PRODUCTION OF KERATINASES FROM NOCARDIOPSIS SP. 28ROR AS A NOVEL IRAQI STRAIN

Objectives: isolate a novel feather- degrading actinobacterial species had the ability to produce wide pH activity keratinases.Methods: Of 23 actinobacterial isolates were recovered from farm soil, poultry farm soil and feather wastes, these isolates were screened for protease and keratinase production on skim milk agar, feather  media, and  antimicrobial production. One potential  isolate was identified depending on phenotypical, physiological and molecular according to partial sequences of 16S r RNA gene analysis and optimized keratinase production. Results:   11 isolates out of 22 protease producer  had the ability to degrade raw chicken feather and some of these  isolates produced  antifungal and antibacterial metabolites.The potential isolate,  Nocardiopsis sp. 28ROR (GenBank: KC702802.1), produced two types of extracellular keratinases in feather meal medium at pH6 (acid type), 30-35°C  for 7d  and  the alkaline keratinase at pH10, 40°C  for 7d.Conclusion: The Nocardiopsis sp. 28ROR was a novel strain produced keratinases using feather meal degradation as a cheap waste medium. The wide tolerance of temperature and pH by keratinase makes it an ideal contender to be investigated further for potential application as a detergent additive.Keywords: Nocardiopsis, Keratinase, Optimization, Feather medium, Antibiotic.Â

Detection of Staphylococcal Species Diversity within Human Skin Microbiome Using a Simple PCR-SSCP Technique

Objective. The aim of this study was to detect the diversity of Staphylococcus species of a healthy human skin using a simple techniquevPCR-SSCP. Methods: Blood samples, saliva, and skin swaps samples were collected from 50 persons from Hilla City  - Iraq. The genomic DNA was extracted from these samples using the Bacteria Genomic DNA Kit. The concentrations and purity of DNA extract estimated by NanoDrop spectrophotometer. Polymerase chain reaction – single-strand conformational polymorphism (PCR–SSCP) technique was performed to detect the diversity between Staphylococcal species in the human skin microbiome using a specific primer of the 16SrRNA gene. Results: The PCR results, indicated that the Staphylococcal species were found within the ski community, but it's not infected blood and mouth of test healthy individuals. SSCP-heteroduplex patterns of PCR products appeared the presence Staphylococcal species diversity within skin microbiome of test healthy individuals. Conclusion: In spite of the PCR-SSCP, heteroduplex method was simple and cheap and appeared the diversity betweenvStaphylococcalspeciesvin the human skin microbiome, but it's not diagnosed the bacterial strains. So these results required to confirm by DNAvsequencingvtechnique

CYTOTOXIC ACTIVITY OF ALKALOIDS EXTRACTED FROM THREE IRAQI PLANTS AGAINST BREAST CANCER CELL LINE

  Objectives: Screening for cytotoxic activity of total alkaloid extracts of Eucalyptus camaldulensis, Aloe vera, and Capparis spinosa against breast cancer cell line Michigan Cancer Foundation-7 (MCF-7) and nontumorigenic fetal hepatic cell line (WRL-68).Methods: The plant powders were extracted separately with 80% methanol and chloroform at pH 2 and 10. Total alkaloids were detected qualitatively by Mayer's, Dragendorff's, and Hager‘s reagents and estimated quantitatively by bromocresol green spectrophotometry depending on the atropine calibration curve. The cytotoxic activity was evaluated by 3-[4, 5-dimethylthiazoyl]-2, 5-diphenyltetrazolium bromide assay.Results: The extract of E. camaldulensis had highest total alkaloid content (24.50±1.70 mg/100 g plant dry weight) than the others. The total alkaloids (400 μg/ml) of E. camaldulensis reduced the cell viability of both cell lines MCF-7 and WRL-68 to 45.25±2.20% and 92.00±1.55%, respectively, and the inhibitory concentration 50% of cells were 375.50 μg/ml for MCF-7. The alkaloids of C. spinosa had effect 79.80±7.08% and 89.50±0.09% against MCF-7and WRL-68, respectively. While the total alkaloids of A. vera had slightly effect on both cell lines.Conclusion: Plant alkaloids appeared variable cytotoxic activity against cancer and normal cell lines depending on the alkaloid contents, concentrations, purity, and cell line types

PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF BIOACTIVE METABOLITES PRODUCED FROM RARE ACTINOBACTERIA PSEUDONOCARDIA ALNI

