Selective Isolation Of Actinomycetes Research Articles

Selective isolation of actinomycetes for inhibition activity against methicillin-resistant Staphylococcus aureus SK1

205 isolates of actinomycetes bacteria were isolated from soil samples collected from Satun province, Thailand. To have more chances to get diverse groups of actinomycetes bacteria, soil samples were treated using three different methods before inoculated on two selective media, humic acid-vitamin agar and coal vitamin agar. After two months of incubation at 30°C, all typically characteristic colonies of actinomycetes were picked and purified then tested for inhibition activity against a virulent pathogen, methicillin-resistant Staphylococcus aureus SK1 (MRSA SK 1) using double-layer agar plate method. There were 49 isolates (23.90%) showed inhibition zone against MRSA SK 1.

Antiproliferative Activity of Philippine Marine Sediment-Derived Actinomycetes

The Philippine archipelago is rich in marine biodiversity and resources that are widely unexplored. Its marine sediments harbor marine microbes that possess secondary metabolites with potent bioactivities. This study aims to determine the antiproliferative activity of the crude extracts of selected Actinomycete isolates (DSD011, DSD017, and DSD042) from Islas de Gigantes, Carles, Iloilo. The antiproliferative screening was done using Saccharomyces cerevisiae as a model organism. Crude extracts of isolates that are active in inhibiting the growth of S. cerevisiae were determined using the broth microdilution method. Afterward, the active extract was tested using antiproliferative and budding yeast assays. With the antiproliferative model, only DSD011 was found to inhibit the growth of S. cerevisiae. The percentage of live and dead cells in DSD011 was comparable to those treated with Triton X (positive control). Further, the budding yeast analysis showed that DSD011 induced G1 cell cycle arrest of nearly 50% of S. cerevisiae cells. Thus, DSD011, a marine sediment-derived Actinomycete, serves as a potential source of naturally occurring bioactive compounds with antiproliferative properties.

A Consolidated Method for Selective Isolation of Actinomycetes Based on Choice of Substrate

Actinomycetes are known as filamentous, Gram positive bacteria. They form the majority of the microbial load in various niches; soils, composts, etc. The study aimed to evaluate the method of isolating slow growing actinomycetes from four different sources: garden soil, cow dung manure compost, floral waste compost, and floral waste vermicompost. In this study, an integrated method consisting of physical and chemical pretreatment of the sample and the use of selective media was used to isolate actinomycetes. Physical treatment includes air drying, sun drying, dry heating in an oven, and moist heat treatment, whereas chemical treatment includes enrichment of the sample with CaCO3 followed by plating on actinomycetes-specific media with the incorporation of antibacterial and antifungal antibiotics. The actinomycetes count on the plate was reported in CFU/gm of dry wt. Morphological and microscopic characteristics of purified isolates were noted. The results were compared, and it was found that the pretreatment method of a particular sample depends on the choice of substrate. Overall, physical treatment followed by chemical enrichment showed relatively higher counts on the plate and better results. Here we also found the dominance of fluorescent Pseudomonas sp. in case samples from vermicompost. The study can be of great importance in isolating novel and rare genera of actinomycetes. These methods can help speed up the isolation and screening of novel actinomycetes which will ultimately be important for the discovery of antibiotics and other industrially vital bioactive compounds.

In vitro antagonism between actinomycete isolates and Fusarium oxysporum f. sp. Ciceri: The causative agent of chickpea vascular wilt

The chickpea is one of the most consumed crops in Algeria. Unfortunately, it is damaged by the phytopathogenic fungus Fusarium oxysporum f. sp. ciceri, the vascular wilt agent. In this study, we highlight the antagonistic activity of actinomycetes which we can use as biological tool to fight this dreaded phytopathogenic fungus. We recovered from the arid soils of southern Algeria, 61 isolates of actinomycetes with different morphological characteristics. Then, we tested the antagonistic activity of these actinomycete isolates, by various in vitro methods, on the growth of the phytopathogenic fungus. The thin layer chromatography was used on the selected actinomycete extract. As results, we obtained 06 isolates of actinomycetes (ACT1, ACT2, ACT3, ACT4, ACT5 and ACT6) presenting potential antagonistic activities against 3 isolates of F. oxysporum f. sp. ciceri (FOC8, FOC13 and FOC15). Preliminary identification using morphological criteria revealed the presence of Streptomyces characteristics for the 6 isolates. The activity of the crude extract from the selected actinomycete isolates against the 3 isolates of F. oxysporum f. sp. ciceri showed that the activity of the isolate ACT2 was greater compared to the others. The thin layer chromatography of the culture filtrate crude extract showed the presence of 5 fractionswith each fraction representing bioactive molecules.

