Imran Sajid

Screening of Rhizospheric Actinomycetes for Various In-vitro and In-vivo Plant Growth Promoting (PGP) Traits and for Agroactive Compounds

In this study 98 rhizospheric actinomycetes were isolated from different wheat and tomato fields, Punjab, Pakistan. The isolates were characterized morphologically, biochemically, and genetically and were subjected to a comprehensive in vitro screening for various plant growth promoting (PGP) traits. About 30% of the isolates screened were found to be the promising PGP rhizobacteria (PGPRs), which exhibited maximum genetic similarity (up to 98–99%) with different species of the genus Streptomyces by using16S rRNA gene sequencing. The most active indole acetic acid (IAA) producer Streptomyces nobilis WA-3, Streptomyces Kunmingenesis WC-3, and Streptomyces enissocaesilis TA-3 produce 79.5, 79.23, and 69.26 μg/ml IAA respectively at 500 μg/ml L-tryptophan. The highest concentration of soluble phosphate was produced by Streptomyces sp. WA-1 (72.13 mg/100 ml) and S. djakartensis TB-4 (70.36 mg/100 ml). All rhizobacterial isolates were positive for siderophore, ammonia, and hydrogen cyanide production. Strain S. mutabilis WD-3 showed highest concentration of ACC-deaminase (1.9 mmol /l). For in-vivo screening, seed germination, and plant growth experiment were conducted by inoculating wheat (Triticum aestivum) seeds with the six selected isolates. Significant increases in shoot length was observed with S. nobilis WA-3 (65%), increased root length was recorded in case of S. nobilis WA-3 (81%) as compared to water treated control plants. Maximum increases in plant fresh weight were recorded with S. nobilis WA-3 (84%), increased plant dry weight was recorded in case of S. nobilis WA-3 (85%) as compared to water treated control plants. In case of number of leaves, significant increase was recorded with S. nobilis WA-3 (27%) and significant increase in case of number of roots were recorded in case of strain S. nobilis WA-3 (30%) as compared to control plants. Over all the study revealed that these rhizospheric PGP Streptomyces are good candidates to be developed as bioferlizers for growth promotion and yield enhancement in wheat crop and can be exploited for the commercial production of different agro-active compounds.

Larvicidal potential of Asteraceae family endophytic actinomycetes against Culex quinquefasciatus mosquito larvae

Pakistan is blessed with plants of Asteraceae family with known medicinal background used for centuries by Hakims (traditional physicians). Keeping in mind the background of their anti-larval potential, a total of 21 endophytic actinomycetes were isolated from four Asteraceae plants and screened against the first and fourth instar stages of Culex quinquefasciatus Say mosquito larvae. Of the 21 isolates, 6 of them gave strong larvicidal activity (80–100% mortality) in the screening results and 4 isolates gave a potent larvicidal activity (100% mortality) at the fourth instar stage. These isolates belonged to different species within the actinomycetes group, namely Streptomyces albovinaceus and Streptomyces badius. This communication reports the larvicidal potential of endophytic actinomycetes residing within the native Asteraceae plants in Pakistan. The study suggests further exploration through large-scale productions leading to the identification of the larvicidal compounds.

Rare actinomycetes Nocardia caishijiensis and Pseudonocardia carboxydivorans as endophytes, their bioactivity and metabolites evaluation

