Recovery of Previously Uncultured Bacterial Genera from Three Mediterranean Sponges

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-017-9766-4) contains supplementary material, which is available to authorized users.

Cultivation of Bacteria From Aplysina aerophoba: Effects of Oxygen and Nutrient Gradients

“…Defining cultivability as the number of taxa that could be cultured divided by the total number of taxa detected in the sponge tissue, we report here a recovery of 2% of the spongeassociated taxa by cultivation. This falls into the range of other sponge-microbe cultivation experiments (Sipkema et al, 2011;Lavy et al, 2014;Keren et al, 2015;Esteves et al, 2016;Versluis et al, 2017), which report a recovery rate of 0.1 to 14%. Even though different sponge species harbor speciesspecific and highly divergent microbiota, it has been shown that their cultivable fractions are comparable (Li et al, 2007;Hardoim et al, 2014) since rare generalists are proliferating under laboratory conditions.…”

“…Plates were divided in quadrants, and kanamycin disks (30 µg Thermo Fisher Scientific, Waltham, MA, United States, Oxoid Kanamycin Antimicrobial Susceptibility Disks) were placed in the approximate center of each of the quadrants, to prevent the growth of Pseudovibrio spp. in limited areas of the plates (Versluis et al, 2017). For each medium and its three dilutions, three replicate plate sets were used for incubation with (AP_positive) and without (AP_negative) aeroplysinin-1, and a negative control.…”

“…Amongst these shared taxa, the alphaproteobacterial Pseudovibrio and Ruegeria OTUs were the most frequently cultivated. Pseudovibrio species represent low abundant sponge symbionts which are known as versatile, opportunistic bacteria capable to adapt to a wide range of cultivation conditions (Muscholl-Silberhorn et al, 2008;Bondarev et al, 2013;Versluis et al, 2017;Fróes et al, 2018). With this study we could furthermore add aeroplysinin-1 to the list of antibiotic resistances exhibited by Pseudovibrio species (Versluis et al, 2017).…”

“…However, detailed disentanglement of prokaryotic functionalities is majorly hindered by the lack of cultured representatives of spongeassociated bacteria and archaea. Despite numerous approaches, none of the predominant sponge associated phylotypes belonging to the Acidobacteria, Chloroflexi, Cyanobacteria, Nitrospirae, Poribacteria or Thaumarchaeota could be cultivated in vitro (Sipkema et al, 2011;Lavy et al, 2014;Steinert et al, 2014;Keren et al, 2015;Versluis et al, 2017). One of the reasons might include the inability to recreate sponge-mesohyl conditions adequately, since sponges and their microbiomes evolved complex networks of cross-feeding and other interactions (Pande and Kost, 2017).…”

“…One example is the antibiotic aeroplysinin-1 (AP), protecting damaged sponge tissue from bacterial infections (Ebel et al, 1997;Thoms et al, 2004;Niemann et al, 2015). Despite various cultivation approaches utilizing antibiotics (Pimentel-Elardo et al, 2003;Sipkema et al, 2011;Versluis et al, 2017), the inclusion of sponge-derived antimicrobials as a selection criterion has remained scarce.…”