Antibiotic-producing Fungi Research Articles

Endophytic Fungal Biodiversity and Antimicrobial Bioprospecting of Penicillium citrinum KIB4 from Khaya ivorensis Bark

Background: The emergence of pathogens that are resistant to antibiotics has raised serious concerns about public health, forcing the hunt for alternative antibacterial substances. Endophytes, particularly those from unscreened plants, may offer new sources of antibacterial substances. Hence, this research aimed to isolate antibiotic-producing fungi from Khaya ivorensis bark, a medicinal plant widely used in Nigeria. Methods: The fungal endophytes were isolated following conventional methods using potato dextrose agar (PDA). They were characterized by morpho-microscopical and sequencing of the internal transcribed spacer (ITS) of the fungal rDNA. The phylogenetic tree was drawn following the Neighbour-joining method and the tree was annotated using the Interactive Tree of Life (iTOL) version 6.0. The agar plug assay method was used to select putative antibiotic-producing isolate for further research. The antimicrobial activity of the fungal fermentation product was assessed by agar well diffusion while the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using the solid agar dilution technique. Results:Atotal of 12 endophytic fungi were isolated. They were noted to be mostly Aspergillus sp. (11 isolates) while only one isolate represented Penicillium species. Only isolate KIB4 showed good inhibitory activity against several bacterial pathogens tested in the preliminary study within 10 mm to 12 mm inhibition zone diameter (IZD) range. Molecular identification proved that strain KIB4 fell into the genus Penicillium and shared the highest ITS gene sequence similarity with Penicillium citrinum D112 (98.83%), and Penicillium sp. TWJL1743 (98.65%). The fermentation extract of Penicillium citrinium KIB4 showed specific inhibition against Escherichia coli ML475, Staphylococcus aureus LN001, Klebsiella pneumoniae ML602, Bacillus subtilis ML225, and Proteus sp. UR979 with an IZD range of 11mm to 23mm. Conclusion: We reported for the first time, the antimicrobial efficacy of fermentation extract from endophytic fungi isolated from Khaya ivorensis against several bacterial pathogens. Further fractionation of the crude extract is needed to isolate specific antimicrobial compounds.

Antibiotic Producing Fungi in Sewage: Inhibitory Effect on 4 Bacterial Test Strains, and Different Fungal Types

Fungi were isolated from raw sewage and sewage that had trickled down soil columns from a waste water treatment plant in Karlsruhe, Germany, using the laboratory techniques.Fusarium sporotrichioides Sherb, Penicillium funiculosum, and Trichoderma harzianum Rafai were named as isolates from raw sewage. P. notatum Westling, P. meleagrinum Biourge, Aspergillus flavus, Link ex Gray, A. repens, A. fumigatus Fresenius, and A. fischeri Wehmer were among the fungi found in the effluent of the soil columns that were isolated under absolutely anaerobic conditions. Fusarium poae (Peck) Wollenw. and Penicillium chrysogenum Thom. were isolated when samples were cultured in anaerobic jars with nitrate. The coloration, smell, and other fungal traits, such as conidial and conidiophore size, etc., were used to identify the organism. The fungi's antibiotic properties against bacteria were investigated. A little amount of the fungal mycelium was plate-plated on new Nutrient agar and Sabouraud agar after isolation. After two days of incubation, bacteria were cross-streaked toward the fungal colonies on the plates. On the plates, six strains of E. coli, Gram-negative Pseudomonas species, and aerobic Gram-positive Enterococci species were streaked in the direction of the fungi. After that, the plates were incubated in an aerobic environment. Similar to this, five anaerobic Gram-positive Bifidobacterium species strains that were isolated from sewage were cross streaked on fungal plates and then further cultured under anaerobic conditions. When compared to A. flavus and A. fumigatus, P. chrysogenum and A. repens were more effective at inhibiting Enterococci. Only P. chrysogenum, A. fumigatus, and A. repens inhibited Pseudomonas sp. A. fumigatus and P. chrysogenum var meleagrinum only little inhibited E. coli, but P. chrysogenum and A. flavus very successfully did so.Pre-growing P. chrysogenum, P. notatum, and P. meleagrinum on plates for two days in an aerobic environment was followed by cross-stripping with test strains of Bifidobacterium and incubation under strictly anaerobic conditions. Both P. chrysogenum and P. notatum had the greatest inhibitory effects on bifidobacterium.

