1.
Proteomic And Genomic Analysis Of Methicillin-Resistant Staphylococcus Aureus And Efficacy Of Indigenous Medicinal Plants Essential Oils
by Sarwat Ali Raja | Prof. Dr. Muhammad Ashraf | Dr. Tayyaba Ijaz | Dr. Aqeel Javeed | Prof. Dr. Aftab Ahmed Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: A Cohort study (prospective and observational) was performed to study the prevalence of Methicillin resistant Staphylococcus aureus from the healthy individuals of community, hospitalized patients and associated health-care workers and indigenous plants essential oils were screened as new, improved & potent antibacterial/s against resistant strains of MRSA.
The method involved isolation and identification of MRSA from surgical wounds of hospitalized patients & associated health care workers in a tertiary care hospital in Lahore and healthy volunteers from the community. Plant essentials oils & extracts were evaluated for their antibacterial activity against selected MRSA isolates. Oils were recovered by steam distillation using an all-glass distillation assembly. Then in vitro sensitivity and MICs of plant essential oils were determined using vancomycin and linezolid as commercial standards. The essential oils were screened further for the active constituents by column chromatography using various solvents and identification of compounds were performed by GC/MS analysis and the fractions which showed prompt results were evaluated for antimicrobial activity against the MRSA isolates in quest to find new therapeutic options. Finally effective essential oils and their active fractions were studied for their toxicity using in vitro Genotoxic assays such as Ames and Comet assays. To further ensure their beneficial effects antimutagenic effect of the essential oils were also studied.
Prevalence of S. aureus among patients was 52.9%, in HCWs 86.5% and in community 74% with an overall percentage of 72.6%. Among S. aureus those declared as MRSA were 91.8% from patients, 50.6% from HCWs and 59.5% from community with an overall percentage of 62.2% MRSA. Among the isolated MRSA overall 90.6% were Coagulase positive and 75.2% were biofilm positive.
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The pattern of MRSA resistance against current antibiotics have shown an overall increase in the resistance with maximum shown for lincomycin followed by tetracycline, ampicillin, fusidic acid, amoxicillin and piperacillin with tazobactam. The most effective options among current regime were tigecyclin, amikacin and meropenem showing an overall least resistance. Resistance against linezolid was observed with an overall percentage of 25.6 % and vancomycin 33.3% by disc diffusion method.
The MRSA isolates resistant to one or more groups of antibiotics were declared as MDRs. Among patients and health-care workers all were declared as MDRs where as in community 93.1% isolates were MDRs.
Upon Protein profiling using whole cell proteins 44 bands of the polypeptides were produced with molecular size 10-200kDa from the three sampling groups and were categorized into 5 clusters showing an overall significance correlation with each other explaining an interesting fact that all these strains were interlinked establishing the fact of flow of hospital acquired MRSA in the community and vice versa. This analysis also gave an insight in explaining the fact of horizontal transmission of infection within the hospital.
Keeping in view the raise in resistance among current available antibiotics indigenous medicinal plants essential oils were screened for active constituents exhibiting anti-bacterial effects against MRSA isolates.
Maximum yield was obtained from Carum copticum followed by Cuminum cyminum and minimum yield was obtained in case of Zingiber officinale. Upon qualitative analysis of all five essential oils Carum copticum essential oil showed zones of inhibition greater than the standards vancomycin and linezolid followed Cuminum cyminum and Zingiber officinale in all three
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sampling groups. Anethum sowa and Myristica fragrans essential oils showed no activity against MRSA.
Minimum inhibitory concentration of the three essential oils determined by micro broth dilution method indicated that Carum copticum showed least value in all three types of MRSA isolates followed by Zingiber officinale and Cuminum cyminum.
Effective essential oils were further fractioned using silica gel gravity columns. All the fractions obtained were screened for the anti-bacterial activity against all three types of MRSA isolates. Only fraction F1 of Carum copticum showed activity greater than pure essential oil and the two commercial standards of vancomycin and linezolid.
