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Role of Type Vi Secretion System in Stress Adaptations and Developing Control Strategies for Campylobacter Jejuni

Thesis Info

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Author

Noreen, Zobia

Program

PhD

Institute

COMSATS University Islamabad

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Bio sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/11814/1/Zobia%20Noreen%20biosciences%202019%20comsats%20isb%20prr.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727126173

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The human Gram-negative enteropathogen Campylobacter jejuni is the leading cause of gastroenteritis, with 25% of the cases worldwide being attributed to this organism. C. jejuni, due to emergence of multidrug resistances, has also been classified as “high priority pathogens” thus making surveillance system as an essential key for the control of disease. However, in developing countries like Pakistan, the prevalence and source prediction of MDR C. jejuni isolates has been under reported due to lack of proper surveillance program. Therefore, the first section of the present study was conducted to determine the prevalence of C. jejuni isolates among pediatric diarrheal cases in Pakistan, their origin through source tracking as well as associated antibiotic resistance patterns. C. jejuni was detected in 54.6% of the total samples and more than 75% of the isolated strains were resistant to panel of 8 out of 13 antibiotics tested however; high level of susceptibility was observed against imipenem (12.2%) and tigecycline (9.7%). Moreover, six isolates (7.3%) were metallo-beta lactamase (MBL) producer and were positive for at least one of the five screened metallo-beta lactamase genes. Source tracking using source predictive PCR, showed that 57.3% of the isolates belonged to livestock associated cluster (C1 to C6) and 42.8% were assigned to nonlivestock/environmental clusters (C7-C9). Isolates belonging to livestock cluster had high MAR index as compared to non-livestock suggesting possible transmission of antimicrobial resistant C. jejuni strains to human population via food chain. Moreover, it also suggests that extensive use of antibiotics for disease control and growth promotion in livestock especially in poultry industry results in the emergence and spread of AMR C. jejuni and the genes associated with resistance. C. jejuni, although being a fastidious microorganism, is able to survive and maintain its pathogenesis in the harsh environments. However, the mechanisms of stress adaptations and pathogenesis is C. jejuni are not fully elucidated. Type VI secretion system among several gram-negative bacteria have been associated with virulence and stress xii adaptations but its role in stress tolerance and pathogenesis of C. jejuni is still unknown. Moreover, in contrast to the other T6SS positive bacteria, C. jejuni has only one hemolysin coregulated protein (Hcp) protein, the hallmark of a functional T6SS system, whose structure and secretory function was not known. Therefore, the second aim of this study was to determine the role of type VI secretion system in biofilm formation (a stress adaptive mechanism) and pathogenesis. Moreover, the structure and function of Hcp protein was also elucidated. Comparative analysis of effect of different sub-lethal stresses on biofilm forming potential of isolates having fully functional T6SS (Hcp+) and those lacking functional T6SS (Hcp-) showed that the biofilm formation was significantly more enhanced in T6SS positive (Hcp+) groups as compared to T6SS negative (Hcp-) group under heat (55ºC) and oxidative stresses (8mM of H2O2). Structural analysis of Hcp showed similarities between the hexameric ring structure of Hcp-Cj and that of Hcp3 from Pseudomonas aeruginosa. Through functional studies, two roles for Hcp-Cj were identified i.e., in cytotoxicity toward HepG2 cells and in biofilm formation in C. jejuni. Structure-based mutational analyses showed that an Arginine to Alanine mutation at position 30 within the extended loop of Hcp-Cj resulted in a significant decrease in cytotoxicity. However, biofilm formation function remained unaffected by this mutation. Collectively, this study supports the dual role of Hcp-Cj as a structural and effector protein in C. jejuni. High prevalence of C. jejuni associated diarrhea in both developed and developing countries can be attributed towards three major factors i) emergence of multidrug resistant C. jejuni isolates making WHO recommended antibiotics ineffective for treatment; ii) lack of control strategies at poultry level (as poultry meat consumption is the major source of infection in human); iii) persistence of C. jejuni as biofilm in poultry processing units and water reservoirs.. Therefore, third part of the present study was to develop control strategies against C. jejuni at three different levels i.e., 1) In-silico drug target identification was done using comparative proteomics and metabolomics approach by which ten essential non-homologous drug targets were identified which can be used in future to develop safe and effective drug against MDR C. jejuni ; 2) pH sensitive alginate coated chitosan nanoparticles were designed for targeted delivery of anti-campylobacter hexane fraction (PE-CANP) of Trachyspermum ammi (ajwain) into the chicken caecum without loss of its activity in gizzard to control C. jejuni at poultry farm level. The PE-CANP nanoparticles were able to reduce C. jejuni load to 6 logs (in CFU/g of caecal content) as compared to control group in chicken after 21 days of post infection and can therefore be used as a good alternative to conventional antibiotics for on-farm control of C. jejuni; 3) Three different metallic nanoparticles i.e., Silver/Graphene/TiO2, Erbium doped Li-Ni Ferrites and ZnO nanoparticle were tested for their antibacterial activity against C. jejuni which showed excellent antibacterial and anti-biofilm activity. All these nanoparticles can serve as an excellent coating material for control of C. jejuni biofilms in food processing setups and hence may control the transmission of C. jejuni to humans. Overall the present study helps in better understanding the disease transmission patterns and associated drug resistance traits as well as provides a better insight into the role of T6SS in C. jejuni in stress adaptation and thereafter developing control strategies for tackling C. jejuni both at farm and food processing level to reduce the burden of campylobacteriosis in humans.
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جہاں گرد کی واپسی

