Production Of Antibiotic Substances Research Articles

Экологические особенности и биотехнологические возможности почвенных актинобактерий (обзор)

Actinobacteria are gram-positive aerobic and facultative anaerobic bacteria that tend to form branching hyphae capable of developing into mycelium. Their main role is in the decomposition of complex polymers in the soil: lignin, cellulose, chitin, humus substances; fertility formation; production of antibiotic substances; accumulation of biologically active compounds. Actinobacteria secrete into the environment a complex of secondary exometabolites of various compositions with aliphatic, carbocyclic, heterocyclic, nitrogenous, oxygen-containing and sulfur-containing compounds. Most of the isolated secondary metabolites have antibiotic, antimicrobial, antiviral, herbicidal, insecticidal properties. Biotechnological capabilities of soil actinobacteria producing biologically active substances with a wide range of environmental effects are used in the development of multifunctional biologics for various industries. Actinobacteria play a crucial role in optimizing the growing conditions of plants, and therefore there is a natural interest in studying the factors that influence the relationship between actinobacteria and various crops in specific soil conditions. The development of this issue is an important link in the ecological characterization of the soil microbial complex and it will help to develop the theoretical foundations for the targeted regulation of the interactions of actinobacteria with phytopathogenic microorganisms and insect pests. The purpose of this work is to analyze the ecological features and biotechnological capabilities of soil actinobacteria. The review describes the distribution of actinobacteria in various types of soils, including those with the presence of pollutant; the composition of the metabolites produced; relationships with other microorganisms, as well as biotechnological properties and the possibility of using actinobacteria for the development of biological products for agroecosystems.

Diversity and antimicrobial potential of bacterial isolates associated with the soft coral Alcyonium digitatum from the Baltic Sea.

It is well recognized that microorganisms associated with marine invertebrates, in particular sponges and hard corals, are an excellent source of new natural products. Therefore, the diversity of bacteria associated with marine invertebrates and their potential to produce bioactive compounds have received much attention in recent years. We report here for the first time on the biodiversity of bacteria associated with the soft coral Alcyonium digitatum, which is abundant in the Baltic Sea. In order to increase the cultured diversity, bacteria were isolated using four different media, identified with support of 16S rRNA gene sequences and screened for antimicrobial activity using two different media. Activity of crude extracts was tested against Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli, and the yeast Candida albicans. A total of 251 coral-associated bacterial isolates were classified and found to belong to 41 species in 14 genera of the Firmicutes, Actinobacteria, Gammaproteobacteria, and Alphaproteobacteria. The genus Bacillus was most abundant and diverse with 17 recognized species. Forty-eight percent of all 251 isolates exhibited antimicrobial activity. All isolates of Bacillus methylotrophicus and Bacillus amyloliquefaciens displayed inhibition of at least three out of the four tested microorganisms. It became obvious during this study that the production of antibiotic substances not only is strain-specific, but in many cases also depends on the media composition and growth conditions. In addition, the antimicrobial potential of bacteria associated with A. digitatum may represent a promising source for antimicrobial substances.

The Early Challenges of Antibiotic Discovery

Professor Kim Lewis' excellent review of the status of antibiotic research (“Challenges of Antibiotic Discovery,” Microbe, September 2015, p. 363–369) unfortunately includes a common microbiological misconception that should be addressed. He comments that Selman Waksman “introduced a simple screen which essentially replicates the accidental discovery of Fleming—a Streptomyces spot-inoculated on a plate with a lawn of the test pathogen, and a zone of growth inhibition indicates the presence of an antibiotic.” However, the variety of complex twists and turns of screening for antibiotics are illustrated by the theses of the following students of Waksman's and not readily apparent from the published papers: H. Boyd Woodruff (actinomycin)(H. B. Woodruff, The production of antibiotic substances by soil microorganisms. Ph.D. Thesis, Rutgers, the State University of New Jersey, New Brunswick, N.J., 1942), Elizabeth Horning (clavacin and fumigacin)(E. S. Horning, Distribution and properties of antagonistic fungi and actinomycetes in nature. Ph.D. Thesis, Rutgers, the State University of New Jersey, New Brunswick, N.J., 1942), Doris Jones (antiviral screen)(D. Jones, The effect of micro-organisms and antibiotic substances upon viruses. M.S. Thesis, Rutgers, the State University of New Jersey, New Brunswick, N.J., 1945), Elizabeth Bugie (antibiotic production)(E. J. Bugie, Production of antibiotic substances by Aspergillus flavus and Chaetomium cochliodes, M.S. Thesis, Rutgers, the State University of New Jersey, New Brunswick, N.J., 1944), and Albert Schatz (streptomycin)(A. Schatz, Streptomycin, an antibiotic agent produced by Actinomyces griseus. Ph.D. Thesis, Rutgers, the State University of New Jersey, New Brunswick, N.J., 1945). In 1952 the Nobel Committee awarded the Nobel Prize in Physiology or Medicine to Selman Waksman for his “ingenious systematic and successful studies of soil microbes that led to the discovery of streptomycin.” Likewise, this year the Nobel Prize in Physiology or Medicine was in part awarded to Satoshi mura for the discovery of Avermectin, and the Nobel Assembly cited Dr. mura's “extraordinary skills in developing unique methods” for the isolation of Streptomyces species. Screening methodology merits focus.