Two strains identified as Nocardia caishijiensis (SORS 64b) and Pseudonocardia carboxydivorans (AGLS 2) were isolated as endophytes from Sonchus oleraceus and Ageratum conyzoides respectively. The analysis of their extracts revealed them to be strongly bioactive. The N. caishijiensis extract gave an LC50 of 570 μg/ml(-1) in the brine shrimp cytotoxicity assay and an EC50 of 0.552 μg/ml(-1) in the DPPH antioxidant assay. Antimicrobial activity was observed against Methicillin resistant Staphlococcus aureus (MRSA) and Escherichia coli ATCC 25922 (14 mm), Klebsiella pneumoniae ATCC 706003 (13 mm), S. aureus ATCC 25923 (11 mm) and Candida tropicalis (20 mm). For the extract of P. carboxydivorans the EC50 was 0.670 μg/ml(-1) and it was observed to be more bioactive against Bacillus subtilis DSM 10 ATCC 6051 (21 mm), C. tropicalis (20 mm), S. aureus ATCC 25923 (17 mm), MRSA (17 mm), E. coli K12 (W1130) (16 mm) and Chlorella vulgaris (10 mm). The genotoxicity testing revealed a 20 mm zone of inhibition against the polA mutant strain E. coli K-12 AB 3027 suggesting damage to the DNA and polA genes. The TLC and bioautography screening revealed a diversity of active bands of medium polar and nonpolar compounds. Metabolite analysis by HPLC-DAD via UV/vis spectral screening suggested the possibility of stenothricin and bagremycin A in the mycelium extract of N. caishijiensis respectively. In the broth and mycelium extract of P. carboxydivorans borrelidin was suggested along with α-pyrone. The HPLC-MS revealed bioactive long chained amide derivatives such as 7-Octadecenamide, 9, 12 octadecandienamide. This study reports the rare actinomycetes N. caishijiensis and P. carboxydivorans as endophytes and evaluates their bioactive metabolites.

Antitumour compounds from a saline soil isolate, Streptomyces griseoincarnatus CTF15

A new actinomycete strain designated as Streptomyces sp. CTF15 was isolated from a saline soil using casein–KNO3 agar medium. The strain Streptomyces sp. CTF15 exhibited promising antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Streptomyces viridochromogens Tü57 and high cytotoxicity (91.2% mortality) against Artimia salina in biological screening. The cultivation of this strain in a 50 L lab fermenter and subsequent isolation and purification by a series of chromatographic techniques and structure elucidation by MS and NMR analysis of the active metabolites revealed that it is a highly stable producer of resistomycin (1), tetracenomycin D (2) and actinomycin D (3), even under non-optimised culture conditions. The morphological, microscopic, biochemical and physiological characterisation suggested that the strain CTF15 belongs to the genus Streptomyces. A partial 16S rRNA gene sequence (1429 bp) from the strain CTF15 was determined and found to have high identity (99%) with Streptomyces griseoincarnatus. As such, this is the first report of a strain of S. griseoincarnatus capable of producing these three bioactive compounds simultaneously.

Effect of crude extracts of selected actinomycetes on biofilm formation of A. schindleri, M. aci, and B. cereus

Actinomycetes are well known group of gram positive bacteria for their potential to produce antibiotics. This study sought to assess the ability of the selected actinomycetes to control biofilm forming bacteria isolated from different dental plaque samples. On the basis of morphological differences three out of ten different dental plaque bacterial isolates were selected for further study. These isolates were biochemically and genetically characterized and were identified as Acinetobacter schinndleri, Moraxella aci, and Bacillus cereus. Antibiotic resistant profile was measured through disc diffusion method and found that all three isolates were moderately sensitive to ofloxacin and erythromycin and resistant to trimethoprim. Antibacterial activity of ten different Streptomyces strains was assessed through an agar plug and well diffusion method against three dental biofilm forming bacteria. Two Streptomyces strains named as S. erythrogriseus and S. labedae showed good antibacterial activity against Moraxella and Acinetobacter strains. Ability of the four active antibiotic producing strains to inhibit biofilm formation was assessed using microtiter biofilm detection assay. It was found that biofilm forming ability of Acinetobacter and Moraxella was inhibited by S. labedae an antibiotic producing strain, while S. macrosporeus can only inhibit biofilm formation by B. cereus.

Purification and Structure Elucidation of the Polyether Antibiotic Alborixin from Streptomyces pulcher CRF17

Aims: To screenStreptomycessp. from saline soil in Pakistan, for its antimicrobial activity and to purify and identify the active metabolites produced by mass spectrometry and NMR spectroscopy. Strain id

Screening, Characterization and Optimization of antibacterial peptides, produced by Bacillus safensis strain MK-12 isolated from waste dump soil KP, Pakistan