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مسافر قاہرہ کے ہوائی مستقر پر کھڑا اجازت لے رہا تھا ۔وقتِ رخصت ان کی آزردہ دلی مجھ سے دیکھی نہیں جا رہی تھی وطن واپسی کے لیے جہاز کے اڑان بھرنے سے چار گھنٹے پہلے مجھے ائیر پورٹ پہنچنا تھا ۔میں دو گھنٹے مزید پہلے پہنچا تھا ۔دکتورہ بسنت ،احمد صاحب ،، دکتورعلی،دکتورہ شائمہ اور باقی سارے دوستوں کے ساتھ مصر میں چھے دن اس قدر مصروف کہ وقت بیتنے کا احساس ہی نہیں ہوا اور اب رخصتی کے یہ چھے گھنٹے جیسے رک سے گئے ہیں ۔ سب کی محبت اور آنسوئوں سے لبریز آنکھیں میرا راستہ روک رہیں تھیں ۔دکتورہ بسنت کا ’’پھر کب آئو گے ‘‘والے سوال سے میرا پیچھا چھڑانا مشکل تھا۔ دکتورہ شائمہ کا مشورہ کہ ’’نیل کا پانی پی کر جائو جس نے بھی پیا واپس ضرور آیا ‘‘مصر سے میرے تعلق اور مراسم کے ایسے کچے دھاگے ہیں جن کی مضبوط گرفت سے خود کو آزاد کرانا مشکل ہو رہا تھا ۔کہا جاتا ہے کہ اہل مصر بے وفا ہوتے ہیں ،خود غرض ہوتے ہیں، حسب ضرورت و سہولت مراسم رکھتے ہیں مگر میں نے مصر اور اہل مصر کو وفادار انسان دوست اور ملنسار پایا ۔ اگر اہل مصر وفا شعار اور انسان دوست نہ ہوتے تو بازارِ مصر میں فروخت شدہ اور زندانِ زاویرا میں مقید رہنے والے حضرت یوسفؑ اپنے والد حضرت یعقوب ؑ اور اپنے اہل و عیال کو کنعان سے مصر مستقل سکونت کے لیے کیوں بلاتے ۔قاہرہ ائیر پورٹ کی انتظار گاہ کی کھڑکی سے روشن چاند واضح نظر آ رہا تھا یو ں لگ رہا تھا جیسے وادی سینا کے عقب سے چاند نے مصر کو منور کر نے کے لیے اپنے دودھیا اجالے کی پھوار پھینکی ہو ۔میں چشمِ تخیل سے ساحلوں ،ٹیلوں ،وادیوں...

الأصول الجامعة بين كبرى الفرق الإسلامية

The Muslim nation shows great diversity in many ways: Ethnical, lingual, tribal, racial, colour, cultural, etc. Nevertheless, such differences have never been supposed to impede their way to their unity. The Muslim nation has far more reasons and mutual foundations to be united. But unfortunately, the Muslim history witnesses squabbles and skirmishes between different sects of this otherwise one Ummah. These disputes led us to the recesses of ignorance and intolerance, which in turn left us lagging behind the world and advancement, and now, we are called the third world, which is not an honour any way. We need to work on the similarities between different sects of the Muslim nation and let them learn how to live together keeping their differences. So that they may get united. All the Muslims sects fundamentally believe in the same set of beliefs, practices, and rituals. The differences in these areas are the differences of diversity and extensions, not of contradiction. This diversity is the beauty of deliberation and discussion, hence a reason for knowledge to grow, it should not be a matter of contentions. We need to highlight the mutual common grounds of Islamic beliefs and religiosity among the sects to unite this nation. We need to teach how to tolerate and respect each other with difference of opinions. We should encourage constructive dialogues between the Muslims sects and let them understand point of views of each other and thus remove the misconception about; each other. We should curb the contentious and discordant voices and build up an atmosphere where a healthy, tolerant and heterogeneous society may emerge and pave the way to Muslim unity and thence to success in this life and the life to come after death. In this perspective, the author of his present study explores to find the teachings and guidance of Islam. This is the theme of the present study.

