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Phylogenetic Characterization, Fermentation and Biological Activities of an Antibiotic Producing Streptomyces Clavuligerus Isolated from KSA

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Phylogenetic Characterization, Fermentation and Biological Activities of an Antibiotic Producing Streptomyces Clavuligerus Isolated from KSA Int.J.Curr.Microbiol.App.Sci (2014) 3(12) xx-xx ISSN: 2319-7706 Volume 3 Number 12 (2014) pp. 519-534 http://www.ijcmas.com Original Research Article Phylogenetic characterization, fermentation and biological activities of an antibiotic producing Streptomyces clavuligerus isolated from KSA Houssam M. Atta1,2* and Ayman M. Yassen2 1Botany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt 2Biotechnology Department, Faculty of Education and Science, Taif University, Al-Khurmah branch, KSA *Corresponding author A B S T R A C T This work was carried out for the biosynthesis of antimicrobial substance that demonstrated inhibitory effects against pathogenic microorganisms from Streptomyces sp. The KSA-T180 isolate has been considered the most potent, this was identified by biochemical, chemotaxonomic, morphological and physiological K e y w o r d s properties consistent with classification in the genus Streptomyces, with the nearest species being Streptomyces clavuligerus. Furthermore, a phylogenetic analysis of Actinomycetes, the 16S rDNA gene sequence and ribosomal database project consistent with Conventional conventional taxonomy confirmed that strain KSA-T180 was most similar to taxonomy, Streptomyces clavuligerus (98%). The active metabolite was extracted using n- Phylogenetic Butanol (1:1, v/v) at pH 7.0. The separation of the active ingredient of the analysis, antibacterial agent and its purification was performed using both thin layer Fermentation, chromatography (TLC) and column chromatography (CC) techniques. The Biological chemical characteristics of the antibacterial agent(s) viz. elemental analysis and activities and spectroscopic characteristics have been investigated. This analysis indicates a clavulanic acid suggested empirical formula of C8H8NO5, ultraviolet (UV) absorption spectrum recorded a maximum absorption peak at 285 nm, Infra-red (IR) spectrum showed characteristic twenty-three bands and Mass spectrum showed that the molecular weight at 200.0. The minimum inhibition concentrations "MICs" of the antibiotic were also determined. The collected data emphasized that the antibiotic was characterized as clavulanic acid Introduction Antibiotics are complex chemical secondary Antibiotics are used to prevent infections metabolites, which are produced by after surgery or at open wounded areas microorganisms and acts against other (Cimochowski et al., 2001). Actinomycetes microorganisms (Singh et al., 2014). are a major source of bioactive natural 519 Int.J.Curr.Microbiol.App.Sci (2014) 3(12) xx-xx products. More than 10,000 substances with produce 21 secondary metabolites (Gouveia bioactivity have been isolated so far from et al., 2001; Ortiz et al., 2007 and Rodríguez terrestrial and marine actinomycetes (Berdy et al., 2008) including holomycin; a member 2005, 2012) and many are clinically used as of the pyrrothine class antibiotics, an antitumor agents, antibiotics, or antibiotic related to tunicamycin; a immunosuppressants. Members of glucosamine-containing antibiotic (Kenig Actinomycete genera are gram positive and Reading, 1979), and -lactam bacteria with high GC content in their DNA metabolites with antibiotic, antifungal (Kieser et al., 2000). They are well known activities, which is why it has been studied for production of a wide range of secondary over three decades (Thai et al., 2001). In the metabolites like antibiotics, antitumor present study were describe the isolation of compounds, immunosuppressants, an actinomycete strain KSA-T180 from Taif herbicides, antiviral and antiparasitic agents. city, KSA, which generates a production the There are 23,000 biologically active bioactive substances that demonstrated secondary metabolites produced by inhibitory affects against microbial microorganisms has been identified up to pathogenic. The identification of this strain now, and 10,000 of them are produced by based on the cultural, morphology, the order of Actinomycetales. Streptomyces physiology and biochemical characteristics, spp. produce 7,600 of these 10,000 as well as 16s rDNA methodology. The secondary metabolites (Sacramento et al., primary bioactive substances were tested 2004 and Olano et al., 2008). Streptomyces against Gram positive and Gram negative spp. are filamentous, spore forming and bacteria and unicellular and filamentous strictly aerobic bacteria which belong to fungi. One major active compound was Actinomycetes order (Paradkar et al., 2003). extracted from the purified fermented broth High GC content genome (more than 70 and chemically characterized as clavulanic mole %) and large linear plasmids (10-600 acid, based on the elemental analysis and kb) are distinctive features of Streptomyces spectroscopic data obtained from the species (Kieser et al., 2000). Mona-Ibrahim application of UV, FT-IR and Mass (2012) reported that, the Streptomyces Spectrum and by comparison with published generally synthesis a sizeable number of data. diverse natural secondary metabolites (Onaka et al., 2001), such as antibiotics, Materials and Methods insecticides, herbicides, immunosuppressive actions (Mao et al., 2007), vitamins, Actinomycete isolate: The actinomycete alkaloids, plant growth factor, enzymes and isolate KSA-T180 was isolated from soil enzyme inhibitors (Augustine et al., 2005). sample collected from Taif city, Saudi Secondary metabolite production of Arabia kingdom. It was purified using the Streptomyces is strictly related and regulated soil dilution plate technique described by with morphological changes (Paradkar et al., (Williams and Davis, 1965). 2003). Streptomyces clavuligerus has been the subject of extensive research in the last Test organisms: The test strains 30 years because of its ability to produce - Staphylococcus aureus, NCTC 7447; lactam metabolites with antibiotic, Bacillus subtilis, NCTC 1040 ; Bacillus antifungal and -lactamase-inhibitory pumilus, NCTC 8214 ; Micrococcus luteus, activities (Thai et al., 2001 and Bibb, 2005). ATCC 9341.Escherichia coli, NCTC 10416; Streptomyces clavuligerus is known to Klebsiella pneumonia, NCIMB 9111; 520 Int.J.Curr.Microbiol.App.Sci (2014) 3(12) xx-xx Pseudomonas aeruginosa, ATCC 10145; DNA Isolation and Manipulation: The Candida albicans, IMRU 3669; locally isolated actinomycete strain was Saccharomyces cervisiae ATCC 9763; grown for seven days on a starch agar slant Aspergillus flavus, IMI 111023, Aspergillus at 30°C. Two ml of a spore suspension were fumigatous, ATCC 16424 ; Fusarium inoculated into the starch- nitrate broth and oxysporum and Penicillium chrysogenum incubated for five days on a shaker was collection, National Research Centre, incubator at 200 rpm and 30°C to form a Dokki-Giza, Egypt. pellet of vegetative cells (pre-sporulation). The preparation of total genomic DNA was Culture media: The seed medium had the conducted in accordance with the methods following composition (in g/L distilled described by (Sambrook et al., 1989). water): glycerol, 15; bacto-peptone, 10; malt extract, 10; yeast extract, 1.0; K2HPO4, 2.5; Amplification and Sequencing of the 16S MgSO4·7H2O, 0.75; MnCl2·4H2O, 0.001; rDNA Gene: PCR amplification of the 16S FeSO4·7H2O, 0.001; and ZnSO4·7H2O, rDNA gene of the local actinomycete strain 0.001. The pH of the medium was adjusted was conducted using two primers, StrepF; to 6.8 with NaOH 5.0 M before autoclaving 5.-ACGTGTGCAGCCCAAGACA-3. and at 121 °C for 15 min. The inoculum medium Strep R; 5.ACAAGCCCTGGAAACGGG used in the cultivations, based on that GT-3., in accordance with the method proposed by (Maranesi et al., 2005), had the described by (Edwards et al., 1989). The following composition (in g/L distilled PCR mixture consisted of 30 pmol of each water): glycerol, 10; soybean, 20; K2HPO4, primer, 100 ng of chromosomal DNA, 200 1.2; MnCl2·4H2O, 0.001; FeSO4·7H2O, M dNTPs, and 2.5 units of Taq 0.001; ZnSO4·7H2O, 0.001, pH 6.8. The polymerase, in 50 l of polymerase buffer. composition of the production medium was Amplification was conducted for 30 cycles similar to that used for the inoculum, except of 1 min at 94°C, 1 min of annealing at for the concentration of glycerol (5.0 g/L). 53°C, and 2 min of extension at 72°C. The PCR reaction mixture was then analyzed via Screening for antimicrobial activity: The agarose gel electro phoresis, and the anti- microbial activity was determined remaining mixture was purified using QIA according to (Kavanagh, 1972). quick PCR purification reagents (Qiagen, USA). The 16S rDNA gene was sequenced Conventional Taxonomy: The cultural, on both strands via the dideoxy chain morphological, physiological and termination method, as described by (Sanger biochemical characteristics of strain KSA- et al., 1977). T180 were assessed following the guidelines adopted by the International Streptomyces Sequence Similarities and Phylogenetic Project (ISP) (Shrilling and Gottlieb, 1966). Analysis: The BLAST program The diaminopimelic acid (LL-DAP) isomers (www.ncbi.nlm.nih. gov/blst) was employed (chemotaxonomy character) in the cell wall in order to assess the degree of DNA were analysed as described by (Lechevalier similarity. Multiple sequence alignment and and Lechevalier, 1980). The media molecular phylogeny were evaluating using composition and the cultivation conditions BioEdit software (Hall, 1999). The were implemented as described by (Shrilling phylogenetic tree was displayed using the and Gottlieb, 1966). Colors characteristics TREE VIEW program. were assessed on the scale developed by (Kenneth and Deane, 1955). 521 Int.J.Curr.Microbiol.App.Sci
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