Laser-assisted microbial culturomics.

Even though metagenomics have revolutionized the characterization of the human microbiome, detailed mechanistic studies are impracticable, as there is a dearth of robust culture collections. We now describe the development and use of a laser-assisted culturomics platform, incorporating the elements of a bioprinter, the culture conditions, the methods to characterize the microorganisms and a biobank. With laser-assisted bioprinting, the microorganisms can be rapidly and precisely transferred from clinical biofilms to highly organized arrays of microbial colonies, which are suitable for co-culturing and molecular analyses. The presented technique has propagated 99 of 100 microbial species and recovered 79% of abundant species from dental plaque in accordance with full 16S rRNA gene profiling of 691,199 sequences. Microscopy, spectroscopy and enzyme assays have been used to guide isolations. Processing of oral biofilms from four individuals has yielded 249 representative isolates, from 14 classes and 124 species in total. Functional profiling with bioprinting has indicated commensals which could potentially contribute to disease development. Isolates from peri-implantitis cover 85.4% of the transcriptionally active clinical biofilms at genus level. Taken together, this work provides the basis for generating on-demand culture collections and biofilms for research and clinical use.