Potential antibiotic-producing fungal strains isolated from pharmaceutical waste sludge

BackgroundAntibiotic resistance and dearth of novel compounds from natural sources warrants the need to search other environments for potential antibiotic-producing microbial species. The study investigated isolation and identification of antibiotic-producing fungi from pharmaceutical waste sludge.ResultsSeven hundred and ninety-seven isolates obtained from sludge of seven pharmaceutical industries in Sango Ota, Ogun State using several growth media, with mould isolates highest (696). Isolated species were from genera Aspergillus (28.55%), Penicillium (18.35%), Trichoderma (13.44%), Rhizopus (10.21%) and Geotrichum (4.01%), and Stachybotrys (0.13%). The CFS of strains named Geotrichum candidum OMON-1, Talaromyces pinophilus OKHAIN-12, and Penicillium citrinum PETER-OOA1 had high reproducible bioactivity against Staphylococcus aureus (32 ± 0.12 mm) and Klebsiella pneumoniae (29 ± 0.12 mm) while P. citrinum MASTER-RAA2 had activity against K. pneumoniae only. Active metabolites were successfully extracted using Diaion HP-20 and methanol:iso-propanol:acetone (6:3:1 v/v). Antibacterial-active fractions of fungal extract successfully eluted with 40–60% NaCl on ion-exchange chromatography using a cation column.ConclusionsThe study successfully screened antibiotic-producing fungal species from pharmaceutical waste storage facilities. Study also showed that similar species from same toxic environment could potentially produce different metabolites.

Evaluation of Secondary Metabolites of Some Fungi Isolated From Beach Soils of Lagos, Nigeria Against Some Pathogens

Three beach soils in Lagos, Nigeria were screened for the presence of antibiotic producing fungi against 8 test pathogenic bacteria & fungi. The physiochemical parameters of the soils were determined following standard procedures. Soil plate dilution method was employed for isolation of marine fungi and they were identified based on cultural and microscopic characteristics. Primary screening of isolated fungi for antibiotic potential was determined by perpendicular streak method against known pathogenic test organisms (Escherichia coli, Saphylococcus aureus, Klebsiella pnuemoniae, Pseudomonas aeruginosa, Candida albicans, Saccharomyces cerevisiae,Aspergillus niger and Aspergillus fumigatus). Further sreening of the product of secondary metabolism [antibiotic] of the active fungi was done by agar disc diffusion assay. The fungus with the highest secondary activity was subjected to molecular analysis for further identification. pH of beach soils was alkaline ranging from 7.6-8.0, while temperature and moisture were normal ranging from 26.333 ± 1.155a-30.333 ± 0.577aand 7.142 ± 1.497a-10.030 ± 3.130a respectively. A total of four fungal species of 2 different genera were isolated from the beaches ;Aspergillus niger,Aspergillus flavus,Penicillium purpurogenum and Penicillium islandicum. Both primary and secondary assays revealed only antibacterial activities against Staphylococcus aureus and Psedomonas aeruginosa with no effect on all test fungi. Highest antibacterial activity[28+0.2a] was exhibited by the fungus identified as Penicillium purpurogenum. Fungi from beaches in Lagos, Nigeria may be a promising source of antibacterial agent useful in the treatment of infections caused by S. aureus and P. aeruginosa.

Solid-liquid Interface Screening SystemーApplication to the Screening of Antibiotic and Cytotoxic Substance-producing Fungi.