Antimicrobial activities of actinomycete isolates from rhizospheric soils in different mangrove forests of Torosiaje, Gorontalo, Indonesia

Retnowati Y, Moeljopawiro S, Djohan TjS, Soetarto ES. 2018. Antimicrobial activities of actinomycetes isolates from rhizospheric soil on different mangrove forests of Torosiaje, Gorontalo, Indonesia. Biodiversitas 19: 2196-2203. Mangrove forests are very productive ecosystems that form unique saline environment very rich in organic matter, containing nitrogen and sulfur available for microorganisms. Mangrove forest as an extreme environment is promising to be sources of antibiotic-producing actinomycetes. The objectives of this study were to analyze the antimicrobial activities of metabolites produced by actinomycete isolates from rhizospheric soil of mangrove forest of Torosiaje, Gorontalo, Indonesia, and identify the active compound for novel antibiotics production. Six isolates from a coastal mangrove forest was selected to produce secondary metabolite. The crude extract of the six selected actinomycete isolates showed antimicrobial activities against pathogenic microbes; the highest antimicrobial activities was indicated by metabolite produced by FUAm2-h1 and FMBg2-x3 isolates. The metabolite crude extracts produced by two potential isolates inhibited growth of pathogenic microbe on MIC value of 0.0625 to 0.5mgmL-1. Bio-autography assay detected an active compound on Rf value of 0.94, especially on extracellular metabolite produced by strain FUAm2-h1. The bioactive compounds were identified by liquid chromatography joined with low-resolution mass spectroscopy (LC/MS) and analysed by MEDINA's database The active compounds composed of 17 substances, and only 3 substances showed a high quantity with molecular weight of 507.37, 344.32 and 563.66 mol G-1, respectively. FTIR analyses identified the functional groups in the active compounds consisted of amide, amine, alkuna, alkane, NO2 nitro compound, alcohol, ether, carboxylic acid, ester and C-H aromatic ring. The biosynthesis of antibiotic on FUAm2-h1 and FMBg2-x3 isolates was regulated by double genes, i.e., PKS-II and NRPS genes. The antimicrobial activities of two actinomycete isolates showed the performance of antibiotics suspected as aromatics polyketides group. The FUAm2-h1 and FMBg2-x3 isolates showed high similarity with Streptomyces qinglanensis strain 172205 and Streptomyces sanyensis strain 219820, respectively, in terms of 16S rRNA gene sequences. The potential of those selected actinomycetes from extreme environments of mangrove forest constitute a source of promising actinomycete strains producing biologically active secondary metabolites.

Selective isolation of Actinomycetes from mangrove sediment of Tanjung Lumpur, Kuantan, Malaysia.

Aims: Mangroves of Tanjung Lumpur, Kuantan, Pahang is considered as a relatively underexplored resource of actinomycetes. Based on the above perspective, a study was conducted on mangrove sediments of Tanjung Lumpur, Kuantan to isolate potential actinomycetes using several pretreatments and various selective media. Methodology and results: Sediments from five different sites at Tanjung Lumpur mangrove were collected and selectively pre-treated. The pretreated sediments were diluted and plated onto eight different selective media. A total of 172 potential actinomycetes were isolated from all the media. Antimicrobial activities of 61 selected strains were checked against 8 test microorganisms using cross streak method. Pretreatment of wet heat with seawater was the most effective method for the isolation of actinomycetes as it yielded a maximum of 105 actinomycete isolates and IM7 was the most suitable medium for actinomycete isolation with highest percentage of recovery (31 %). Forty three isolates (70.5 %) showed antimicrobial activities against one or more test microorganisms. Isolates IIUM B21 and IIUM B31 showed antimicrobial activity against all test microorganisms. Seven isolates showed antifungal activity as they inhibited only C. albicans. Ten isolates were randomly selected for identification based on partial sequences of 16S rRNA gene. Six isolates were found belong to the genus Streptomyces, two isolates belong to the genus Micromonospora and two isolates were identified as Rhodococcus spp. Conclusion, significance and impact of study: These findings revealed the potential of mangrove sediment of Tanjung Lumpur as an important source of actinomycetes with biosynthetic capabilities which might be beneficial to pharmaceutical industries.

