Centre of Biotechnology and Microbiology

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Biochemical and Biomolecular Homology Modeling (Structural Bioinformatics) of Biologically Important Toxin Proteases from Vibrio Cholerae
(Center of Biotechnology & Microbiology, University of Peshawar, 2009) Farhat Amin; Supervised by Prof Dr. Ghosia Lutfullah
At the molecular level, the pathogenesis of cholera is a multifactorial process. Several genes encoding virulence factors aid the pathogen in its colonization, coordinated expression of virulence factors, and toxin action such as cholera toxin (CT) and the toxin-coregulated pilus (TCP). Cholera Toxin is primarily responsible for the profuse secretory diarrhea while the toxin-coregulated pilus is an essential colonization factor and receptor for the phage encoding CT. In addition to potent CT, there are other putative toxic factors such as accessory cholera enterotoxin (Ace), zonula occludens toxin (Zot), hemolysin/cytolysin and hemagglutinin/protease (Vibriolysin). Neuraminidase, a sialidase, is also considered a virulence factor. It is a key glycoside hydrolase that catalyzes the conversion of higher-order gangliosides to monosialoganglioside, which is the cholera toxin receptor. It has been hypothesized that neuraminidase may locally produce high concentration of the GM1 receptor for CT, thereby enhancing the binding and internalization of CT and resulting in greater efflux of water and electrolytes. The present study comprises the analysis of toxin proteins of V. cholerae in order to have a better understanding of pathogenesis using molecular biology and bioinformatics tools as the signaling cascades involved in the induction and regulation of mucosal inflammatory responses to infection by V. cholerae are still largely unknown. The proposed work is carried out in two phases. The first phase deals with the mRNA expression of proinflammatory, anti-inflammatory and various other cytokines in intestinal tissue and bone marrow cells upon infection with purified Cholera Toxin (CT) alone and in combination with purified neuraminidase from Vibrio cholerae in order to elucidate the mechanisms the V. cholerae induced proinflammatory response as well as…
Biochemical Studies & Biomolecular Homology Modeling (Structural Bioinformatics) of Biologically Important Toxin Proteases from Vibrio Cholerae
(Center of Biotechnology & Microbiology, University of Peshawar, 2009) Farhat Amin
At the molecular level, the pathogenesis of cholera is a multifactorial process. Several genes encoding virulence factors aid the pathogen in its colonization, coordinated expression of virulence factors, and toxin action such as cholera toxin (CT) and the toxin-regulated pilus (TCP). Cholera Toxin is primarily responsible for the profuse secretory diarrhea while the toxin-coregulated pilus is an essential colonization factor and receptor for the phage encoding CT. In addition to potent CT, there are other putative toxic factors such as accessory cholera enterotoxin (Ace), zonula occludens toxin (Zot), hemolysin/cytolysin and hemagglutinin/protease (Vibrioysin). Neuraminidase, a sialidase, is also considered a virulence factor. It is a key glycoside hydrolase that catalyzes the conversion of higher-order gangliosides to monosialoganglioside, which is the cholera toxin receptor. It has been hypothesized that neuraminidase may locally produce high concentration of the GM1 receptor for CT, thereby enhancing the binding and internalization of CT and resulting in greater efflux of water and electrolytes. The present study comprises the analysis of toxin proteases of V. cholerae in order to have a better understanding of pathogenesis using molecular biology and bioinformatics tools as the signaling cascades involved in the induction and regulation of mucosal inflammatory responses to infection by V. cholerae are still largely unknown. The proposed work is carried out in two phases. The first phase deals with the mRNA expression of proinflammatory, anti-inflammatory and various other cytokines in intestinal tissue and bone marrow cells upon infection with purified Cholera Toxin (CT) alone and in combination with purified neuraminidase from Vibrio cholerae in order to elucidate the mechanisms of the V. cholerae induced proinflammatory response as well as
BIOCHEMICAL, ENVIRONMENTAL AND MEDICINAL EXPLORATION OF EUPHORBIA PROSTRATA AITON AND EUPHORBIA GRANULATA FORSSK
(Center of Biotechnology & Microbiology, University of Peshawar, 2014) Mohammad Parvez; Prof.Dr.Farrukh Hussain
Euphorbia granulata Forssk and Euphorbia prostrata Aiton are two medicinal plants belonging to family euphorbiaceae. The present study was aimed at biochemical, environmental and medicinal exploration of these two plants. Biochemically the two plants and their parts were investigated for proximate composition, important phytochemicals and mineral elements. Nutritionally both plants were found with almost identical and excellent quantities of crude fibers (18.3 ±0.68 to 20 ± 0.49), proteins (17.5 ±0.52 to 18.3 ± 0.18) and carbohydrates (39.64 ±0.29 to39.9 ±1.32) however E. prostrata was comparatively more nutritious than E. granulata. Similarly more lipids were noted for E. granulata compared to E. prostrata. Vertical distribution in plant parts showed comparatively more protein and lipids in seeds, more carbohydrates in stem and roots and more fibers in roots than other parts. Phytochemicals are medicinally important secondary metabolites. The composition of the two plants showed the presence of total phenolics, flavonoids, alkaloids, tannins, glycosides and saponins in good quantity. Comparing the two plants, showed high amount of phenolics, flavonoids and alkaloids in E. granulata however tannins and glycosides were found more in E. prostrata. Both plants contained equal amount of saponins. Leaves were found the highest contributor to the total phytochemicals quantified in whole plant. Macro and micro minerals in plants are important for the health of its consumers. The composition of E. granulata and E. prostrata revealed that the two plants were good source of most minerals. The plants contained micronutrients Fe, Mg, Mn, Zn, Mo and Se in sufficient amount to supply them well above the recommended dietary allowances (RDA). Only Cu was present in small amount. On the other hand only Cl was present in small amount while the rest of macronutrients (S, Ca, P, Na, N and K) were present in good dietary quantity.