For the identification of active constituents GC/MS analysis was performed on all three essential oils and their respective fractions. In case of fraction F1 the most dominant constituents were Carvacrol, p-Cymene, Ʈ-Terpinene and Apiol. In other two plants none of the fractions were effective. Therefore it was concluded to use pure essential oils in case of Zingiber officinale and Cuminum cyminum rather than their individual fractions and incase of Carum copticum Fraction F1 has shown superior activity.
Finally these essential oils were tested for possible mutagenic effect using bacterial reversion mutation assay and Comet assay. No mutagenic effects were observed at MIC and above doses. These effective essential oils were also evaluated for possible antimutagenic effect. Both Carum copticum and Zingiber officinale essential oils showed strong antimutagenic effects and weak antimutagenic effect by Cuminum cyminum. Upon analysis of nuclear damage none of the plants essential oils and fraction F1 of Carum copticum showed genotoxic effects and indicated to be safe. Thus from the study it was concluded that Carum copticum essential oil and its fraction F1 were the most effective to be further investigated as an alternative treatment for MRSA infections. Availability: Items available for loan: UVAS Library [Call number: 2410-T] (1).
2.
Chemical, Microbiological And Toxicological Evaluation Of Textile Dyeing Industry Wastewater
by Muhammad Furqan Akhtar (2011-VA-265) | Prof. Dr. Muhammad Ashraf | Dr. Aqeel Javeed | Prof. Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Exposure to complex mixtures like textile effluent poses risks to animal and human health such as mutations, genotoxicity, pathological lesions and oxidative damage. The aim of the present study was to quantify metals and identify organic pollutants in untreated textile dyeing industry wastewater, to determine the bacterial load of wastewater, isolate and identify heavy metals tolerant bacteria and to determine its mutagenic, genotoxic and cytotoxic potential, influence on normal physiology and effects on oxidative stress biomarkers in effluent exposed rats.
Metal analysis through AAS revealed presence of high amounts of zinc, copper, chromium, iron, arsenic and mercury in industrial effluent. Various organic pollutants such as chlorpyrifos, cucurbitacin-b and phthalates were identified by screening through GC-MS.
Microbiological evaluation of textile dyeing industry wastewater revealed a high bacterial load. Different bacteria isolated from wastewater such as Staphylococcus aureus, Pseudomonas aeruginosa, Corynebacterium xerosis, Bacillus megaterium, Staphyoloccus epidermidis and Micrococcus varians exhibited resistance to Cr and Cu salts and antibiotics to varying degree.
Ames test with/without enzyme activation and MTT assay showed strong association of industrial effluent with mutagenicity and cytotoxicity respectively. Bacterial reverse mutation assay revealed that the mutagenicity of textile dyeing industry wastewater decreased with increase in dilution of wastewater.
In-vitro comet assay revealed the evidence of high oxidative DNA damage induced by textile wastewater. Wastewater exhibited concentration dependent genotoxicity in sheep
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peripheral lymphocytes. When Wistar rats were exposed to industrial effluent in different dilutions for 60 days, then activities of total superoxide dismutase and catalase and hydrogen peroxide concentration were found to be significantly lower in kidney, liver and blood/ plasma of effluent exposed rats than control. Vitamin C at a dose of 50mg/Kg/day significantly reduced oxidative effects of effluent in rats. Industrial effluents may decrease activities of T-SOD and CAT and concentration of H2O2 in liver, kidney and blood/plasma of Wistar rats. Vitamin C may have a possible ameliorating effect on industrial effluent induced oxidative stress in Wistar rats.
Wastewater exposed rats exhibited necrosis of epithelial cells of nephron, pulmonary emphysema, and inflammation of the lungs, degradation and infiltration of cardiac myocytes, fibrosis of the liver, damage to the intestinal mucosa and sloughing off epithelial cells from the intestinal lumen.