ABSTRACTObjectives: Pseudonocardia alni exhibits antimicrobial activity against tested pathogenic Staphylococcus aureus, Microsporum canis, and Trichophytonmentagrophyte. The present paper aimed to optimize various cultural conditions for antimicrobial metabolite production, purification, andcharacterization of the active substance.Methods: The effects of various parameters such as culture media, carbon and nitrogen sources, phosphate concentration, pH, temperature, incubationperiod, and agitation rate on bioactive metabolite production were studied using a flask scale with varying single parameter. The active substanceswere purified by adsorption chromatography using Silica gel column and Sephadex LH 20 column, and the physical, chemical, and biological propertieswere characterized.Results: The metabolite production by P. alni was greatly influenced by various cultural conditions. It produced high levels of the antimicrobialsubstance in International Streptomyces project-2 broth compared with that in potato dextrose broth. The optimum parameters for antimicrobialproduction from the actinobacterium occurred in the production medium consisting of glucose (1%) and tryptone (1%), 0.001 M of K2HPO4 and0.05M glycine at initial pH 8.5 and incubated at 30°C for 4 d in stand incubator. The higher concentration of phosphate buffer salts (˃0.01M) repressedthe bioactive production. The purified active substance had relative factor Rf=0.53 in the mobile phase of a thin layer chromatography system andthe maximum absorbance (λ max) at 216 nm. The results of infrared spectra Fourier transform-infrared spectroscopy analysis indicate that it may beregarded to glycopeptide antibiotic. The purified substance had antibacterial and antifungal activities as well as cytotoxic activity in breast cancer cellline Michigan Cancer Foundation-7 and normal hepatic cell line (WRL-68) at a percentage up to 23.7% and 7.64%, respectively.Conclusions: The actinobacterium P. alni was a novel strain having the ability to produce antimicrobial and anticancer substances.Keywords: Pseudonocardia alni, Antifungal–antibacterial, Anticancer optimization, Production, Purification, Characterization

OPTIMIZATION PRODUCTION CONDITIONS OF ANTIBACTERIAL METABOLITE FROM STREPTOMYCES SP.

  Objectives: The paper aimed to isolate Streptomyces strain having the ability to produce antibacterial metabolites and optimize some environmental parameters for excellent antibiotic production.Methods: Different soil samples were collected from extreme environments of desert regions at Karbala Province, Iraq. Actinomycetes were isolated using different media. The primary screening for antibacterial production was accomplished, and the antibacterial activities were tested against pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Pseudomonas aeruginosa. The most potent strain was chosen for optimizing some of environmental parameters to increase the bioactive metabolite production. Different parameters were studied such as culture media, temperature, pH, and agitation rate.Results: About eight Streptomyces strains were isolated from soil samples. All isolates appeared variable levels of antibiotic productions against Gram-positive and negative pathogenic bacteria, and the best one was Streptomyces sp. LHR 9. The antibacterial metabolite production from Streptomyces sp. LHR 9 was affected by various cultural parameters. Glucose soybean meal broth as a fermentation medium at pH 7 yielded the highest antibiotic production under the optimal fermentation conditions, including the temperature at 35°C with 200 rpm (revolution/min) agitation rate and 7 days incubation period.Conclusion: The Streptomyces sp. LHR 9 showed antibacterial activity against both Gram-positive and negative pathogenic bacteria. It may consider as a potential source of drug production. Further study needs to purification and characterization of antibiotic and analyzes the mechanism for the antimicrobial activity of this bioactive compound