BIODEGRADATION OF SOME MODEL POLYCYCLIC AROMATIC HYDROCARBONS BY BACTERIAL STRAINS ISOLATED FROM SOIL AND WATER
(Center of Biotechnology & Microbiology, University of Peshawar, 2015) Nadia Bibi; Supervisor Dr Sumaira Afzal Khan
Biodegradation of polycyclic aromatic hydrocarbons (PAHs), using soil microorganism is the most efficient, cost effective and environment friendly remediation strategy. In the present study, bacterial strains were isolated from four different sites including rhizospheric soil of three native species and sewage waste water using serial dilution and spread plate technique in order to isolate wild strains. Initial screening was done on simple nutrient agar via streak and spray method. The isolates were adapted to biodegradation through changing PAHs concentration, different media and physical mutation. It was observed that exposure to UV-light for a short period of 15 minutes results in enhanced growth rate but, some isolates lose the degrading ability of a certain compound. A total of 360 isolates were initially screened for growth on nutrient agar medium containing up to 1100 and 1200 ppm on selected PAHs (anthracene, naphthalene, acenaphthene, phenanthrene and pyrene). Among which 101 of these isolates with best growth, were then screened on PNRG and PNR medium for the utilization of PAHs and selection of best degrading isolates was done. Eight bacterial isolates were selected on the basis of best growth in liquid static media condition, namely; B4, B30, B36, S13, S21, S51, M3 and W2. These were characterized on the basis of morphology and biochemical characterization. S13 was most closely related to Bacillus cereus and M3 to Mycobacterium sp. on the basis of key provided by Bergey’s manual classification. Identification was finalized on the basis of molecular analysis i.e., 16S rRNA analysis and construction of phylogenetic tree. All the selected isolates except M3 were identified as Bacillus sp., isolates that were closely related to one another, and probably were sub-species of Bacillus cereus.
Bioinspired Synthesis, Characterization and Biological Evaluation of Silver Nanoparticles from aqueous leaves extract of hippophae rhamnoides linn
(Center of Biotechnology & Microbiology, University of Peshawar, 2014) Javid Ali; Supervisor Prof.DR.Bashir Ahmad
In this study we have evaluated the physiochemical, minemls contents, phytochemicals and biological acti. ity of Hippophae rhamnoides Li/.n \H. rhamnoides L.) leaves. The physiochemical and mineral contents ofll rhamnoides L leaves revealed that fiber was 18.0*2.6470, protein was 10.45+0.88% and carbohydrate value was found 68.'15+1%. Sodium was 30000+l ppm, calcium 7800+1 ppm and potassium 6200+2 ppm as high concenhation. While qualitative phytochemicals investigations showed that tannins, phenols and flavonoids are present in large quandry. The Fouder tansform infrared spectroscopy (FTIR) spectra of all extmcts revealed, the presence of different functional goups like OH stretching for hydroxyl group, alkanes, alkenes, aromatic rings, carboxylic and amides (aromatic). The highest antibacterial zone of inhibition was observed in aqueous and methanolic extracts against S. ar,"er.t and V. cholerde wilh 21!l mm and lowest Zone of inhibition measured 07+0 mm against ,a cholerue (ethyl acetate extracts). The antibacterial minimum inhibitory concentration values of extracts were determined ranging between 40 to 120 mg/ml and minimum bactedcidal concentration values of the extracts ranged between 50 and 135 mg/ml. The highest antifungal zone of inhibition was calculated against l/ternatia ahernatd (18+0 mm) followed by Aspergillus parasiticus (17,!l mm) of methanolic extact, while l/le maria alternata (07+ I mm) and Penicillium digitdtum (07L 0 mm) was the least found in chloroform and aqueous extract respectively. Further antifungal minimum inhibitory concentration and minimum tmgicidal concentration are between 40 135 mg/ml and 50 - 180 mg/ml respectively.
