) medium, prolonged incubation time, and dilution of the moonmilk suspension were found to most effectively improve colony forming units. Taxonomic analyses of the 40 isolates revealed new representatives of the Agromyces, Amycolatopsis, Kocuria, Micrococcus, Micromonospora, Nocardia, and Rhodococcus species, as well as additional new streptomycetes. The applied methodologies allowed the isolation of strains associated with both the least and most abundant moonmilk-dwelling actinobacterial operational taxonomic units. Finally, bioactivity screenings revealed that some isolates displayed high antibacterial activities, and genome mining uncovered a strong potential for the production of natural compounds.

Keywords: Amycolatopsis; Cave microbiology; Streptomyces; antibiotics; rare Actinobacteria; secondary metabolite; siderophore; unculturability.

Figure 1

Total number of colony forming units (CFUs) according to the different media and methods used. The different media were inoculated with a diluted moonmilk suspension (10⁻² in blue and 10⁻³ in red) prepared according to the rehydration-centrifugation method and incubated for up to two months. Data obtained after 16 days (16 d) and 44 days (44 d) of incubation are displayed. The “separated” label indicates that the autoclaving of agar and phosphate was performed separately from the other components of the media. Abbreviations: Fe, 20 μM of FeCl₃; DFB, 1 μM of desferrioxamine B.

Figure 2

Effect of reactive oxygen species on bacterial growth in International Streptomyces Project (ISP) medium number 5 (ISP5) medium. Growth in (a) ISP5 medium prepared according to the standard protocol, in (b) the same medium but with catalase injected on the paper disc in the center of the plate, and in (c) the ISP5 medium prepared with autoclaving of agar and phosphate separate from the other media components. (d) CFUs on (a), (b) and (c).

Figure 3

Phenotypes of actinobacterial strains isolated in this study. For each isolate, the front and back of the Petri dish-grown bacteria are presented together with the phenotype of a single colony. The phylotype number for Streptomyces isolates is indicated in parentheses.

Figure 3

Phenotypes of actinobacterial strains isolated in this study. For each isolate, the front and back of the Petri dish-grown bacteria are presented together with the phenotype of a single colony. The phylotype number for Streptomyces isolates is indicated in parentheses.

Figure 4

16S rRNA-based phylogenetic tree of moonmilk-dwelling Streptomyces strains. Strains isolated using the rehydration-centrifugation (RC) method (MMun) are shown in grey, and strains isolated in our previous study (MM) are in black. Phylotype affiliations are indicated in parentheses. The alignment of nearly complete 16S rRNA sequences had 1538 unambiguously aligned positions for 92 strains, but only the 1342 positions without missing nucleotides were used to infer the tree. The evolutionary model was GTR + Γ₄ and bootstrap values are based on 100 pseudoreplicates (bootstrap values <50% are not displayed). Saccharopolyspora erythraea was used as outgroup. The scale bar represents 0.02 substitutions per site.

Figure 5

Membership of MMun strains isolated in this study to the actinobacterial operational taxonomic units (OTUs) identified by the high-throughput sequencing (HTS) metagenetic study. OTUs in blue boxes are those for which we isolated a first representative strain in this study. Note that for MMun149 and MMun172, the search of their associated OTU was performed at the family level (Micrococcaceae and Micromonosporaceae) and not at the genus level, as the HTS study [13] did not allow us to unambiguously assign the sequences of the 16S rRNA V6–V7 amplicons to the Micrococcus and Micromonospora genera. Symbol: *, due to the phylogenetic divergence of its V6–V7 region and based on the full sequence of its 16S rRNA gene (extracted from the genome sequence), MMun149 was manually affiliated to OTU23 even though its “closest” OTU in the tree was OTU141 (Figure S2).

Figure 6

Heatmap illustrating antibacterial activities of the moonmilk actinobacteria isolated in this study. The size of the inhibition zone (in cm) is given for each strain.

Figure 7

Selected representative examples of the cross-streak results. MMun strain numbers, media, and tested cultures are mentioned.

Figure 8

Number of strains with antibacterial activity, broken down by actinobacterial genus and test conditions.

Figure 9

Illustration of the paradoxical zone phenomenon (or Eagle effect) observed with Klebsiella pneumonia and Citrobacter freundii. MMun156 was chosen as a control to show non-affected growth of K. pneumoniae on the ISP7 medium. All conditions where an Eagle effect was observed are marked with a “star” symbol in Figure 6.