English

One promising strain Actinomadura sp. AC170, isolated from Algerian Saharan soil, with strong antifungal activity against pathogenic and toxinogenic fungi, was selected for further studies. The 16S rRNA results showed a distinct phylogenetic lineage from the other species within the Actinomadura genus. The production of antibiotic substances was investigated using GYEA solid medium. The butanolic extract contained four bioactive spots detected on thin layer chromatography plates. Among these antibiotics, a complex called 170A, which showed the more interesting antifungal activity, was selected and purified by reverse-phase HPLC. This complex is composed of four compounds. Ultraviolet-visible, infrared, mass and H nuclear magnetic resonance spectroscopy studies showed that these molecules contain an aromatic ring substituted by aliphatic chains. These compounds differ from the known antibiotics produced by Actinomadura species.   Key words: Actinomadura, taxonomy, antifungal compounds, Saharan soil.

Suppression of Pythium ultimum root rot of sorghum by rhizobacterial isolates from Ethiopia and South Africa

Bacteria isolated from the rhizosphere of sorghum in Ethiopia and from different grass species within the Nylsvlei Nature Reserve in South Africa were evaluated for their in-vitro and in-vivo antagonistic activity against Pythium ultimum that causes root rot in sorghum. Root rot caused by this pathogen in sorghum is characterized by blackened discolorations with white mycelial growth on the surface of the roots. Statistically significant disease suppression was achieved by 15 isolates from sorghum rhizosphere in Ethiopia (⩾50% disease suppression), whereas eight isolates from the rhizosphere of grasses at Nylsvlei Nature Reserve in South Africa resulted in ⩾80% disease suppression. The isolates maintained themselves at a level ⩾105cfu/g indicating that they are also highly competent in sorghum rhizosphere. Isolates which showed the best overall performance in-vitro and in-vivo were identified by means of the API system and sequencing of the bacterial 16S rDNA genes. The majority of these isolates were identified as members of the genus Bacillus, 56% of which were B. cereus. Three other isolates were identified as Brevibacterium laterosporus, Pseudomonas fluorescens and Serratia marcescens. Selected modes of action studies indicated that production of antibiotic substances and siderophores as well as induction of systemic resistance in sorghum were exhibited by some of the most effective isolates. The results of this study offer a significant impetus to the application of plant growth promoting rhizobacteria for use as biological control agents against P. ultimum in sorghum.

Protection of Oilseed Rape (Brassica napus) Toward Fungal Pathogens by Strains of Plant-associated Bacillus amyloliquefaciens

In this report, four Bacillus strains were tested for effects on plant fitness and disease protection of oilseed rape (Brassica napus). The strains belonged to newly discovered plant-associated Bacillus amyloliquefaciens and a recently proposed species, Bacillus endophyticus. The fungal pathogens tested represented different infection strategies and included Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. The B. amyloliquefaciens strains showed no or a weak plant growth promoting activity, whereas the B. endophyticus strain had negative effects on the plant as revealed by phenological analysis. On the other hand, two of the B. amyloliquefaciens strains conferred protection of oilseed rape toward all pathogens tested. In vitro experiments studying the effects of Bacillus exudates on fungal growth showed clear growth inhibition in several but not all cases. The protective effects of Bacillus can therefore, at least in part, be explained by production of antibiotic substances, but other mechanisms must also be involved probably as a result of intricate plant-bacteria interaction. The protective effects observed for certain Bacillus strains make them highly interesting for further studies as biocontrol agents in Brassica cultivation.

