Rhizocarpon geographicum Lichen Discloses a Highly Diversified Microbiota Carrying Antibiotic Resistance and Persistent Organic Pollutant Tolerance

Abstract

As rock inhabitants, lichens are exposed to extreme and fluctuating abiotic conditions associated with poor sources of nutriments. These extreme conditions confer to lichens the unique ability to develop protective mechanisms. Consequently, lichen-associated microbes disclose highly versatile lifestyles and ecological plasticity, enabling them to withstand extreme environments. Because of their ability to grow in poor and extreme habitats, bacteria associated with lichens can tolerate a wide range of pollutants, and they are known to produce antimicrobial compounds. In addition, lichen-associated bacteria have been described to harbor ecological functions crucial for the evolution of the lichen holobiont. Nevertheless, the ecological features of lichen-associated microbes are still underestimated. To explore the untapped ecological diversity of lichen-associated bacteria, we adopted a novel culturomic approach on the crustose lichen Rhizocarpon geographicum. We sampled R. geographicum in French habitats exposed to oil spills, and we combined nine culturing methods with 16S rRNA sequencing to capture the greatest bacterial diversity. A deep functional analysis of the lichen-associated bacterial collection showed the presence of a set of bacterial strains resistant to a wide range of antibiotics and displaying tolerance to Persistent Organic Pollutants (POPs). Our study is a starting point to explore the ecological features of the lichen microbiota.

Keywords: antibiotic resistance; culturomics; holobiont; lichen microbiome.

Figure 1

Rhizocarpon geographicum populations. (a) Locations where populations were sampled across France (north and south). Colored dots represent the R. geographicum populations (6 geographic sites), and the color gradient within the same localities indicates the different harvest locations. Four populations per site were sampled. (b) MONT populations, (c) ITXA populations, and (d) CARO populations, which are examples of the diversity of the terrestrial and maritime habitats harboring R. geographicum.

Figure 2

Number of bacterial strains isolated from each medium. The bar plot is based on the abundance of each bacterial strain (expressed in %) reported in the y-axis and calculated by dividing the number of strains recovered on each medium by the number of the total strains. The x-axis indicates the isolation medium. The class color scale indicates the overall isolated bacteria (light blue) and the unique bacterial clusters (dark blue) based on their 16S sequences after CD-Hist analysis.

Figure 3

Abundance of the bacterial classes and families obtained on all isolation media. (a) The bar plot on the frequency (N° of each bacterial class/N° of total isolated strains) of the bacterial classes found on Rhizocarpon geographicum. (b) Bar plot on the frequency of the bacterial families included in the α-Proteobacteria class. (c) Bar plot on the frequency of the bacterial families included in the Actinomycetia class. (d) Bar plot on the frequency of the bacterial families included in the β-Proteobacteria class.

Figure 4

Heatmaps on the antibiotic resistance coefficient. Heatmaps were performed by lsmeans obtained by running a liner-mixed model on the antibiotic resistance coefficient and by controlling for the block and replication effect. (a) Heatmap on the antibiotic coefficients obtained when strains were inoculated in the stationary phase; (b) heatmap on the antibiotic coefficients obtained when strains were inoculated in the exponential phase. The names of the tested strains are reported in the y-axis; antibiotics are reported in the x-axis. Abbreviations: Genta = Gentamicin, Kana = Kanamycin, Vanco = Vancomycin, Chlora = Chloramphenicol, Tetrac = Tetracycline, Rifam = Rifampicin, Strep = Streptomycin, Coli = Colistin, Pen = Penicillin G, Carbe = Carbenicillin, Amp = Ampicillin, Cefa = Cefalexin. The color scale from dark red to light yellow represents high antibiotic resistance (dark red) and no resistance (light yellow).