EXPLOITING MARINE BIODIVERSITY: THE POTENTIAL OF UNCULTIVABLE MICROORGANISMS FOR THE IDENTIFICATION OF NOVEL ANTIMICROBIAL COMPOUNDS

Antimicrobial resistance has spread dramatically in last 60 years leading to an increase in the number of deaths due to infection diseases. The excessive and often inappropriate use of antimicrobial drugs has led to the development of a new group of microorganisms, the Multidrug Resistant (MDR) bacteria, which show resistance toward the most common antibiotics. This phenomenon is becoming a serious threat to the public health and the economy. The bioprospecting of marine and extreme environments has yielded a noteworthy number of novel molecules with biotechnological applications from a wide range of macro and microorganisms, representing a very promising strategy to counteract MDR bacteria. The main gap is to have access to the real microbial biodiversity because less than 1% of microorganisms is cultivable in the laboratory conditions. In this project, new antimicrobial compounds have been discovered from bacteria and fungi by different strategies. In the first work, following a bioprospecting pipeline, Antarctic shallow water sediments were used to isolate microorganisms that were screened for their capability to inhibit the growth of selected MDR bacteria. A bioassay-guided purification approach allowed the identification of rhamnolipids (a class of glycolipids well known as biosurfactants) produced by a Pseudomonas gessardii strain able to strongly inhibit MDR strains, in particular Gram-positive bacteria. These molecules have many biotechnological applications, especially in bioremediation field and, over last years, as antimicrobial compounds. The second work focuses the attention on the improvement of cultivation methods, exploiting a new device, the Miniaturized Culture Chip (MCC), for the isolation of “not-common” or novel bacteria. The innovation of this system is the possibility to grow microorganisms directly in their natural habitat simulating environmental conditions. By using the MCC an unexplored Antarctic strain, Aequorivita sp., was isolated. A genome mining approach on Aequorivita sp. was applied identifying the main biosynthetic gene clusters. The evaluation of its bioactive potential led to the discovery of three new intracellular aminolipids showing antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) and anthelmintic activity against the nematode Caenorhabditis elegans. This was the first report that demonstrated the bioactivity of this strain and stimulates the research of new cultivation method by which obtain new and unexplored sources of compounds. The third work instead, gave more attention to marine fungi, well-known producers of secondary metabolites. Extracts of nine fungi isolated from the green alga Flabellia petiolata, collected from the Mediterranean Sea, resulted to be active against some pathogen bacteria. In particular, the chemical profiling of three marine fungi, Beauveria bassiana (MUT 4865), Knufia petricola (MUT 4979) and a new fungal species (MUT 4861) expressed a high compounds variability, novelty and activity. The successful results of this project that combines the bioprospecting of hostile environments with the optimisation of the critical step of the “biodiscovery pipeline” confirmed the huge potential of microorganisms as producers of novel bioactive compounds and demonstrated that this research field is far from to be fully exploited

Enhanced production of biobased, biodegradable, Poly(3-hydroxybutyrate) using an unexplored marine bacterium Pseudohalocynthiibacter aestuariivivens, isolated from highly polluted coastal environment.

The production and disposal of plastics from limited fossil reserves, has prompted research for greener and sustainable alternatives. Polyhydroxyalkanoates (PHAs) are biocompatible, biodegradable, and thermoprocessable polyester produced by microbes. PHAs found several applications but their use is limited due to high production cost and low yields. Herein, for the first time, the isolation and characterization of Pseudohalocynthiibacter aestuariivivens P96, a marine bacterium able to produce surprising amount of PHAs is reported. In the best growth condition P96 was able to reach a maximum production of 4.73 g/L, corresponding to the 87 % of total cell dry-weight. Using scanning and transmission microscopy, lab-scale fermentation, spectroscopic techniques, and genome analysis, the production of thermoprocessable polymer Polyhydroxybutyrate P(3HB), a PHAs class, endowed with mechanical and thermal properties comparable to that of petroleum-based plastics was confirmed. This study represents a milestone toward the use of this unexplored marine bacterium for P(3HB) production. [Abstract copyright: Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Linear Aminolipids with Moderate Antimicrobial Activity from the Antarctic Gram-Negative Bacterium Aequorivita sp.

The combination of LC-MS/MS based metabolomics approach and anti-MRSA activity-guided fractionation scheme was applied on the Gram-negative bacterium Aequorivita sp. isolated from shallow Antarctic sea sediment using a miniaturized culture chip technique. This methodology afforded the isolation of three new (1⁻3) and four known (4⁻7) N-terminal glycine- or serine-bearing iso-fatty acid amides esterified with another iso-fatty acid through their C-3 hydroxy groups. The chemical structures of the new compounds were elucidated using a set of spectroscopic (NMR, [α]D and FT-IR) and spectrometric (HRMS, HRMS/MS) methods. The aminolipids possessing an N-terminal glycine unit (1, 2, 4, 5) showed moderate in vitro antimicrobial activity against MRSA (IC50 values 22⁻145 μg/mL). This is the first in-depth chemistry and biological activity study performed on the microbial genus Aequorivita

Isolation and characterization of strain exiguobacterium sp. Krl4, a producer of bioactive secondary metabolites from a Tibetan glacier

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 ◦C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds

Antibiotics from Deep-Sea Microorganisms : Current Discoveries and Perspectives

Funding: This research was funded by the MarPipe project: Improving the flow in the pipeline of the next generation of marine biodiscovery scientists, funded through the European Commission H2020-MSCA-ITN-ETN scheme, GA721421.Peer reviewedPublisher PD

Isolation by Miniaturized Culture Chip of an Antarctic bacterium Aequorivita sp. with antimicrobial and anthelmintic activity

Microbes are prolific sources of bioactive molecules; however, the cultivability issue has severely hampered access to microbial diversity. Novel secondary metabolites from as-yet-unknown or atypical microorganisms from extreme environments have realistic potential to lead to new drugs with benefits for human health. Here, we used a novel approach that mimics the natural environment by using a Miniaturized Culture Chip allowing the isolation of several bacterial strains from Antarctic shallow water sediments under near natural conditions. A Gram-negative Antarctic bacterium belonging to the genus Aequorivita was subjected to further analyses. The Aequorivita sp. genome was sequenced and a bioinformatic approach was applied to identify biosynthetic gene clusters. The extract of the Aequorivita sp. showed antimicrobial and anthelmintic activity towards Multidrug resistant bacteria and the nematode Caenorhabditis elegans. This is the first multi-approach study exploring the genomics and biotechnological potential of the genus Aequorivita that is a promising candidate for pharmaceutical applications

Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus: a New Polyextremophilic Marine Fungus from Barents Sea

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillusprotuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 μg/mL. © 2018, Springer Science+Business Media, LLC, part of Springer Nature

Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica)

Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains

Identification Of A Sorbicillinoid-Producing Aspergillus Strain With Antimicrobial Activity Against Staphylococcus Aureus: A New Polyextremophilic Marine Fungus From Barents Sea

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillusprotuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 μg/mL