Cytotoxic Compounds Derived from Marine Algicolous and Spongicolous Endophytic Fungi: A Review

Endophytes have gained particular interest in the search of potential pharmaceutical candidates for a long time due to their diversity, species richness and bioprospecting nature. They generally produce the essential metabolites for their expansion inside the plant which is involved in various biotransformation processes of utilizing host nutrients and cell components to continue microbial growth, sustenance, and reproduction. In above processes, they produce a huge amount of both structurally and functionally diverse secondary metabolites for maintaining an effective symbiosis with hosts. These compounds are proven to have significant bioactive properties like antibacterial, antifungal, antiviral, anti-inflammatory, antioxidants, antitumor activities. Despite the proven significance, a little is exploited so far about endophytes. Particularly marine fungal endophytes which are the centre of attention in this review have gained much less importance. Due to unique environmental feature, fungal endophytes derived from marine environment offer vast diversity in different bioactive secondary metabolites. This review has focused on algicolous endophytes and bioactive secondary metabolites discovered during the last two decades. Particular importance has been given to cytotoxic and antimicrobial metabolites. Due to intensive studies during last several years, an extensive number of publications are now available on cytotoxic compounds derived from endophytic fungi of marine algicolous and spongicolous origin that have been summarized in this review. Dhaka Univ. J. Pharm. Sci. 20(2): 247-265, 2021 (December)

Exploring the Nature of the Antimicrobial Metabolites Produced by Paenibacillus ehimensis Soil Isolate MZ921932 Using a Metagenomic Nanopore Sequencing Coupled with LC-Mass Analysis

The continuous emergence of multidrug-resistant (MDR) pathogens poses a global threat to public health. Accordingly, global efforts are continuously conducted to find new approaches to infection control by rapidly discovering antibiotics, particularly those that retain activities against MDR pathogens. In this study, metagenomic nanopore sequence analysis coupled with spectroscopic methods has been conducted for rapid exploring of the various active metabolites produced by Paenibacillus ehimensis soil isolate. Preliminary soil screening resulted in selection of a Gram-positive isolate identified via 16S ribosomal RNA gene sequencing as Paenibacillus ehimensis MZ921932. The isolate showed a broad range of activity against MDR Gram-positive, Gram-negative, and Candida spp. A metagenomics sequence analysis of the soil sample harboring Paenibacillus ehimensis isolate MZ921932 (NCBI GenBank accession PRJNA785410) revealed the presence of conserved biosynthetic gene clusters of petrobactin, tridecaptin, locillomycin (β-lactone), polymyxin, and macrobrevin (polyketides). The liquid chromatography/mass (LC/MS) analysis of the Paenibacillus ehimensis metabolites confirmed the presence of petrobactin, locillomycin, and macrobrevin. In conclusion, Paenibacillus ehimensis isolate MZ921932 is a promising rich source for broad spectrum antimicrobial metabolites. The metagenomic nanopore sequence analysis was a rapid, easy, and efficient method for the preliminary detection of the nature of the expected active metabolites. LC/MS spectral analysis was employed for further confirmation of the nature of the respective active metabolites.

Six New Antimicrobial Metabolites from the Deep-Sea Sediment-Derived Fungus Aspergillus fumigatus SD-406

Six new metabolites, including a pair of inseparable mixtures of secofumitremorgins A (1a) and B (1b), which differed in the configuration of the nitrogen atom, 29-hydroxyfumiquinazoline C (6), 10R-15-methylpseurotin A (7), 1,4,23-trihydroxy-hopane-22,30-diol (10), and sphingofungin I (11), together with six known compounds (2–5 and 8–9), were isolated and identified from the deep-sea sediment-derived fungus Aspergillus fumigatus SD-406. Their structures were determined by detailed spectroscopic analysis of NMR and MS data, chiral HPLC analysis of the acidic hydrolysate, X-ray crystallographic analysis, J-based configuration analysis, and quantum chemical calculations of ECD, OR, and NMR (with DP4+ probability analysis). Among the compounds, 1a/1b represent a pair of novel scaffolds derived from indole diketopiperazine by cleavage of the amide bond following aromatization to give a pyridine ring. Compounds 1, 4, 6, 7, 10 and 11 showed inhibitory activities against pathogenic bacteria and plant pathogenic fungus, with MIC values ranging from 4 to 64 μg/mL.

PROBIOTIC CHARACTERIZATION OF BACILLUS SUBTILIS STRAIN ISOLATED FROM INFANT FECAL MATTER REVEALED BY 16S rRNA GENE AND PHYLOGENETIC ANALYSIS

Objective: The rationale of our study was to isolate and identify the putative probiotic strain from infant fecal matter exhibiting a broad range of antimicrobial activity and to analyze the effect of different culturing conditions on its probiotic properties and the production of antimicrobial metabolites. Methods: In the present study, bacterial strains were screened for probiotic properties and antimicrobial activity from infant fecal matter (6 months–2 years). The effect of varying culture conditions such as tolerance to acid, bile salt, phenol, NaCl, pH, incubation period, and temperature along with autoaggregation assay, hydrophobicity, and hemolysis was studied. The characterization of the potent strain was studied by morphological, biochemical, and 16S rRNA gene sequencing along the phylogenetic affiliation of the strain was studied. Results: Two putative probiotic bacteria (DAM and IFM) were isolated, identified, characterized, and predicted at pH 2.0, 3.0, and 4.0, the isolate IFM had 50%, 60%, and 70% survivability, while isolate DAM had 55%, 63%, and 75% survivability, respectively. At a bile salt concentration of 0.5%, both isolates had a 75% survival rate. The isolates exhibited a high percentage of hydrophobicity and autoaggregation. The isolates also had non-hemolytic activity and were susceptible to many clinical tested antibiotics (tetracycline, erythromycin, ampicillin, gentamycin, penicillin, etc.). The isolate showed antimicrobial activity against enteric pathogens such as Staphylococcus aureus, Escherichia coli, and Shigella dysenteriae. The accession number of Bacillus subtilis MT279753 and MK453362 was submitted to NCBI. Conclusion: The result revealed that isolates have potent probiotic properties and possess a direct influence on the production of antimicrobial metabolites. These parameters can be modified for the improvement of the potentiality of the isolates.