This study concludes that untreated textile dyeing wastewater being a complex mixture of inorganic and organic pollutants may be highly eco-toxic and may contaminate of the environment via continuous release of various organic and inorganic pollutants. Availability: Items available for loan: UVAS Library [Call number: 2580-T] (1).
3.
Chemical Microbiological And Toxicological Evaluation Of Pharmaceutical Effluent Wastewater
by Ali Sharif (2011-VA-266) | Prof. Dr. Muhammad Ashraf | Dr. Aqeel Javeed | Prof. Dr. Aftab Ahmad Anjum .
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Pharmaceutical effluent being a complex mixture of drugs and heavy metals may affect human health exhibiting a strong potential of mutagenicity, carcinogenicity, cytotoxicity and oxidative stress induction along with pathological changes in various organs of the body. The current study was focused to quantify the presence of heavy metals, detection of various drugs, determining the bacterial load along with isolation and identification of different bacteria and assessment of the mutagenic and genotoxic, cytotoxic and oxidative stress induction of pharmaceutical effluent wastewater when exposed to sheep lymphocytes, Salmonella typhimurium strains, cell lines and rats respectively.
Atomic absorption spectrophotometer was used to quantify heavy metals and showed the presence of arsenic, chromium, lead and iron in concentrations above the normal limits recommended by WHO and EPA. Gas Chromatograph mass spectrophotometer analysis shown the presence of digitoxin, lignocaine, caffeine and trimethoprim and various other organic pollutants.
Microbiological evaluation showed a high bacterial load in the pharmaceutical waste water. Several bacteria were also found in PEW in the presence of different drugs and heavy metals. Aeromonas sobria, Micrococcus varians, Staphyoloccus epidermidis, Staphylococcus aureus, Bacillus megaterium showed tolerance to potassium di chromate and copper sulphate and resistance to various antibiotic discs.
Ames assay revealed a strong mutagenic potential with and without the presence of metabolic activation mixtures. A concentration dependent effect was observed when samples were tested with increasing dilution factor.
MTT assay and comet assay also showed a concentration dependent effect. The BHK-21 cell line was used to evaluate cytotoxicity and cell viability decreased with increasing concentration of PEW. Sheep lymphocytes used in comet assay exhibited a concentration dependent DNA damage.
Different antioxidant enzymes were also evaluated. Rats were exposed to PEW at different concentrations and following 60 days oral exposure, rats were evaluated for the presence of total superoxide dismutase, catalase and hydrogen peroxide in kidney, liver and plasma. Exposure to Pharmaceutical waste water significantly decreased the (TSOD), (CAT) and (H2O2) levels in plasma, liver and kidney. Treatment with Vitamin E significantly ameliorated the levels of enzymes.
Exposed rats were also evaluated for any pathological changes. Coagulative necrosis of renal epithelial cells were observed along with severe degeneration and cellular swelling in hepatocytes of hepatic cord.
Availability: Items available for loan: UVAS Library [Call number: 2600-T] (1).
4.
Effect Of Colchicine On Cellular And Humoral Immune Responses In Mice
by Shahzada Khurram Syed (2007-VA-444) | Dr. Aqeel Javeed | Prof. Dr. Muhammad Ashraf | Dr. Jawad Nazir | Dr. Shahbaz Yousaf.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Colchicine is a medication that treats gout. It is a natural product and secondary metabolite, originally extracted from plants Colchicum autumnale .It causes modulation of chemokine and prostanoid production and inhibition of neutrophil and endothelial cell adhesion molecules by which it interferes with the initiation and amplification of the joint inflammation.