A useful tool for the screening of fungi producing biologically active secondary metabolites such as antibiotics and cytotoxic substances has been developed. An agar plate-organic solvent interface cultivation (A/S-IFC) system, which comprised a hydrophobic organic solvent (upper phase) , a fungal mat (middle phase) and an agar plate (lower phase) , was constructed. The metabolite profiles were compared among the A/S-IFC, a traditional submerged cultivation (SmC) and an extractive liquid surface immobilization (Ext-LSI) system consisted of a hydrophobic solvent (upper phase) , a fungal cells-ballooned microspheres (middle phase) and a liquid medium (lower phase) , with high-performance liquid chromatography-photodiode array detector (HPLC-PDA) . In the A/S-IFC, many hydrophobic metabolites vastly different from those in the SmC were accumulated in the organic phase as with the Ext-LSI. For example, a valuable azaphilone, sclerotiorin, was remarkably produced into the organic phase in the A/S-IFC. The A/S-IFC was applied to the screening of antibiotic-producing fungi. As a result of paper disk method, it was found that 321 isolated among 811 strains produced antifungal metabolites (hit rate, 39.6%) . Furthermore, 8, 23, and 30 strains also produced cytotoxic metabolites against SKOV-3 (human ovary adenocarcinoma) , MESO-1 (human malignant pleural mesothelioma) , and Jurkat cells (immortalized human T lymphocyte) .

Discovery of secondary metabolites in an extractive liquid-surface immobilization system and its application to high-throughput interfacial screening of antibiotic-producing fungi.

An extractive liquid-surface immobilization (Ext-LSI) system, which consists of a hydrophobic organic solvent (an upper phase), a fungal cell-ballooned microsphere layer (a middle phase) and a liquid medium (a lower phase), is a unique interfacial cultivation system for fungi. The fungal cells growing at the interface between the organic and aqueous phases efficiently produce hydrophobic metabolites, which are continuously extracted into the organic phase, and/or hydrophilic metabolites that migrate into the aqueous phase without carbon catabolite repression and product and/or feed-back inhibitions. Application of the system to fermentation of Penicillium multicolor IAM 7153 and Trichoderma atroviride AG2755-5NM398 afforded remarkably different profiles of secondary metabolites in the organic phase compared with those in an aqueous phase in traditional submerged cultivation (SmC). Various hydrophobic metabolites exhibiting unique UV-visible spectra were accumulated into the organic phase. The system was applied to a novel interfacial screening system of antibiotic-producing fungi. Compared with the SmC, the interfacial cultivation system exhibited some interesting and important advantages, such as the higher accumulation of hydrophobic secondary metabolites, the lack of requirement for shaking and troublesome solvent extraction, and the small scale of the vessels (medium, 5 ml; dimethylsilicone oil, 1 ml), as well as the significantly different metabolite profiles. The interfacial screening system yielded a high incidence of antimicrobial activity, with 21.9% of the fungi tested exhibiting antifungal activity against Pichia anomala NBRC 10213. This novel interfacial high-throughput screening approach has the potential to discover new biologically active secondary metabolites even from strains previously found to be unproductive.

New method for isolating antibiotic-producing fungi

A convenient and efficient method was established for isolating antifungal antibiotic-producing fungi from soil samples. In this method, soil samples were diluted and directly plated in agar medium by the standard fungi-isolating method, and the plates were cultured at 27 °C for 2-3 days to permit the growth of fungal colonies. Then, the suspension of pathogenic Candida albicans in saline (40 μl, 5-10 × 10⁵ CFU ml⁻¹) was overlaid by spraying on the plates under controlled conditions in the safety cabinet. After 1-day incubation, fungal colonies showing an antagonistic effect with the inhibition zone against sprayed C. albicans were selected. Among 151 isolates, 26 strains were found to reproduce anti-C. albicans activity in liquid medium, yielding a higher selection rate (17.2%) than that (3.1%) by the traditional method. This new method can be applied for isolation of microorganisms (fungi and actinomycetes) that produce antibiotics active against pathogenic microorganisms.