CHARACTERIZATION OF ANTIBIOTIC PRODUCING SOIL MICROORGANISMS AGAINST HUMAN PATHOGENIC BACTERIA, ISOLATED FROM HOSPITILIZED PATIENTS IN KHYBER PAKHTUNKHWA
(Center of Biotechnology & Microbiology, University of Peshawar, 2013) Riaz Muhammad; Supervisor Prof.Dr.Bashir Ahmad
Pathogenic bacteria are medically very important as they caused various diseases, develop resistance against antibiotics and that is why require great concern worldwide. Most prominent antibiotic resistant pathogens are Methicillin resistant Staphylococcus aureus (MRSA), Vancomycin intermediate Staphylococcus aureus (VISA), Vancomycin resistant Staphylococcus aureus (VRSA), multi drugs resistant Pseudomonas aeroginosa, extended spectrum beta-lactamases producing Escherichia coli and Enterobacteraciae spp. which cause hospital acquired and community acquired infections. Methicillin and Oxacillin were considered as potent antibiotics against S. aureus infection but unfortunately more than 50% S. aureus resistance to these drugs have been reported from different countries recently. Now Vancomycin is the drug of choice for the treatment of MRSA infection but unfortunately due to the misuse of antibiotics VRSA have been recently evolved. In Khyber Pakhtunkhwa pathogenic bacteria have developed resistance to mostly prescribed antibiotics. According to our survey mostly Oxacillin, Meropenem, Amikacin, Ciprofloaxacin, Ofloaxacin, Piperacillin/Tazobactam and Vancomycin antibiotics are prescribed against bacterial infections in hospitalized patients. Bacterial resistance to these antibiotics has also been reported from different geographical locations of Pakistan. Drug resistance is a serious problem which is often a consequence of the failure of control strategies. In the current scenario our options to deal with the antibiotic resistant bacterial pathogens limited or in-effective. As the resistance evolved comparatively rapidly due to the irrational antibiotic use, therefore we expect a much faster evolutionary rate in a third world country like Pakistan. The problem of resistance could be tackled in several ways including synthetic modification in the existing drugs or looking for novel compounds for neutralizing the threat.
EXPLOITATION OF ASPERGILLUS AND CLADOSPORIUM SPECIES FOR BIOLOGICALLY ACTIVE SECONDARY METABOLITES
(Center of Biotechnology & Microbiology, University of Peshawar, 2013) Abid Ali Khan; Supervisor Dr.Nafees Bacha
Aspergillus carbonarius (NRRL–369) and Aspergillus oryzae from Aspergillus genus as well as Cladosporium carrionii and Cladosporium resinae (NRRL–6437) from Cladosporium genus were selected for the present study. Nutrient media were optimized for the growth and production of secondary metabolites. Out of five different media used, A. carbonarius and A. oryzae produced relatively more metabolites in Czapek–dox (Glucose and Starch) broth media (CGSB). Whereas; C. carrionii and C. resinae produced relatively more metabolites in Czapek yeast extracts broth (CYB). To further increase secondary metabolites productivity, two additional chemical compounds (suberoyl anilide hydroxamic acid; SAHA and 5–azacytidine; 5–AZA) were also used as chemical inducers for all fungi except C. carrionii. A dose of 10 μM/100 mL of SAHA resulted in higher secondary metabolites production from Aspergillus species and 15 μM/100 mL of SAHA resulted in higher secondary metabolites production from C. resinae. While a dose 15 μM/100 mL of 5–AZA resulted in higher secondary metabolites production from all the species. Secondary metabolites produced were then studied for its respective biological activities. In antibacterial assay a dose of 500 μg/mL of ethyl acetate extracted from A. carbonarius inhibited the growth of B. subtilis (64.5%), while for antifungal testing a dose of 1000 μg/mL ethyl acetate extract inhibited the linear growth of C. glabrata (58.5%). Whereas, in cytotoxic activities, dose of 1000 μg/mL of ethyl acetate extract showed 94% mortality against brine shrimps, while for phytotoxic activities, a dose 1000 μg/mL showed 90% mortality against Lemna.