CYTOTOXIC ACTIVITY OF ALKALOID EXTRACTS OF DIFFERENT PLANTS AGAINST BREAST CANCER CELL LINE

Objectives: To study in vitro cytotoxic activity of total alkaloid extracts of Pinus sabiniana L., Phoenix dactylifera L. and Ferocactus sp. L. against breast cancer cell line Michigan Cancer Foundation-7 (MCF-7) and non-tumorigenic fetal hepatic cell line (WRL-68). Methods: Plant powder of each P. sabiniana L. leaves, P. dactylifera L. pollen grains, and Ferocactus sp. L. The leaves were extracted separately with 80% methanol, chloroform at pH 2 and pH 10 and the chloroform portion was dried to obtain the total alkaloid extracts. The total alkaloids were detected qualitatively by Mayer's, Dragendorff's and Hager's reagents and estimated quantitatively by bromocresol green spectrophotometry depending on the atropine calibration curve. The cytotoxic activity was evaluated by 3-[4, 5-dimethylthiazoyl]-2, 5-diphenyltetrazolium bromide assay. Results: The extract of P. sabiniana L. had highest total alkaloid content (164.62±2.8 mg/100 g dry weight of plant) than the other plants (P. dactylifera l., Ferocactus sp. L.), the total alkaloids of Ferocactus sp. L. and P. dactylifera L., reduced the cell viability of both cell lines, the highest reduction occurred in the concentration 400 μg/ml was 46±2.20% (MCF-7) and 56.2±2.2% (WRL-68) for Ferocactus sp. L., followed by 56.2±2.2% (MCF-7) and 57.5±3.2% (WRL-68) for P. dactylifera L. The alkaloids of P. sabiniana was very lower effects on both cell lines MCF-7, and WRL-68 was 89.3±3.44% and 90.16±2.7%, respectively, at the same concentration. Conclusion: Plant alkaloids had variable effects against cancer and normal cell lines depending on the type of alkaloid compounds and their concentration in the extract

Antimicrobial Activity of Nigella Sativa Extract Against some Bacterial and Fungal Species.

استخدمت بذور الحبة السوداء منذ آلاف السنين كتوابل ومواد حافظة للأغذية كما استخدمت هذه البذور في تعزيز الصحة ومكافحة الأمراض وخاصة في الشرق الأوسط. وفي هذه الدراسة استخلصت بذور الحبة السوداء مع 96 ٪ من الإيثانول وتم تنقيته كروماتوجرافيا بالسيليكا جيل مع مذيبات مختلفة. وأظهرت ألنتائج لمستخلصات الحبة السوداء على الفطر F. Solani أن كلا من مستخلص الهكسان والإيثانول من زيت بذور الحبة السوداء فعالية عالية مضادة للفطريات وكان قطر النمو 24 ± 2.1mm  و 28 ± 1.5  mm ،على التوالي  في حين لم يظهر F.Solani  أي حساسية لمستخلص ألأسيتون ، اثيل أسيتيت  و المائي. كما تم اختبار جميع المستخلصات كمضاد للبكتيريا  E.coli ، S.aureus K.pneumonia و Enterobacter aerogenes .  كما وجد ان مستخلص الكلوروفورم كان ساما لل E.coli مع منطقة تثبيط 18.3 ± 4.3mm  و 19.3 ± 3.5 ملم اضافة الى ان لها نفس التأثير على K.pneumonia و  Enterobacter aerogenes في حين كان تأثيرها ضعيفا على S.aureus ، في نفس الوقت الاسيتون، أثيل اسيتيت والماء لم تظهر أي تأثيرات على انواع  البكتيريا الاخرىSeeds of Nigella sativa have been employed for thousands of years as spice and food preservative these seeds have been used to promote health and fight disease especially in the Middle East. In this study black seed extracted with 96% ethanol and purified chromatographically by using silica gel column with different solvents. The purpose of this study is to evaluate effect of Nigella sativa purified oil fractions on some fungal and bacterial species. The antifungal results on Fusarium Solani showed that both hexane and ethanol fractions of black seed oil revealed high antifungal properties and the diameter of growth were 24 ± 2.1mm  and 28 ± 1.5 mm, while chloroform and methanol revealed moderate effect on Fusarium Solani, the diameter of growth were 30 ± 2.5mm and 37 ± 2.9mm. Fusarium Solani did not show any sensitivity for acetone, ethyl acetate and water fractions and the diameter of growth was between 40 to 44 mm. All seven fractions tested as antibacterial with Escherichia coli, Staphylococcus aurous, Klepsiella pneumonia and Enterobacter aerogene. Hexane and chloroform fractions were toxic to the Escherichia coli with inhibition zone 18.3 ± 4.3mm and 19.3±3.5 mm also these both fractions have the same effect on Klepsiella pneumonia and Enterobacter aerogenes while having the weak effect on Staphylococcus aurous with inhibition zone ranged between 8.6 - 3.3mm. Staphylococcus aurous revealed high sensitivity to ethanol fraction with inhibition zone 22.3 ± 5.4 mm in the same time acetone, ethyl acetate and water did not show any effect on bacterial spices

OPTIMIZATION OF MEDIUM COMPOSITION FOR ANTIBACTERIAL METABOLITE PRODUCTION FROM STREPTOMYCES SP.

