Recent methods for discovering novel bioactive metabolites, specifically antimicrobial agents, from marine-associated micro-organisms.

Marine micro-organisms are a promising source for novel natural compounds with many medical and biotechnological applications. Here, we demonstrate limitations and recent strategies for investigating the marine microbial community for novel bioactive metabolites, specifically those of antimicrobial potential. These strategies include culture-dependent methods such as modifying the standard culture media, including changing the gelling agent, dissolving vehicle, media supplementation and preparation to access a broader range of bacterial diversity from marine samples. Furthermore, we discuss strategies such as in situ cultivation, dilution-to-extinction cultivation and long-term incubation. We are presenting recent applications of culture-independent methods such as genome mining, proteomics profiling and the application of metagenomics as a novel strategy for structure confirmation in the discovery of the marine micro-organism for novel antimicrobial metabolites. We present this review as a simple guide and a helpful resource for those who seek to enter the challenging field of applied marine microbiology.

Schumannella soli sp. nov., a novel actinomycete isolated from mangrove soil by in situ cultivation.

A novel actinobacterial strain, designated 10F1D-1T, was isolated from soil sample collected from Futian mangrove nature reserve, China using of the in situ cultivation technique. Preliminary analysis based on the 16S rRNA gene sequence revealed that strain 10F1D-1T was the member of genus Schumannella with sharing highest sequence similarity (99.7%) to Schumannella luteola DSM 23141T. Phylogenetic analyses based on 16S rRNA gene sequences and core proteome consistently exhibited that strain 10F1D-1T formed a monophyletic clade with Schumannella luteola DSM 23141T. Comparative genomic analyses clearly separated strain 10F1D-1T from the only species of the genus Schumannella based on average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values below the thresholds for species delineation. The genome of strain 10F1D-1T contains the biosynthetic gene clusters for osmoprotectants to adapt to the salt environment of mangrove. Strain 10F1D-1T also contains the biosynthetic gene clusters for bioactive compounds as secondary metabolites. On the basis of the polyphasic analysis, strain 10F1D-1T is considered to represent a novel species of the genus Schumannella, for which the name Schumannella soli sp. nov. (type strain 10F1D-1T = CGMCC1.16699T = JCM 33146T) is proposed.

Emcibacter congregatus sp. nov., isolated from sediment cultured in situ.

A Gram-negative, aerobic, motile, rod-shaped, pale-yellow bacterial strain, designated as ZYLT, isolated from a cultured in situ sediment sample collected from the East China Sea coast, was studied using a polyphasic taxonomic approach. Strain ZYLT grew at 4-30 °C (optimum, 25 °C), at pH 6.0-8.5 (pH 7.0) and with 0-7.0 % (w/v) NaCl (2.0 %). Results of phylogenetic analysis based on 16S rRNA gene sequences clearly showed that strain ZYLT and Emcibacter nanhaiensis HTCJW17T, which was most closely related to strain ZYLT with 93.6 % sequence similarity, clustered together. The genomic DNA G+C content was 51.5 % (genome sequence). The quinone system was composed only of ubiquinone-10. Strain ZYLT possessed C18 : 1ω7c and/or C18 : 1ω6c (summed feature 8), iso-C15 : 0 2-OH and/or C16 : 1ω7c (summed feature 3), C14 : 0 2-OH and C14 : 0 as the major fatty acids. The content of summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c) in strain ZYLT was far greater than that in E. nanhaiensis. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminophospholipids, three unidentified phospholipids, one unidentified aminolipid and four unidentified lipids. One unidentified aminophospholipid and two unidentified lipids present in strain ZYLT were not found in E. nanhaiensis in this research. On the basis of phenotypic, phylogenetic and chemotaxonomic data, strain ZYLT (=KCTC 62328T=JCM 32378T=MCCC 1K03526T) represents a novel species of the genus Emcibacter for which the name Emcibacter congregatus sp. nov. is proposed.

Application of the filter plate microbial trap (FPMT), for cultivating thermophilic bacteria from thermal springs in Barguzin area, eastern Baikal, Russia.

Hot springs are regarded as treasury of valuable thermophiles. Like other bacteria, thermophiles are not easily cultivated using conventional culture methods. We used an advanced cultivation method, the filter plate microbial trap (FPMT), to isolate bacteria from thermal springs. In total, 184 isolates were obtained from five thermal springs using the FPMT and standard agar plate method, and their 16S rRNA gene sequences were analyzed. FPMT allowed us to obtain a culture collection that was larger, richer, and more novel than that obtained by standard cultivation. Seven novel species were obtained using the FPMT technique, whereas only one was isolated using a standard cultivation. We also found clear differences in the patterns of phylogenetic diversity and physiological properties between isolates from two cultivation methods. The results have encouraged us to apply the FPMT method in other extreme environments and offer further support for fostering the development of new cultivation methods.