Exploitation of Soil and Rhizosphere Inhabiting Fungal Species for Biologically Active Secondary Metabolites by
(Center of Biotechnology & Microbiology, University of Peshawar, 2015) Saeed ullah Khattak; Supervisor Prof.Dr.Ghosia Lutfullah
In quest for new bioactive compounds the present study deals with isolation, identification and bioassay screenings of fungi and purification and structural elucidation of pure compounds isolated from fungal strains under study. For production of fungal bioactive secondary metabolites fungal strains were isolated from soil and rhizosphere of Mentha piperita using Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA) media. After preliminary antimicrobial testing, two fungal strains were selected for metabolites production, their bioassays and subsequent structural characterization. The rhizospheric strain was identified as Aspergillus specie while soil isolate was identified to be Penicillium specie. For secondary metabolites production both fungal strains were cultured in Czapec Yeast Broth (CYB) medium at 28 oC for 14 days at 150 rpm. Crude secondary metabolites produced by fungal strains were then extracted and tested for their respective bioassays. In our study fungal metabolites were tested for five different activities i.e. antibacterial, antifungal, cytotoxic, phytotoxic and herbicidal activities. In antibacterial bioassay crude extracts of Aspergillus and Penicillium species were tested against eight pathogenic bacterial strains i.e. Xanthomonas oryzae, Klebsiella pneumonia, Bacillus subtilus, Proteus vulgaris, Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella flexeneri. A dose concentration from 1 to 500 μL mL-1 of the crude extracts was tested against pathogenic bacterial strains and zone of inhibition was recorded in millimeters (mm). A dose concentration of 500 μL mL-1 of ethyl acetate extract of Aspergillus sp. inhibited growth of all test bacterial strains but the highest zones of inhibition were recorded against B. subtilus (47.5 mm) and S. flexeneri (45 mm) while lowest zone of inhibition was recorded against X. oryzae (9 mm). At 500 μg/mL of ethyl acetate extract of Penicillium sp growth of P. xxii vulgaris, E. coli, S. aureus, B. subtilus and S. flexeneri was inhibited by forming zones of 34, 28, 24, 23 and 21 mm respectively. Lowest zone of inhibition was recorded against S. typhi (9.5 mm) while considerable inhibition was noted against K. pneumoniae (16 mm) and X. oryzae (13 mm) at 500 μL mL-1 of Penicillium sp. extract.
GENETIC STUDIES OF OIL CONTENT IN MUTANT POPULATION OF RAPESEED (Brassica Napus L.) USING MOLECULAR MARKERS
(Center of Biotechnology & Microbiology, University of Peshawar, 2014) Sher Mohammad; Supervisor prof.Dr. Bashir Ahmad
The current study titled “genetic studies of oil content in mutant population of rapeseed (Brassica napus L.) using molecular markers” was partially conducted at Nuclear Institute for Food and Agriculture (NIFA), Peshawar in 2008-13. Main objective of the study were to intimate and study the influence of induce mutation on the genetics of oil and oil related components in advance induce mutant line of oilseed rape (Brassica napus). Two contrast mutant lines with high and low oil contents were crossed with each other and F1 hybrids were developed. These F1 hybrids were grown in the cropping season 2009-10 and BCF1 were developed by crossing back the F1 with their parents. These BCF1 hybrids were grown in the next cropping season 2010- 11 and selfed to develop BCF2. The parent and the BCF2 hybrids were used for molecular assessment to confirm mutation in the genetics of the advance mutant lines. Thirty five advance mutant lines were utilized for agronomical and biochemical study. Five plants were randomly selected from each mutant line and the parent. Data on days to 50% flowering (DF), plant height (PH), 1000- seed weight (SW), seed yield (SY) and oil yield (OY) were recorded. Seeds were analyzed at for biomechanical parameter viz. Oil content (OC), protein content (PC), glucosinolates (GSL), oleic acid (OA), linolenic acid (LA), and erucic acid (EA).