  Objectives: This paper aimed to optimize some essential nutritional components (carbon, nitrogen, and phosphate) of fermentation medium necessary for the production of antibacterial metabolites from Streptomyces sp.Materials and Methods: Streptomyces sp. LH9 previously isolated from desert soil in Karbala Province, Iraq. This strain produced antibiotic against 4 pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Streptococcus agalagtiae, and Pseudomonas aeruginosa. For optimizing, the essential nutritional requirements such as carbon, nitrogen, and phosphate in fermentation media different concentrations of these sources were used to improve the antibacterial metabolite production.Results: All the studied nutritional parameters were had impacts on the antibacterial metabolite production from Streptomyces sp. LH9. The actinobacterial strain produced a highest antibiotic metabolites when was grown in the fermentation medium supplemented with 2% dextrose (as a sole carbon source), 0.05% peptone (as a sole nitrogen source), and 0.05% K2HPO4 at pH 7 and incubated under optimal conditions; at 30°C with 250 rpm (revolutions/min) agitation for 7 days.Conclusion: Streptomyces sp. LH9 was a good producer for antibacterial against Gram-positive and Gram-negative bacteria, which required simple nutritional supplements in the fermentation medium. Furthermore, could be utilized the industrial waste for improving the production in the most economic manner

DETECTION SINGLE NUCLEOTIDE POLYMORPHISMS IN UROMODULIN PROMOTER REGION ASSOCIATED WITH RENAL DISEASES USING SINGLE-STRAND CONFORMATION POLYMORPHISM-POLYMERASE CHAIN POLYMORPHISMS TECHNIQUE

  Objective: The uromodulin, a glycoprotein, expressed and secreted by epithelial kidney cells lining the thick ascending limb of the Henle's loop. It is encoded by the UMOD gene in humans. Our objective was to analyze single nucleotide polymorphisms (SNPs) in the UMOD promoter region in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD).Methods: The blood samples were collected from 100 patients with CKD (50) and ESRD (50), who admitted at Merjan Teaching Hospital in Babylon Province, Iraq (February-July 2016). In addition, 50 blood samples of healthy control. The SNPs of UMOD promoter region was investigated using single-strand conformation polymorphism-polymerase chain polymorphisms (SSCP-PCR) and DNA sequencing techniques.Results: UOMD promoter region polymorphisms using PCR-SSCP and sequencing DNA appeared three different conformational haplotypes, including A\G 49 haplotype (5 bands), A\G 49 and C\A 247 haplotype (5 bands), and C\G 45 and A\G 49 haplotype (6 bands) distributed among CKD and ESRD cases, due to the presence of three SNPs. There was no association between band numbers of PCR-SSCP with ESRD and CKD compared with a control group.Conclusion: SSCP-PCR is a good screening method to detect genetic variations in an uromodulin promoter region

INVESTIGATION OF Helicobacter pylori VIRULENCE GENOTYPE IN GASTRIC BIOPSIES BY PCR

  Background: Helicobacter pylori infections has been associated with the genetic diversity of their virulence factors, the virulence genotypes are valuable as molecular marker in the diagnosis of patients with bacterial infections . Our main objective was to analyze the frequency and allelic genotype of vacA , cagA also investigate another virulence genes of H. pylori. Methods: 75 biopsies of  patients with gastritis and peptic ulcer diseases were selected to investigate the presences of H. pylori and collected from them antrum biopsies, then  genomic DNA was extracted from  antrum biopsies using genomic DNA kit .Subsequently, the virulence genes of H. pylori   were amplified using specific primers including vacA , cagA, cagE and oipA and iceA by PCR in 49 cases that positive to 16SrRNA which previously investigated. Results: A high prevalence of genes cagA  (28.6%), vacAs1bm2 (56.8%), iceA2 (30.6%)  and oipA  (42.9%) was found, while  vacA s2m1  and iceA1 genotypes  was not found in our study.  There was significant correlation between the presence of cagA and cagE genotypes (p = 0.02), suggesting that these two genes almost used together as cag PAI integrity marker. The  presence of cagA gene was significantly associated with peptic ulceration (p ≤ 0.001), whereas different vacA genotypes or iceA2 genotype were no statistically significant  with clinical outcome.  Patients with peptic ulcer disease more likely to have oipA gene (61.9% ) than those with gastritis (38.1%), P = 0.037, also the presence oipA gene was statistically significant with presence iceA2. Conclusion : Most H. pylori genotypes which associated with peptic ulcer and gastritis were moderate virulent strains. Â