Phylogeny and intercontinental distribution of the pneumocandin-producing anamorphic fungus Glarea lozoyensis

Glarea lozoyensis is an anamorphic ascomycete that produces pneumocandin B0, the starting molecule for the synthesis of the antifungal drug caspofungin (CANCIDAS™). Glarea lozoyensis was first isolated in 1985 from a water sample from Madrid, Spain. Until now, only the original strain was known, but we have discovered new strains from Argentina and the USA. Molecular phylogenetic reference to a 28S rDNA database of antibiotic-producing fungi quickly identified these strains as being conspecific with G. lozoyensis. Bayesian inference phylogeny of ITS, 28S rDNA and α-actin gene fragments revealed that G. lozoyensis is related to species of the genus Cyathicula (Helotiales). Glarea lozoyensis was not conspecific with any of the Cyathicula species sequenced, although it appears to share a common ancestor. Glarea lozoyensis and Cyathicula strains were fermented on nutritional microarrays in 96-well plates. Cyathicula extracts did not show antifungal activity and did not produce pneumocandins, whereas potent antifungal activity and pneumocandin A0 production were confirmed for the four G. lozoyensis isolates. Also, culture morphology differed, with G. lozoyensis strains producing a dark brown, profusely sporulating mycelium with pigmented multicellular conidia accumulating in conidial masses, while all Cyathicula species tested in culture formed hyaline to light brown mycelia and lacked conidia. The chemistry and taxonomic distribution of the echinocandin class of antifungals is comprehensively reviewed.

Characterization of a novel peroxisome membrane protein essential for conversion of isopenicillin N into cephalosporin C

The mechanisms of compartmentalization of intermediates and secretion of penicillins and cephalosporins in β-lactam antibiotic-producing fungi are of great interest. In Acremonium chrysogenum, there is a compartmentalization of the central steps of the CPC (cephalosporin C) biosynthetic pathway. In the present study, we found in the 'early' CPC cluster a new gene named cefP encoding a putative transmembrane protein containing 11 transmembrane spanner. Targeted inactivation of cefP by gene replacement showed that it is essential for CPC biosynthesis. The disrupted mutant is unable to synthesize cephalosporins and secretes a significant amount of IPN (isopenicillin N), indicating that the mutant is blocked in the conversion of IPN into PenN (penicillin N). The production of cephalosporin in the disrupted mutant was restored by transformation with both cefP and cefR (a regulatory gene located upstream of cefP), but not with cefP alone. Fluorescence microscopy studies with an EGFP (enhanced green fluorescent protein)-SKL (Ser-Lys-Leu) protein (a peroxisomal-targeted marker) as a control showed that the red-fluorescence-labelled CefP protein co-localized in the peroxisomes with the control peroxisomal protein. In summary, CefP is a peroxisomal membrane protein probably involved in the import of IPN into the peroxisomes where it is converted into PenN by the two-component CefD1/CefD2 protein system.

Antibiotic-Producing Fungi: Current Status of Nomenclature

Publisher Summary This chapter focuses on nomenclature of antibiotic-producing fungi, and discusses the problem of nomenclature for fungi used in industrial fermentations. Only three groups of antibiotics are produced by fungi in the United States: cephalosporins, penicillins, and fumagillin. Fungi, including Myxomycetes, Phycomycetes, Ascomycetes, Basidiomycetes, Lichens, and Fungi Imperfecti, are covered by the International Code of Botanical Nomenclature. This code covers all plants. It originated in 1906, although a set of rules for nomenclature was published in 1867. Since then, the rules have been improved and revised in 1912, 1935, 1947, 1950, 1952, 1956, 1961, 1966, and finally 1972. The Botanical Code arose to satisfy a need for an orderly manner of naming taxa. The fundamental requirements of a satisfactory nomenclature are uniformity, stability, exact application, and convenience. The chapter provides a comparison between the Botanical Code and the Bacterial Code. It further discusses deposit of cultures, especially those in connection with patent applications.