Green Synthesis Characterization and Biological Evaluation of Zinc and Copper Nanoparticles from Aqueous Leaves Extract of Eriobotrya Japonica and Ficus Carica
(Center of Biotechnology & Microbiology, University of Peshawar, 2012) Aamir Aziz; Supervisor Prof. Dr. Bashir Ahmed
The main aim of the study was to screen the methanolic, ethanolic and ethyl acetate (EtOAc)fractions of Eriobotrya japonica and Ficus carica leaves and their Zinc (ZnNPs) and Copper nanoparticles (CuNPs), respectively, for various pharmacological activities such as, antibacterial, antifungal, phytotoxic, insecticidal, anti-termite, hemagglutination and their comparison. The synthesis of ZnaNPs and CuNPs was carried out using aqueous leaf extracts of E. japonica and F. carica, standardizing conditions for their synthesis like temperature, pH and incubation. The characterization of these nanoparticles was done using UV-Vis spectrophotometry, Fourier Transform Infrared spectroscopy, Scanning Electron Microscope (SEM), Energy Dispersive X-ray (EDX), X-ray diffraction (XRD) and Thermogravimetric/Differential Thermal Analysis (TGA/DTA). The UV-Visible absorption spectra of ZnNPs and CuNPs showed peaks at 455 and 426 nm,respectively. The FTIR spectra of ZnNPs and CuNPs confirmed the presence ofvarious functional groups suchas, - hydroxyl, amide, carbonyl, aldehyde, and alkynes. The SEM analysis showed that the particle size was between 99 and 96 nm with spherical shape. The presence of zinc and copper along with other elements, such as C, O, S, Si, and K, was confirmed by EDX analysis. The plant extract contributed the last four elements that stabilize and cap ZnNPsand CuNPs. The mean size for ZnNPs and CuNPs in XRD spectra according to Debye-Scherer’s equation was 33 and 32 nm,respectively. A clear antibacterial activity of the methanolic, ethanolic and EtOAcfractions of E. japonica was observed in terms of percentinhibition: E. coli (62, 59.2 and 44.4%), S. aureus (69.2, 46.1 and 50%), P. aeruginosa (44.4, 40.7 and 37%), M. morganii (39, 40.9 and 68 %), A. baumannii (52.1, 43.4 and 0 %), P. vulgaris (60, 52 and 64%) and Methicillin Resistant Staphylococcus Aureus (MRSA)(73.6, 52.6 and 0 %). Similar results were also observed for methanolic, ethanolic and EtOAcfractions of F. carica against E. coli (51.8, 40.7 and 48.8), S. aureus (65.3, 46.1 and 65.3),
INTERFERON BASED THERAPY OF CHRONIC HEPATITIS C PATIENTS. A CASE STUDY OF KHYBER PAKHTUNKHWA
(Center of Biotechnology & Microbiology, University of Peshawar, 2012) Sajid Ali; Supervisor Prof.Dr. Bashir Ahmad
Hepatitis C is the inflammation of the Liver caused by Hepatitis C virus (HCV), the leading cause of the liver cirrhosis and hepatocellular carcinoma. About 3% of the people have been affected by HCV world wide and in Pakistan being an underdeveloped country; an estimated 10 million people have Hepatitis C infection (WHO). As hepatitis C infection is asymptomatic and due to untimely diagnosis it leads to severe liver diseases and anuualy a lot of affected individuals lose their lives. In order to cure the infection Food and Drug Agency (FDA) has approved the use of Interferon (IFN) as the treatment remedy. In Pakistan National Institute of Health (NIH) has also given the recommendation for the use of IFN as the therapeutic agent In KhyberPakhtunKhwa (KPK) mostly conventional IFN and Ribavirin combination therapy is considered due to the prevalence of responsive genotypes 2 and 3. Earlier no study has been conducted to sort out IFN response among chronic HCV patients in different districts of KPK province, therefore we attempted to find out response of conventional IFN combination therapy at districts level in KPK. Samples were collected from chronic HCV patients referred by clinician/laboratories of different regions of KPK. The samples were analyzed for screening by ICT (Immunochromatographic Technique) and ELISA (Enzyme Linked Immunosorbant Asaay) followed by confirmation through polymerase chain reaction (PCR). We have also done genotyping for some of the chronic HCV patients. PCR confirmed positive patients were given IFN and Ribavirin combination therapy keeping in mind the therapy exclusion criteria. The dose of IFN and Ribavirin was 3 Million Units thrice a week and 800-1200mg daily depending on the age of the patients, respectively. This therapy was continued for six months with repeated testing of ALT (Alanin Transaminase) and CBC (Complete Blood Count), during and after therapy. At the end of six months of therapy, PCR test was done for all course completed patients.
ISOLATION, OPTIMIZATION AND CHARACTERIZATION OF SECONDARY METABOLITES FROM SOIL BORNE FUNGI SCRELOTIUM AND ASPERGILLUS SPECIES
(Center of Biotechnology & Microbiology, University of Peshawar, 2016) Muhammad Rizwan; Supervisor Dr. Bashir Ahmad
In the present dissertation, our research findings on the production of bioactive secondary metabolites by soil borne fungi are discussed. The application of microorganisms for the welfare of human beings is the main goal of biotechnology. In this study, two phytopathogenic fungi, Sclerotium rolfsii and Aspergillus flavus were isolated from soil samples collected from Malakand Division, Khyber Pakhtunkhwa, Pakistan. Growth parameters (nutrient media, temperature, pH, incubation period, and static/shaking intervals) were optimized for achieving maximum production of bioactive secondary metabolites. Five nutrient media were used for fungal growth. S. rolfsii produced maximum amounts of metabolites in Czapek yeast broth media (CYB), whereas maximum formation of metabolites by A. flavus was found in potato dextrose media (PDB). Furthermore, the crude secondary metabolites in ethyl acetate (EtOAc) and n-hexane extract obtained from each medium were screened for their activities against different pathogenic bacteria. The EtOAc and n-hexane fractions obtained from the CYB medium were more active against pathogenic bacteria as compared to the crude metabolites obtained from the other media in the case with S. rolfsii, whereas PDB was the media, in which A. flavus yielded the largest quantities of secondary metabolites, showed significant results against pathogenic bacteria. EtOAc and n-hexane fractions of both fungi were screened for their in vitro effects, including antifungal, phytotoxic, and insecticidal properties and brine shrimp lethality. The in vivo activities of the metabolites, such as acute toxicity, analgesic, and sedative action, were also studied. The results showed that the ethyl acetate (EtOAc) fraction was more active as compared to the n-hexane fraction due to the presence of polar constituents. The results obtained from the examinations of the biological activities indicated that both fungi produced bioactive secondary metabolites which have antimicrobial, phytotoxic, insecticidal, cytotoxic, analgesic, and sedative effects.