The present study is designed to evaluate the effects of colchicine on cellular and humoral immunity in mice. There were five groups for each assay i.e. group I (negative control), positive control and three colchicine treated group II (40μg/kg), group III (80μg/kg) and group IV (160μg/kg). The number of mice in each group was five to eight. All these groups were administered doses intraperitoneally. To determine the effect of colchicine on cell mediated immunity , delayed type hypersensitivity (DTH) assay, macrophage engulfment assay, cyclophosphamide induced neutropenic test and nitric oxide production was performed .DTH was performed by measuring skin thickness. DTH showed significant difference (P<0.001) of negative control to colchicine treated groups 40μg/kg, 80μg/kg and 160μg/kg. With increasing dose, there was decrease in skin thickness of the mice. Highest reduction of skin was found at 160μg/kg. Macrophage engulfment assay was performed to evaluate the effect of macrophage induced phagocytosis. There was significant ( P <0.001) difference of engulfment of SRBCs by macrophages with negative control to colchicine treated group II (40μg/kg), group III(80μg/kg) and group IV(160μg/kg) groups. There was significant difference of engulfment of macrophages at 45 and 90 minutes.
Cyclophosphamide induced neutropenic test was performed to assess the effect of colchicine on total leukocyte count (TLC) and differential leukocyte count (DLC). There was
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reduction of TLC to about 45.3% in control to 48.3%, 54.68% and 65.42% in group II (40μg/kg), group III (80 μg/ kg) and group IV (160μg/kg) respectively when these were compared with primary values of TLC. There was significant difference of reduction in the neutrophil count of negative control 1057 (±120) to 902 (±67) in group II (40μg/kg), 734(±69) in group III (80 μg/ kg) and 609 (±71) in group IV (160μg/kg) of doses of colchicine. This test showed that with the increasing dose of colchicine, there was significant (P<0.001) difference of TLC count and neutrophil count.
Nitric oxide (NO) production by macrophages was performed for measuring different concentrations of nitric oxide produced. There was significant difference (P<0.001) in NO production by macrophages alone and LPS stimulated between negative control to group II (40 μg /kg), group III (80μg/kg), group IV (160μg/kg) of colchicine. With increasing dose, there was significant reduction in production of NO. There was significant P<0.0001 reduction in body weight andspleen weight difference of mice in different groups of colchicine treated 40μg/kg, 80μg/kg and 160μg/kg from negative control after treatment. There was difference of weight of Thymus of group II (40 μg/kg), group III (80μg/kg) and group IV (160μg/kg) but difference was statistically not significant. There were no histopathological changes observed in spleen and Thymus at 40μg/kg and 80μg/kg doses of colchicine. At 160μg/kg dose, increase in thickness of trabecular was seen .due to edema in the spleen. For evaluation of colchicine effect on humoral immunity, haemagglutination assay, mice lethality test and Jerne hemolytic plaque formation were performed. Haemagglutination assay (HA) was performed by using red blood cells injected intraperitoneally in mice to measure antibody titer. There was significant difference of (P >0.001) to colchicine treated group II (40μg/kg), group III (80μg/kg) and group IV (160μg/kg)with group I (negative control).With the increasing dose, there was reduction in the
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HA titer. Mice lethality test was performed by testing immune response of the mice to the challenge infection of P.multocida. It was performed by comparing mortality ratio of mice after administration of drug. There was no death of mice in the negative control group in which there was administration of PBS and vaccine. At 40μg/kg dose of colchicine, there was 50% mortality ratio. At 80μg/kg dose of colchicine 75% mortality ratio was observed. Maximum mortality ratio was observed at the 160μg/kg colchicine dose i.e. 100%.
Jerne plaque formation test was performed and plaques formed was enumerated and recorded as the number of plaque forming cells (PFCs) per million cells. There was significant difference (P<0.001) of reduction in number of plaques from negative control to all doses of colchicine 40 μg/kg, 80 μg/kg and 160μg/kg. Antibody formation was decreased with increasing the dose of colchicine. Therefore, it is concluded that colchicine suppresses the cellular and humoral responses in mice. Availability: Items available for loan: UVAS Library [Call number: 2650-T] (1).
