Dennis Versluis

Background: Members of the bacterial familyFlavobacteriaceaeare widely distributed in the marine environment and often found associated with algae, fish, detritus or marine invertebrates. Yet, little is known about the characteristics that drive their ubiquity in diverse ecological niches. Here, we provide an overview of functional traits common to taxonomically diverse members of the familyFlavobacteriaceaefrom different environmental sources, with a focus on the Marine clade. We include seven newly sequenced marine sponge-derived strains that were also tested for gliding motility and antimicrobial activity.Results: Comparative genomics revealed that genome similarities appeared to be correlated to 16S rRNA gene- and genome-based phylogeny, while differences were mostly associated with nutrient acquisition, such as carbohydrate metabolism and gliding motility. The high frequency and diversity of genes encoding polymer-degrading enzymes, often arranged in polysaccharide utilization loci (PULs), support the capacity of marineFlavobacteriaceaeto utilize diverse carbon sources. Homologs of gliding proteins were widespread among all studiedFlavobacteriaceaein contrast to members of other phyla, highlighting the particular presence of this feature within theBacteroidetes. Notably, not all bacteria predicted to glide formed spreading colonies. Genome mining uncovered a diverse secondary metabolite biosynthesis arsenal ofFlavobacteriaceaewith high prevalence of gene clusters encoding pathways for the production of antimicrobial, antioxidant and cytotoxic compounds. Antimicrobial activity tests showed, however, that the phenotype differed from the genome-derived predictions for the seven tested strains.Conclusions: Our study elucidates the functional repertoire of marineFlavobacteriaceaeand highlights the need to combine genomic and experimental data while using the appropriate stimuli to unlock their uncharted metabolic potential.

BackgroundMembers of the bacterial family Flavobacteriaceae are widely distributed in the marine environment and often found associated with algae, fish, detritus or marine invertebrates. Yet, little is known about the characteristics that drive their ubiquity in diverse ecological niches. Here, we provide an overview of functional traits common to taxonomically diverse members of the family Flavobacteriaceae from different environmental sources, with a focus on the Marine clade. We include seven newly sequenced marine sponge-derived strains that were also tested for gliding motility and antimicrobial activity. ResultsComparative genomics revealed that genome similarities appeared to be correlated to 16S rRNA gene phylogeny, while differences were mostly associated with nutrient acquisition, such as carbohydrate metabolism and gliding motility. The high frequency and diversity of genes encoding polymer-degrading enzymes support the capacity of marine Flavobacteriaceae to utilize diverse carbon sources. Homologs of gliding proteins were widespread among all studied Flavobacteriaceae in contrast to members of other phyla, highlighting the particular presence of this feature within the Bacteroidetes. Notably, not all gliding bacteria formed spreading colonies. Genome mining uncovered a diverse secondary metabolite biosynthesis arsenal of Flavobacteriaceae with high prevalence of gene clusters encoding pathways for the production of antimicrobial, antioxidant and cytotoxic compounds. Antimicrobial activity tests showed, however, that the phenotype differed from the genome-derived predictions for the seven tested strains.ConclusionsOur study elucidates the functional repertoire of marine Flavobacteriaceae and highlights the need to combine genomic and experimental data while using the appropriate stimuli to unlock their uncharted metabolic potential.