Molecular Epidemiology of Antibiotic Resistant Genes in Pathogenic Bacteria ( A Case Study of Khyber Pukhtunkhwa, Pakistan)
(Center of Biotechnology & Microbiology, University of Peshawar, 2013) Johar Jamil; supervisor Prof. Dr.Bashir Ahmad
This study was conducted from 2009 to 2012 on isolates collected from two tedary hospitals of Peshawar, I(iyber Pakhtunkhwa, Pakistan. The aim ofthis study was to find out the resistance profile of different antibiotics and biocides, prevalence of extendedspectum flactamases (ESBLs) and caxbapenemases ilt ,t aureus, E. coli, Klebsiella spp_ eJtd P. aeruginosa, rcspectively. Among 133 S. aureus, 4'7yo werc Methicillin resistant S azreus (MRSA). Cefoxitin was found to be the most efficient and effective method for the detection of MRSA. In S. aureus, 'tal]lcomycir' (VAN) and linzolid (LZD) showed best activity against both MRSA and MSSA. High level of antibiotic resista[ce was found in MRSA isolates. Most of our ,S. arrem isolates were susceptible to biocides. The genes including sea and pvl were the most dominant genes in S. a,rezs isolates. Carbapenems including meropenem and imipenem showed very good activity against E coli and Klebsiella spp. and P aeruginosa. ESBL and carbapenem.tse prcducers were also detected inE' coli arrd Klebsiella spp
Molecular Epidemiology of Staphylococcus A Case Study of Khyber Pakhtunkhwa, Pakistan
(Center of Biotechnology & Microbiology, University of Peshawar, 2015) FARMAN ULLAH; Supervisor Dr. Bashir Ahmad
The present study was conducted from June 2010 to June 2014 in Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC), Peshawar, Pakistan to know about the prevalence of resistance to antibiotics and molecular epidemiology of Staphylococcus aureus. The specimens include pus, blood and urine received for culture and sensitivity. S. aureus were identified at molecular level using polymerase chain reaction (PCR). The identified strains were subjected to antimicrobial testing by disc diffusion method and minimum inhibitory concentration (MIC) method according to the guide lines ofclinical laboratory and standards institute (CLSI). Cefoxitin, 30 pg discs were also applied in the culture sensitivity to find methicillin resistant S. aureus (MRSA) strains. Majority of S. aureus wereisolated from skin infections. A total of 273 (61.34%) isolates were found to be MRSA out of 445. The samples collected from KTH’s clinical laboratory were named as KLS (n=167), KTH’s environmental samples as KE (n=64), KTH’s blood isolates as KLBS (n=15), KTH’s currency notes as CRN (n=18), KTH’s burn ward samples as KBRN (n=89) and the isolates collected from Hayatabad medical complex were named as HMC (n=92). As a whole, amongtheantibiotics tested against S. aureus, vancomycin was most potent among alLantibiotics to which 89.89% isolates were susceptible. Next to vancomycin were fusidic acid, rifampicin, linezolid and chloramphenicol inhibiting 84.94, 83.37, 83.15 and 81.12 % of the isolates respectively. Among the 445 isolates, 73.03% were susceptible to doxycycline, 62.47 % to amikacin and 60% to meropenem. Gentamicin, clarithromycin and ciprofloxacin had average activity, ie. 53.93, 44.04 and 41.57 % respectively. Rest of the antibiotics had below 40 % activities. MRSA strains were found more resistant as compared to MSSA. Thereason for erythromycin resistance was genetically investigated.