Culturable bioactive actinomycetes from the Great Indian Thar Desert

The present study was attempted to determine the antimicrobial potential of actinomycetes from the Great Indian Thar Desert. A total of 100 different morphotypes based on phenotypic characterization were isolated from desert ecosystems located in the northwest of India and tested for their antimicrobial activity by the cross-streak method. Among the strains tested, 13 actinomycetes exhibiting strong antimicrobial activities against several test organisms, including multidrug resistant bacteria (MRSA) were chosen for a phylogenetic diversity study. The results of 16S rRNA gene sequencing showed their affiliation to actinobacterial genera: Actinomadura, Nocardia, Nonomuraea, Spirillispora, and Streptomyces. Three of these isolates were considered to be new members of the Streptomyces genus and another strain also seemed to be a new species of the genus Spirillispora. Among these strains, five were chosen to study the bioactive products using Q-Tof-MS because of their broad spectrum activity against the panel of test pathogens used. The results showed that they produce many known compounds and might produce few unknown compounds as well. This is the first such report on the selective isolation of actinomycetes from the Great Indian Thar desert, and their screening for antibacterial potential. This ecosystem has never before been explored to this extent.

In vitro antimicrobial activity and characterization of mangrove isolates of streptomycetes effective against bacteria and fungi of nosocomial origin

The study aimed at determining the in vitro antimicrobial activity of alkaliphilic and halotolerant actinomycetes isolated from a mangrove ecosystem and identification of a potent strain. Twenty five isolates of actinomycetes were isolated from the sediment samples of Valapattanam mangrove swamp in Kerala, India. Antimicrobial activity of four selected actinomycete isolates was determined against bacterial and fungal pathogens of nosocomial origin by agar well diffusion method. Molecular characterization of the potent isolate was performed by 16S rDNA sequencing. Isolate no I-1 significantly inhibited Staphylococcus aureus ATCC 25923 (12 mm), S. aureus (15±0.05 mm), S. citreus (20±0.5 mm), Bacillus cereus (17±0.2 mm) and Serratia marcescens (12 mm). It also demonstrated effective antifungal action against Penicillium sp. (12±0.2 mm), Candida albicans (20±0.5 mm), C. parapsilosis (12 mm) and Cryptococcus neoformans (12 mm). Morphological study revealed that all the isolated actinomycetes belonged to the genus Streptomyces. Based on 16S rDNA sequence data, the selected isolate I-1 was shown to be closely related to Streptomyces xiamenensis. The results revealed that the mangrove ecosystem of Valapattanam harboured a rich consortium of many potent actinomycetes, which could synthesize novel bioactive compounds of pharmacological significance.

Efficiency of some actinomycete isolates in biological treatment and removal of heavy metals from wastewater

The main focus of studies and research in the field of wastewater treatment is treating wastewater without causing environmental hazards as well as getting benefits from the treated waste materials. In this regard, the aim of the current study was to isolate some actinomycete strains from Beni-Suef Wastewater Treatment Plant to study their capacities for biological treatment and for removal of heavy metals from the wastewater. For this purpose, 17 actinomycete strains were isolated from 3 wastewater samples representing the main stages of treatment in the plant, namely: the influent, the secondary sedimentation tank and the effluent. Then, 10 morphologically dissimilar isolates were selected. The 10 selected actinomycete isolates were characterized by their morphological characteristics and were found to belong to the genera Nocardia, Streptomyces, Rhodococcus, Gordonia and Nocardiopsis. The ability of the selected actinomycetes for biological treatment of the wastewater was evaluated by measuring the biochemical oxygen demand (BOD), the chemical oxygen demand (COD) and the total suspended solids (TSS); and their ability for removal of some heavy metals (Cu, Fe, Mn, Pb and Zn) was also determined. The results showed that most of the selected actinomycetes were effective in the biological treatment of the wastewater and have the ability to decrease the values of BOD, COD and TSS; and also to reduce the concentrations of the tested heavy metals (Cu, Fe, Mn, Pb and Zn) markedly. The Streptomyces strain C11 was found to be the most efficient organism in respect to biological treatment of the wastewater and removal of the heavy metals from the raw wastewater. Key words: Actinomycetes, wastewater, biological treatment, heavy metals removal.

Evaluation of actinomycete isolates obtained from herbal vermicompost for the biological control of Fusarium wilt of chickpea

