Recovery of Previously Uncultured Bacterial Genera from Three Mediterranean Sponges

Sponges often harbour a dense and diverse microbial community. Presently, a large discrepancy exists between the cultivable bacterial fraction from sponges and the community in its natural environment. Here, we aimed to acquire additional insights into cultivability of (previously uncultured) bacteria from three sponge species, namely Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis, by studying bacterial growth on five media in the form of 60 communities scraped from plates without antibiotics, as well as in the form of individual isolates that were grown on these media supplemented with antibiotics. We applied (double-)barcoded 16S ribosomal RNA (rRNA) gene amplicon sequencing for species identification. We show that previously uncultured bacteria can be cultivated using conventional plating and that application of antibiotics in the media can serve to capture a greater bacterial diversity. Moreover, we present criteria to address an important caveat of the plate scraping method whereby bacteria may be detected that did not actually grow. Fourteen out of 27 cultivated novel taxa (<95% identity of the 16S rRNA gene amplicon to reported species) belong to Actinobacteria, which indicates the presence of a large untapped reservoir of bioactive compounds. Three Flavobacteriaceae spp. were isolated that potentially constitute two new genera and one new species.