Selection of Bacteriocinogenic Bacillus spp. from Traditional Fermented Korean Food Products with Additional Beneficial Properties

Two Bacillus spp. isolated from kimchi, Bacillus tequilensis ST816CD and Bacillus subtilis ST830CD, were characterized for their antimicrobial properties and safety. The proteinaceous nature of their inhibitory metabolites was confirmed after exposure to proteolytic enzymes, resulting in partial loss of the antimicrobial effect. This indicated that different non-proteinaceous antimicrobial substances may also be produced by these strains. This hypothesis was later confirmed when genes associated with the production of surfactants were detected in their DNA. The expressed antimicrobial metabolites were not affected by treatment at different temperatures and pH levels, including exposure to selected chemicals. Their strong adherence to susceptible pathogens was not significantly affected by different temperatures, chemicals, or pH values. Both Bacillus strains showed inhibitory activity against clinical and food-associated pathogens, including Listeria monocytogenes ATCC 15313, and some Staphylococcus species. Several genes associated with the production of antimicrobial metabolites were detected, but key virulence and beneficial genes were not present in these strains. Even though only B. tequilensis ST816CD displayed γ-hemolysin production, both selected strains were found to produce gelatinase and biogenic amines, which are considered as either potential virulence- or health-related factors. Moreover, the strains were susceptible to a variety of antibiotics except for the penicillin G [1 IU/disc] resistance of B. tequilensis ST816CD. Both strains showed proteolytic activity. Additionally, both strains showed low hydrophobicity based on bacterial adherence measured by hydrocarbons (n-hexadecane).

Antifungal activity of medicinal plants, Adathoda vasica and Andrographis paniculata against Colletotrichum capsici, the chilli fruit rot pathogen

Aim: The objective was formulated to screen the extracts of medicinal plants for tapping the antimicrobial activity against Collectotrichum capsici. Further, the work was planned to characterize and identify the nature of antimicrobial compounds and their functional groups. Methodology: Extracts of eleven medicinal plants were tested against the mycelial growth and spore germination of C. capsici under in-vitro conditions. Based on these results, the potential plant extracts of A. vasica and A. paniculata found effective against C. capsici were assayed for the presence of antimicrobial metabolites through TLC, GC-MS and FTIR analysis. Results: Among the medicinal plants screened, the crude extracts from Adathoda vasica and Andrographis paniculata inhibited mycelial growth and spore germination of C. capsici by 53.33% and 38.14%, respectively, under in-vitro conditions. GC-MS analysis of ethyl acetate extracts of A. vasica indicated antimicrobial compound, 1H-Pyrrolo[2,1-b]quinazolin-9-one,3-hydroxy-2,3-dihydro- and A. paniculata showed the presence of two compounds, docosahexaenoic acid and oleic acid. Similarly, FTIR analysis revealed esters, alcohols, and halide groups, which are known antimicrobials. Interpretation: The medicinal plants, A. paniculata and A. vasica possessed antimicrobial metabolites, which was responsible for inhibiting the mycelial growth and spore germination of C. capsici.

A Metagenomic Nanopore Sequence Analysis Combined with Conventional Screening and Spectroscopic Methods for Deciphering the Antimicrobial Metabolites Produced by Alcaligenes faecalis Soil Isolate MZ921504

The continuous development of multidrug resistance pathogens with limited therapeutic options has become a great problem globally that impose sever health hazards. Accordingly, searching for of new antimicrobials became an urgent demand and great challenge. Soil significantly have been associated with several species that are antibiotic producers. In this study, combination of conventional screening methods with Liquid chromatography- Mass spectroscopy (LC/MS) and metagenomic nanopore sequence analysis have been conducted for the deciphering the active metabolites produced by soil isolate(s). Preliminary soil screening resulted in a Gram-negative isolate identified via 16S ribosomal RNA as Alcaligenes faecalis isolate MZ921504 with promising antimicrobial activities against wide range of MDR gram-positive and gram-negative pathogens. The LC/MS analysis of the metabolites of A. faecalis isolate MZ921504 confirmed the presence of ectoine, bacillibactin, quinolobactin and burkholderic acid. Metagenomics sequence analysis of the soil sample (NCBI GenBank accession PRJNA771993) revealed the presence of conserved biosynthetic gene clusters of ectoine, bacteriocin, bacillibactin, quinolobactin, terpene and burkholderic acid of A. faecalis. In conclusion, A. faecalis isolate MZ921504 is a promising source for antimicrobial metabolites. LC/MS spectral analysis and third generation sequencing tools followed by secondary metabolite gene clusters analysis are useful methods to predict the nature of the antimicrobial metabolites.

Phenazine-1-carboxylic Acid Produced by Pseudomonas chlororaphis YL-1 Is Effective against Acidovorax citrulli

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.