A total of 137 actinomycetes cultures, isolated from 25 different herbal vermicomposts, were characterized for their antagonistic potential against Fusarium oxysporum f. sp. ciceri (FOC) by dual-culture assay. Of the isolates, five most promising FOC antagonistic isolates (CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90) were characterized for the production of siderophore, cellulase, protease, hydrocyanic acid (HCN), indole acetic acid (IAA) and antagonistic potential against Rhizoctonia bataticola, which causes dry root rot in chickpea (three strains viz. RB-6, RB-24 and RB-115) and sorghum (one strain). All of the five FOC antagonistic isolates produced siderophore and HCN, four of them (except KAI-90) produced IAA, KAI-32 and KAI-90 produced cellulase and CAI-24 and CAI-127 produced protease. In the dual-culture assay, three of the isolates, CAI-24, KAI-32 and KAI-90, also inhibited all three strains of R. bataticola in chickpea, while two of them (KAI-32 and KAI-90) inhibited the tested strain in sorghum. When the FOC antagonistic isolates were evaluated further for their antagonistic potential in the greenhouse and wilt-sick field conditions on chickpea, 45–76% and 4–19% reduction of disease incidence were observed, respectively compared to the control. The sequences of 16S rDNA gene of the isolates CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90 were matched with Streptomyces tsusimaensis, Streptomyces caviscabies, Streptomyces setonii, Streptomyces africanus and an identified species of Streptomyces, respectively using the BLAST searching. This study indicated that the selected actinomycete isolates have the potential for biological control of Fusarium wilt disease in chickpea.

Isolation and characterization of a Nocardiopsis sp. from honeybee guts

Although actinomycetes are the plant-associated environmental bacteria best known for producing thousands of antibiotics, their presence in the guts of flower-feeding honeybees has rarely been reported. Here, we report on the selective isolation of actinomycetes from the gut microbiota of healthy honeybees, and their inhibitory activity against honeybee indigenous bacteria. More than 70% of the sampled honeybees (N>40) in a season carried at least one CFU of actinomycete. The isolates from bees of one location produced inhibitory bioactivities that were almost exclusively against several bee indigenous Bacillus strains and Gram-positive human pathogens but not Escherichia coli. An antibiotic-producing actinomycete closely related to Nocardiopsis alba was isolated from the guts in every season of the year. A DNA fragment encoding a homologous gene (phzD) involved in phenazine biosynthesis was identified in the isolate. Expression of the phzD detected by reverse transcription-PCR can explain the survival of this organism in anaerobic environments as some redox-active extracellular phenazines are commonly regarded as respiratory electron acceptors. The results raise important questions concerning the roles of the antibiotic-producing actinomycetes and the phenazine-like molecules in honeybee guts and honey.

Plant growth promotion and biological control ofPythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes

To evaluate the potential of Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis endophytic in cucumber roots, to promote plant growth and to protect seedlings and mature plants of cucumber from diseases caused by Pythium aphanidermatum, under greenhouse conditions. Three endophytic isolates, out of 29, were selected through tests aimed at understanding their mechanisms of action as biocontrol agents and plant growth promoters. When applied individually or in combination, they significantly promoted plant growth and reduced damping-off and crown and root rot of cucumber. The combination of the three isolates resulted in significantly better suppression of diseases and plant growth promotion, than where the plants were exposed to individual strains. The three selected actinomycete isolates colonized cucumber roots endophytically for 8 weeks, promoted plant growth and suppressed pathogenic activities of P. aphanidermatum on seedling and mature cucumber plants. The results clearly show that the endophytic, glucanase-producing actinomycetes used, especially as a combined treatment, could replace metalaxyl, which is the currently recommended fungicide for Pythium diseases in the United Arab Emirates. These endophytic isolates also have the potential to perform as plant growth promoters, which is a useful attribute for crop production in nutrient impoverished soils.

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.

Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord, Norway: isolation, diversity and biological activity

Actinomycete bacteria produce a wide variety of secondary metabolites with diverse biological activities, some of which have been developed for human medicine. Rare actinomycetes are promising sources in search for new drugs, and their potential for producing biologically active molecules is poorly studied. In this work, we have investigated the diversity of actinomycetes in the shallow water sediments of the Trondheim fjord (Norway). Due to the use of different selective isolation methods, an unexpected variety of actinomycete genera was isolated. Although the predominant genera were clearly Streptomyces and Micromonospora, representatives of Actinocorallia, Actinomadura, Knoellia, Glycomyces, Nocardia, Nocardiopsis, Nonomuraea, Pseudonocardia, Rhodococcus and Streptosporangium genera were isolated as well. To our knowledge, this is the first report describing isolation of Knoellia and Glycomyces species from the marine environment. 35 selected actinomycete isolates were characterized by 16S rDNA sequencing, and were shown to represent strains from 11 different genera. In addition, these isolates were tested for antimicrobial activity and the presence of polyketide synthase and non-ribosomal peptide synthetase genes. This study confirms the significant biodiversity of actinobacteria in the Norwegian marine habitats, and their potential for producing biologically active compounds.