Frondibacter mangrovi sp. nov., a member of the family Flavobacteriaceae isolated from seawater by in situ cultivation, and emended description of Frondibacter aureus.

A Gram-stain-negative, rod-shaped, non-motile, strictly aerobic, chemoheterotrophic, light-yellow-pigmented bacterium, designated strain 02OK1/10-76T, was isolated from a mangrove estuary in Japan by use of an in situ cultivation technique. Preliminary analysis based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (96.9 %) to Frondibacter aureus A5Q-67T. The DNA G+C content of strain 02OK1/10-76T was 35 mol%; MK-6 was the only menaquinone; and iso-C15 : 0, iso-C17 : 0 3-OH and iso-C16 : 0 3-OH were the major (>10 %) cellular fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, three unidentified aminolipids, an unidentified aminophospholipid and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain represents a novel species of the genus Frondibacter, for which the name Frondibacter mangrovi sp. nov. is proposed. The type strain is 02OK1/10-76T (= KCTC 52666T = NBRC 112695T). An emended description of F. aureus is also provided.

New approaches to antibiotic discovery.

New antibiotics are urgently required by human medicine as pathogens emerge with developed resistance to almost all antibiotic classes. Pioneering approaches, methodologies and technologies have facilitated a new era in antimicrobial discovery. Innovative culturing techniques such as iChip and co-culturing methods which use 'helper' strains to produce bioactive molecules have had notable success. Exploiting antibiotic resistance to identify antibacterial producers performed in tandem with diagnostic PCR based identification approaches has identified novel candidates. Employing powerful metagenomic mining and metabolomic tools has identified the antibiotic'ome, highlighting new antibiotics from underexplored environments and silent gene clusters enabling researchers to mine for scaffolds with both a novel mechanism of action and also few clinically established resistance determinants. Modern biotechnological approaches are delivering but will require support from government initiatives together with changes in regulation to pave the way for valuable, efficacious, highly targeted, pathogen specific antimicrobial therapies.

In-situ rapid cultivation of aerobic granular sludge in A/O bioreactor by using Ca(ClO)2 pretreating sludge.

The efficient utilization of residual sludge and the rapid cultivation of aerobic granular sludge in continuous-flow engineering applications present significant challenges. In this study, aerobic granular cultivation was fostered in a continuous-flow system using Ca(ClO)2-sludge carbon (Ca-SC). Ca-SC retained the original sludge properties, contributing to granular growth in an A/O bioreactor. By day 40, the granule diameters increased to 0.8 mm with the SVI30 decreased by 2.7 times. Moreover, Ca-SC facilitated protein secretion, reaching 98.06 mg/g VSS and enhanced the hydrophobicity to 68.4 %. The continuous-flow aerobic granular sludge exhibited a nutrient removal rate above 90 %. Furthermore, Tessaracoccus and Nitrospira were enriched to promote granular formation and nitrogen removal. The residual sludge was carbonized and reused in the traditional wastewater treatment process to culture granular sludge in situ, aiming to achieve "self-production and self-consumption" of sludge and promote the innovative model of "treating waste with waste" in urban sewage environmental restoration.

Improved Loading Capacity and Viability of Probiotics Encapsulated in Alginate Hydrogel Beads by In Situ Cultivation Method.

The objective of this research was to encapsulate probiotics by alginate hydrogel beads based on an in situ cultivation method and investigate the influences on the cell loading capacity, surface and internal structure of hydrogel beads and in vitro gastrointestinal digestion property of cells. Hydrogel beads were prepared by extrusion and cultured in MRS broth to allow probiotics to grow inside. Up to 10.34 ± 0.02 Log CFU/g of viable cell concentration was obtained after 24 h of in situ cultivation, which broke through the bottleneck of low viable cell counts in the traditional extrusion method. Morphology and rheological analyses showed that the structure of the eventually formed probiotic hydrogel beads can be loosed by the existence of hydrogen bond interaction with water molecules and the internal growth of probiotic microcolonies, while it can be tightened by the acids metabolized by the probiotic bacteria during cultivation. In vitro gastrointestinal digestion analysis showed that great improvement with only 1.09 Log CFU/g of loss in viable cells was found after the entire 6 h of digestion. In conclusion, the current study demonstrated that probiotic microcapsules fabricated by in situ cultivation method have the advantages of both high loading capacity of encapsulated viable cells and good protection during gastrointestinal digestion.

Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle.

BACKGROUND: Ruminants can utilize urea as a dietary nitrogen source owing to their ability to recycle urea-N back to the rumen where numerous ureolytic bacteria hydrolyze urea into ammonia, which is used by numerous bacteria as their nitrogen source. Rumen ureolytic bacteria are the key microbes making ruminants the only type of animals independent of pre-formed amino acids for survival, thus having attracted much research interest. Sequencing-based studies have helped gain new insights into ruminal ureolytic bacterial diversity, but only a limited number of ureolytic bacteria have been isolated into pure cultures or studied, hindering the understanding of ureolytic bacteria with respect to their metabolism, physiology, and ecology, all of which are required to effectively improve urea-N utilization efficiency. RESULTS: We established and used an integrated approach, which include urease gene (ureC) guided enrichment plus in situ agarose microsphere embedding and cultivation under rumen-simulating conditions, to isolate ureolytic bacteria from the rumen microbiome. We optimized the dilutions of the rumen microbiome during the enrichment, single-cell embedding, and then in situ cultivation of microsphere-embedded bacteria using dialysis bags placed in rumen fluid. Metabonomic analysis revealed that the dialysis bags had a fermentation profile very similar to the simulated rumen fermentation. In total, we isolated 404 unique strains of bacteria, of which 52 strains were selected for genomic sequencing. Genomic analyses revealed that 28 strains, which were classified into 12 species, contained urease genes. All these ureolytic bacteria represent new species ever identified in the rumen and represented the most abundant ureolytic species. Compared to all the previously isolated ruminal ureolytic species combined, the newly isolated ureolytic bacteria increased the number of genotypically and phenotypically characterized ureolytic species by 34.38% and 45.83%, respectively. These isolated strains have unique genes compared to the known ureolytic strains of the same species indicating their new metabolic functions, especially in energy and nitrogen metabolism. All the ureolytic species were ubiquitous in the rumen of six different species of ruminants and were correlated to dietary urea metabolism in the rumen and milk protein production. We discovered five different organizations of urease gene clusters among the new isolates, and they had varied approaches to hydrolyze urea. The key amino acid residues of the UreC protein that potentially plays critical regulatory roles in urease activation were also identified. CONCLUSIONS: We established an integrated methodology for the efficient isolation of ureolytic bacteria, which expanded the biological resource of crucial ureolytic bacteria from the rumen. These isolates play a vital role in the incorporation of dietary nitrogen into bacterial biomass and hence contribute to ruminant growth and productivity. Moreover, this methodology can enable efficient isolation and cultivation of other bacteria of interest in the environment and help bridge the knowledge gap between genotypes and phenotypes of uncultured bacteria. Video abstract.

Application of culturomics in fungal isolation from mangrove sediments.

BACKGROUND: Fungi play a crucial role in ecosystems, and they have been widely considered a promising source for natural compounds that are crucial for drug discovery. Fungi have a high diversity, but about 95% of them remain unknown to science. The description rate of fungi is very low, mainly due to the inability of most fungi to grow in artificial media, which could not provide a sufficiently similar environment to their natural habitats. Moreover, many species in nature are in a state of low metabolic activity which cannot readily proliferate without proper resuscitation. Previously developed culturomics techniques are mostly designed and applicable for bacteria, with few attempts for fungal isolation because of their significantly larger cell size and hyphal growth properties. RESULTS: This study attempted to isolate previously uncultured and rare fungi from mangrove sediments using newly developed fungal enrichment culture method (FECM) and fungal isolation chips (FiChips). Comparison of fungal community composition at different enrichment stages showed that FECM had great influence on fungal community composition, with rare taxa increased significantly, thus improving the isolation efficiency of previously uncultured fungi. Similarly, in situ cultivation using FiChips has a significant advantage in detecting and culturing rare fungi, as compared to the conventional dilution plate method (DPM). In addition, based on morphological comparisons and phylogenetic analyses, we described and proposed 38 new ascomycetous taxa, including three new families, eight new genera, 25 new species, and two new combinations (presented in additional file 1). CONCLUSIONS: Our study demonstrated that mangrove sediments harbor a high diversity of fungi, and our new isolation approaches (FECM and FiChips) presented a high efficiency in isolating hitherto uncultured fungi, which is potentially usable for fungal isolation in other similar environments. Video Abstract.