In order to minimize re-discovery of already known anti-infective compounds, we focused our screening approach on understudied, almost untapped marine environments including marine invertebrates and their associated bacteria. Therefore, two sea cucumber species, Holothuria leucospilota and Stichopus vastus, were collected from Lampung (Indonesia), and 127 bacterial strains were identified by partial 16S rRNA-gene sequencing analysis and compared with the NCBI database. In addition, the overall bacterial diversity from tissue samples of the sea cucumbers H. leucospilota and S. vastus was analyzed using the cultivation-independent Illumina MiSEQ analysis. Selected bacterial isolates were grown to high densities and the extracted biomass was tested against a selection of bacteria and fungi as well as the hepatitis C virus (HCV). Identification of putative bioactive bacterial-derived compounds were performed by analyzing the accurate mass of the precursor/parent ions (MS¹) as well as product/daughter ions (MS²) using high resolution mass spectrometry (HRMS) analysis of all active fractions. With this attempt we were able to identify 23 putatively known and two previously unidentified precursor ions. Moreover, through 16S rRNA-gene sequencing we were able to identify putatively novel bacterial species from the phyla Actinobacteria, Proteobacteria and also Firmicutes. Our findings suggest that sea cucumbers like H. leucospilota and S. vastus are promising sources for the isolation of novel bacterial species that produce compounds with potentially high biotechnological potential.

The emergence of bacterial pathogens that are resistant to clinical antibiotics poses an increasing risk to human health. An important reservoir from which bacterial pathogens can acquire resistance is the human gut microbiota. However, thus far, a substantial fraction of the gut microbiota remains uncultivated and has been little-studied with respect to its resistance reservoir-function. Here, we aimed to isolate yet uncultivated resistant gut bacteria by a targeted approach. Therefore, faecal samples from 20 intensive care patients who had received the prophylactic antibiotic treatment selective digestive decontamination (SDD), i.e. tobramycin, polymyxin E, amphotericin B and cefotaxime, were inoculated anaerobically on porous aluminium oxide chips placed on top of poor and rich agar media, including media supplemented with the SDD antibiotics. Biomass growing on the chips was analysed by 16S rRNA gene amplicon sequencing, showing large inter-individual differences in bacterial cultivability, and enrichment of a range of taxonomically diverse operational taxonomic units (OTUs). Furthermore, growth of Ruminococcaceae (2 OTUs), Enterobacteriaceae (6 OTUs) and Lachnospiraceae (4 OTUs) was significantly inhibited by the SDD antibiotics. Strains belonging to 16 OTUs were candidates for cultivation to pure culture as they shared ≤95% sequence identity with the closest type strain and had a relative abundance of ≥2%. Six of these OTUs were detected on media containing SDD antibiotics, and as such were prime candidates to be studied regarding antibiotic resistance. One of these six OTUs was obtained in pure culture using targeted isolation. This novel strain was resistant to the antibiotics metrodinazole and imipenem. It was initially classified as member of the Ruminococcaceae, though later it was found to share 99% nucleotide identity with the recently published Sellimonas intestinalis BR72^T. In conclusion, we show that high-throughput cultivation-based screening of microbial communities can guide targeted isolation of bacteria that serve as reservoirs of antibiotic resistance.

Wide application of antibiotics has contributed to the evolution of multi-drug resistant human pathogens, resulting in poorer treatment outcomes for infections. In the marine environment, seawater samples have been investigated as a resistance reservoir; however, no studies have methodically examined sponges as a reservoir of antibiotic resistance. Sponges could be important in this respect because they often contain diverse microbial communities that have the capacity to produce bioactive metabolites. Here, we applied functional metagenomics to study the presence and diversity of functional resistance genes in the sponges Aplysina aerophoba, Petrosia ficiformis and Corticium candelabrum. We obtained 37 insert sequences facilitating resistance to D-cycloserine (n=6), gentamicin (n=1), amikacin (n=7), trimethoprim (n=17), chloramphenicol (n=1), rifampicin (n=2) and ampicillin (n=3). Fifteen of 37 inserts harboured resistance genes that shared <90% amino acid identity with known gene products, whereas on 13 inserts no resistance gene could be identified with high confidence, in which case we predicted resistance to be mainly mediated by antibiotic efflux. One marine-specific ampicillin-resistance-conferring β-lactamase was identified in the genus Pseudovibrio with 41% global amino acid identity to the closest β-lactamase with demonstrated functionality, and subsequently classified into a new family termed PSV. Taken together, our results show that sponge microbiota host diverse and novel resistance genes that may be harnessed by phylogenetically distinct bacteria.

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.