Molecular Mechanism of Respiration in High Altitude Birds Primary Structure and Homology Modeling of Hemoglobin Components from Common Crane (Grus grus Gruiforms.pdf)
(Center of Biotechnology & Microbiology, University of Peshawar, 2011) Zahid khan; Supervisor Dr. Ghosia Lutfullah
MOLECULAR STUDIES AND BIOINFORMATIC ANALYSIS OF INDUSTRIALLY IMPORTANT PROTEASE CODING GENES IN METAGENOMIC EXTRACTS AND PURE ISOLATES FROM EXTREME ENVIRONMENT
(Center of Biotechnology & Microbiology, University of Peshawar, 2012) Noreen Azhar
Proteases are large group of ubiquitous enzymes having small size and are compact structures. They are very important industrial enzymes possessing wide range of applications in the detergent, food, leather and pharmaceutical industries. Microbial proteases are known for their key role in biotechnological applications. Metagenomic techniques set a new dimension in exploring novel genes. To maximize industrial enzyme production, the host organism is generally manipulated to carry multiple copies of protease gene. This can be accomplished by cloning the gene on a replicating plasmid, but preferentially this is achieved by amplification of the gene on the chromosome, as such amplification offers the more stable alternative. Current studies deal with the study of biodiversity contained in indigenous soil of varied nature for the exploration of industrially important protease gene. Moreover, protease genes have been PCR amplified, cloned and sequenced from various cultured microorganisms and metagenomic soil samples, molecular aspects of these genes were studied using different molecular biology techniques, prior to this they were tested for protease activity on casein agar plates. Various bioinformatics tools have been employed for the characterization of these genes. Also protein structures prediction was performed to know the structural and functional features of sequenced protease gene.
Phytochemical and Bioassay Screening for Fruits of Sarcococca Saligna (D.Don) Muel and Aerial Parts of Astragalus Anisacanthus
(Center of Biotechnology & Microbiology, University of Peshawar, 2016) Saima Naz; Supervisor Prof.Dr.Bashir Ahmad
Crude methanolic extract (Crd. MeOH Ext.) and different fractions ofS. saligna fruits were investigated for various pharmacological / biological activities; Antimicrobial (Antibacterial and Antifungal) Brine-shrimplethality, Phytotoxic, Heamagglutination, Insecticidal, Anti-termites, Diphenyl Picryl Hydrazine (DPPH) free radical scavenging assay, Acute toxicity, Analgesic, locomotr, Antidepressant and Gastro Intestinal Tract Motility (GIT). These pharmacological activities revealed that the plant possess good antibacterial, anti-termite, analgesic activity and GIT Motility. Moderate phytotoxic activity was shown bythe test samples at higher concentration (1000p:g/ml). Low antifungal, insecticidal, cytotoxic, locomotor and antidepressant activity was shown by various fractions of this plant. A concentration dependent DPPH free radical scavenging activity was shownby the test samples and moderate ECso (58.61pg/ml). Fruits of the plant lack phytolectin as no heamagglutination activity was observed against human erythrocytes. The S. saligna fruits extract significantly inhibited the germination of wheat seeds (20g/72hrs). S. saligna fruits showed the presence of carbohydrates, proteins, alkaloids, sterols and triterpenoids, phenols, flavonoids, tannins and saponins. Elemental analysis showed the presence of Sodium (Na), Magnesium (Mg), Zinc (Zn), Potassium (K), Nickel (Ni), Copper (Cu), Lead (Pb), Manganese (Mn), Cobalt (Co), Cadmium (Cd), Iron (Fe) and Chromium (Cr ). The proximat analysis showed carbohydrate, proteins, fibers, fats and moisture content in fairly large amounts. The Scanning Electron Microscopy (SEM) technique was used to determine the morphology ofthe S. saligna fruits powder. The Energy Dispersive X-rays (EDX) technique was utilized to determine elemental composition. Maximum number of oil component were identified through GC-MS analysis.
Phytochemical evaluation bioassay screening and standization of Zizy jujuba and Acacia modesta
(Center of Biotechnology & Microbiology, University of Peshawar, 2011) Ibrar Khan; supervisor Prof. Dr.Bashir Ahmad
The present PhD dissertation comprises of two Parts; Part A and Part B Part A describes the phytochemical and biological investigations of Acacia modesta. The phytochemical investigations include the isolation of four compounds from the ethyl acetate fraction i.e. Nonaeicosanol (1) [P # 70], Palmitone (2) [P # 73], benzoic acid (3) [P # 76] and Lupeol (4) [P # 78]. Acacia modesta was a new source for Nonaeicosanol (1) and Palmitone (2). The oils isolated from the n-hexane fraction were subjected to GC-MS analysis. The GC-MS analysis of the oils revealed 38 components. The pharmacological / biological investigations include the antibacterial, antifungal, phytotoxic, insecticidal, brine shrimp lethality, haemagglutination, anti-termite, Nitric Oxide (NO) free radical scavenging and spasmolytic activities of the crude methanolic extract and various fractions of the plant. The oils obtained from the plant were also screened for antibacterial, antifungal, phytotoxic, insecticidal and brine shrimp lethality bioassays. The pharmacological / biological investigations revealed that the