Antimicrobial compounds produced byActinomadurasp. AC104 isolated from an Algerian Saharan soil

During a search for nonpolyenic antifungal antibiotics, an actinomycete designated AC104 was isolated from a Saharan soil sample by a dilution agar plating method using a chitin - vitamins B medium supplemented with rifampicin. Isolate AC104 presented the morphological and the chemical characteristics of the genus Actinomadura. On the basis of 76 physiological tests and 16S rDNA analysis, this isolate was determined to be quite different from the known species of Actinomadura. It is active against filamentous fungi and both Gram-positive and Gram-negative bacteria. The production of antibiotic substances was investigated using several culture media. The highest antimicrobial activities were obtained on ISP2 medium. The benzenic extract contained five bioactive spots detected on thin layer chromatography plates. Among these antibiotics, a complex called 104A, which showed the more interesting antifungal activity, was selected and purified by reverse-phase high-pressure liquid chromatography. This complex is composed of four compounds. Ultraviolet-visible, infrared, mass, and 1H nuclear magnetic resonance spectroscopy studies showed that these molecules contain an aromatic ring substituted by aliphatic chains. These compounds differ from the known antibiotics produced by Actinomadura species.

Enhancement of Biocontrol Efficacy of Serratia plymuthica A21-4 Against Phytophthora Blight of Pepper by Improvement of Inoculation Buffer Solution

The production of antibiotic substances by Serratia plymuthica A21-4 was greatly enhanced by modifying components of a growth medium. When the minimal medium containing 0.7%, 0.2%, 0.1%, 0.01% was used as basal medium, the best carbon source for antibiotic production was glycerol and the most favorable nitrogen source was ammonium sulfate. The modified medium for antibiotic production also increased colonization ability of A21-4 on pepper root and in the rhizosphere soil. When the cells of A21-4 were suspended in modified medium, the population density of A21-4 on pepper root was 10-100 times higher than that suspended in 0.1 M . The population density of A21-4 on root did not decrease under cfu/groot up to 21 days after treatment although the inoculum of A21-4 was reduced to cell/ml. Similar tendency was also observed in the rhizosphere soil. Consequently, Phytophthora blight of pepper was successfully controlled by A21-4 with cell/ml suspended in the modified buffer solution instead of cfu/ml suspended in 0.1 M .

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae.

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode's development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae (Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode’s development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae ( Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.

Induction of antibacterial activity by α- d-oligogalacturonides in Nephrolepis sp. (pteridophyta)

This paper presents data on the induction by α- d-oligogalacturonides (OG) of antibiotic activity in vitro by the fern Nephrolepis sp. The extracts from the fern grown aseptically, partly in a medium containing a mixture of OG and partly in a medium lacking OG, as control, were tested against several bacterial strains. The results show that the OG mixture promotes the production of antibiotic compounds. Comparing the present results with those on the antimicrobial properties of the same fern grown in a greenhouse, we discuss the hypothesis that the production of antibiotic substances can be elicited by different factors, such as products of synthesis or degradation of the biotic component of the soil or by OG (in axenic culture) that can mimic the effect of natural elicitors.

Taxonomic Revision of the Genus Saccharomonospora and Description of Saccharomonospora glauca sp. nov.

Representatives of Saccharomonospora viridis (type strain DSM 43017), Saccharomonospora caesia (type strain DSM 43044), and Saccharomonospora internatus (type strain DSM 43671), together with 52 Saccharomonospora isolates from compost, manure, hay, and soil, were characterized by determining morphological, biochemical, and physiological properties, phage sensitivity, antibiotic activity, enzyme and protein patterns (polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and restriction patterns of chromosomal deoxyribonucleic acids. The type strain of S. internatus proved to be identical to S. viridis, and thus S. internatus should be regarded as a synonym of S. viridis. A total of 16 strains differed significantly from S. viridis and S. caesia and are proposed as members of a new species, Saccharomonospora glauca (type strain, DSM 43769). Since the three species differ in phage sensitivity, production of antibiotic substances, and enzyme, protein, and deoxyribonucleic acid restriction patterns, these properties can be used for reliable species identification.

Ectomycorrhizae of Pinus patula as Biological Deterrents to Phytophthora cinnamomi

SUMMARY The protective effect of ectomycorrhizae of Pinus patula Schlecht. et Cham against the root pathogen Phytophthora cinnamomi was investigated. Three methods were employed to evaluate this effect. Firstly, extracts were prepared from the root systems of mycorrhizal plants and tested for antibiotic activity against P. cinnamomi. Secondly, fungal antagonism between two mycobionts Amanita muscaria and Boletus edulis, and the pathogen was tested for in vitro; and thirdly, in situ studies were conducted to determine the protective effect afforded under natural conditions. No fungal antagonism was observed in vitro, but significant protection was obtained in situ and when the pathogen was exposed to the extracts. The mechanism involved could thus probably be attributed to the production of antibiotic substances by the macrobiont and mycobiont, and the formation of a physical barrier, the Hartig-net and the fungal mantle.