Antibacterial potential of antagonistic Streptomyces sp. isolated from marine sponge Dendrilla nigra

The role of Streptomyces sp. (BTL7) in synthesis of antibacterial agents reported from the marine sponge Dendrilla nigra was evaluated. Selective isolation of actinomycetes was performed on the newly developed selective media, Sponge Agar (SA) 1 and SA 2. The growth rate and antibiotic production were increased on the media supplemented with sponge extract. The chosen isolate BTL7 showed inhibitory interaction with Micrococcus luteus and the extracellular products contained potent antibacterial agents. The minimum inhibitory concentration of BTL7 against M. luteus was 44 μg protein/ml and the minimum bactericidal concentration was 88 μg protein/ml. Peak antibacterial activity was observed at 72 h in batch culture. Based on the findings, it could be inferred that bacterial endosymbionts sponges could form a reliable source for bioprospecting of next generation pharmaceutical agents.

The application of succession analysis in combination with EHF irradiation to the selective isolation of actinomycetes from soil

A new method employing succession analysis and extremely high frequency (EHF) irradiation is proposed for the selective isolation of actinomycetes from soil. Total actinomycetes were efficiently isolated from soil suspensions irradiated in the wavelength band 4.6–5.8 mm on the 14th and 45th days of succession initiated by soil wetting and from soil suspensions irradiated in the wavelength band 8–11.5 mm on the 7th day of succession. The rare actinomycete genera Actinomadura, Micromonospora, Nonomuraea, Microbispora, Amycolatopsis, Pseudonocardia, Saccharothrix, Streptosporangium, Actinosynnema, Nocardioides, and Saccharopolyspora were isolated by either of the two approaches (succession analysis and EHF irradiation); however, the range of isolated rare actinomycetes was considerably wider when a combination of the two approaches was used. For instance, actinomycetes of the rare genera Actinocorallia, Promicromonospora, Actinoplanes, and Kibdelosporangium were isolated only when EHF irradiation was employed at the early stages of succession.

Some Approaches to the Selective Isolation of Actinomycetes of the Genus Actinomadura from Soil

Some approaches to the selective isolation of actinomycetes of the genus Actinomadura from soil are described. The approach that involves the thermal treatment of soil samples and their plating onto Gauze 1 medium with the antibiotics nystatin, nalidixic acid, and rubomycin provides for an increased amount of actinomaduras isolated from the soil actinomycete complex and for a decreased amount of streptomycetes.

Isolation of actinomycetes from soil using extremely high frequency radiation

A new method employing extremely high frequencies (EHFs) is proposed for the selective isolation of actinomycetes from soil. The pretreatment of soil suspensions with EHF wavelengths of 5.6 and 7.1 mm led to a nonselective isolation of actinomycetes. At the same time, the irradiation of soil suspensions within wavelength bands of 3.8-5.8 and 8-11.5 mm considerably augmented the total number of isolated actinomycetes and increased the fraction of the isolated rare genera by 2 and 7 times, respectively. The rare actinomycete genera were represented by Actinomadura, Microtetraspora, Nonomuraea, Micromonospora, Amycolatopsis, Pseudonocardia, Saccharotrix, and Streptosporangium.

Geomicrobiological Study of the Grotta dei Cervi, Porto Badisco, Italy

Speleothems (active stalactites, wall concretions), rock walls, ceiling, and soils from the galleries of Grotta dei Cervi, Porto Badisco, Italy, were sampled to investigate the culturable heterotrophic microbial communities present in this cave. Sampling was carried out in a transect of about 230 m from the entrance to the central gallery where numerous Gram-positive bacteria were isolated from all studied sites. Members of the genera Agromyces, Arthrobacter, Rhodococcus , and particularly Streptomyces , of the order Actinomycetales, are spread throughout the whole cave. The ability of actinomycetes, and particularly of Nocardiopsis , to colonize salt-stressed environments is favored by the presence of ectoine, a compatible solute for osmotic adaptation. Selected actinomycete isolates were tested for the formation of crystals. Strains from all tested genera, except isolates of Gordonia and Nocardia , produced vaterite and/or calcite. Production of Mgcalcite was restricted to strains of Brachybacterium, Rhodococcus , and Streptomyces , whereas struvite was only precipitated by an unidentified isolate. These findings indicate that actinomycetes may play a role in the formation of mineral deposits in caves.

A new method for the selective isolation of actinomycetes from soil

A coal-vitamin medium was developed to isolate actinomycetes from soil, which was superior to other currently used media. It increased the number of actinomycetes and inhibited the growth of other soil bacteria. The pretreatment of soil suspension with peptone (6%) and lauryl sulfate (0.05%) at 50°C for 10 min, also greatly increased the number of actinomycetes from soil prior to incubation with new medium.