Aims The current study was designed to isolate, screen and identify the indigenous soil antibacterial exhibiting bacteria (AEB) and effect of various parameters on growth of AEB and antibacterial peptides production. Methods and results The soil isolates were screened for antagonistic activity against a set of ATCC and local MDR human pathogenic bacterial strains. The antibacterial compound was protein in nature, exhibited no haemolysis and molecular weight was less than 20 KDa. The potential AEB isolate was identified by morphology, biochemical testing and by 16S rRNA gene sequencing as B. safensis MK-12. Growth and antibacterial activity was optimized for B. safensis strain MK-12, exhibited maximum growth as well as antibacterial activity after 48 hours of incubation at pH 8, 30 °C in shaking incubator when fermented in optimized medium. Conclusion The current study results indicate that indigenous soil is rich source of AEB and could be a promising source of antimicrobial compounds to fight against MDR bacteria in future. Significance and impact This is the first scientific report on soil bacteria from northern region of Pakistan as per our knowledge. Therefore, screening of soil bacteria for antibacterial activity from unexplored area may contribute towards new antibiotic. Selected soil strain in the current study exhibited promising antibacterial activity against human pathogenic MDR bacterial strains.

PURIFICATION AND IDENTIFICATION OF BIOACTIVE ANGUCYCLINONES FROM Streptomyces matensis BG5, ISOLATED FROM THE RHIZOSPHERE OF Rosa indica L.

A newly isolated strain Streptomyces sp. BG5 was investigated for the production of bioactive compounds. The strain exhibited broad-spectrum activity against an array of nine test organisms including gram-positive bacteria, gram-negative bacteria, and fungal and microalgal pathogens, along with a moderate cytotoxic response (28.9% mortality) in a microwell cytotoxicity assay against the brine shrimp Artimia salina. The morphological, physiological, and biochemical characterization of the Streptomyces sp. BG5 strongly suggested it to be a member of the genus Streptomyces. The nucleotide sequence of 16S rRNA gene (1433 pb) of the Streptomyces sp. BG5 (Gene bank accession number EU301836) exhibited high similarity (98%) with Streptomyces matensis. The large-scale fermentation of Streptomyces sp. BG5 and subsequent extraction, isolation, and purification of the crude extract afforded three pure compounds. The structures of these compounds were identified as ochromycinone (1a), emycin D (2), and 1-acetyl-β-carbolin (3), based on nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and by comparison with reference data from the literature. Supplemental materials are available for this article. Go to the publishers online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Screening for type I polyketide synthases genes of endophytic Streptomycetes isolated from Parthenium hysterophorus L.

Polyketides are a vital group of secondary metabolites comprising of antifungal, antibacterial as well as anticancer agents. The multifunctional enzymes responsible for the biosynthesis of these secondary metabolites are polyketide synthases (PKS) in which polyketide synthases type I (PKS-I) is mainly responsible for highly assorted group of metabolites with substantial medical importance. In this study molecular screening was done using six sets of degenerate primers to determine the presence of PKS-I gene cluster in endophytic Streptomyces isolated from the weed Parthenium hysterophorus L. A total of 40 endophytes were isolated and identified by morphological, biochemical and physiological characterization as belonging to the genus Streptomyces. The 16S rRNAgene sequencing of the selected isolates exhibited maximum similarity with different species of Streptomyces such as Streptomyces rochei (99%), Streptomyces litmocidini (99%), Streptomyces enissocaesili (99%), Streptomyces djakartensis (99%), Streptomyces olivaceus (99%), Streptomyces spp. (99%), Streptomyces plicatus (99%), Streptomyces geysiriensis (99%) and Streptomyces vinaceusdrappus (99%). The molecular screening revealed the presence of PKS-I gene with a PCR amplification products of size ∼300 bps, ∼320 bps and ∼700 bps in the isolates RT-13, RT-43, RT-47, RT-49, RT-54, RT-56, RT-57, RT-58, RT-59, RT-61 and RT-65 through all the six sets of primers used. To the best of our understanding no previous study has been carried out reporting the molecular screening of PKS-I genes in endophytic Streptomyces from Parthenium hysterophorus L. The results provide an insight into an unexplored environment containing endophytic Streptomyces harboring the polyketide synthases gene which if further investigated may lead to a new source of antimicrobial agents.