plant possesses significant antibacterial, insecticidal and anti-termite activities. Moderate NO free radical scavenging and haemagglutination activity was also observed. The plant showed low antifungal, phytotoxic, brine shrimp lethality and spasmolytic activities. The oils of the plant possessed significant brine shrimp lethality activity, moderate antifungal and phytotoxic and no antibacterial and insecticidal activity. Part B describes the phytochemical and biological investigations of Zizyphus jujuba. The phytochemical investigations include the isolation of compounds from the ethyl acetate, nhexane and chloroform fractions. Two compounds i.e. 4-methoxydalbergeione (5) [P # 178] and betulinic acid (6) [P # 180] were isolated from the ethyl acetate fraction. Stigmasterol (7) xii [P # 184] was isolated from the chloroform and β –sitosterol (8) [P # 188] from the n-hexane fraction of the plant. The pharmacological / biological investigations include antibacterial, antifungal, phytotoxic, insecticidal, brine shrimp lethality, haemagglutination, anti-termite, NO free radical scavenging and spasmolytic activities of the crude methanolic extract and various fractions of the plant. The oils obtained from the plant were also screened for antibacterial, antifungal, phytotoxic, insecticidal and brine shrimp lethality bioassays. The pharmacological / biological investigations revealed that the plant possesses significant antibacterial and anti-termite activities. Moderate phytotoxic, insecticidal and brine shrimp lethality activities were observed. The plant showed low antifungal, NO free radical scavenging and spasmolytic activities. No haemagglutination activity was shown by the crude methanolic extract and various fractions of the plant. The oils of the plant possessed moderate brine shrimp lethality and low phytotoxic activities but did not show antibacterial, antifungal and insecticidal activities.
PHYTOCHEMICAL, PHARMACOGNOSTIC AND PHARMACOLOGICAL INVESTIGATIONS OF VITEX AGNUS CASTUS AND MYRSINE AFRICANA
(Center of Biotechnology & Microbiology, University of Peshawar, 2011) Sadiq Azam; Suoervisor Prof. Dr. Muhammad Iqbal Chaudhary
The present Ph.D dissertation comprises of two parts, part A and part B. The part A describes extraction, isolation and structure elucidation of isolated compounds. Pharmacological and biological activities of crude extract, different fractions thereof and some compounds isolated in reasonable quantity from Vitex agnus castus have been investigated. As regarded phytochemical study three new compounds namely vitexcarpan (01) vitexcarpan 1 (02) and Vitexoide (03) were isolated from the aerial parts of the plant in amount of 10.0, 16.4 and 5.0 mg respectively. Other known compounds (04, 05 and 06) were also isolated from this plant along with. Crude methanol extract, different fractions thereof and some compounds isolated in fair quantity were investigated for various pharmacological / biological activities including, Antibacterial, Antifungal, Brine-shrimp lethality, Phytotoxic, Heamagglutination, Insecticidal, Anti-termites, Anti-inflammatory, Spasmolytic, Nitric oxide free radical scavenging assay and Enzyme inhibition assay.
SEQUENCING AND ANALYSIS OF A cDNA ENCODING A PUTATIVE COCLAURINE N-METHYLTRANSFERASE (CNMT) FROM Aristolochia fimbriata, A BASAL ANGIOSPERM by Roshan Ali
(Center of Biotechnology & Microbiology, University of Peshawar, 2010) Roshan Ali; supervisor Dr Ghosia lutfullah
Alkaloids are produced in plants through various pathways involving several enzymes that lead to diverse alkaloids. One of the most important alkaloid biosynthetic enzymes is coclaurine N-methyltransferase (CNMT) which is an S-adenosyl-L-methionine-dependent methyltransferase (SAM-MTase). SAM-MTases utilize S-adenosyl-L-methionine (SAM) as a cofactor to methylate other molecules. CNMT catalyzes the methylation of coclaurine. Crystal structures of more than hundred SAM-MTases have been investigated. Several O-methyltransferases have been characterized at the molecular as well as structural levels, but there have been very few molecular studies of N-methyltransferases especially about CNMTs. In this study, the amino acids sequence of Aristolochia fimbriata putative CNMT has been determined by isolating and translating the full-length cDNA. In order to investigate the mechanism of methylation by this putative CNMT, three-dimensional homology model has been built and the ligand (SAM) as well as the substrate (S-Coclaurine) has been docked into its active site. Phylogenetic analyses were performed using the MEGA 4.0 software. The phylogenetic relationship of A. fimbriata putative CNMT with their homologs has also been analyzed. In order to identify the putative CNMT gene and determine its function, online similarity searches were performed by BLAST program using the cDNA sequence as well as the putative protein that could be encoded by the gene. All the methods, applied, predicted that the gene identified might be involved in the production of CNMT. The predicted homology model consists of two domains: the N-terminal catalytic core domain and the C-terminal domain. The catalytic core domain has a central sheet of β-strands surrounded by α helices. The catalytic core domain contains binding site for VI SAM.

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