Genome analysis of a coral-associated bacterial consortium highlights complementary hydrocarbon degradation ability and other beneficial mechanisms for the host.

Here we report the oil degradation genetic potential of six oil-degrading bacteria (ODB), previously used as a bioremediation consortium, isolated from the hydrocoral Millepora alcicornis and seawater. The strains were identified as Halomonas sp. (LC_1), Cobetia sp. (LC_6), Pseudoalteromonas shioyasakiensis (LC_2), Halopseudomonas aestusnigri (LC_3), Shewanella algae (LC_4), and Brucella intermedia (LC_5). The taxonomic identification differed from that of the original paper when we used whole genome gene markers instead of just 16S rRNA gene. Genes responsible for the degradation of aromatic hydrocarbons and n-alkanes were found in all genomes, although different (and complementary) steps of the metabolic pathways were unique to each strain. Genes for naphthalene and toluene degradation were found in various strains. We annotated quinate degradation genes in LC_6, while LC_3 and LC_5 presented genes for biosurfactant and rhamnolipid biosynthesis. We also annotated genes related to beneficial mechanisms for corals, such as genes involved in nitrogen and DMSP metabolism, cobalamin biosynthesis and antimicrobial compounds production. Our findings reinforce the importance of using bacterial consortia for bioremediation approaches instead of single strains, due to their complementary genomic arsenals. We also propose a genome-based framework to select complementary ODB that can provide additional benefits to coral health.

Shedding light on the composition of extreme microbial dark matter: alternative approaches for culturing extremophiles.

More than 20,000 species of prokaryotes (less than 1% of the estimated number of Earth's microbial species) have been described thus far. However, the vast majority of microbes that inhabit extreme environments remain uncultured and this group is termed "microbial dark matter." Little is known regarding the ecological functions and biotechnological potential of these underexplored extremophiles, thus representing a vast untapped and uncharacterized biological resource. Advances in microbial cultivation approaches are key for a detailed and comprehensive characterization of the roles of these microbes in shaping the environment and, ultimately, for their biotechnological exploitation, such as for extremophile-derived bioproducts (extremozymes, secondary metabolites, CRISPR Cas systems, and pigments, among others), astrobiology, and space exploration. Additional efforts to enhance culturable diversity are required due to the challenges imposed by extreme culturing and plating conditions. In this review, we summarize methods and technologies used to recover the microbial diversity of extreme environments, while discussing the advantages and disadvantages associated with each of these approaches. Additionally, this review describes alternative culturing strategies to retrieve novel taxa with their unknown genes, metabolisms, and ecological roles, with the ultimate goal of increasing the yields of more efficient bio-based products. This review thus summarizes the strategies used to unveil the hidden diversity of the microbiome of extreme environments and discusses the directions for future studies of microbial dark matter and its potential applications in biotechnology and astrobiology.

Species-specific relationships between deep sea sponges and their symbiotic Nitrosopumilaceae.

Sponges thrive in the deep, dark and nutrient-depleted ocean and may rely on microbial symbionts for carbon acquisition and energy generation. However, these symbiotic relationships remain largely unexplored. In this study, we analyze the microbiome of deep-sea sponges and show that ammonia-oxidizing archaea (AOA) of the family Nitrosopumilaceae make up at least 75% of the microbial communities of the sponges Aphrocallistes sp., Farrea sp. and Paratimea sp.. Given the known autotrophic metabolism of AOAs, this implies that these sponge holobionts can have the capacity for primary production in the deep-sea. We also show that specific AOA lineages are highly specific towards their hosts, hinting towards an unprecedent vertical transmission of these symbionts in deep-sea sponges. Our results show that the ecology and evolution of symbiotic relationships in deep-sea sponge is distinct from that of their shallow-water counterparts.

In situ devices can culture the microbial dark matter of corals.

Most microorganisms found in environmental samples have never been cultured and can often only be explored through molecular or microscopic approaches. Here, we adapt the use of in situ diffusion-based devices to culture "yet-to-be-cultured" microorganisms associated with coral mucus and compare this with a traditional culturing method. The culturability of microorganisms associated with mucus of the coral Pocillopora damicornis increased by 420% and 570% with diffusion growth chambers and microwell chip devices, respectively, compared with the traditional method tested. The obtained cultures represent up to 64.4% of the total diversity of amplicon sequence variants (ASVs) found in the mucus of the coral P. damicornis. In addition, some previously uncultured microorganisms, such as members of the family Nitrosopumilaceae and halophilic/halotolerant bacteria were cultured. Our results validate alternative microbial culturing strategies to culture coral-associated microorganisms, while significantly increasing the culturability of previous microbial dark matter.

Methods and Strategies to Uncover Coral-Associated Microbial Dark Matter.

The vast majority of environmental microbes have not yet been cultured, and most of the knowledge on coral-associated microbes (CAMs) has been generated from amplicon sequencing and metagenomes. However, exploring cultured CAMs is key for a detailed and comprehensive characterization of the roles of these microbes in shaping coral health and, ultimately, for their biotechnological use as, for example, coral probiotics and other natural products. Here, the strategies and technologies that have been used to access cultured CAMs are presented, while advantages and disadvantages associated with each of these strategies are discussed. We highlight the existing gaps and potential improvements in culture-dependent methodologies, indicating several possible alternatives (including culturomics and in situ diffusion devices) that could be applied to retrieve the CAM "dark matter" (i.e., the currently undescribed CAMs). This study provides the most comprehensive synthesis of the methodologies used to recover the cultured coral microbiome to date and draws suggestions for the development of the next generation of CAM culturomics.

Fluopsin C for Treating Multidrug-Resistant Infections: In vitro Activity Against Clinically Important Strains and in vivo Efficacy Against Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of multidrug-resistant (MDR) organisms in hospital infections is causing a global public health crisis. The development of drugs with effective antibiotic action against such agents is of the highest priority. In the present study, the action of Fluopsin C against MDR clinical isolates was evaluated under in vitro and in vivo conditions. Fluopsin C was produced in cell suspension culture of Pseudomonas aeruginosa LV strain, purified by liquid adsorption chromatography and identified by mass spectrometric analysis. Bioactivity, bacterial resistance development risk against clinically important pathogenic strains and toxicity in mammalian cell were initially determined by in vitro models. In vivo toxicity was evaluated in Tenebrio molitor larvae and mice. The therapeutic efficacy of intravenous Fluopsin C administration was evaluated in a murine model of Klebsiella pneumoniae (KPC) acute sepsis, using six different treatments. The in vitro results indicated MIC and MBC below 2 µg/mL and low bacterial resistance development frequency. Electron microscopy showed that Fluopsin C may have altered the exopolysaccharide matrix and caused disruption of the cell wall of MDR bacteria. Best therapeutic results were achieved in mice treated with a single dose of 2 mg/kg and in mice treated with two doses of 1 mg/kg, 8 h apart. Furthermore, acute and chronic histopathological studies demonstrated absent nephrotoxicity and moderate hepatotoxicity. The results demonstrated the efficacy of Fluopsin C against MDR organisms in in vitro and in vivo models, and hence it can be a novel therapeutic agent for the control of severe MDR infections.