Studies in Actinomycetes in Egyptian Soils: III. The Antagonistic Activity of Actinomycetes

Summary The antimicrobial activity of 359 antibiotic-producing actinomycetes, isolated from Egyptian soils, was investigated. The results revealed that isolation of antagonists requires the use ot several media. Organisms of wide spectrum are less frequent than those of narrow ones. Some actinomycetes seemed to possess a selective antimicrobial effect against one organism or a similar group of micro-organisms. The production of antibiotic substance, active against a particular test organism, depends on the antagonist, the chemical composition, and the physical state of the medium.

STUDIES ON MARINE MICROORGANISMS. II

In all, 136 strains of actinomycetes were isolated from 37 samples collected at three inlets in Sagami Bay. The salt tolerance of terrestrial actinomycetes was examined. Most of actinomycetes tested were able to grow in media containing 3.0-3.5% NaCl eqivalent to the average salt concentration of sea water, with the exception of two strains of Microbispora, which failed to grow in media containing less than 2% NaCl. A strain of Actinomyces aureoverticillus, of Chainia violens and of Nocardia asteroides were able to grow in media containing more than 10% NaCl. The production of antibiotic substances by four strains of these isolated actinomycetes was studied in various media with varied temperatures. It was remarkable that the antibacterial spectra changed with varied cultural conditions. In all, 27% of the isolates had antimicrobial activity and 17% showed inhibition against YOSHIDA sarcoma cell.

Biological control of cotton wilt

Using the antagonistic actinomycetes isolated from theFusarium “wilt-sick” and “wilt-free” cotton field soils it was possible to confirm production of antibiotic substance(s) by them inin vitro studies. They inhibited spore germination and radial growth of the cotton wilt pathogenFusarium vasinfectum Atk. In addition, various adverse morphological changes were brought about by them on the fungal mycelium. Most of the actinomycetes were seen to lyse both live and heat-killed bud-cells of the pathogen. Similar effect was also observed in soilin situ.

Ultraviolet-induced mutation in Streptomyces induces

The wild-type strain Streptomyces indicus Chakrabarty and 4 mutant strains derived from it were irradiated by UV rays of 253.7 nm wavelength at 100 erg/mm 2/sec, for 20, 40, 60 and 80 sec. The survival curve was exponential, and the rate of mutation was parallel with the increasing dosage and was followed later by a decline. The mutants differed from the wild parent in reduced growth rates, changes in the shape and size of the colony, sporulation level, antibiotic activity and variation in the colour of the mycelium. Biochemical mutants were obtained that required histidine, glycine, aspartic acid, lysine, valine, methionine, ornithine, arginine, tyrosine, cysteine, proline, leucine, alanine, threonine, riboflavin, adenine, guanine, inositol, nicotinic acid, hypoxanthine and thymine, either singly, doubly or alternatively. The number of histidine-requiring mutants was maximal. There was marked variation in the antibiotic activity of biochemical mutants: increase, decrease or complete absence of such activity were seen. In some of the auxotrophs, a linking of genetic characters controlling the biosynthesis of amino acids and the production of antibiotic substances was observed.

Antibiotic activity of marine microorganisms

1. 41 sea water samples vollected between 18°00'N – 72°00'E and 18°52'N – 72°85'E were screened for marine bacteria possessing antagonistic properties againstStaphylococcus aureus andSalmonella typhosa. 2. Of 60 cultures elaborating antibiotic principles, a majority (45) were aerobic spore forming bacilli; the rest included gram-positive cocci (11), gram-negative bacilli (2) and streptomycetes (2). 3. The majority of the isolates showed higher activity against the gram-negative test organism. 4. Eleven different media were used to observe the effect of nutrients on the production of antibiotic substances.

Effects of nutrition of Marasmius urens upon the production of antibiotic substances.

IT has earlier been found that species of the genus Marasmius produce antibiotic substances1,2. Recent work on antibiotics has shown that Marasmius urens (Bull). Fr. is highly active against several pathogenic bacteria (Oblom and Wallmark, unpublished). The present investigation deals with some nutritional effects upon the production of the active substances.

Strain specificity and production of antibiotic substances. IX. Bacteriostatic and bactericidal properties of neomycin and development of resistant strains.

Strain specificity and production of antibiotic substances. IX. Bacteriostatic and bactericidal properties of neomycin and development of resistant strains.