Spatio-Temporal Trends and Integrated Management of Sugarcane Whitefly, Aleurolobus Barodensis Mask. , Aleyrodidae: Homoptera

The experiments were conducted to study the spatio temporal trend and integrated management of whitefly, Aleurolobus barodensis (Mask.) (Aleyrodidae: Homoptera) during 2008 to 2009 in sugarcane growing areas of various districts of the Punjab. The results are abstracted under the following sub headings: Spatio-Temporal Trend of Whitefly Three farmer’s fields surrounding each of the Chinar Sugar Mills, Tandlianwala, Faisalabad; Ashraf Sugar Mills, Bahawalpur and Jamal Din Wali Sugar mills, Raheem Yar Khan were selected. The population of whitefly (nymphs and adults) were recorded at fortnight interval during the crop season 2008. The data were also correlated with the ambient weather factors. The results revealed that population of whitefly (nymphs + pupae) differed significantly among dates of observation, districts and in interaction between dates of observation and districts. The population was appeared during 2 nd fortnight of June in district Faisalabad and Bahawalpur while in district Rahim Yar Khan it was appeared during 1 st fortnight of July. The maximum peak population was recorded on 1 st fortnight of November each in district Faisalabad (7.96/leaf) and Rahim Yar Khan (14.94/leaf) while on 2nd fortnight of October in district Bahawalpur (15.81/leaf). On an average basis, the district Rahim Yar Khan showed maximum population of whitefly (4.61/leaf) while district Faisalabad possessed minimum population (2.48/leaf). All the weather factors viz. Maximum temperature, minimum temperature, average temperature, relative humidity and rainfall showed non-significant correlation with the population of whitefly in all the districts. On an average basis relative humidity had positive and significant (P ≤ 0.05) correlation with the whitefly population. Relative humidity was found to be the most important factor that contributed maximum in population fluctuation of the pest in all the districts as well as on cumulative basis by exerting 42.3%, 43.8%, 35.9% and 18.3% role in population fluctuation of the pest, respectively. Host Plant Resistance Twenty (20) genotypes of sugarcane viz. BF-16, BF-129, BF-162, CMP-13, CO- 132, COJ-84, COJ-1148, CP-43/33, CP-72/2086, CP-77/400, CPF-237, I-62-96, NIA-98, CPF-243, HSF-240, HSF-242, SPF-213, SPF-234, SPF-245 and SPSG-26 sown in the farmer’s Jhang following RCBD with three replications were tested for their resistancesusceptibility against whitefly during 2008 as a preliminary screening experiment. From these studies, three varieties showing comparatively susceptible trend (HSF-242, COJ-84 and SPSG-26), three intermediate (CO-132, NIA-98 and CP-72/2086) and three showing resistant response (CPF-237, CPF-243 and CPM-13) were selected for final screening trial during 2009. The selected varieties of sugarcane were sown under the same ecological conditions following RCBD with three replications. The data on whitefly population were recorded at fortnightly interval at the appearance of the pest. The results revealed that in the preliminary screening trial during 2008, significant variations were found to exist among dates of observation, varieties and interactions between dates of observation and varieties. The variety HSF-242 was found to be comparatively susceptible showing maximum population of whitefly (nymphs + pupae) per leaf (15.48) while CPF-243 appeared to be comparatively resistant with minimum population of whitefly (5.63/leaf). In the final screening trial during 2009, the trend was found to be almost similar as that of observed in the preliminary screening experiment. The variety CPF-243 was found to be relatively resistant showing minimum population of whitefly (4.01/leaf) while HSF-242 appeared as susceptible with maximum population of the pest (11.09/leaf). The descending order of other genotypes based on population density count per leaf are COJ-84 (9.68), SPSG-26 (8.76), CO-132 (7.29), NIA-98 (6.70), CP-72/2086 (6.12), CMP-13 (5.43) and CPF-237 (4.82). The 1 st fortnight of the month of October during 2008 showed maximum abundance of the pest i.e. 22.85/leaf and there was only one peak in the season. Similarly during 2009 the 1 st week of October showed 16.75/leaf population of whitefly followed by 2 nd week of October i.e. 14.40/leaf. Thus it was concluded that the 1 st fortnight of October is the most suitable period for the development of the pest. Relative humidity during 2009 showed positive and significant correlation (P < 0.01) with r-value of 0.754 while maximum temperature on cumulative basis exerted negative and significant correlation with r-values of 0.419 at P < 0.05. The impact of relative humidity during 2008 and during 2009 was maximum on the fluctuation of the pest and showed 10.5 and 47.3% contribution. On cumulative basis minimum temperature was found to be an important factor and contributed maximum role i.e. 16.01% in population fluctuation of the pest.Role of Morpho-physical and chemical Plant Characters towards Resistance against Whitefly on Sugarcane Various morpho-physical (plant height, cane girth, leaf area, hair density, thickness of leaf seed and moisture in leaves) and chemical plant characters ( total minerals. nitrogen, phosphorus, potassium, calcium, magnesium, carbohydrates, fat, ferrous, manganese, copper and zinc) were studied in various selected genotypes of sugarcane and correlated with the population density of whitefly. POL, brix, CCS and fiber contents in the samples of sugar from each selected genotype were also determined. The selected genotypes differed significantly in all plant characters except moisture percentage, total minerals and magnesium in the leaves. Cane girth, leaf area and hair density on leaf sheath resulted negative and significant correlation with the pest population whereas, plant height and thickness of leaf sheath showed positive and significant correlation. Cane girth had negative and significant impact on the pest population fluctuation and contributed maximum role i.e. 35.2 percent. nd plant height were the 2 Leaf area and most important factors and showed 19.2 and 18.9% role in population fluctuation of the pest. All the regression equation was found to be fitted the best. Nitrogen, potassium, magnesium and zinc contents had positive and significant correlation with the pest population while phosphorus showed negative and significant correlation. Nitrogen percentage in the leaves of sugarcane plants showed positive and significant impact with maximum role i.e. 96.3% in population fluctuation of the pest. CCS in sugar samples had positive and significant correlation while fiber percentage showed negative and significant correlation with the pest population. CCS showed positive and significant role with 49.2% impact while fiber contents in the samples of sugar had negative and significant role with 25.8% impact on the population fluctuation of whitefly. Integrated Pest Management Studies Various control methods such as cultural (de-trashing during July and September) biological (release of 2 nd instars larva per plant, chemical (application of imidachloprid 200 SL @ 568 ml/ha and botanical (application of neem seed kernel extract 5% @ 6.17 litre/ha twice in the season on August 26 and September 27, 2009) were applied singly and in their possible combinations for the control of whitefly on a resistant and asusceptible genotypes of sugarcane. There were sixteen treatments in total including control. The experiment was sown in two sets (one for resistant genotype and the other for susceptible) at three localities viz. in the farmer’s field near Shakarganj Sugar Mills, Jhang, Kamalia Sugar Mills Toba Tek Singh and Chinar Sugar Mills Tandlianwala, Faisalabad, following randomized complete block design with three replications. The data on the population of whitefly (pupae + nymphs) were recorded at fortnightly interval from each locality. The results revealed that the population of whitefly in the farmer’s field near Shakarganj Sugar Mills, Jhang and Kamalia Sugar Mills did not reach to a desired level, therefore, no plant protection measure were taken. The results obtained from the farmer’s field near Chinar Sugar Mills, Tandlianwala, Faisalabad revealed significant variation among dates of observation, treatments and between genotypes. The maximum control of whitefly was recorded to be 1.57 per leaf and 3.49 per leaf with maximum population reduction of 72.45 and 65.68% in susceptible (HSF-242) and resistant (CPF-243) genotypes of sugarcane, respectively in those plots where all the control methods were applied in combinations. The release of 2 nd instars larva per leaf of Chrysoperla carnea showed minimum control of whitefly with 4.40 and 8.22 individuals per leaf with minimum reduction in population of whitefly i.e. 22.80 and 19.17% in resistant and susceptible genotypes of sugarcane, respectively. The population of whitefly was higher in susceptible genotype of sugarcane as compared to resistant genotype in all the treatments including control. Cane yield also showed significant difference between genotypes, among treatments and between the interaction of genotypes and treatments. The application of all the control methods in combination resulted in maximum cane yield i.e. 2857.79 and 3061.15 maund/ha in susceptible and resistant genotypes of sugarcane with maximum increase in yield over control i.e. 24.27 and 20.17%, respectively. The release of C. carnea showed discouraging results with minimum cane yield i.e. 2310.27 and 2674.19 maund/ha with minimum increase over control i.e. 0.46 and 4.97% in susceptible and resistant genotypes, respectively. The application of imidachloprid 200 SL @ 568 ml/ha twice in the season when applied alone showed maximum cost benefit ratio i.e. 1: 50.80 and 1: 59.63 in susceptible and resistant genotypes of sugarcane, respectively. De-trashing of older leaves twice in the season showing CBR of 1: 37.09 and 1: 40.98 in susceptible and resistant genotypes ofsugarcane, respectively. The minimum CBRs were recorded to be 1: 8.26 and 1: 8.85 in those treatments where all the control methods were combined together in susceptible and resistant genotypes of sugarcane, respectively" xml:lang="en_US